4 Discussion
NO, IL-1β, IL-6, and TNF-α participate in the pathogenesis of
inflammatory diseases[13, 14]. We found that Tube inhibited the
expression of IL-1β, IL-6, and iNOS and suppressed NO production but did
not affect TNF-α expression in LPS-induced RAW264.7 cells. We also
measured NF-κB and MAPK signaling pathway-related proteins (e.g., NF-κB
p65 and MAKP p38) and found that Tube had no significant effect on the
phosphorylation levels[15-20]. These findings suggest that Tube may
not inhibit inflammation through the classical NF-κB signaling pathway.
Small molecules of bacterial origin can be recognized by cell surface
receptors and activate relevant signal transduction, leading to the
reprogramming of macrophage energy metabolism from mitochondrial
oxidative phosphorylation to a high rate aerobic glycolysis; this
reprogramming regulates the synthesis and accumulation of macrophage
immune metabolites, which in turn affects the expression of inflammatory
factors.
Glucose transporter protein (GLUT1) and lactate dehydrogenase (LDH) play
pivotal roles in glycolysis. They convert glucose into pyruvate and
lactate, which then enter the tricarboxylic acid (TCA)[21, 22]. Tube
increased the mRNA levels of fructose-2,6-bisphosphatase 3, HK2, and
GLUT1, the rate-limiting enzyme genes of glycolysis, but decreased mRNA
levels of LDH. These findings suggest that Tube causes cellular
accumulation of pyruvate; therefore, we measured the pyruvate content in
RAW264.7 cells and found thatTube did increase it. Notably, we found
little effect of Tube on basal and maximal respiration in RAW264.7
cells; therefore, we tentatively concluded that Tube increases the
production of pyruvate, an intermediate product of glycolysis, and does
not increase levels of mitochondrial oxidative respiration. Pyruvate is
involved in energy metabolism by entering the TCA cycle; therefore,Tube
might exert its anti-inflammatory effect by regulating the energy
metabolic process.
Macrophage metabolic pathways are altered during inflammatory responses,
known as “metabolic reprogramming”[23]. We found that metabolic
reprogramming participates in the development of inflammatory responses
by regulating the expression of macrophage inflammatory factors[24].
Moreover, we observed an intermediate product, itaconic acid. During LPS
stimulation, macrophages upregulate expression of immune response gene
1, a mitochondria-related enzyme that catalyzes the conversion of
cis-aconitate into itaconate and inhibits succinate dehydrogenase
through competitive binding, creating a metabolic breakpoint in the TCA
cycle and blocks the TCA cycle[17, 25] . Itaconic acid inhibits the
electron transport chain in mitochondria[25, 26], and it regulates
the transcription of IL-1β and IL-6 through the ATF3-IκBζ axis; however,
it has no significant effect on the transcription level of TNF-α[8,
10]. Therefore, we measured expression levels of downstream effector
molecules of itaconic acid using RT-PCR experiments and found that Tube
was associated with the regulatory role of itaconic acid.
IκBζ is a transcriptional regulator of the non-classical NF-κB signaling
pathway. It binds to the NF-κB p50 subunit and promotes the
transcription of pro-inflammatory factors, including IL-6, IL-1β, and
IL-1α[27, 28]. Electrophilic stress induced by itaconic acid
inhibited IκBζ by upregulating activating transcription factor 3 (ATF3),
reducing the production of the pro-inflammatory factor IL-6[8].
Western blot showed that Tube increased the ATF3 protein level and
inhibited IκBζ protein levels. These findings suggest that Tube exerts
its anti-inflammatory effect by reprogramming glucose metabolism in
macrophages, affecting the metabolism of the small molecule itaconic
acid. Finally, to confirm the necessity of the anti-inflammatory effect
of itaconic acid on Tube, we used an inhibitor of itaconic acid,
citraconate (that competitively inhibits aconitate decarboxylase
1)[29]. Citraconate impaired the protective effect of Tube in the
LPS-induced acute inflammation model. Citraconate also attenuated the
downregulation of IL-1β and IL-6 by Tube. These results suggest that
Tube requires the involvement of itaconic acid to exert its inflammatory
inhibitory effects.
In summary, tubeimoside Ⅲ regulates the reprogramming of macrophage
glucose metabolism, increasing the content of small metabolic molecule
itaconic acid, inhibiting the expression of IL-1β, IL-6, and iNOS, and
reducing the inflammatory response.