## 3 Results
### Wnt/\(\beta\)-catenin Signaling during Adipogenic Differentiation of hBM-MSCs  
The effect of loliolide on the Wnt/\(\beta\)-catenin pathway was determined by detecting Wnt 10b and \(\beta\)-catenin protein levels in adipo-induced hBM-MSCs (Figure 4). The results show that adipogenic differentiation of hBM-MSCs notably inhibited the levels of Wnt 10b whereas loliolide treatment (10 uM) increased Wnt 10b levels significantly. Next, the downstream effector of Wnt activation, \(\beta\)-catenin, was evaluated. Adipogenic differentiation decreased the Ser675 phosphorylated levels of \(\beta\)-catenin along with Axin protein, which is a part of the \(\beta\)-catenin degradational complex, indicating that adipogenesis resulted in increased degradation of \(\beta\)-catenin. The results show no changes in Axin levels following loliolide treatment; however, p-(Ser675) \(\beta\)-catenin levels were increased substantially (Figure 4A). The effect of loliolide on stimulating Wnt signaling was also confirmed by immunostaining of Wnt 10b in adipo-induced hBM-MSCs. Fluorescence staining of Wnt 10b protein displayed that loliolide treatment significantly attenuated adipogenesis-induced decrease in Wnt 10b levels (Figure 4B). Further support for the \(\beta\)-catenin activity stimulatory effect of loliolide came from analyzing nuclear and cytosolic fractions of the cells by Western blot. Loliolide treatment decreased the p-\(\beta\)-catenin levels in cytosol while increasing its levels in nuclear fractions (Figure 4C). In addition, decreased PPAR\(\gamma\) levels in nuclear fractions of adipo-induced hBM-MSCs indicated that loliolide treatment suppressed the adipogenic differentiation through enhancement of the Wnt/\(\beta\)-catenin pathway activation, which resulted in inhibited PPAR\(\gamma\) levels.  
Figure 4: The effect of loliolide treatment on the Wnt/\(\beta\)-catenin pathway. The hBM-MSCs were differentiated into adipocytes in six-well plates with or without loliolide treatment (10 uM). One group was left untreated and non-differentiated as  
the negative control. The hBM-MSCs at Day 10 of differentiation were used for the analysis of total and phosphorylated (p-) Wnt, \(\beta\)-catenin, and Axin (**A**) using immunoblotting. \(\beta\)-actin was used as housekeeping protein. The inhibitor was compound C, an AMPK activation inhibitor. Different letters (a-c) above the sample groups indicate that the difference between each group is statistically significant (\(p<0.05\)) compared to the untreated differentiated control. (**B**) Images of fluorescence-stained hBM-MSCs at incubation Day 10 after adipogenesis inducement (except undifferentiated untreated group which was fed growth medium only), stained with FITC conjugated anti-Wnt 10b antibody (green) and DAPI (blue) to highlight the nuclei. Scale bar, 100 \(\upmu\)m. (**C**) Cell lysates were separated between cytosolic (c) and nuclear (n) fractions, and protein levels of p- \(\beta\)-catenin and PPAR\(\gamma\) were detected using immunoblotting. Lamin-B1 was used as the internal control. Different letters (a-c) above the sample groups indicate that the difference between each group is statistically significant (\(p<0.05\)) compared to the untreated differentiated control.