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\begin{document}

\title{Major ginsenosides from Panax ginseng promote aerobic cellular
respiration and SIRT1-mediated mitochondrial biosynthesis in
cardiomyocytes and neurons}



\author[1]{Qingxia Huang}%
\author[1]{Tingting Lou}%
\author[2]{Jing Lu}%
\author[2]{Manying Wang}%
\author[2]{Xuenan Chen}%
\author[2]{Linyuan Xue}%
\author[2]{Xiaolei Tang}%
\author[1]{Wenxiu Qi}%
\author[2]{Zepeng Zhang}%
\author[1]{Hang Su}%
\author[2]{Wenqi Jin}%
\author[2]{Chenxu Jing}%
\author[1]{Daqing Zhao}%
\author[2]{Liwei Sun}%
\author[1]{Xiangyan Li}%
\affil[1]{Changchun University of Chinese Medicine}%
\affil[2]{First Affiliated Hospital to Changchun University of Chinese Medicine}%


\vspace{-1em}



  
  \date{May 10, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Aerobic cellular respiration provides chemical energy, adenosine
triphosphate (ATP), to maintain multiple cellular functions. Sirtuin 1
(SIRT1) can deacetylate peroxisome proliferator-activated receptor gamma
coactivator 1 alpha (PGC-1\selectlanguage{greek}α\selectlanguage{english}) to promote mitochondrial biosynthesis.
Targeting energy metabolism is a potential strategy for the prevention
and treatment of various diseases, such as cardiac and neurological
disorders. Ginsenosides, one of the major bioactive constituents of
Panax ginseng, have been extensively used due to their diverse
beneficial effects on healthy subjects and patients with different
diseases. However, the underlying molecular mechanisms of total
ginsenosides (GS) on energy metabolism remain unclear. In this study,
oxygen consumption rate, ATP production, mitochondrial biosynthesis,
glucose metabolism, and SIRT1-PGC-1\selectlanguage{greek}α \selectlanguage{english}pathways in untreated and
GS-treated different cells, fly, and mouse models were investigated. GS
pretreatment enhanced mitochondrial respiration capacity and ATP
production in aerobic respiration-dominated cardiomyocytes and neurons,
and promoted tricarboxylic acid metabolism in cardiomyocytes. Moreover,
GS clearly enhanced NAD+-dependent SIRT1 activation to increase
mitochondrial biosynthesis in cardiomyocytes and neurons, which was
completely abrogated by nicotinamide. In addition, GS had protective
effects against hypoxia- or oxygen-glucose deprivation-induced
cardiomyocyte damage through activation of the SIRT1-PGC-1\selectlanguage{greek}α \selectlanguage{english}pathway.
Importantly, ginsenoside monomers, such as Rg1, Re, Rf, Rb1, Rc, Rh1,
Rb2, and Rb3, were found to activate SIRT1 and promote energy
metabolism. This study may provide new insights into the extensive
application of ginseng for cardiac and neurological protection in
healthy subjects and patients with ischemic disorders.%
\end{abstract}%



\sloppy


\textbf{Major ginsenosides from\emph{Panax ginseng} promote aerobic
cellular respiration and SIRT1-mediated mitochondrial biosynthesis in
cardiomyocytes and neurons}

\textbf{Short title: Major ginsenosides promotes mitochondrial function}

Qingxia Huang\textsuperscript{1,2}, Tingting Lou\textsuperscript{2},
Jing Lu\textsuperscript{2}, Manying Wang\textsuperscript{2}, Xuenan
Chen\textsuperscript{2}, Linyuan Xue\textsuperscript{2}, Xiaolei
Tang\textsuperscript{2}, Wenxiu Qi\textsuperscript{1}, Zepeng
Zhang\textsuperscript{2}, Hang Su\textsuperscript{3}, Wenqi
Jin\textsuperscript{2}, Chenxu Jing\textsuperscript{2}, Daqing
Zhao\textsuperscript{1*}, Liwei Sun\textsuperscript{2*}, Xiangyan
Li\textsuperscript{1*}

\textsuperscript{1}Jilin Ginseng Academy, Key Laboratory of Active
Substances and Biological Mechanisms of Ginseng Efficacy, Ministry of
Education, Jilin Provincial Key Laboratory of Bio-Macromolecules of
Chinese Medicine, Changchun University of Chinese Medicine, Changchun,
Jilin, China

\textsuperscript{2}Research Center of Traditional Chinese Medicine, the
First Affiliated Hospital to Changchun University of Chinese Medicine,
Changchun, Jilin, China

\textsuperscript{3}Practice Innovations Center, Changchun University of
Chinese Medicine, Changchun, Jilin, China

*Corresponding authors:

Daqing Zhao, Xiangyan Li: Jilin Ginseng Academy, Key Laboratory of
Active Substances and Biological Mechanisms of Ginseng Efficacy,
Ministry of Education, Jilin Provincial Key Laboratory of
BioMacromolecules of Chinese Medicine, Changchun University of Chinese
Medicine, Changchun, Jilin, China.

Liwei Sun: Research Center of Traditional Chinese Medicine, First
Affiliated Hospital to Changchun University of Chinese Medicine,
Changchun, Jilin, China. Tel.: +86-431-86177630, Fax: +86-431-86177630.
Email: zhaodaqing1963@163.com; sunnylilwei@163.com;
xiangyan\_li1981@163.com.

\textbf{Acknowledgments:}

We thank you for the kind supports on mouse model from Shuang Liu and
Guan Wang, College of Life Science, Jilin University. We appreciate the
comments for data organization from Tong Li, School of Medicine, Johns
Hopkins University.

\textbf{Conflict of interest statement:}

All authors declare that they have no conflict of interest.

\textbf{Abstract}

\emph{Background and Purpose} : Aerobic cellular respiration provides
chemical energy, adenosine triphosphate (ATP), to maintain multiple
cellular functions. Sirtuin 1 (SIRT1) can deacetylate peroxisome
proliferator-activated receptor gamma coactivator 1 alpha (PGC-1\selectlanguage{greek}α\selectlanguage{english}) to
promote mitochondrial biosynthesis. Targeting energy metabolism is a
potential strategy for the prevention and treatment of various diseases,
such as cardiac and neurological disorders. Ginsenosides, one of the
major bioactive constituents of \emph{Panax} ginseng, have been
extensively used due to their diverse beneficial effects on healthy
subjects and patients with different diseases. However, the underlying
molecular mechanisms of total ginsenosides (GS) on energy metabolism
remain unclear. \emph{Experimental Approach} : In this study, oxygen
consumption rate, ATP production, mitochondrial biosynthesis, glucose
metabolism, and SIRT1-PGC-1\selectlanguage{greek}α \selectlanguage{english}pathways in untreated and GS-treated
different cells, fly, and mouse models were investigated. \emph{Key
Results} : GS pretreatment enhanced mitochondrial respiration capacity
and ATP production in aerobic respiration-dominated cardiomyocytes and
neurons, and promoted tricarboxylic acid metabolism in cardiomyocytes.
Moreover, GS clearly enhanced NAD\textsuperscript{+}-dependent SIRT1
activation to increase mitochondrial biosynthesis in cardiomyocytes and
neurons, which was completely abrogated by nicotinamide. In addition, GS
had protective effects against hypoxia- or oxygen-glucose
deprivation-induced cardiomyocyte damage through activation of the
SIRT1-PGC-1\selectlanguage{greek}α \selectlanguage{english}pathway. Importantly, ginsenoside monomers, such as Rg1,
Re, Rf, Rb1, Rc, Rh1, Rb2, and Rb3, were found to activate SIRT1 and
promote energy metabolism. \emph{Conclusion and Implications} : GS
promotes aerobic cellular respiration and SIRT1-mediated mitochondrial
biosynthesis, which may provide new insights into the extensive
application of ginseng for cardiac and neurological protection in
healthy subjects and patients with ischemic disorders.

\textbf{Key Words} : ginsenosides, aerobic cellular respiration,
mitochondrial biosynthesis, SIRT1, PGC1\selectlanguage{greek}α, \selectlanguage{english}cardiomyocytes, neurons

\textbf{Abbreviations:}

ATP adenosine triphosphate

HK-I/II hexokinase I/II

PFK-1 phosphofructokinase-1

PK pyruvate kinase

MPC mitochondrial pyruvate carrier

PDH pyruvate dehydrogenase

TCA tricarboxylic acid

NADH nicotinamide adenine dinucleotide

FADH2 flavin adenine dinucleotide

ETC electron transport chain

OGD oxygen-glucose deprivation

NAD\textsuperscript{+} Oxidized nicotinamide adenine dinucleotide

SIRT1 sirtuin 1

PGC-1\selectlanguage{greek}α \selectlanguage{english}peroxisome proliferator initiated receptor gamma and coactivator
1 alpha

Nrf-1 nuclear respiratory factor-1

I/R ischemia/reperfusion

GS total ginsenosides

OCR oxygen consumption rate

MRC mitochondrial respiration capacity

CS citrate synthase

IDH1/2 isocitrate dehydrogenase 1/2

DLST dihydrolipoamide S-succinyltransferase

SDHA succinate dehydrogenase complex, subunit A

ACO2 aconitase 2

GAPDH glyceraldehyde-3-phosphate dehydrogenase

PDH pyruvate dehydrogenase

PFKP platelet-type phosphofructokinase

HPLC high-performance liquid chromatography

NAM Nicotinamide

LC-MS liquid chromatography-mass spectrometry

BMSCs bone marrow mesenchymal stem cells

HUVECs human umbilical vein endothelial cells

qPCR Quantitative PCR

\textbf{Bullet points:}

\textbf{What is already known:} Ginsenosides, one of the major bioactive
constituents of \emph{Panax} ginseng, have been extensively used due to
their diverse beneficial effects on healthy subjects and patients with
different diseases.

