4. DISCUSSION
The number of elderly patients in need of treatment related to bone
formation has increased, and they may have a decreased capability of
bone remodeling. Therefore, stimulating bone and cartilage formation
from natural sources that selectively enhance osteogenesis and
chondrogenesis without stimulating adipogenesis are desirable.
Andrographis paniculata has long been used in traditional
medicine in Asia. In Thailand, this medicinal plant was selected by the
Ministry of Public Health as one of the medicinal plants to be included
in “The National List of Essential Drugs A.D. 1999” (List of Herbal
Medicinal Products(Pholphana et al., 2004)). Several studies have shown
that its main bioactive component, AG, has a broad range of beneficial
pharmacological effects, such as antiviral, anti-mararial,
immunostimulatory, and anti-inflammatory. Moreover, it contains
bioactive molecules like estrogen. In the present study, we have
significant findings. We have demonstrated that AG did not influence
cell viability when it was used at concentrations ranging from 1.56 to
12.5 μM. This indicates that AG did not have an effect on human
mesenchymal stem cells. In addition, we also originally demonstrated for
the first time that AG promoted osteogenic and chondrogenic
differentiation of SPFP-MSCs and suppressed their adipogenic
differentiation.
There are two major modes of osteogenesis, and both involve the
transformation of a preexisting mesenchymal tissue into chondroblast,
chondrocytes, and osteoblasts. The mesenchymal stem cells differentiate
into chondroblasts and chondrocytes and this cartilage is later replaced
by bone. The important markers for osteogenic differentiation,Runx2 and OPN , where Runx2 is a master regulator in
the late stage marker of osteogenesis and OPN is the important
intermediate stage marker during the differentiation into mature
osteoblasts for osteogenic differentiation followed by matrix maturation
and matrix mineralization(Jensen et al., 2011; Lee et al., 2000;
Lindfors, Heikkilä, & Aho, 2008; Long, 2001). Zhai et al. also showed
that AG exhibits the inhibitory effects on osteoclastogenesis and
osteoclast function in vitro and in vivo through the
suppression of nuclear factor-kappaB (NF-κB) and
extracellular-signal-regulated kinase (ERK) signaling pathways (Zhai et
al., 2014). After 21 days of
culture with
SPFP-MSCs,
AG stimulated the expression of Runx2 , OPN and increased
the calcium deposition activity in
a dose-dependent manner, suggesting that AG could enhance the osteogenic
ability of SPFP-MSCs.
In order to investigate whether AG
could inhibit adipogenic differentiation of SPFP-MSCs, the
adipocyte-specific markers includingPPAR-γ2 and LPL and
also qualitative of adipocytes number (Oil Red O staining) were
examined. The PPAR-γ2 which has been shown to play an important
role in mature adipocyte differentiation as a terminal differentiation
marker, lipid storage, insulin sensitization and can be activated by
fatty acids(Auwerx, 1999; Xu et al., 1999). LPL is thought to be
an early marker of adipogenesis and highly expressed during adipogenic
differentiation(Shaughnessy, Smith, Kodukula, Storch, & Fried, 2000).
As shown in this study, after 21 days of culture treatment with AG. AG
suppressed both PPAR-γ2 and LPL mRNA expression by
inhibiting adipocyte cells formation. These results suggest that AG
could suppress the adipogenic differentiation both qualitatively and
quantitatively.
In this study, we used an in vitro cell pellet culture of chondrogenesis
for 21 days derived from SPFP-MSCs and found the different
concentrations of AG were able to significantly enhance differentiation
in a dose-dependent manner by upregulating a number of genes associated
with chondrogenesis such as Sox9 and Aggrecan under
chondrogenic plus andrographolide extract conditions(de Crombrugghe et
al., 2000). Moreover, different concentrations of AG revealed a
significant higher cell growth rate than the control. In addition,
qualitative study exhibited increased synthesis of matrix proteoglycans
and visible formation of glycosaminoglycan when stained with Toluidine
blue and Alcian blue(Chamberlain, Fox, Ashton, & Middleton, 2007). The
resulting pellet was fixed with H&E stain for the identification of
chondrocyte morphology and revealed a significant dark stain in doses
dependent of AG. The intensities of the stain were dependent on the dose
of AG.
In conclusion, this study is the first to demonstrate that
Andrographolide in the concentration where cell viability was more than
90% could promote cell proliferation coupled. Moreover, Andrographolide
could promote osteogenic and chondrogenic differentiation in a
dose-dependent manner whereas inhibit their adipogenesis of SPFP-MSCs.
Furthermore, Andrographolide is associated with regulation of
osteocyte-specific markers including Runx2 , OPN and
chondrocyte-specific markers including Sox9 and Aggrecanactivity, and its anti- adipogenic effect is associated with blockingPPAR-γ2 and LPL signaling. Our data also strongly
suggested that Andrographolide
could be developed and expected for the possibility to translate the
results to human clinical practice
as a treatment for regenerative medicine for cartilage and bone
regeneration.