## 4 Discussion  
Excessive adipose tissue, which is one of the easily conformable markers of obesity progression, is mainly a result of uncontrolled formation of new adipocytes. The formation of adipose tissue occurs via increased lipid storage via adipocytes differentiated from mesenchymal stromal cells in either adipose tissue or bone [26]. This uncontrolled formation of new adipocytes is often linked with other diseases such as osteoporosis as an underlying cause or stimulatory factor [27]. The obesity-linked complications and deterioration that affects the behaviors of bone-marrow MSCs are the main cause of several other metabolic syndrome-related complications. Therefore, the current study tested the effects of loliolide on adipogenesis using hBM-MSC models. To the best of our knowledge, this is the first report of loliolide-aided inhibition of adipogenesis in MSCs. Loliolide has been reported a bioactive phytochemical with various health benefits due to its antioxidant, antitumor, antiaging, and anti-inflammatory properties [17; 20]. Although its effect on inhibiting adipogenesis was reported in murine pre-adipocytes, there has been no report of loliolide indicating its effect on differentiation of any type of MSCs. As a part of ongoing research to develop anti-obesity natural products, this study employed hBM-MSCs as the model to screen the effect of loliolide on adipogenesis and provide comparison data on whether loliolide's effect on murine pre-adipocytes could be observed in human MSCs, which are the source of adipose tissue in the human body. Lipid accumulation as intracellular lipid droplets is a key characteristic of adipocytes and observed during the maturation of adipocytes [28]. Loliolide treatment decreased the lipid accumulation in adipo-induced hBM-MSCs, indicating its inhibitory effect in early stages of adipogenic differentiation or stimulatory effect on lipolysis. To determine whether loliolide inhibits adipogenic differentiation, the PPAR\(\gamma\) pathway was examined. PPAR\(\gamma\) is a well-known and intensely studied transcription factor for adipogenesis along with its upstream effector C/EP\(\alpha\)[29]. Loliolide-treated hBM-MSCs exerted a dose-dependent drop at the levels of mRNA and protein expression for PPAR\(\gamma\) and C/EBP\(\alpha\). Treatment with loliolide also suppressed the expression of SREBP1c, which is an adipocyte-specific gene [30]. These results suggest that loliolide inhibited the adipogenic differentiation with an influence on PPAR\(\gamma\) signaling by modulating early stages of adipogenesis via PPAR\(\gamma\) and C/EBP\(\alpha\) expression. To determine the underly mechanisms behind the anti-adipogenic effect of loliolide, signaling pathways that regulate PPAR\(\gamma\) activation were analyzed. The AMPK and MAPK signaling pathways are such pathways with vital roles in adipogenesis. MAPK-mediated regulation of adipogenesis is primarily dependent on the cell type, experimental conditions, and extracellular stimuli [24]. Reports have shown that, while ERK phosphorylation may be involved in both stimulation and inhibition of adipogenesis, p38 and JNK phosphorylation is in mostly involved in adipogenesis stimulation [31; 32]. Nevertheless, the current results show that adipogenesis in hBM-MSCs came with elevated activation of MAPKs. Loliolide was able to prevent adipogenesis-induced phosphorylation of p38 and JNK but was ineffective against ERK. Although being closely related with MAPK activation, the role of AMPK in adipogenesis is readily reported. It is known that AMPK activation playsvarious roles in cellular mechanisms against obesity, diabetes, and metabolic diseases [33]. In addition to its roles in energy and lipid metabolisms, AMPK signaling is involved in adipogenesis by being suppressed concurrently during PPAR\(\gamma\) activation [34]. Inhibiting AMPK activation has been shown to increase the expression of adipogenic markers including PPAR\(\gamma\).