\textbf{What this study adds:} Total ginsenosides enhances mitochondrial
respiration capacity, ATP production and promotes
NAD\textsuperscript{+}-dependent SIRT1 activation to increase
mitochondrial biosynthesis in cardiomyocytes and neurons. Rg1, Re, Rf,
Rb1, Rc, Rh1, Rb2, and Rb3 are major ginsenoside monomers for SIRT1
activation and enhanced energy metabolism.

\textbf{Clinical significance:} This study may provide new insights into
the extensive application of ginseng for cardiac and neurological
protection in healthy subjects and patients with ischemic disorders.

\textbf{Introduction}

Aerobic cellular respiration refers to the process by which organisms
convert nutrients into chemical energy, adenosine triphosphate (ATP),
using oxygen, which is dominated in highly aerobic tissues, such as
cardiac and neural systems (Hall, Klein-Flugge, Howarth \& Attwell,
2012; Hickey et al., 2009; Neubauer, 2007). During glucose aerobic
metabolism, glucose with 6-carbon is broken down into 3-carbon pyruvate
by multiple key enzymes such as hexokinase-I/II (HK-I/II),
phosphofructokinase-1 (PFK-1), and pyruvate kinase (PK) in the
cytoplasm. Then pyruvate is transported into the mitochondria through
the mitochondrial pyruvate carrier (MPC) (Bricker et al., 2012) and
metabolized to synthesize acetyl-coenzyme A (CoA) by pyruvate
dehydrogenase (PDH) (Sugden \& Holness, 2003). After the metabolism of
tricarboxylic acid (TCA) cycle, nicotinamide adenine dinucleotide (NADH)
and flavin adenine dinucleotide (FADH2) from acetyl-CoA can pass their
electrons into the mitochondrial electron transport chain (ETC) to
produce ATP molecules, with the help of oxygen (Fernie, Carrari \&
Sweetlove, 2004). During this process, respiratory chain protein
complexes I-V embedded in the inner mitochondrial membrane is involved
in ATP production (Wallace, Fan \& Procaccio, 2010). Abnormal energy
metabolism is a critical target during the progression of disease,
including myocardial ischemia, heart failure (Fillmore \& Lopaschuk,
2013), stoke (Narne, Pandey \& Phanithi, 2017), or neurodegenerative
diseases (Butterfield \& Halliwell, 2019). During the exposure to
hypoxia, cells display decreased carbon flux into the TCA cycle and
diminished electron flux through ETC (Papandreou, Cairns, Fontana, Lim
\& Denko, 2006; Wheaton \& Chandel, 2011). Moreover, oxygen-glucose
deprivation (OGD) can cause mitochondrial damage and ATP depletion in
cardiomyocytes and neurons (Almeida, Delgado-Esteban, Bolanos \& Medina,
2002; Kalogeris, Baines, Krenz \& Korthuis, 2012). Therefore, the
enhancements of glucose oxidation and mitochondrial bioenergy have been
potential strategies against various disorders, such as ischemic
conditions.

Oxidized nicotinamide adenine dinucleotide (NAD\textsuperscript{+}) is a
well-known small molecule that drives energy metabolism through its
electron transfer function (Hopp, Gruter \& Hottiger, 2019). Increased
NAD\textsuperscript{+} level promotes the activity of sirtuin 1 (SIRT1),
a strong deacetylase of class III histone deacetylase, which heavily
implicates in a number of physical and pathological processes, such as
aging-related diseases (Imai \& Guarente, 2014). SIRT1 deacetylates its
substrate, peroxisome proliferator initiated receptor gamma and
coactivator 1 alpha (PGC-1\selectlanguage{greek}α\selectlanguage{english}), to regulate mitochondrial biosynthesis and
energy metabolism (Aquilano, Baldelli, Pagliei \& Ciriolo, 2013). PGC-1\selectlanguage{greek}α
\selectlanguage{english}can bind and co-activate different transcription factors, such as
nuclear respiratory factor-1 (Nrf-1) and Nrf-2, in the promoter of
mitochondrial transcription factor A (TFAM), which increases the
transcription of key mitochondrial enzymes, such as cytochrome c oxidase
subunits (COXII, COXIV) and ATP synthase \selectlanguage{greek}β \selectlanguage{english}subunit, and drives
mitochondrial DNA transcription and replication through the interaction
of TFAM (Irrcher, Adhihetty, Sheehan, Joseph \& Hood, 2003; Wu et al.,
1999). Previous studies have shown that the activation of SIRT1-PGC-1\selectlanguage{greek}α
\selectlanguage{english}pathway plays a protective role in hypoxia or OGD-induced injuries (Ma
et al., 2017; Zhou, Wang, Li, Yu \& Zhao, 2017), which improves
mitochondrial function to prevent and treat ischemic heart or brain
disorders (Yang, Mukda \& Chen, 2018; Zhao et al., 2019).

Ginsenosides are major active components of \emph{Panax ginseng C.A.
Meyer} , a medicinal herb as new-resource health food approved by the
Ministry of Health, China. They are extensively used for diverse
beneficial effects on myocardial infarction, stroke, and
ischemia/reperfusion (I/R) injury (Aravinthan et al., 2015; Jia, Zhang,
Huang \& Leung, 2012; Liu, Anderson, Fernandez \& Dore, 2019).
Currently, about 250 ginsenosides have been isolated and identified,
which are mainly divided into three groups, protopanaxadiol (PPD, such
as Rb1, Rb2, Rc, Rd, Rg3), protopanaxatriol (PPT, such as Re, Rf, Rg1,
Rh1), and oleanolic acid (OA, such as Ro), based on their chemical
structures (Wu, Kwaku, Li, Yang, Ge \& Xu, 2019). Proteomic analysis has
shown that total ginsenosides (GS) can increase the expression of
proteins in the TCA cycle to enhance cardiac energy metabolism in the
ischemic rat (Wang, Zhou, Yi, Jiang \& Liu, 2012). Meanwhile,
ginsenoside Rb1, Rb3, Rg3, Rg1, Rg5, and compound K exert
cardioprotective and neuroprotective effects by increasing energy
metabolism (Chen et al., 2019b; Xu, Ma, Fan, Chen, Zhang \& Tang, 2019;
Yang et al., 2017), improving mitochondrial dynamics and quality (Dong
et al., 2016; Sun et al., 2013), or inhibiting mitochondrial apoptosis
(Huang et al., 2020; Li et al., 2018). Importantly, several
ginsenosides, such as Rb1, Rc, F1, Rg3, Rh2, and their metabolites M1-M4
have been identified as SIRT1 activators (Ma, Zhou \& Yang, 2015; Wang,
Liang, Chen \& Zhao, 2016; Yang, Ma, Zhou, Xu \& Zhang, 2017), which
play protective effects on a series of pathological injuries, such as
oxidative stress, inflammation, senescence, hypoxia, or OGD (Cheng et
al., 2019; Song et al., 2014; Wang et al., 2019). However, the molecular
mechanism underlying the effects of GS and each ginsenoside monomer on
mitochondrial aerobic metabolism and biosynthesis in cardiomyocytes and
neurons remains unclear and needs to be further investigated.

In this study, we first investigated the effect of GS on mitochondrial
oxygen consumption rate (OCR) in a series of cell lines, including
aerobic respiration-dominated cardiomyocytes, neuronal cells, and
skeletal myoblasts (Di Lisa, Canton, Menabo, Kaludercic \& Bernardi,
2007; Zeiger, Stankowski \& McLaughlin, 2011), as well as anaerobic
respiration-dominated vascular endothelial, epithelial, and stem cells
(De Bock et al., 2013; Tang et al., 2016). Then ATP production and
mitochondrial respiration capacity (MRC) were examined in untreated and
GS-treated cardiomyocytes and neurons. Targeted metabolomics and Western
blot analysis were performed to observe the effect of GS on the contents
of major metabolites and the levels of key multiple enzymes from
glycolysis and TCA cycle. Furthermore, NAD\textsuperscript{+} level,
SIRT1-PGC-1\selectlanguage{greek}α \selectlanguage{english}pathway, and mitochondrial biosynthesis in the cell,
Drosophila melanogaster, and mice models were further evaluated to
explore the molecular mechanism of GS. In addition, we observed the
effect of GS on SIRT1-mediated mitochondrial function in hypoxia- and
OGD-induced cell models. Of note, the ginsenoside monomer in GS for
promoting SIRT1 activation and mitochondrial energy production was
preliminarily identified in cardiomyocytes. This is the first study to
explore the molecular mechanism by which GS promote mitochondrial energy
metabolism, which may provide new insights into the clinical application
of ginseng for the prevention and treatment of cardiac and neurological
disorders.

