| Objective: To study the effect of in vitro apoptosis on activated rat hepatic stellate cells(HSCs)by Ferulic acid(FA)combined with Adipose-Derived Mesenchymal Stem Cells(ADMSCs).To further explore if the mechanism of apoptosis acceleration by FA and ADMSCs can be achieved by regulating the TGF-β/Smad signaling pathway of HSCs.Methods: 1.Detection of the best concentration of FA:(1)HSCs were cultured with complete medium till the cellular fusion rate was 60%-70% first,and then these cells were changed into serum-free medium and cultured for 12 h,24h,6h,36 h and 48 h respectively.Methyl thiazolyl blue(MTT)assay was used to measure the OD value of HSCs and the growth curve of HSCs was drawn to confirm the optimum time for cell culture.(2)HSCs were cultured with complete medium till the cells density was60%-70%,and were processed with different concentration of FA(25-800μg/m L)in serum-free mediums.The cell proliferation inhibition rate of HSCs was measured by MTT assay and IC50 was calculated to determine the optimal concentration of FA.(3)Cells culture of ADMSCs was processed in the same way as HSCs.The effect of FA on the survival rate of ADMSCs was also measured with the MTT assay.2.Experimental grouping:In our study,upper and lower indirect co-culture system of ADMSCs and HSCs at 1:5 ratio was established by Transwell.ADMSCs and HSCs were cultured till the density wasabout 60%-70%,afterwards the two kinds of cells were cultured with serum-free medium.Then divide HSCs into 4 groups:the blank control group(HSCs alone),the FA control group(HSCs was processed by adding FA),the ADMSCs control group(HSCs was cocultured with ADMSCs)and the FA/ADMSCs experimental group(FA was added in co-culture system of ADMSCs and HSCs).3.the effect of apoptosis on HSCs by FA combined with ADMSCs: the apoptosis rate of HSCs in each group was examined by flow cytometry;the concentration of collagen type I(Collagen I)in supernate of each group was examined by Enzyme-linked immunosorbent assay(ELISA)4.The expression of TGF-β1,Smad2,Smad3 and Smad7 m RNA were examined by q RT-PCR;the protein levels of TGF-β1,Smad7 and phosphorylated-Smad2/3(p-Smad2/3)were detected by Western blot analysis.Results:1.MTT results showed that the optimum time for cell culture was 24 h after they were changed into serum-free medium;After processed with different concentration of FA for 24 h,the cell proliferation inhibition rate of HSCs in experimental groups all increases observably(P<0.05)compared with the control group without FA.The best concentration of FA is 243μg/ml calculated with Chinese version of SPSS22.0.Combined with previous experiments in this project,the final concentration of FA was determined to be 200μg/ml;MTT experiment results suggest that this concentration of FA has no obvious effect on the survival rate of ADMSCs.2.Meanwhile,the levels of TGF-β1,Smad2 and Smad3 m RNA were significantly decreased,and the Smad7 m RNAincreased in experimental group.3.Moreover,the results of Western blot analysis showed that the expression levels of TGF-β1 protein and p-Smad2/3were significantly decreased,while the Smad7 protein was significantly increased in experimental group.Conclusion: The mechanism that the combination of FA and ADMSCs promoted apoptosis of HSCs was probably the expression of TGF-β1 down-regulated in HSCs,which led to a decrease of activity in downstream Smad2/3 phosphorylation and an increase of expression in Smad7,accelerating the apoptosis of HSCs ultimately. |
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