| Objective:By studying the effect of modified Dahuang Zhechong Pill on hepatic stellate cells in patients with primary biliary cholangitis(PBC)under the injury state of LncRNA GAS5/miR-21 and TGF-β1/Smads.The mechanism of the treatment of PBC with modified Dahuang Zhechong Pill is clarified at the protein molecular level,providing a practical and reliable experimental basis for the prevention and treatment of PBC with traditional Chinese medicine.Methods:1.Twenty healthy male SPF SD rats were randomly divided into experimental group and control group.The 10 rats in the experimental group were given the modified rhubarb Zhechong pill suspension by gavage,while the control group was given the same amount of normal saline by gavage.After 7 days of continuous gavage,the rats fasted for 24 hours,and blood was collected through the abdominal aorta to prepare serum.2.HSC-T6 cells were divided into six groups:The blank group was added with culture medium without cells,and the cells in the fetal bovine serum group were not treated.The blank serum group was added with 5%and 10%blank serum,and the drug-containing serum group was added with 5%and 10%drug-containing serum,respectively.After culture,the activity of HSC-T6 cells was detected by CCK8 method.3.The normal growth HSC-T6 cells were divided into blank group,normal control group,normal drug-containing serum group,lactic acid model group,and lactic acid drug-containing serum group.The cell activity was analyzed to evaluate whether the model was successful.4.The LncRNA GAS5 of the above groups of cells were silenced and cultured for 72 hours respectively.the relative expression of LncRNA GAS5 and miR-21 in each group was detected by PCR and TGF in each group was determined by Western Blot-β1.The relative expression of Smad3,Smad7 and their phosphorylated proteins.Results:1.HSC-T6 cell activity in each group:Compared with the blank control group,the activity of HSC-T6 cells in fetal bovine serum group,5%blank serum group,10%blank serum group,5%medicated serum group and 10%medicated serum group increased significantly(P<0.01).2.Comparison of HSC-T6 cell viability in each group after adding lactic acid:Compared with the normal control group,there was no significant difference in the activity of HSC-T6 cells in the normal drug-containing serum group(P>0.05);The activity of HSC-T6 cells in the lactic acid model group and the si-LncRNA GAS5 transfection group increased significantly(P<0.01).Compared with the si-LncRNA GAS5 transfected group,the activity of HSC-T6 cells in the si-LncRNA GAS5 transfected lactic acid model group increased significantly(P<0.01).3.Comparison of the relative expression of LncRNA GAS5 and miR-21 in HSC-T6 cells in each group:Compared with the lactic acid model group,the relative expression of LncRNA GAS5 in the lactic acid drug-containing serum group increased significantly(P<0.05).Compared with the si-LncRNA GAS5 transfected group,the expression of LncRNA GAS5 in the si-LncRNA GAS5 transfected lactic acid model group was significantly lower(P<0.01);The content of LncRNA GAS5 in the group of si-LncRNA GAS5 transfected with lactic acid medicated serum decreased significantly(P<0.05).Compared with the si-LncRNA GAS5 transfected lactic acid model group,the relative expression of LncRNA GAS5 in the si-LncRNA GAS5 transfected lactic acid drug-containing serum group was significantly higher(P<0.01).Compared with the lactic acid model group,the relative expression of miR-21 in the lactic acid drug-containing serum group decreased significantly(P<0.05).Compared with the si-LncRNA GAS5 transfected group,the expression of miR-21 in the si-LncRNA GAS5 transfected lactic acid model group was significantly higher(P<0.01);The content of miR-21 in the group of si-LncRNA GAS5 transfected with lactic acid medicated serum increased significantly(P<0.05).Compared with the si-LncRNA GAS5 transfected lactic acid model group,the relative expression of miR-21 in the si-LncRNA GAS5 transfected lactic acid drug-containing serum group was significantly lower(P<0.01).4.TGF of HSC-T6 cells in each group-β1.Comparison of Smad3/Smad7 and its phosphorylated protein:Compared with the lactic acid model group,the lactic acid drug-containing serum group TGF-β1.The expression level of phosphorylated Smad3 protein and phosphorylated Smad7 protein decreased significantly(P<0.05).Compared with si-LncRNA GAS5 transfected with lactic acid model group,si-LncRNA GAS5 transfected with lactic acid drug-containing serum group TGF-β1.The level of phosphorylated Smad3 protein decreased and the level of phosphorylated Smad7 protein increased,both of which were statistically significant(P<0.05).Compared with other groups,Smad3 and Smad7 protein levels did not change significantly,and the difference was not statistically significant(P>0.05).Conclusion:1.HSC-T6 cells can grow normally in drug-containing serum.2.Low concentration(0.05 mmol/L)of lactic acid can activate HSC-T6 cells in large quantities and successfully create PBC cell injury model.3.Further verified the effect of modified rhubarb Zhechong pill on the signal factor TGF-β1.4.The mechanism of the treatment of PBC with modified Dahuang Zhechong Pill may be the inhibition of LncRNA GAS5/miR-21,thus inhibiting TGF in the downstream pathway-β1.The expression of Smad3 and its phosphorylated protein can promote the expression of Smad7 and its phosphorylated protein,inhibit the excessive proliferation and activation of HSC-T6 cells,and achieve the goal of slowing down liver fibrosis. |