\textbf{Materials and Methods}

\textbf{Materials and reagents}

Total ginsenosides (GS, purity [?] 80\%) and ginsenoside monomers (purity
[?] 98\%), such as Rg1, Re, Rf, Rb1, Rc, Rh1, Rb2, Rb3, Rd, F1, F3, Rk3,
S-Rg3, R-Rg3, PPT, Rk1, Rg5, and Rh2, were purchased from Shanghai
Yuanye Bio-Technology (Shanghai, China). Carbonyl cyanide
4-(trifluoromethoxy) phenylhydrazone (FCCP, C2920), and rotenone (R8875)
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oligomycin B
(ab143424); antimycin (ab141904); antibodies against oxdiative
phosphorylation (OXPHOS, ab110413; CV-ATP5A, CIII-UQCRC2, CIV-MTCO1,
CII-SDHB, and CI-NDUFB8); SIRT1 (ab189494); PGC-1\selectlanguage{greek}α \selectlanguage{english}(ab54481); Nrf-1
(ab175932); and Nrf-2 (ab137550) were obtained from Abcam (Cambridge,
MA, USA). Glycolysis Antibody Sampler Kit (\#8337) containing antibodies
against HK-I/II, PKM1/2, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), PDH, lactate dehydrogenase isozyme A, and platelet-type
phosphofructokinase (PFKP), and Tricarboxylic Acid Cycle Antibody
Sampler Kit (\#47767) containing antibodies against citrate synthase
(CS), isocitrate dehydrogenase 1/2 (IDH1/2), MPC1/2, dihydrolipoamide
S-succinyltransferase (DLST), succinate dehydrogenase complex, subunit A
(SDHA), aconitase 2 (ACO2), fumarase, and \selectlanguage{greek}β-\selectlanguage{english}Actin (\#3700) were
purchased from Cell Signaling Technology (Beverly, MA, USA). p2E2
(Q86LE9) and JLA20 (P68139) were obtained from Developmental Studies
Hybridoma Bank (Iowa City, IA, USA). Nicotinamide (NAM, HY-B0150) was
purchased from MedChemExpress (Shanghai, China).

\textbf{High-performance liquid chromatography and liquid
chromatography-mass spectrometry analysis}

The ginsenoside composition of the GS sample was analyzed by
high-performance liquid chromatography (HPLC; Agilent Technologies,
Billerica, MA, USA) with a diode-array detector (Ultimate3000, DIONEX,
Sunnyvale, CA, USA) using the Elite Kromasil C18 column (4.6\selectlanguage{ngerman}×250 mm, 5
\selectlanguage{greek}µ\selectlanguage{english}m). The mobile phase condition was ultrapure water (solvent A) and
acetonitrile (solvent B; Sigma-Aldrich), and the column temperature was
set at 35\selectlanguage{ngerman}°C. The flow rate was 1.0 mL/min, and the chromatograms were
obtained using a UV/VIS detector at 203 nm. According to the retention
time and chromatographic peak area of ginsenoside standards, 18
ginsenosides were identified and quantified as Rg1 (11.23\%), Re
(18.32\%), Rf (4.37\%), Rb1 (5.96\%), Rc (10.34\%), Rh1 (1.40\%), Rb2
(3.15\%), Rb3 (0.57\%), F1 (0.22\%), Rd (7.42\%), F3 (2.83\%), Rk3
(6.30\%), S-Rg3 (2.46\%), R-Rg3 (1.00\%), PPT (0.16\%), Rk1 (1.72\%),
Rg5 (2.31\%), and Rh2 (1.68\%) in the GS sample\textbf{(Supplementary
Fig. 1A and 1B)} . Based on mobile phase and elution gradient from HPLC,
LC-mass spectrometry (LC-MS) was performed on the Thermo LTQ XL system
(Thermo Fisher Scientific, Carlsbad, CA, USA) with the Agilent ZORBAX
SB-C18 column (2.1×100 mm, 1.7 \selectlanguage{greek}µ\selectlanguage{english}m) to obtain the chromatogram of
different ginsenosides in the negative ion mode. MS analysis conditions
were set as follows: gas temperature 270 \selectlanguage{ngerman}°C, electric voltage 3.5 kV and
scan range from 165 to 2000 \emph{m/z.} The MS chromatogram and
properties of different ginsenosides from GS are shown in
\textbf{Supplementary Fig. 2A and 2B} .

\textbf{Animal and drug administration}

Pregnant (16-day-old), neonatal (3-day-old), or 4-week-old Sprague
Dawley rats were purchased from the Jilin University Experimental Animal
Center (Changchun, China), which were used for the isolation of primary
cells such as cardiomyocytes, cortical neurons, or bone marrow
mesenchymal stem cells (BMSCs). Five-week-old Kunming mice were
purchased from Liaoning Changsheng Biotechnology Co., Ltd (Benxi,
China). All animal procedures were performed in accordance with the
guidelines approved by the Animal Ethics Committee of Changchun
University of Chinese Medicine. After acclimatization under temperature
of 23-25°C and 60\%-70\% humidity with 12 h-day/night cycle, the mice
were randomly divided into two groups as control (sterilized distilled
water) and GS (100 mg/kg), which were orally administered every day for
3 weeks. General health, food/water intake, and body weight of all mice
with free access to food and water were monitored and recorded every
day. At the end of the experiment, mice were euthanatized to collect
brain and heart tissues for further analysis.

\textbf{Cell culture}

H9c2 rat cardiomyocyte, PC12 rat adrenal pheochromocytoma, L6/C2C12
rat/mouse skeletal muscle myoblast, human umbilical vein endothelial
cells (HUVECs), THP-1 human acute monocytic leukemia cell lines were
purchased from the American Type Culture Collection (Manassas, VA, USA).
The 16-HBE human bronchial epithelial cell line was kindly provided by
Procell Life Science \& Technology Co., Ltd. (Wuhan, China). Primary
neonatal rat cardiomyocytes, cortical neurons, or BMSCs were isolated,
established, and identified as our previous protocols (Huang et al.,
2018; Jin et al., 2020; Louch, Sheehan \& Wolska, 2011). BMSCs were kept
in medium with 10 mM \selectlanguage{greek}β-\selectlanguage{english}glycerophosphate, 50 \selectlanguage{greek}μ\selectlanguage{english}g/mL ascorbate, and 100 nM
dexamethasone for 14 days to differentiate into the osteoblasts (Jin et
al., 2020). All cells were cultured in appropriate medium containing
10\% fetal bovine serum (CLARK Bioscience, Claymont, DE, USA) and 1\%
penicillin-streptomycin (Biosharp, Hefei, China) and incubated at 37\selectlanguage{ngerman}°C
in a water-saturated atmosphere of 5\% CO\textsubscript{2}. All cell
lines tested negative for mycoplasma contamination. Detailed information
for cell culture is described in \textbf{Supplementary Table 1} .

\textbf{OCR assay}

As previously reported, OCR was measured using the MitoXpress® Xtra
probe or Cell Mito Stress Test Kit (Agilent Technologies) by a
time-resolved fluorescence (TR-F) plate reader (Cytation 5; BioTek,
Winooski, VT, USA) or Seahorse XFp analyzer (Agilent Technologies),
respectively (Yepez et al., 2018). After incubation with GS or
ginsenoside monomer for 48 h, each well was equilibrated in a
CO\textsubscript{2}-free incubator at 37°C for 1 h and measured every 2
min over 2 h to obtain TR-F intensity for calculating the initial slopes
(\selectlanguage{greek}μ\selectlanguage{english}s min\textsuperscript{-1}), according to the phosphorescence lifetime
(\selectlanguage{greek}τ\selectlanguage{english}) values. To further analyze mitochondrial respiration, oligomycin (1
\selectlanguage{greek}µ\selectlanguage{english}M), FCCP (5 \selectlanguage{greek}μ\selectlanguage{english}M), rotenone (Rot, 1 \selectlanguage{greek}μ\selectlanguage{english}M), and antimycin A (AA, 1 \selectlanguage{greek}μ\selectlanguage{english}M) were
sequentially added to each well with XF base medium (Agilent, pH 7.4)
supplemented with 2 mM L-glutamine, 1 mM pyruvate, and 25 mM glucose.
After normalization to the intensity of Hoechst 33342 nuclear staining,
basal OCR, ATP production, maximal respiratory capacity (MRC), and spare
respiratory capacity (SRC) were calculated.

\textbf{Metabolite measurements}

Major intermediates for glycolysis, TCA cycle, and
oxidative-phosphorylation were analyzed with the assistance of Applied
Protein Technology Co., Ltd. (Shanghai, China), as previously described
(Mitsuishi et al., 2012). Briefly, 10 samples of untreated and treated
cells (H9c2 and HUVECs) were stored in liquid nitrogen and centrifuged
at 14,000 rpm for 20 min to collect the supernatants, which were dried
by vacuum. Then, cold acetonitrile/H\textsubscript{2}O (1:1, v/v) was
added to each sample and centrifuged at 14,000 rpm for 20 min to obtain
the supernatants, which were separated and analyzed using a UHPLC (1290
Infinity LC system; Agilent Technologies) coupled to the Triple Quad
5500 Mass Spectrometer (QTRAP/MS; AB SCIEX, Framingham, MA, USA). The
raw MS data were processed using Multiquant software based on
chromatographic peak area and retention time to identify different
energy metabolism-related metabolites.

\textbf{Measurement of PDH activity}

PDH activity was measured using a commercial assay kit (ab109902;
Abcam), according to the manufacturer's instruction (Baccelli et al.,
2019). Briefly, cell lysates from different groups were loaded onto the
96-well plates coated with an anti-PDH monoclonal antibody and incubated
for 3 h at room temperature for the measurement of optical density at
450 nm (OD 450 nm) for 30 min with 1 min internal in a kinetic mode. The
relative PDH activity was calculated as the rate (mOD/min) = (absorbance
2-absorbance 1)/time (min).

\textbf{Quantification of ATP and NAD/NADH levels}

According to the manufacturer's instructions, the contents of ATP,
NAD\textsuperscript{+}, and NADH in the cells of different groups were
detected using the ATP Assay System Bioluminescence Detection Kit
(FF2000; Promega, Madison, WI, USA) and NAD/NADH-Glo Assay Kit (G9071;
Promega). Meanwhile, the levels of cellular ATP, and
NAD\textsuperscript{+} were measured by the multiple reaction monitoring
MS (MRM-MS).

