ABSTRACT
【Objective】 To investigate the effect and mechanism of achyranthes bidentata polysaccharides （ABPS） on the differentiation of bone mesenchymal stem cells （BMSCs） into nucleus pulposus-like cells. 【Methods】 Five 4-week-old SD rats were sacrificed and their BMSCs were isolated and purified by repeated adherent method. The third-generation BMSCs were treated with different concentrations of ABPS for 48 h. The cell viability was detected by MTT method. The highest concentration of ABPS was selected for subsequent experiments. According to different treatment methods， cells were divided into control group （conventional culture）， ABPS group （200 mg/L of ABPS）， Notch1 group （transfected with Notch1）， Notch1 negative control group （transfected with Notch1 negative control）， and ABPS+Notch1 group （200 mg/L of ABPS was added after transfection with Notch1）. After 14 days of induction， the cell survival rate was measured by MTT method. CollagenⅡ was detected by immunohistochemical staining. The mRNA and protein expression levels of collagenⅡ， aggrecan， Notch1 and Hes1 were detected by qPCR and Western Blotting respectively. The glucosaminoglycan （GAG） was detected by alcian blue method on the 1st， 4th， 7th， 10th and 14th day of cell culture. 【Results】 After BMSCs were treated with 400 mg/L of ABPS for 48 h， the cell survival rate was significantly lower than that in the control group （P<0.05）. When the concentration was ≤200 mg/L， ABPS had no significant toxic effect on the growth of BMSCs. After 14 days of induction， compared with the control group， A value， GAG content in the medium， the expressions of collagen II and aggrecan mRNA and protein in the ABPS group were increased， and the expressions of Notch1 and Hes1 mRNA and protein were decreased （P<0.05）. The relative cell viability， GAG content in the medium， the expressions of collagen II and aggrecan mRNA and protein in the Notch1 group were decreased， and the expressions of Notch1 and Hes1 mRNA and protein were increased （P<0.05）. ABPS+Notch1 could partially reverse the inhibitory effect of Notch1 overexpression on the differentiation of BMSCs into nucleus pulposus-like cells. 【Conclusion】 ABPS can promote the differentiation of BMSCs into nucleus pulposus-like cells， and the mechanism may be related to the inhibition of Notch1 pathway.
ABSTRACT
@# Objective: To explore the role of Thy-1 cell surface antigen（Thy-1）in promoting epithelial-mesenchymal transition (EMT) in liver cancer HepG2 and MHCC-97 cells by regulating Notch1 pathway. Methods: MHCC-97 cells with high metastatic characteristics and HepG2 cells with low metastatic characteristics were selected as subjects. WB was used to detect the expression levels of Thy-1 and Notch1 in cells. MHCC-97 and HepG2 cells were transfected with lentivirus to construct cells with high and low expression of Thy-1 protein. Cells were treated with Notch1 agonist rhNF-κB (1 gsu/ml) and Notch1 inhibitor MW167 (100 μmol/L) for 24 h respectively. Transwell assay was used to detect the effect of Thy-1 expression on cell invasion; qPCR was used to detect the effect on Notch1 mRNA expression; WB was used to detect the effect on intracellular EMT-related protein expression. Results: The expression levels of Thy-1 and Notch1 in MHCC-97 cells were higher than those in HepG2 cells (P<0.05). Thy-1 overexpressing HepG2 cells and Thy-1 low expressing MHCC-97 cells were successfully constructed. Compared with HepG2 cells, the invasion ability of Thy-1 overexpressing HepG2 cells was significantly enhanced (183.23±55.34 vs 475.78±80.37, P<0.05), vimentin expression was significantly increased (P<0.05), epithelial cadherin protein expression was significantly decreased (P<0.05), and the expression level of Notch1 mRNAwas significantly increased (P<0.05). Compared with MHCC-97 cells, the invasion ability of Thy-1 silenced MHCC97 cells was significantly decreased (543.56±77.94 vs 237.44±62.18, P<0.05), the expression of vimentin was significantly decreased (P<0.05), epithelial cadherin protein expression was significantly increased (P<0.05), and Notch1 mRNA expression level was significantly decreased (P<0.05). Treatment of liver cancer cells with Notch1 activators or inhibitors can reverse the changes caused by Thy-1 silencing or overexpression. Conclusion: Thy-1 can affect the EMT process of HepG2 and MHCC-97 cells by regulating the expression of Notch1.