| 1.Obj ectiveTo elucidate the mechanism of miRNA-193a-3p（miR-193a-3p）regulating transfor ming growth factor beta-2（TGF-β2）to inhibit the activation of hepatic stellate cells（HSCs）and the intervention effect of shuganjianpi formula（SGJPF）to provide experimental basis for the treatment of liver fibrosis（LF）.2 Method2.1 Experiment 1:The effect of miR-193a-3p on the function of JS-1 cellsThe homology of miR-193a-3p was first exa mined by miRBase（https://www.mirbase.org/）online website,and then miR-193a-3p mimics and inhibitors were constructed and transfected into mouse hepatic stellate cells JS-1,and cell counting kit-8(cell counting kit-8.The viability of JS-1 cells was measured using cell counting kit-8（CCK8）;the migration ability of JS-1 cells was measured by scratch assay;the proliferation ability of JS-1 cells was measured by thymidine（d-TMP）analog（5-ethynyl-2’-deoxyuridine,EdU）;the proliferation ability of JS-1 cells was measured by real-time fluorescent quantitative PCR(real time quantitative PCR（RT-qPCR）to detect the expression levels of miR-193a-3p,α-SMA and Collagen Ⅰ;western blot（WB）to detect the protein expression of α-SMA and Collagen Ⅰ.2.2 Experiment 2:miR-193a-3p target gene screeningUsing miRDB（https://mirdb.org/）,TargetScan 8.0（https://www.targetscan.org/vert80/）and miRWalk（http://mirwalk.umm.uni-heidelberg.de/）databases,the The binding fraction and binding power between miRNA and mRNA were calculated to screen miR-193a-3p target genes,while the CCL4（carbon tetrachloride,CCL4）induced liver fibrosis mouse model was replicated in male SPF grade C57BL/6 mice for high-throughput RNA-Seq（Quantification）sequencing,combining the RNA-Seq results with the database screening results,followed by the literature research method to finally obtain the miR-193a-3p target gene TGF-β2.Then,the expression levels of miR-193a-3p and the target gene TGF-β2 in JS-1 cells were detected by PCR and WB experimental techniques.2.3 Experiment 3:Study on the effect of liver sparing and spleen strengthening formula on JS-1 cell function and related gene expressionSGJPF-containing serum was prepared and the optimal serum concentration was screened by CCK8;the viability of SGJPF-containing serum on JS-1 cells was exa mined by CCK8;the proliferation ability of SGJPF-containing serum on JS-1 cells was exa mined by EdU;the migratory ability of SGJPF-containing serum on JS-1 cells was exa mined by scratch assay;the effect of SGJPF-containing serum on JS-1 cells was exa mined by real-time fluorescence quantitative PCR.The effect of SGJPF-containing serum on the expression of α-SMA,Collagen I,TGF-β2 and miR-193a-3p in JS-1 cells was exa mined by real-time fluorescence quantitative PCR;finally,the effect of SGJPFcontaining serum on the proliferation,viability,migration and gene expression of JS-1 cells was further investigated by revertant assay.2.4 Experiment 4:Intervention effects of liver-sparing and spleen-strengthening formula on LF mice and the effects on related gene expressionA mouse model of liver fibrosis was established,and high,medium and low doses of SGJPF were ad ministered by gavage;the changes of liver elasticity index were investigated by ultrasound,and the effects of SGJPF on liver pathology histology of mice were investigated by liver histopathological sections and staining;the effects of SGJPF on the expression levels of α-SMA,Collagen I and TGF-β2 in liver tissues of LF mice were investigated by immunohistochemistry;the effects of SGJPF on the expression of α-SMA,Collagen I,miR-193a-3p and TGF-β2 in liver tissues of mice were detected by RT-qPCR.3 Results3.1 Experiment 1:Study of the effect of miR-193a-3p on JS-1 cell functionThe miR-193a-3p mimics were mimics,the optimal transfection concentration was 0.5 nmol/L,and the transfection time was 48 h.The miR-193a-3p inhibitor was inhibitor,the optimal transfection concentration was 50 nmol/L,and the transfection time was 48 h.After miR-193a-3p overexpression,the cell viability,proliferation ability and migration power of HSCs cells were reduced.Conversely,after miR-193a-3p inhibition,HSCs cell viability,proliferation ability and mobility were increased,and αSMA and Collagen I expression was increased.3.2 Experiment 2:miR-193a-3p target gene screeningIn the miRDB database,273 miR-193a-3p downstream target genes were obtained,and in the TargetScan database,233 miR-193a-3p downstream target genes were obtained.936 differential mRNAs upregulated in the liver fibrosis model were screened by high-throughput RNA-Seq sequencing,and after taking the intersection,7 common target genes were obtained,which are Rnf222,Igfbp5,Tgf-β2,Plau,Etvl,Kcnj2,Stmn1.Using TargetScan and miRWalk database,the binding of miR-193a-3p to the seven target genes was analyzed,and in addition,combined with literature research,TGF-β2 was identified as the target gene for the follow-up study.RT-qPCR and WB results showed that the mRNA and protein expression of TGF-β2 were significantly higher in the model group than in the control group;after ad ministration of miR-193a-3p inhibitor,TGF-β2 mRNA and protein expression were significantly higher compared to the model group;on the contrary,after ad ministration of miR-193a-3p mimic,TGF-β2 mRNA and protein expression were significantly decreased compared to the model group.3.3 Experiment 3:Study on the effect of liver sparing and spleen strengthening formula on JS-1 cell function and related gene expressionThe optimal SGJPF-containing serum concentration was 20%and the duration of action was 48h,which showed the best inhibitory effect on HSCs.Compared with the LF model group,SGJPF-containing serum significantly reduced the cell viability of HSCs and inhibited the expression of α-SMA and Collagen I.In addition,TGF-β2 expression was decreased and miR-193a-3p expression was increased after SGJPF intervention.In response experiments,SGJPF-containing serum inhibited HSCs cell viability,proliferation and migration in miR-193a-3p-interfered state,and decreased the expression of α-SMA,Collagen I and TGF-β2.3.4 Experiment 4:Effects of liver-sparing and spleen-healthy formula on the expression of miR-193a-3p and TGF-β2 in LF miceUltrasound results showed that compared with the control group,the elasticity index of liver in the model group was significantly higher,and after the ad ministration of SGJPF,the elasticity index gradually decreased with the increase of the ad ministered dose;the pathological histological results showed that the liver hepatocytes in the LF model group were degenerated and damaged,the structure of liver lobules was blurred,the arrangement of hepatocyte cords was disordered,and there was obvious collagen fiber deposition,and different dose groups of SGJPF could improve the liver of mice The different doses of SGJPF could improve the damaged state of mouse liver and reduce the deposition of collagen fibers.Compared with the LF model group,different doses of SGJPF significantly reduced the expression levels of α-SMA,Collagen I and TGF-β2,and up-regulated the expression level of miR-193a-3p in the liver tissues of LF mice.4 ConclusionmiR-193a-3p regulates the expression of TGF-β2 and inhibits the activation of HSCs.SGJPF-containing serum upregulates the expression of miR-193a-3p,downregulates the expression of TGF-β2 as well as activation markers,and inhibits the activation of HSCs,thus exerting a therapeutic effect on LF. |
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