\textbf{Mitotracker staining and transmission electron microscopy
analysis}

Cells were placed in 35 mm glass bottom dish with 10 mm micro-well
(Cellvis, Mountain View, CA, USA) and treated with or without GS for 48
h. After staining with Mitotracker (Beyotime Biotechnology, Shanghai,
China) at 200 nM diluted in pre-warmed culture media for 30 min, cells
were stained with Hoechst 33342 (Sigma-Aldrich) to mark the nuclei as
previously described (Lin et al., 2015). Twenty visual fields in each
well from three dishes at 400\selectlanguage{ngerman}× magnification were observed by the Nikon
C2 confocal microscope with ZEN software (Nikon, Tokyo, Japan), and
mitochondria quantity was analzyed. For transmission electron microscopy
(TEM) analysis, cells treated with or without GS were fixed in 2.5\%
glutaraldehyde for 1 h and then incubated with 1\% osmium tetroxide.
After dehydration and embedding in Durcupan Water-Soluble Embedding
Medium, the specimens were sectioned into 60 nm by a diamond knife and
mounted on copper grids. Micrographs were viewed by TEM (FEI Tecnai G2
20; TWIN, Hillsboro, OR, USA) to analyze and quantify the mitochondrial
size and amount in each cell from four chambers by ImageJ software (NIH,
Bethesda, MD, USA) (Liu et al., 2018).

\textbf{Cell viability, scratch assay and reactive oxygen species
measurement}

After the treatment with different concentrations of GS for 48 h, cells
were incubated with Cell Counting Kit 8 (CCK8) solution (10 \selectlanguage{greek}μ\selectlanguage{english}l/well,
Glpbio, Montclair, CA, USA) at 37\selectlanguage{ngerman}°C for 40 min. The absorbance at 450 nm
for different wells was detected on a microplate reader (Infinite
M200pro, TECAN, Zurich, Switzerland) to analyze cell viability,
according to our previous study (Li et al., 2018). For the scratch
assay, a straight scratch was made using a P200 pipet tip in cells
treated with GS for 48 h. After washing with phosphate-buffered saline,
the images of scratch area at 0 and 24 or 72 h were captured using the
Olympus Viva View FL microscope (Tokyo, Japan) and analyzed to calculate
the rate of scratch closure for four fields per dish from three
independent experiments using ImageJ software (Graham et al., 2018).
Intracellular and mitochondrial reactive oxygen species (ROS) generation
of cells treated with various concentrations of GS for 48 h were
detected by carboxy-H2DCFDA fluorescent probes (Beyotime Biotechnology)
or MitoSOX mitochondrial superoxide indicator (Thermo Fisher Scientific)
using the FACS-Calibur\selectlanguage{english} flow cytometer (BD Biosciences, San Jose, CA,
USA).

\textbf{Establishmentof hypoxia- and OGD-induced cell models}

As previously reported, H9c2 cells were kept in 800 \selectlanguage{greek}μ\selectlanguage{english}M cobalt chloride
(CoCl\textsubscript{2}) medium or in serum and glucose-free 800 \selectlanguage{greek}μ\selectlanguage{english}M
CoCl\textsubscript{2} medium for 9 h to establish hypoxia- or
OGD-induced cell injury models (Huang et al., 2020).

\textbf{Drosophila melanogaster activity and climbing distance assays}

Wild-type Drosophila melanogaster was maintained on standard medium (50
g/L yeast, 50 g/L glucose, 0.8\% agar, and 1\% soy flour) at 25-26\selectlanguage{ngerman}°C in
50\% relative humidity under a 12:12 h light-dark cycle. The 7-day-old
flies were loaded into plastic tubes containing standard food or with GS
(1 mg/mL) for 7 days. Then, the sleep-waking activity of 32 flies was
consecutively monitored for 24 h using the Drosophila Activity
Monitoring System (Trikinetics, Waltham, MA, USA) and recorded as
beam-breaks in 1-min bins for generating the number of activities. For
the climbing ability assay, 30 flies in the vertical vials (110×10 mm)
were acclimatized for 15 min and simultaneously tapped to the bottom of
the vial for 5 sec. The climbing distance of each fly from control or GS
pretreatment was recorded to obtain the average of the distance
(Algarve, Assmann, Aigaki \& da Cruz, 2020).

\textbf{Immunohistochemical staining}

For immunohistochemical (IHC) staining, the heart and brain tissues from
different groups were fixed in 4\% paraformaldehyde, paraffin-embedded,
and sectioned into 5 \selectlanguage{greek}μ\selectlanguage{english}m slides, which were analyzed for SIRT1 (1:100)
expression as previously described (Huang et al., 2018).

\textbf{Quantitative PCR analysis}

Quantitative PCR (qPCR) was performed as previously described (Jin et
al., 2020). Briefly, total RNA was extracted using TRizol (Life
Technologies, Frederick, MD, USA), followed by ethanol extraction. qPCR
was conducted in triplicate using SYBR Green PCR reagent (Bio-Rad,
Hercules, CA, USA) on the CFX96 Real-Time PCR system (Bio-Rad). PCR
conditions included a denaturation step of 95\selectlanguage{ngerman}°C for 5 min, followed by
40 cycles consisting for 95°C for 15 sec, 60°C for 30 sec, and 72°C for
30 sec. The primer sequences are described in Supplementary Table 2. The
relative expression of each target gene was calculated by the
2\selectlanguage{greek}\textsuperscript{-ΔΔCt}\selectlanguage{english} method after normalization to \selectlanguage{greek}β-\selectlanguage{english}Actin.

\textbf{Western blot analysis}

Cells or fly brain tissues were lysed in RIPA buffer (Beyotime
Biotechnology) for 1 h on ice and centrifuged at 14,000 rpm to obtain
the supernatants, which were measured to detect protein concentration
quantified with a BCA protein assay kit (Beyotime Biotechnology). Total
protein (30\selectlanguage{greek}μ\selectlanguage{english}g) was resolved on 10\% or 12\% sodium dodecyl sulfate
polyacrylamide gel electrophoresis to separate different sizes of
proteins, followed by eletrotransfer onto nitrocellulose membranes. The
membranes were blocked in 5\% non-fat milk and probed with primary
antibodies overnight at 4\selectlanguage{ngerman}°C. Proteins were detected using a
chemiluminescent imaging system (FluorChem, ProteinSimple, San Jose, CA,
USA), after incubating with the appropriate secondary antibodies for 1 h
at room temperature (Huang et al., 2018).

\textbf{Statistical analysis}

Data are presented as the mean ± standard deviation or mean ± stanard
error of the mean from at least three independent experiments. The
unpaired Student's \emph{t} - test for comparing two groups or one-way
analysis of variance for comparing multiple groups was used to analyze
statistical significance using GraphPad Prism 8 (GraphPad Software, San
Diego, CA, USA). Hierarchical cluster analysis of different metabolites
was performed by Python. The raw metabolomic intensity of each
metabolite from two groups was transformed into the
Log\textsuperscript{2} value of normalized intensity. For all
statistical analyses, a significant difference was accepted at
\emph{P}\textless{} 0.05.

\textbf{Results}

\textbf{GS enhances MRC and ATP production in cardiomyocytes and
neurons}

To explore the molecular mechanism of GS on extensive cell protection,
we first investigated the effects of GS on mitochondrial function by
examining OCR, as a direct measure of mitochondrial respiration
(Diepart, Verrax, Calderon, Feron, Jordan \& Gallez, 2010). Oxygen
consumption probes were used to assess the effect of GS on the rate of
basal oxygen consumption in a variety of cell lines, including H9c2,
primary neonatal cardiomyocytes, differentiated PC12, primary cortical
neurons, L6, C2C12, HUVECs, BMSCs, osteoblast, 16HBE, and THP-1 cells.
As expected, GS pretreatment for 48 h increased basal oxygen consumption
by 2.0-fold in H9c2 cells, 2.8-fold in PC12 cells, 1.5-fold in neurons
\textbf{(Fig. 1A and Supplementary Fig. 3A)} , 1.6-fold in C2C12 cells,
and 1.5-fold in L6 cells\textbf{(} \textbf{Supplementary Fig. 3B)} .
Meanwhile, we found that GS had no effect on basal OCR in HUVECs, BMSCs,
osteoblast, 16HBE, and THP-1 cells \textbf{(Fig. 1B,Supplementary Fig.
3B-3F)} . To further assess the potential function of GS in
mitochondrial oxidative capacity, we conducted a mitochondrial stress
test in H9c2, PC12, and HUVECs. The pretreatment of GS at 5 \selectlanguage{greek}μ\selectlanguage{english}g/mL for 48
h led to increases in basal OCR, MRC, and SRC in H9c2 and PC12
cells\textbf{(Fig. 1C and Fig. 1D)} . The similar results for
mitochondrial oxidative capacity were detected by Luxcel oxygen
consumption probes in H9c2 and PC12 cells \textbf{(Supplementary Fig.
4A-4B)} . In HUVECs, GS pretreatment had no effects on basal OCR, MRC,
and SRC\textbf{(Supplementary Fig. 4C)} . Furthermore, intracellular ATP
content was examined in H9c2, PC12, and primary neurons after GS
pretreatment. As shown in \textbf{Figure 1E} , GS pretreatment
significantly increased ATP production in cardiomyocytes and neurons in
the same manner as OCR. LC-MS analysis showed that GS at 5 \selectlanguage{greek}μ\selectlanguage{english}g/mL
promoted relative ATP intensity and the ratio of ATP/ADP in H9c2 cells
\textbf{(Fig. 1F)} . In HUVECs, GS slightly increased ATP content and
had no effect on ATP/ADP ratio\textbf{(Supplementary Fig. 4D)} . To
further identify the time-dependent effect of GS on energy production,
the levels of basal OCR and ATP in H9c2 cells at different time points
were measured after GS pretreatment for 48 h. The results in Figure 1G
and 1H demonstrated that GS pretreatment for 6 h enhanced basal OCR and
ATP content at concentrations of 5 and 10 \selectlanguage{greek}μ\selectlanguage{english}g/mL GS. In particular, basal
OCR was gradually increased by GS pretreatment in time- and
dose-dependent manners. Collectively, GS pretreatment enhanced MRC and
ATP production in aerobic respiration- dominated cardiomyocytes and
neurons, and had no obvious effect on anaerobic respiration- dominated
other cell lines such as HUVECs and BMSCs.

