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.