| Background:The activation,proliferation,secretion of cytokines and extracellular matrix of hepatic stellate cells?HSC?are important links in the process of hepatic fibrosis,and promoting apoptosis of hepatic stellate cells is one of the therapeutic strategies for anti-hepatic fibrosis.Evidence shows that 17?estradiol plays a protective role in hepatic fibrosis.However,the specific mechanism of 17?estradiol against fibrosis and the related signaling pathways still need to be further explored.Objective:This study used PDGF-BB to activated mouse hepatic stellate cells JS1 as a model to study the effect of 17?estradiol on the apoptosis of mouse hepatic stellate cells JS1 and the regulatory effect of Wnt signaling pathway,and to explore the potential mechanism of 17?-estradiol against hepatic fibrosisMethods:1.Expanded the mouse hepatic stellate cells JS1 were cultured in vitro.10-9-10-3mol/L 17?-estradiol was used to stimulate the proliferation of mouse hepatic stellate cells JS1 stimulated by PDGF-BB of 0?g/L,10?g/L and 20?g/L respectively.The OD values of 24,48 and 72h cells were measured by CCK-8 method to select the optimal concentration of mouse hepatic stellate cells JS1 to inhibit the activity of mouse hepatic stellate cells.2.Cells were divided into five groups:drug group(10-7 mol/L,10-8 mol/L,10-9mol/L 17?estradiol+PDGF-BB 20?g/L),PDGF group?PDGF-BB 20?g/L?and blank group?PBS?.The apoptosis rate of mouse hepatic stellate cells JS1 was detected by cell flow cytometry which use Annexin V-PE/7-AAD staining.Cells were divided into three groups:drug group,PDGF group and blank group.Western blot was used to detect the expression of?–SMA,PARP,and Bax protein in mouse hepatic stellate cells JS1 under the intervention of 17?estradiol.3.Cells were divided into three groups:drug group(10-7 mol/L17?estradiol+PDGF-BB 20?g/L),PDGF group?PDGF-BB 20?g/L?and blank group?PBS?.Western blot was used to detect the expression of Wnt/?-catenin signaling pathway protein?Wnt-3a,?-Catenin?in mouse hepatic stellate cells JS1 under the intervention of 17?estradiolResult:1.In the experimental groups with different PDGF-BB,there was no statistically significant difference in the OD value between the DMSO group and the 0 mol/L group 17?estradiol group at 24,48,and 72h?all P>0.05?,and the OD values of the DMSO group and the 0 mol/L 17?estradiol group were significantly higher than other drug groups?all P=0.000?.Tukey method was used for group-by-group comparison,In the experimental group without PDGF-BB,Among 10-9-10-7 mol/L group,10-9mol/L group had the highest OD value,10-8 mol/L group had the second OD value,10-7 mol/L group had the lowest OD value,and the difference is statistically significant?all P=0.000?.The OD value difference in the 10-7-10-4 mol/L group were not statistically significant?all P>0.05?.In the experimental group of 10?g/L PDGF-BB,among the 10-9-10-7 mol/L group,the 10-9mol/L group also showed the highest OD value,followed by the 10-8 mol/L group,10-7 mol/L group was the lowest,and the difference was statistically significant?all P=0.000?.There was no statistically significant difference in the OD value of the 10-7-10-4 mol/L group?all P>0.05?.In the experimental group of 20?g/L PDGF-BB,among the 10-9?10-7 mol/L group,the 10-9mol/L group also had the highest OD value,followed by 10-8 mol/L group,10-7 mol/L group was the lowest?all P=0.000?.10-7-10-4 mol/L group had no statistically significant difference in OD value?all P>0.05?.10-3 mol/L 17?estradiol significantly inhibits the activity of mouse hepatic stellate cells JS1.2.The results of flow cytometry,using Tukey method for group-by-group comparison,showed that there was no statistically significant difference in the total apoptosis rate between the blank group and the PDGF group?P=0.779?,while the total apoptosis rates of the three drug groups were all higher than the blank group and PDGF group,the difference was statistically significant?all P<0.05?.The total apoptosis rate in the10-7 mol/L 17?estradiol group was higher than that in the 10-8 mol/L group and the10-9 mol/L group,the difference was statistically significant?all P<0.05?;The total apoptosis rate in the 10-8 mol/L 17?estradiol group was higher than that in the 10-9mol/L 17?estradiol group,the difference was statistically significant?P<0.05?.3.Western blot detection of apoptosis-related proteins showed that compared with the blank group,the expression of?-SMA in the PDGF group was significantly up-regulated,and there was no significant difference in the expression of apoptosis-related proteins PARP and Bax;compared with the PDGF group,the expression of?-SMA in the drug group Significantly down-regulated,PARP,Bax protein expression was significantly up-regulated.4.Western blot detection of Wnt/?-catenin signaling pathway protein results showed that compared with the blank group,Wnt-3a of the mouse hepatic stellate cell line JS1in the PDGF group was significantly up-regulated and?-catenin expression was significantly down-regulated;compared with the PDGF group,the drug group The expression of Wnt-3a and?-catenin protein in mouse hepatic stellate cell JS1 was significantly up-regulated.Conclusion:17?estradiol can significantly inhibit the proliferation of mouse hepatic stellate cells JS1,and its mechanism may be caused by promoting the apoptosis of mouse hepatic stellate cells JS1,and within a concentration gradient of 10-9?10-7mol/L,with the increase of 17?estradiol concentration,the apoptosis rate of mouse hepatic stellate cells JS1 increased,showing a dose-effect relationship.Its potential mechanism may be regulating the Wnt/?-catenin signaling pathway. |
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