\textbf{GS significantly promote aerobic glucose metabolism in
cardiomyocytes}

Increased OCR and ATP production are the manifestations of enhanced
energy metabolism; thus, we performed targeted metabolomic analysis of
glucose metabolites by LC-MS/MS in H9c2 and HUVECs untreated or treated
with GS for 48 h. A total of 15 different intermediates of glycolysis
and TCA cycle, such as pyruvate, lactate, citrate, and succinate were
identified in these two cell lines. In H9c2 cells, GS led to decreases
of seven metabolites and increases of eight metabolites, compared with
the control group \textbf{(Fig. 2A)} . The analysis of relative
intensity for each metabolite showed that GS decreased the levels of
fructose 6-phosphate, fructose 1, 6-bisphosphate, 3-phosphoglycerate,
dihydroxyacetone phosphate, and phosphoenolpyruvate, and increased the
levels of glucose 6-phosphate and pyruvate in H9c2 cells, suggesting
that GS increased the conversion of glucose to pyruvate. Moreover, key
metabolites of TCA cycle such as citrate, succinate, fumarate, malate,
oxaloacetate, and glycolytic metabolite, lactate were clearly increased
by GS pretreatment \textbf{(Fig. 2B)} , suggesting that GS mainly
accelerated TCA metabolism in the cardiomyocytes. In HUVECs, only six
metabolites were upregulated by GS pretreatment, including
glucose-6-phosphate, fructose-6-phosphate, pyruvate, succinate, malate,
and fumarate. There were no obvious differences in the other nine
metabolites between the control and GS groups \textbf{(Fig. 2C,
Supplementary Fig. 5A)} . These data suggest that GS mainly increased
the conversion from glucose to fructose-6-phosphate and pyruvate, and
enhanced the metabolic process from succinate to malate in HUVECs.
Collectively, GS had more obvious effects on promoting glucose oxidation
in cardiomyocytes than endothelial cells.

Multiple key enzymes regulate glucose breakdown and TCA metabolism
reaction, which were examined by Western blot analysis in the control
and GS-treated H9c2 cells. GS pretreatment upregulated the levels to the
rate-limiting enzymes such as HK-II, PFKP, PKM2, and PDH, and did not
change GAPDH expression in H9c2 cells \textbf{(Fig. 2D, Supplementary
Fig. 5B)} . Mitochondrial pyruvate carriers, MPC1 and MPC2, which play a
potential role in the transport of pyruvate to mitochondria, were
increased by GS compared with the control group \textbf{(Fig. 2D,
Supplementary Fig. 5B)} . Furthermore, CS, the first and rate-limiting
enzyme of the TCA cycle; DLST, one of the core enzymes of
\selectlanguage{greek}α-\selectlanguage{english}ketoglutarate dehydrogenase complex; and fumarase, a mitochondrial
metabolic enzyme, were significantly higher in GS-pretreated H9c2 cells
than the control group \textbf{(Fig. 2D, Supplementary Fig. 5B)} . Two
isocitrate dehydrogenase isoforms in cytoplasm (IDH1) and mitochondria
(IDH2) were not regulated by GS. In addition, ACO2, which is responsible
for catalyzing the isomerization of citrate to isocitrate and SDHA, were
decreased by GS pretreatment \textbf{(Fig. 2D, Supplementary Fig. 5B)} .
Importantly, the activity of PDH, as a rate-limiting metabolic step for
glucose oxidation was measured in untreated- and GS-pretreated H9c2 and
HUVECS. We found a significant induction in PDH activity in GS-treated
H9c2 cells and no change of PDH activity in GS-treated HUVECs compared
with control cells \textbf{(Fig. 2E)} . Taken together, our findings
suggest that GS pretreatment mainly accelerates glucose conversion to
pyruvate, and increases pyruvate transporting to promote TCA metabolism
in the cardiomyocytes. In future experiments, we will focus on the
effect and molecular mechanism of GS on cardiomyocytes and neurons
dominated by aerobic respiration.

\textbf{GSincrease mitochondrial biogenesis incardiomyocytes and
neurons}

To investigate the role of GS in mitochondrial biogenesis, we first
examined mitochondrial count in cardiomyocytes and neurons using the
Mitotracker probe. We found that pretreatment with GS induced a
significant increase in mitochondrial content in a dose-dependent manner
in H9c2 cells \textbf{(Fig. 3A-3B)} and primary neurons \textbf{(Fig. 3A
and 3C)} . Moreover, high-resolution micrographs of mitochondria were
obtained in control and GS-pretreated H9c2 cells using TEM. As shown
in\textbf{Fig. 3D-3E} , GS pretreatment at 5 \selectlanguage{greek}μ\selectlanguage{english}g/mL for 48 h increased
the number of mitochondria in the cardiomyocytes and had no effect on
the size of myocardial mitochondria, compared with control cells. To
determine if the observed changes in mitochondrial respiration and
biogenesis translate into altered mitochondrial function, we performed
immunoblotting with antibodies against five complexes of the oxidative
phosphorylation. Compared with the control group, GS induced increases
in complex I-IV levels in H9c2 cells and mainly increased the levels of
CII-SDHB and CI-NDUFB8 in PC12 cells \textbf{(Fig. 3F-3G)} . The
enhancement of mitochondrial function can increase ROS generation, which
damages the cells, so we examined ROS level in untreated- and GS-treated
H9c2 cells. Flow cytometry analysis showed that GS had no significant
effect on the production of intracellular and mitochondrial ROS in H9c2
cells \textbf{(Supplementary Fig. 6A)} . Additionally, mitochondrial
amount and complex protein level were further investigated in HUVECs. We
did not observe the effect of GS pretreatment on mitochondrial mass in
HUVECs \textbf{(Supplementary Fig. 6B-6C)} . Collectively, GS
preferentially promote the mitochondrial biosynthesis of cardiomyocytes
and neurons, and not that of endothelial cells.

\textbf{GS increase NAD\textsuperscript{+}-dependent SIRT1 activation in
cardiomyocytes and neurons}

NAD\textsuperscript{+} level and its dependent deacetylase, SIRT1, play
key roles in regulating mitochondrial function (Katsyuba et al., 2018).
Based on the findings above, we determined if GS could alter the
intracellular level of NAD\textsuperscript{+} and regulate SIRT1
activation in cardiomyocytes and neurons. Consistent with our
hypothesis, the level of NAD\textsuperscript{+} luminescence was
obviously higher in GS-treated H9c2 and primary neurons than in the
control group \textbf{(Fig. 4A)} . Moreover, the targeted metabolomic
analysis showed that NAD\textsuperscript{+} level was significantly
increased by GS pretreatment in H9c2 cells \textbf{(Fig. 4B)} . As
reported, NAD\textsuperscript{+}-dependent SIRT1 can deacetylate and
activate PGC-1\selectlanguage{greek}α \selectlanguage{english}to stimulate mitochondrial biogenesis (Aquilano,
Baldelli, Pagliei \& Ciriolo, 2013). As shown in \textbf{Fig. 4C} , qPCR
analysis showed that GS incubation for 8 h increased SIRT1 and PGC-1\selectlanguage{greek}α
\selectlanguage{english}mRNA levels in H9c2 and primary neurons. Western blot analysis
demonstrated that GS pretreatment for 48 h led to increases in SIRT1 and
its targets PGC-1\selectlanguage{greek}α, \selectlanguage{english}Nrf1, and Nrf2 in H9c2 cells \textbf{(Fig. 4D)} .
Furthermore, similar findings in cardiomyocytes were observed in
GS-treated PC12 cells \textbf{(Fig. 4E)} . In addition, the
wound-healing ability in H9c2 cells and neurons was increased by GS
pretreatment, suggesting that GS promoted cell migration
\textbf{(Supplementary Fig. 7A-7C)} .

Intriguingly, GS were administered to the flies and mice to further
validate the function of GS in NAD\textsuperscript{+}-dependent SIRT1
activation. Consistent with the \emph{in vitro} results, GS
administration for 7 days led to a significant increase in the intensity
of NAD\textsuperscript{+} luminescence and the ratio of
NAD\textsuperscript{+}/NADH in the drosophila brain compared with the
control group \textbf{(Fig. 5A)} . The increased SIRT1 in response to
NAD\textsuperscript{+} was confirmed in the brain of drosophila treated
with GS \textbf{(Fig. 5B)} . Meanwhile, the number of activities and
climbing distance of 30 flies fed with GS were greatly increased,
compared to the control group with common food \textbf{(Fig. 5C)} . In
addition, IHC staining showed that SIRT1 expression was significantly
increased in the heart and brain tissues of mice after 21 days of GS
administration \textbf{(Fig. 5D)} . Together, these \emph{in vitro} and
\emph{in vivo} results indicate that GS obviously increase
NAD\textsuperscript{+}-dependent SIRT1 activation in cardiomyocytes and
neurons.

