| Objective: To explore the molecular mechanism of Hic-5 in the hepatic stellate cells(HSCs)regulating liver regeneration on the basis of previous studies.Methods: 1.Mice models of 2/3 hepatectomy were established by using wild type(WT)and Hic-5 KO C57BL/6 mice.Liver samples were collected at different time points after operation.Immunofluorescence technique was used to detect the expression of Ki-67 in mice liver tissue to determine the effect of Hic-5 on liver regeneration;Reverse transcription PCR(RT-PCR)was used to detect the expression of Cyclin E1 and Cyclin D1 mRNA in mice liver tissue to further clarify the effect of Hic-5 on liver regeneration;The expression of Collagen 1a1 and Collagen 3a1 mRNA in mice liver tissue was detected by RT-PCR to determine the effect of Hic-5 on the expression of major components in extracellular matrix(ECM).2.The purchased human HSCs(LX-2)were cultured and the expression of Hic-5 in LX-2 cell line was detected by IF to determine whether Hic-5 was expressed in human HSCs.The vitro co-culture system was established by purchased LX-2 cell line and normal human hepatocyte cell line(HL-7702).Hic-5 gene was silenced in human HSCs by siRNA and normal control group(co-culture group of normal LX-2 cell line and HL-7702 cell line),negative transfected group(co-culture group of siRNA-NC transfected LX-2 cell line and HL-7702 cell line),transfected group(co-culture group of si RNA-Hic-5 transfected LX-2 cell line and HL-7702 cell line)were set.Cultured cells or culture media were harvested at 0 h,24 h,48 h and 72 h.Cell Counting Kit 8(CCK-8),a common method for measuring cell proliferation,was used to detect the viability of hepatocyte cell lines,then we plot cell proliferation curves to understand the effect of Hic-5 in HSCs on hepatocyte proliferation;The expression of Hic-5 and Cyclin D1 mRNA at different time points was detected by RT-PCR to clarify the change of Hic-5 in HSCs with culture time and to further verify its effect on hepatocyte proliferation;The expression of Collagen 1a1 and Collagen 3a1 at the protein level was detected by Western Blot(WB)to clarify the effect of Hic-5 in HSCs on the expression of major components of ECM.Enzyme linked immunosorbent assay(ELISA)was used to detect the levels of ALT,AST and ALB in the culture media at different time points to determine the effect of Hic-5 in HSCs on hepatocyte function;The expression of JNK1,p-JNK1 and p-MKK4 at the protein level in different time periods was detected by WB to clarify whether MKK4/JNK signaling pathway may involve in Hic-5 regulating hepatocytes proliferation and the specific molecular mechanisms of Hic-5 regulating hepatocytes proliferation;Finally,we pretreated HSCs with JNK signaling pathway-specific blockers and blocker vehicle,and established co-culture system(normal group and transfection group).After 24 h and 48 h,we detected the expression of p-JNK1,Cyclin D1,Collagen 1a1 and p-MKK4 at the protein level to further determine whether JNK signaling pathway involved in Hic-5 regulating hepatocytes proliferation.Results: 1.The expression of Ki-67 in liver tissue of WT and Hic-5 KO mice at 48 h and 72 h after hepatectomy was detected by IF,then the results showed that Ki-67 positive cells in Hic-5 KO mice group were significantly higher than that in WT group at 72 h,and the difference was statistically significant(P<0.05);The expression of Cyclin E1 and Cyclin D1 mRNA in WT and Hic-5 KO mice after 2/3 hepatectomy was detected by RT-PCR,then it was suggested that the expression of Cyclin D1 and Cyclin D1 mRNA in liver tissue of Hic-5 KO mice was significantly higher than that in WT group at 72 h after 2/3 hepatectomy,and the difference was statistically significant(P<0.05);We detected the expression of Collagen 1a1 and Collagen 3a1 mRNA in liver tissue of WT and Hic-5 KO mice at 48 h after 2/3 hepatectomy by RT-PCR,then the results suggested that the expression of Collagen 1a1 and Collagen 3a1 mRNA Compared with Hic-5 KO group,the level in WT group was significantly increased,and the difference was statistically significant(P<0.05).2.We cultured purchased LX-2 cell line and detected the expression of Hic-5 and ?-SMA by IF,the results showed that Hic-5 and ?