The Effect And Mechanism Of Sodium Ferulate On The Apoptosis Of Hepatic Stellate Cells Induced By Rat Adipose-derived Stem Cells

Objective: To promote the activation of hepatic stellate cells hepatic(HSCs)apoptosis is an important research direction in the treatment of liver fibrosis.Apoptosis of HSCs can be induced by co-culture of adipose derived stem cells(ADSCs).On the basis of this,the research explore whether sodium ferulate(SF)can enhance the apoptosis inducing effect,and discuss the underlying mechanism.To provide the experimental basis for the united application effect of SF and ADSCs in the treatment of liver fibrosis.Methods : 1.The isolation,culture and identification of ADSCs :Inguinal adipose tissue were gotten from SD Rats,to remove the blood vessels and fascia,then cut into pieces.To obtain the Primary cellof ADSCs with collagenase digestion method,and observing cell growth state and morphological changes.After passage and purification of cells,the 3rd generation ADSCs were collected.Detection of CD29,CD44,CD45,CD90 expression by flow cytometry.2.Recovery of HSC-T6 cell line,then the expression of ?-SMA was detected by immunocytochemistry to demonstrate whether the cells are in activated state.3.Co-cultureexperiment withsodium ferulate:Using transwell co-culture system of ADSCs and HSCs were trained.Experiment is divided into four groups,each group five wells,as followed.Group A: ADSCs+HSCs,group B: SF+ADSCs+HSCs,group C:SF + ADSCs,group D: HSCs.After 48 hours,collect each hole HSCs,Annexin V PI staining and flow cytometry to test and compare apoptosis rate of HSCs in each group.4.SF pretreatment of ADSCs and co-culture experiment: SF pretreatment of ADSCs,then co-culture with HSCs as the experimental group.ADSCs without any processing co-culture with HSCs as control group.After 48 hours Annexin V PI staining and flow cytometry to test and compare the two groups of HSCs apoptosis rate.5.Effects of sodium ferulate treatment on Characteristics of ADSCs:500?g/mLSFeffect on ADSCs48 h,evaluate the effect of sodium ferulate(SF)on morphology,growth curve andsurface markers of ADSCs.6.Hepatocyte growth factor(HGF)and nerve growth factor(NGF)concentration changes in ADSCs culture supernatant which were treated with SF: The ADSCs were treated with different concentrations of SF(0?g/mL,100?g/mL,500?g/mL,1000?g/mL)for 48 hours.The concentration of HGF and HGF were measured by using ELISA kits.7.HGFmRNA and NGFmRNA expression changes in ADSCs which were treated with SF: The ADSCs were treated with different concentrations of SF(0?g/mL,100?g/mL,500?g/mL,1000?g/mL).After 48 hours,total RNA was extracted.Real-time PCR detection of HGFmRNA and NGFmRNA expression.Results:1.In the 3rd generation,the expression of surface markers was consistent with the expression of ADSCs surface markers in the literatures.2.HSC-T6 cells grow well after recovery.Immunocytochemical result sindicate HSC-T6 as the activation of HSCs.3.After 48 hours,SF joined the co-culture system: Apoptosis rate of Group B is higher than that of group A,group C and group D(P<0.05 or P<0.01).Apoptosis rate of Group A,group B and group C is higher than group D(P<0.05).4.Pretreatment of ADSCs with SF after 48 hours,then co-culture with HSCs.The apoptosis rate of the experimental group is higher than that of the control group(P<0.01).5.After 500?g/mLSF processed,cells in morphology,growth curve and surface markers are similar tounprocessed ADSCs.6.HGF concentration of the five experimental groups was higher than the control group(P < 0.05 or P <0.01).And NGF level increased with increasing the concentration of SF except for 50?g/mL group(P < 0.01).7.there was no Statistically significance compared with 100?g/mL group and control group with HGFmRNA(P>0.05),While the 500?g/mL and 1000?g/mL groups increased significantly(P<0.01).NGFmRNA of the three experimental groups was higher than that of the control group(P<0.05 or P<0.01).Conclusion: 1.Pretreatment of ADSCs by sodium ferulate could enhance the apoptosis effect on HSCs.2.SF may increase the secretion of HGF and NGF,promote the expression of HGFmRNA and HGFmRNA in ADSCs,binding of c-met receptor and P75 receptor,respectively,to promote the apoptosis of HSCs.3.The use of a certain concentration of sodium ferulate,ADSCs did not have a clear differentiation.