\textbf{GS increase mitochondrial function through the SIRT1 pathway in
cardiomyocytes subjected to hypoxia and OGD injuries}

Generally, ischemia in the heart can cause mitochondrial dysfunction,
which consequently reduces energy metabolism to cause further cardiac
damage (Kalogeris, Baines, Krenz \& Korthuis, 2012). To further explore
the protective effect of GS against hypoxia and OGD injuries, cell
viability, OCR, ATP production, mitochondrial respiration complexes, and
the SIRT1-PGC-1\selectlanguage{greek}α \selectlanguage{english}pathway were investigated in control, injured model,
and GS-treated H9c2 cells. As shown in \textbf{Fig. 6A} , hypoxia and
OGD led to decreases in H9c2 cell viability, which were slightly
recovered by GS. Consistent with current results, we found that hypoxia
induced by Cocl\textsubscript{2} for 9 h significantly diminished the
rate of oxygen consumption and ATP production of H9c2 cells, which were
lower during the process of glucose and oxygen deprivation\textbf{(Fig.
6B-6C)} . GS pretreatment for 48 h reversed the decreases of OCR and ATP
level induced by hypoxia and OGD in H9c2 cells \textbf{(Fig. 6B-6C)} .
Meanwhile, the levels of five mitochondrial respiratory complexes,
CV-ATP5A, CIII-UQCRC2, CIV-MTCO1, CII-SDHB, and CI-NDUFB8, were
decreased by hypoxia for 9 h, which were obviously recovered by GS at 10
\selectlanguage{greek}μ\selectlanguage{english}g/mL \textbf{(} \textbf{Fig. 6D-6E)} . In addition, Western blot
analysis demonstrated that hypoxia and OGD led to decrease in SIRT1 and
PGC-1\selectlanguage{greek}α \selectlanguage{english}levels in H9c2 cells and the levels of SIRT1 and PGC-1\selectlanguage{greek}α \selectlanguage{english}in
GS-pretreated H9c2 cells were higher than those in the hypoxia- or
OGD-induced model group \textbf{(Fig. 6F-6G)} . Taken together, these
results clearly demonstrate the protective effect of GS to increase
mitochondrial function against the hypoxia- or OGD-induced cardiomyocyte
damage through activation of the SIRT1-PGC-1\selectlanguage{greek}α \selectlanguage{english}pathway.

\textbf{SIRT1 pathway is involved in GS-enhanced mitochondrial function
in cardiomyocytes}

To further confirm that GS regulate mitochondrial function by the
activation of SIRT1, we tested whether mitochondrial respiration, ATP
production, and the NAD\textsuperscript{+}-dependent SIRT1 pathway were
affected by a SIRT1 inhibitor, nicotinamiede (NAM) combined with GS.
Consistent with the results above, we observed increased basal or
maximal mitochondrial respiration in H9c2 cells pretreated with GS for
48 h, which was significantly blocked by NAM \textbf{(Fig. 7A-7B)} .
Similarly, GS-mediated increase of ATP content in H9c2 cells was
inhibited by NAM\textbf{(} \textbf{Fig. 7C)} . For respiratory chain
complexes, higher expression of three complexes, CIII-UQCRC2, CIV-MTCO1,
and CII-SDHB, induced by GS was inhibited by the combination of GS with
NAM \textbf{(Fig. 7D-7E)} . Importantly, GS combined with NAM
significantly reduced GS-induced NAD\textsuperscript{+} level in H9c2
cells \textbf{(Fig. 7F)} . Then, to further determine whether increased
mitochondrial function by GS is mediated by the
NAD\textsuperscript{+}-dependent SIRT1 pathway, we measured protein
levels of SIRT1 and its targets in control, GS-, NAM-, or GS+NAM-treated
H9c2 cells by Western blot analysis. As expected, GS-mediated activation
of SIRT1 and its targets, PGC-1\selectlanguage{greek}α, \selectlanguage{english}Nrf1, and Nrf2, were completely
abrogated by the pretreatment of GS and NAM, which was similar to the
control or NAM group \textbf{(Fig. 7G-7H)} . These findings suggest that
the SIRT1 pathway is involved in GS-enhanced mitochondrial function in
cardiomyocytes

\textbf{Ginsenoside monomers increase mitochondrial function in
cardiomyocytes by activating the SIRT1 pathway}

As we showed, GS are a complex mixture of ginsenoside monomers of
different proportions \textbf{(Supplementary Fig. 1-2)} . To further
gain insight into which ginsenoside in GS promotes mitochondrial
function through increased glucose oxidation and SIRT1 activation, we
performed a series of experiments to investigate the effects of
different ginsenosides on basal OCR, ATP content, and key protein levels
in H9c2 cells. Similar to that of GS, different ginsenoside monomers,
such as Rg1, Re, Rf, Rb1, Rc, Rh1, Rb2, Rb3, Rd, S-Rg3, R-Rg3, and Rk1,
at individual concentrations of GS increased basal OCR in H9c2 cells
after 48 h treatment compared with the control group \textbf{(Fig. 8A)}
. Moreover, ATP content was higher in Re-, Rf-, Rb1-, Rc-, Rh1-, Rb2-,
or Rb3-treated H9c2 cells than the control or GS group \textbf{(Fig.
8B)} . These results confirm that these ginsenosides can promote
mitochondrial energy metabolism. Additionally, key proteins of the
glucose oxidation process, HK-II and MPC1, were increased by Rg1, Re,
Rf, and Rc, which was similar to GS \textbf{(Fig. 8C-8D)} . For SIRT1
expression, we found that the level of SIRT1 was upregulated by most of
the ginsenoside monomers, with the exception of Rk3 \textbf{(Fig.
8C-8D)} . Especially, ginsenosides Rg1, Re, Rc, Rh1, Rb2, and Rb3 had a
similar role in increasing SIRT1 expression as GS \textbf{(Fig. 8C-8D)}
. Taken together, these results identified the major ginsenosides in GS,
including Rg1, Re, Rf, Rb1, Rc, Rh1, Rb2, and Rb3, which promote aerobic
cellular respiration and SIRT1-mediated mitochondrial biosynthesis to
protect cardiomyocytes.

\textbf{Discussion}

Here, we revealed the novel mechanism by which GS promote mitochondrial
energy metabolism in cardiomyocytes and neurons, aerobic respiration-
dominated cells. In this study, GS pretreatment increased mitochondrial
aerobic respiration, ATP production, and cellular
NAD\textsuperscript{+}level. Moreover, GS activated the SIRT1-PGC-1\selectlanguage{greek}α
\selectlanguage{english}pathway to promote mitochondrial biosynthesis and ETC complex II-IV
levels, which was inhibited by NAM. In hypoxia- and OGD-induced cell
injuries, GS also increased ATP content and OXPHOS protein levels by
promoting the activation of the SIRT1-PGC-1\selectlanguage{greek}α \selectlanguage{english}pathway. Finally, major
ginsenoside monomers in GS were identified for increasing HK-II, MPC1,
and SIRT1 levels to promote mitochondrial energy promotion. These
findings confirmed the potential role of GS for energy metabolism, which
supported the clinical application of ginseng for the benefit of human
health and ischemic disorders \textbf{(Fig. 9)} .

As previously reported, ginsenosides have potential health benefits in
improving immunity and energy, and combating cardiovascular,
cerebrovascular, and metabolic diseases (Ratan et al., 2020), which
might be associated with the enhancement of glucose energy metabolism.
In individual cell types, glycolysis and OXPHOS are utilized to maintain
energy metabolic needs. Several studies have demonstrated that neurons
depend on mitochondria, and endothelial cells rely on glycolysis,
whereas skeletal cells utilize both OXPHOS and glycolysis for energy
supply (Chang, Singh, Gross \& Kioussi, 2019; De Bock et al., 2013;
Zeiger, Stankowski \& McLaughlin, 2011). For the first time, we chose a
couple of types of cell lines such as cardiomyocytes (H9c2, primary
cardiac myocytes), neurons (differentiated PC12, primary neurons), bone
marrow-derived stem cells, endotheliocytes (HUVECs), and stem cells to
determine the effect of GS on energy metabolism. The analysis of basal
OCR demonstrated that GS preferably increased oxygen consumption in
cells, cardiomyocytes, and neurons, which were highly dependent on
mitochondria, not those cell lines depdendent on glycolysis for energy
production, such as endothelial cells, or stem cells. Furthermore, ATP
measurement and mitochondrial stress assay confirm the preferable effect
of GS on mitochondrial respiration-dominant cardiomyocytes and neurons,
similar to previous findings that ginseng extract containing Rg1, Re,
Rb1, and Rd can promote the OCR and ATP production in healthy
cardiomyocytes (Huang et al., 2019).

Glucose 6-phosphate is an essential metabolite in the first step of
glucose metabolism, which is converted from glucose by hexokinases
(John, Weiss \& Ribalet, 2011). During the early stage of glycolysis,
the content of glucose-6-phosphate was increased by GS in both cell
lines. However, other major metabolites before pyruvate were obviously
decreased in cardiomyocytes and not in endothelial cells. It suggests
that the rapid metabolic process from fructose 6-phosphate to pyruvate
was induced by GS in cardiomyocytes, which may be related to GS-mediated
high expression of key rate-limiting enzymes, HK-II, PFKP, and PKM2.
Meanwhile, pyruvate transport to mitochondria medicated by MPC1/2 and
its conversion into acetyl-CoA by PDH were enhanced by GS in
cardiomyocytes, similar to the functions of Rb1 (Li et al., 2017) and
Rg5 (Peng et al., 2019). PDH is a vital regulatory enzyme for the
connection of glycolysis to the aerobic TCA cycle, which was activated
by GS in cardiomyocytes, not in endothelial cells. As previously
reported from heart tissue proteome and urine metabolome, total
ginsenosides or ginseng extract can modulate the levels of TCA cycle
proteins, such as pyruvate dehydrogenase complex in rat heart tissues
(Wang, Zhou, Yi, Jiang \& Liu, 2012; Yang et al., 2016), which was
deeply confirmed by our study. In addition, multiple metabolites in
cardiomyocytes from succinate to citrate were increased by GS, but GS
only enhanced the metabolic process from succinate to malate in
endothelial cells. Compared with current reports, our findings clearly
explore the role of GS on energy metabolism that GS pretreatment
preferably promotes glucose metabolism into the TCA cycle for providing
energy in cardiomyocytes, and has a slight function in endothelial cells
through regulating different key enzymes in a series of steps of glucose
metabolism. During the OXPHOS, for the first time, we found that GS
mainly promoted the expressions of ETC complex proteins I-IV to increase
the transfer of electrons in the inner membrane of mitochondria, which
were not changed in endothelial cells. Currently, only one previous
report has shown that ginsenoside Re can rescue complex IV deficiency in
PINK1 null neuronal cells (Kim, Song, Yoon, Shehzad, Kim \& Son, 2012).
However, we need to perform the\textsuperscript{13}C-based metabolic
flux analysis to identify the accurate mapping of GS-mediated aerobic
glucose oxidation in the future.

