| Aim:The hepatic stellate cell（HSC）JS1 undergoes autophagy reaction under the intervention of lipopolysaccharide（LPS）and study the regulating effect of17β-estradiol（17β-E2）on cell autophagy and protein kinase B（AKT）/rapamycin（MTOR）pathway,which provide a new theoretical basis for the study of the mechanism and clinical application of 17β-E2in inhibiting the autophagy of hepatic stellate cells.Method:1.Mouse’s HSC JS1 were cultivated in high-sugar medium containing 10%fetal bovine serum（FBS）with double-antibodies.The medium was cultivated in constant temperature cultivator with 37℃,5%concentration of CO2.The serious sub-cultivation was started when cell grow to range of 80%cell fusion.2.Different concentrations of LPS were used to intervene in JS1,where the LPS concentration was 0,0.01,0.05,0.1,0.25,0.5,1,2μg/ml.CCK-8 method was used to determine the cell activity（OD value）and draw a curve.3.Combining 0.1μg/ml LPS and different concentrations of 17β-E2to intervene in JS1,the concentration of 17β-E2is 0,10-3,10-4,10-5,10-6,10-7,10-8and 10-9mol/L and setting containing DMSO reagent group（to eliminate the effect of 17β-E2solvent DMSO）,CCK-8 method to measure cell activity and draw a curve.4.Western blot The study was set to LPS group,blank group and 10-6,10-7,10-8mol/L17β-E2groups.The protein of each group of cells was extracted and measured,and Western blot was applied to detect expression of AKT/MTOR pathway protein and autophagy-related protein respectively.5.RTQ-PCR method The study was set to LPS group,blank group and 10-6,10-7,10-8mol/L17β-E2groups.The whole RNA of each group were extracted and reversed transcribed into c DNA,and using c DNA as a template,the expression of autophagy related genes were detected by performing real-time fluorescent quantitative PCR reaction.6.Statistical Analysis Statistical analysis was performed by SPSS 25.0 software.The measurement data was expressed as mean±standard deviation（x±S）,the difference between groups was analyzed by one-way ANOVA,and the Tukey test was used for pairwise comparison.P<0.05 was considered statistically significant.Result:1.The activity of JS1 under the intervention of LPS:The results showed that the activity of JS1 significantly increased under the intervention of LPS at concentrations of 0.05,0.1 and 0.25μg/ml,（x±S）were 2.82±0.15,2.91±0.08 2.76±0.01,which were statistically significant compared with the blank group（P=0.001,0.000,0.027）,and the JS1 activity under the intervention of 0.1μg/ml of LPS had the best value-adding effect.2.The results of JS1 activity under the combined intervention of 17β-E2and 0.1μg/ml of LPS at different concentrations indicated that and there was no significant difference in OD value in all groups（P>0.05）on the frist day;On the 2nd day,the OD value of the LPS group were significantly higher than that of the blank group and the17β-E2groups of 10-6,10-7,10-8mol/L（P=0.000,0.000,0.003,0.005）,which was not statistically significant with 10-9mol/L（P>0.05）;on the 3rd day,the OD value of LPS group was significantly higher than that of blank group and 17β-E2groups of 10-6,10-7,10-8mol/L（P=0.000,0.012,0.005,0.011）,which was no statistical significance with the 10-9mol/L 17β-E2group（P>0.05）;on the 4th day,the OD value of the LPS group was significantly higher than that of the blank group and 10-6,10-7,10-8mol/L17β-E2groups（all P=0.000）,which was no statistical significance with the 10-9mol/L17β-E2group（P>0.05）;the OD value of blank group was significant higher than 10-3,10-4,10-5mol/L17β-E2groups（all P-value=0.000）.3.The results of Western Blot assay for autophagy-related proteins and AKT/MTOR signaling pathway proteins showed that the expression of Beclin1 in LPS group was significant higher than blank group and 10-6,10-7,10-8mol/L 17β-E2groups（all P=0.000）and the expression of Beclin1 in blank group was significant lower than10-6,10-7mol/L17β-E2groups（all P=0.000）,which not statistically significant with 10-8mol/L 17β-E2group;the expression of LC3Ⅱ/LC3Ⅰin LPS group was significantly higher than blank group（P=0.007）and lower than 10-6,10-7mol/L17β-E2groups（all P=0.000）,which not statistically significant with 10-8mol/L17β-E2group（P>0.05）.In the expression of pathway protein,The p-AKT/AKT protein expression of the blank group and 10-6,10-7,10-8mol/L 17β-E2groups were significantly higher than that of the LPS group（P=0.001,0.001,0.001,0.005）.p-AKT/AKT protein expression of17β-E2groups were not statistically significant compared with the blank group（all P>0.05）.The p-MTOR/MTOR protein expression of the blank group and 10-6,10-7,10-8mol/L 17β-E2groups were significantly higher than the LPS group（P=0.000,0.043,0.010,0.000）,the expression of p-MTOR/MTOR protein in the 17β-E2groups of 10-6,10-7mol/L were significantly lower than that of the blank group（all P=0.000）,and there was no significant difference between the 10-8mol/L17β-E2and the blank group（P>0.05）.4.RTQ-PCR show that the level of LC3,Beclin1 m RNA in LPS were significant higher than blank group and 10-6,10-7,10-8mol/L17β-E2groups（all P=0.000）,the level of LC3、Beclin1 m RNA in each 17β-E2groups were significant higher than blank group（all P=0.000）.Conclusion:1.10-6,10-7,10-8mol/L 17β-E2can inhibit the autophagy of JS1cells induced by LPS.2.The autophagy of JS1 cells induced by 0.1μg/ml LPS is related to inhibition of AKT/MTOR signal pathway.3.The effect of 10-6,10-7,10-8mol/L 17β-E2on the inhibition of 0.1μg/ml LPS-induced autophagy in JS1 cells is related to activation of AKT/MTOR signal pathway. |
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