NAD\textsuperscript{+} can modulate glycolysis in the cytoplasm and the
TCA cycle/OXPHOS in the mitochondria, and activate sirtuins to maintain
energy homeostasis (Canto, Menzies \& Auwerx, 2015). In our study,
NAD\textsuperscript{+} levels in the cardiomyocytes, primary neurons,
and fly brains were increased by GS treatment, which can activate SIRT1
to match cellular energy requirements (Canto \& Auwerx, 2012).
SIRT1-mediated deacetylation leads to higher levels of PGC-1a to induce
the nuclear transcription of Nrf1/2 for mitochondrial biogenesis (Canto
\& Auwerx, 2012). In different models, we found that SIRT1 and its
downstream pathway were obviously activated by GS, which may be the
molecular mechanism of GS on the induction of mitochondrial mass. As we
showed, GS-mediated mitochondrial respiration, ATP production, and
higher levels of ETC proteins were abolished by NAM, which confirm that
GS promote mitochondrial biosynthesis and function through the
activation of SIRT1 pathway. In the stress conditions, such as hypoxia
or OGD, we obtained similar findings that GS can activate the
SIRT1-PGC-1\selectlanguage{greek}α \selectlanguage{english}pathway to increase the levels of all five complexes and
mitochondrial bioenergetics to maintain energy homeostasis in
cardiomyocytes, which may be one of the potential mechanisms of GS
against ischemic disorders. Many studies demonstrate that ginsenoside
Rg3 or a prescription containing ginseng could enhance mitochondrial
respiration in healthy and unhealthy cells (Lin et al., 2015; Sun et
al., 2013). Our results together with previous findings can verify the
beneficial functions of ginsenosides on mitochondrial energy metabolism
without ROS injury.

For glucose oxidation, very few ginsenosides, such as compound K, Rg3,
or Rg5, can regulate HK-II expression to inhibit tumor growth or protect
cardiomyocyte ischemic injury (Chen et al., 2019a; Yang et al., 2017).
In our study, we found that other ginsenosides Rg1, Re, Rf, and Rc can
upregulate HK-II level. For SIRT1 activation, ginsenoside monomers,
including Rb1, Rb2, Rc, and Rg3, have been identified as potential SIRT1
activators (Wang, Liang, Chen \& Zhao, 2016; Yang, Ma, Zhou, Xu \&
Zhang, 2017). Ginsenoside Rb2, Rc, and F1 can promote SIRT1 activation
to increase ATP production and inhibit oxidative stress in
cardiomyocytes (Wang, Liang, Chen \& Zhao, 2016). Another study showed
that ginsenosides Rg1, Rb1, Re, Rh2, Rg3, and Rg5 can protect ischemic
brain injury, which is associated with STIRT1 activation and Toll-like
receptor 4/myeloid differentiation factor 88 signaling pathways (Cheng
et al., 2019). In our study, we found that most ginsenosides can
activate SIRT1 and enhance ATP content, similar to previous findings.
According to the content percentage of each ginsenoside in GS, we found
that Rh1, Rb3, Rb2, or Rf at the concentration of lower than 0.2 \selectlanguage{greek}μ\selectlanguage{english}g/mL
had a good effect on SIRT1 activation and mitochondrial respiration.
Moreover, the analysis from published findings speculated that two
glucopyranosyl groups on the C-3 position of ginsenosides are critical
for SIRT1 activity (Ma, Zhou \& Yang, 2015; Wang, Liang, Chen \& Zhao,
2016; Yang, Ma, Zhou, Xu \& Zhang, 2017). Therefore, the binding
mechanism of ginsenosides on the SIRT1 needs to be well studied to
explore the sites of key amino acids in SIRT1 proteins using molecular
docking, protein interaction, and mutation techniques. More importantly,
the synergistic effects of different ginsenosides targeting potential
molecules of glucose metabolism and SIRT1 pathway could be investigated
in the future.

\textbf{Conclusion}

In summary, \emph{in vitro} and \emph{in vivo} data provided evidence
that GS from \emph{Panax} ginseng promoted aerobic cellular respiration
and SIRT1-mediated mitochondrial biosynthesis in cardiomyocytes and
neurons. Major ginsenoside monomers, such as Rb1, Rb2, Rb3, Rc, Rd, Re,
Rf, or Rg1, in GS were found to activate mitochondrial respiration and
SIRT1 pathway to promote energy metabolism. This study provides new
insights into the extensive application of ginseng for cardiac and
neurological protection in healthy subjects and patients with ischemic
disorders.

\textbf{Source of funding}

This study was supported by the National Key Research and Development
Program of China (2017YFC1702103), National Natural Science Foundation
of China (U19A2013) and the Science and Technology Development Plan
Project of Jilin Province (202002053JC, 20190101010JH, and
20180623041TC), the Project for Science and Technology Bureau of
Changchun (18YJ013) and Jilin Provincial Administration of Traditional
Chinese Medicine (2020168).

\textbf{Author contributions}

DZ and XL: conceived and designed this study. LS, DZ, and XL:
participated in the study design and interpreted data. QH and XL:
prepared the draft manuscript. QH, TL, JL, MW, XC, LX, XT, WQ, ZZ, HS,
WJ, and CJ: participated in the data collection and analysis. DZ and XL:
funding support. All authors have read and approved final manuscript.

\textbf{Declaration of transparency and scientific rigour}

This Declaration acknowledges that this paper adheres to the principles
for transparent reporting and scientific rigour of preclinical research
as stated in the \emph{BJP} guidelines for Natural Products Research,
Design and Analysis, Immunoblotting and Immunochemistry, and Animal
Experimentation, and as recommended by funding agencies, publishers and
other organisations engaged with supporting research.

\textbf{Data availability statement}

The data that support the findings of this study are available from the
corresponding author upon reasonable request.

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Fig. 1. GS enhanced mitochondrial respiration capacity and ATP
production in cardiomyocytes and neurons. (A-B)} Basal oxygen
consumption rate (OCR) in H9c2, NGF-induced PC12, neuron, HUVECs and
BMSCs treated with different concentrations of total ginsenosides (GS)
for 48 h was measured using a MitoXpress\selectlanguage{ngerman}® Xtra probe normalized to cell
number. \textbf{(C)}OCR was analyzed at basal condition and sequential
injections of oligomycin (1 \selectlanguage{greek}µ\selectlanguage{english}M), FCCP (5 \selectlanguage{greek}μ\selectlanguage{english}M), rotenone (Rot, 1 \selectlanguage{greek}μ\selectlanguage{english}M) and
antimycin A (AA, 1 \selectlanguage{greek}μ\selectlanguage{english}M) in control and GS-pretreated H9c2 cells by the
MitoStress Test Kit. After the normalization to cell number, basal OCR,
ATP production, maximal respiratory capacity (MRC), and spare
respiratory capacity (SRC) were calculated and shown. \textbf{(D)}
Similar to the panel C, basal OCR, ATP production, MRC, and SRC were
analyzed in NGF-induced PC12 cells, after GS pretreatment for 48 h.
\textbf{(E)} After pretreatment with GS at 2.5, 5, or 10 \selectlanguage{greek}μ\selectlanguage{english}g/mL for 48 h,
ATP levels in H9c2, PC12, and neurons were measured by the
Bioluminescence Detection Kit. \textbf{(F)}The intensities of ATP and
ADP in control and GS-treated H9c2 cells were analyzed by the LC-MS/MS
method based on MRM detection. The ratio of ATP and ADP was calculated
and shown. \textbf{(G-H)} Basal OCR and ATP content in H9c2 cells
treated with different concentrations (2.5, 5, or 10 \selectlanguage{greek}μ\selectlanguage{english}g/mL) of GS and
incubated for 3, 6, 12, 24, or 48 h were measured as described as the
methods above. Ctrl: control group. *\emph{P} \textless{} 0.05,
**\emph{P}\textless{} 0.01, and ***\emph{P} \textless{} 0.001
\emph{versus} Ctrl group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Fig. 2.} \textbf{Effect of GS on key metabolites and enzymes of
glucose metabolism in H9c2 and HUVECs.(A)} The heatmap represents the
targeted metabolomic analysis of 15 key metabolites of glucose
metabolism in control and GS-pretreated H9c2 cells, after 48 h
incubation.\textbf{(B)} Bar graph depicts the quantification of 15
metabolites in H9c2 cells in control and GS-pretreated H9c2 cells.
\textbf{(C)} The heatmap showing 15 metabolites of glucose metabolism in
control and GS-pretreated HUVECs.\textbf{(D)} After GS pretreatment for
48 h, key enzymes of glycolysis and TCA cycle and mitochondrial pyruvate
carriers were detected by Western blot analysis. \textbf{(E)} Pyruvate
dehydrogenase (PDH) activities in H9c2 and HUVECs cells pretreated with
GS for 48 h were measured. Ctrl: control group. *\emph{P} \textless{}
0.05, **\emph{P}\textless{} 0.01, and ***\emph{P} \textless{} 0.001
\emph{versus} Ctrl group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Fig. 3.} \textbf{GS increase mitochondrial biogenesis in
cardiomyocytes and neurons. (A)} After GS pretreatment for 48 h, H9c2
and neurons were co-stained with MitoTracker Green probe and Hoechst
33342 to analyze mitochondria amount by confocal
microscopy.\textbf{(B-C)} Quantitative analysis of mitochondrial
fluorescence intensity in control and GS-pretreated H9c2 and neurons
from (A).\textbf{(D-E)} Mitochondrial structure in control and
GS-treated H9c2 cells was observed by a transmission electron
microscope. The number and size of mitochondria in H9c2 cells from
different groups were analyzed by ImageJ. \textbf{(F)} After incubation
with GS at 5 \selectlanguage{greek}μ\selectlanguage{english}g/mL for 48 h, the levels of mitochondrial complexes I-V
in H9c2 and PC12 cells were detected by Western blot analysis.
\textbf{(G)} Relative expressions of complexes I-V in H9c2 and PC12
cells from (F) were quantified. Ctrl: control group. *\emph{P}
\textless{} 0.05, **\emph{P} \textless{} 0.01, ***\emph{P} \textless{}
0.001 \emph{versus} Ctrl group. CI-NDUFB8: NADH dehydrogenase 1 beta
subcomplex subunit 8, CII-SDHB: succinate dehydrogenase iron-sulfur
subunit, CIII-UQCRC2: ubiquinol-cytochrome-c reductase complex core
protein 2, CIV-MTCO1: mitochondrially encoded cytochrome C oxidase I,
CV-ATP5A: ATP synthase F1 subunit \selectlanguage{greek}α.

\selectlanguage{english}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Fig. 4.} \textbf{GS promote NAD\textsuperscript{+}-dependent
SIRT1 activation in cardiomyocytes and neurons} . \textbf{(A)} The
NAD\textsuperscript{+} levels in H9c2 and neurons treated with different
concentrations of GS for 48 h were measured by a luminescence -based
kit. \textbf{(B)} The NAD level in control and GS-treated H9c2 cells was
analyzed by the LC-MS/MS method. \textbf{(C)} After GS treatment for 8
h, SIRT1 and PGC-1\selectlanguage{greek}α \selectlanguage{english}mRNA levels in H9c2 cells and primary neurons were
analyzed by qPCR analysis. \selectlanguage{greek}β-\selectlanguage{english}Actin is used as internal
control.\textbf{(D-E)} The levels of SIRT1, PGC-1\selectlanguage{greek}α, \selectlanguage{english}Nrf1, and Nrf2 in
H9c2 and PC12 cells pretreated with different concentrations of GS for
48 h were examined by Western blot analysis. \selectlanguage{greek}β-\selectlanguage{english}Actin is loading control.
Semi-quantitative analysis of relative expression for each protein is
shown on the right. Ctrl: control group. *\emph{P} \textless{} 0.05,
**\emph{P} \textless{} 0.01, and ***\emph{P} \textless{}
0.001\emph{versus} Ctrl group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Fig. 5.} GS increase NAD\textsuperscript{+}-dependent activation
of SIRT1 in drosophila and mice. \textbf{(A)} The
NAD\textsuperscript{+}level and NAD\textsuperscript{+}/NADH ratio in the
brain tissue of 10 flies from control and GS treatment groups were
analyzed by a bioluminescent detection kit. \textbf{(B)} The dSir2
(SIRT1) level in the drosophila brain of control and GS groups was
detected by Western blot and quantified as the relative expression on
the right. JLA20 is the loading control. \textbf{(C)} The activities and
climbing distance of 30 flies per group were investigated after
treatment with GS at 1 mg/mL for 7 d. \textbf{(D)} IHC staining of SIRT1
in the heart and brain tissues from control and GS-treated mice (10
mg/kg, 21 days) was performed. Scale bar=50 \selectlanguage{greek}μ\selectlanguage{english}m. Ctrl: control group.
*\emph{P} \textless{} 0.05, **\emph{P} \textless{} 0.01, and
***\emph{P}\textless{} 0.001 \emph{versus} Ctrl group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Fig. 6.} GS increase mitochondrial function against the hypoxia-
and OGD-induced injuries of the cardiomyocytes through the activation of
SIRT1. \textbf{(A)} H9c2 cells were pretreated with GS for 48 h and kept
in the medium with 800 \selectlanguage{greek}μ\selectlanguage{english}M CoCl\textsubscript{2} (hypoxia) or serum and
glucose-free and 800 \selectlanguage{greek}μ\selectlanguage{english}M CoCl\textsubscript{2} (OGD) for 9 h. Cell
viability was analyzed by the CCK8 assay kit and showed as the
percentages. \textbf{(B-C)} Basal OCR and ATP content were measured in
H9c2 cells, after GS pretreatment for 48 h and hypoxia/OGD incubation
for 9 h. \textbf{(D)} H9c2 cells treated with GS for 48 h and incubated
with hypoxia exposure for 9 h were collected and lysed to analyze the
levels of complex I-V by Western blot. \textbf{(E)} Relative levels of
CV-ATP5A, CIII-UQCRC2, CIV-MTCO1, CII-SDHB, and CI-NDUFB8 from (D) were
calculated by Image J. \textbf{(F)} After GS treatment for 48 h, the
expressions of SIRT1 and PGC-1\selectlanguage{greek}α \selectlanguage{english}in H9c2 cells subjected to hypoxia and
OGD injury were examined by Western blot. \textbf{(G)} Bar graphs show
relative levels of SIRT1 and PGC-1\selectlanguage{greek}α \selectlanguage{english}from (F). \selectlanguage{greek}β-\selectlanguage{english}Actin is loading
control. Ctrl: control group, CoCl\textsubscript{2}: cobalt chloride.
*\emph{P} \textless{} 0.05, **\emph{P} \textless{} 0.01, and
***\emph{P}\textless{} 0.001 \emph{versus} Ctrl group;
\textsuperscript{\#}\emph{P}\textless{} 0.05 and
\textsuperscript{\#\#}\emph{P} \textless{} 0.01\emph{versus} hypoxia or
OGD group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Fig. 7.} The effect of GS on mitochondrial function was
abrogated by NAM, a SIRT1 inhibitor. \textbf{(A-B)} Basal OCR and MRC in
H9c2 cells were measured, after the treatment by GS and/or NAM for 48
h.\textbf{(C)} H9c2 cells were treated with GS and/or NAM for 48 h to
measure ATP level by a bioluminescence detection kit. \textbf{(D)} The
expressions of CV-ATP5A, CIII-UQCRC2, CIV-MTCO1, CII-SDHB, and CI-NDUFB8
in H9c2 cells treated with GS and/or NAM for 48 h were detected by
Western blot. \textbf{(E)} Relative levels of five mitochondrial
complexes from (D) are shown, after the quantification by
ImageJ.\textbf{(F)} The effect of GS combined with NAM on the
NAD\textsuperscript{+} level was analyzed by a NAD/NADH-Glo assay
kit.\textbf{(G-H)} Total proteins of H9c2 cells treated with GS and/or
NAM for 48 h were prepared and followed by Western blot analysis using
antibodies against SIRT1, PGC1\selectlanguage{greek}α, \selectlanguage{english}Nrf1, Nrf1, and \selectlanguage{greek}β-\selectlanguage{english}Actin. Relative
expression of these proteins was quantified with ImageJ and shown on the
right. \selectlanguage{greek}β-\selectlanguage{english}Actin is loading control. Ctrl: control group, OCR: oxygen
consumption rate, MRC: maximal respiration capacity.
\textsuperscript{*}\emph{P} \textless{} 0.05
and\textsuperscript{***}\emph{P} \textless{} 0.001 \emph{versus} Ctrl
group; \textsuperscript{\#}\emph{P} \textless{} 0.05
and\textsuperscript{\#\#}\emph{P} \textless{} 0.01 \emph{versus} GS+NAM
group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Fig. 8.} The effects of main ginsenosides monomers on
SIRT1-mediated mitochondrial function in H9c2 cells. \textbf{(A-B)}Basal
OCR and ATP production were detected in H9c2 cells treated with GS or
different ginsenoside monomer for 48 h as described in Materials and
Methods. \textbf{(C-D)} After the pretreatment with different
ginsenosides monomers at the composition percentage of GS (5\selectlanguage{greek}μ\selectlanguage{english}g/mL) for
48 h, the levels of HK-II, MPC1 and SIRT1 in H9c2 cells were detected by
Western blot. Relative HK-II, MPC1, and SIRT1 expressions were
quantified after the normalization to \selectlanguage{greek}β-\selectlanguage{english}Actin. \selectlanguage{greek}β-\selectlanguage{english}Actin is the loading
control for Western blot analysis. Ctrl: control group, OCR: oxygen
consumption rate. HK-II: Hexokinase-II, MPC1: mitochondrial pyruvate
carrier 1. *\emph{P} \textless{} 0.05, **\emph{P} \textless{} 0.01, and
***\emph{P} \textless{} 0.001 \emph{versus} Ctrl group.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Fig. 9.} Scheme indicating the molecular mechanism of total
ginsenosides from Panax ginseng for promoting aerobic cell respiration
and SIRT1 mitochondrial biosynthesis in cardiomyocytes and neurons. GS
can increase the level of key metabolic enzymes and mitochondrial
protein complexes II-IV to enhance TCA cycle and oxidative
phosphorylation. Meanwhile, GS can promote the
NAD\textsuperscript{+}level and activate the SIRT1-PGC-1\selectlanguage{greek}α \selectlanguage{english}pathway to
increase mitochondrial biosynthesis. GS-induced increased level (Red)
and decreased level (Blue).

\selectlanguage{english}
\FloatBarrier
\end{document}