-SMA were co-expressed in human HSCs.Hic-5 gene was silenced in human HSCs by siRNA and the results of siRNA transfection efficiency indicated that the expression of Hic-5 mRNA in LX-2 transfected with siRNA-Hic-5 was significantly lower than that in LX-2 transfected with siRNA-NC,and the difference was statistically significant(P<0.05);Using CCK-8 to detect the hepatocyte proliferation suggested that hepatocyte proliferation in each experimental group gradually increased with the increasing of culture time within the set time point,and the hepatocyte proliferation rate was the fastest in 0-24 h,in 24-48 h hepatocyte proliferation rate was still fast,but slower than that in 0-24 h,and after 48 h the hepatocyte proliferation rate was more slower,while at the same time hepatocyte proliferation in the co-culture group of LX-2 cell line transfected with siRNA-Hic-5 and HL-7702 cell line was significantly higher than that in the co-culture group of LX-2 cell line transfected with siRNA-NC and HL-7702 cell line,and the difference was statistically significant(P<0.05);The expression of Hic-5 and Cyclin D1 mRNA in each group at different time points was detected by RT-PCR and the results indicated that in the same experimental group,the expression of Hic-5 and Cyclin D1 mRNA increased with the culture time,while the expression of Hic-5 mRNA in the co-culture group of LX-2 cell line transfected with siRNA-Hic-5 and HL-7702 cell line was significantly lower than that in the co-culture group of LX-2 cell line transfected with siRNA-NC and HL-7702 cell line,but the expression of Cyclin D1 mRNA was significantly higher at the same time point,and the difference was statistically significant(P<0.05);The expression of Collagen 1a1 and Collagen 3a1 at the protein level was detected by WB and the results indicated that in the same experimental group,the expression of Collagen 1a1 and Collagen 3a1 increased with the culture time,while the expression of Collagen 1a1 and Collagen 3a1 in the co-culture group of LX-2 cell line transfected with siRNA-Hic-5 and HL-7702 cell line was significantly lower than that in the co-culture group of LX-2 cell line transfected with siRNA-NC and HL-7702 cell line at the same time point,and the difference was statistically significant(P<0.05);We used ELISA to detect the level of ALT,AST and ALB in culture medium at different time points and the results indicated that in the same experimental group,the level of of ALT,AST and ALB gradually increased with the culture time,while the level of ALT,AST in the co-culture group of LX-2 cell line transfected with siRNA-Hic-5 and HL-7702 cell line was significantly lower than that in the co-culture group of LX-2 cell line transfected with siRNA-NC and HL-7702 cell line,but the level of ALB was higher at the same time point,and the difference was statistically significant(P<0.05);The expression of JNK1,p-JNK1 and p-MKK4 at the protein level in each group at different time points was detected by WB and the results indicated that the expression of p-JNK1 and p-MKK4 increased with the culture time,while the expression of p-JNK1 and p-MKK4 in the co-culture group of LX-2 cell line transfected with siRNA-Hic-5 and HL-7702 cell line was significantly higher than that in the co-culture group of LX-2 cell line transfected with siRNA-NC and HL-7702 cell line at the same time point,and the difference was statistically significant(P<0.05),but there was no significant difference in the expression of JNK1 at protein level(P>0.05);After pretreatment with JNK blockers and blocker vehicle,we used WB to detect the expression of p-JNK1,Cyclin D1,Collagen 1a1 and p-MKK4 at the protein level and the results indicated that at the same time point,the expression of p-JNK1 and Cyclin D1 significantly decreased,while the expression of Collagen 1a1 significantly increased in the blocker group compared with the blocker vehicle group,and the difference was statistically significant(P<0.05),but the expression of p-MKK4 silightly decreased and there was no significant difference in the reduction of p-MKK4(P>0.05).Conclusion: 1.the deletion of Hic-5 gene can promote ALT,AST,increase synthesis of ALB,and improve hepatocyte function while promoting hepatocyte proliferation;2.Deletion of Hic-5 gene may regulate hepatocyte proliferation by remodeling extracellular matrix proteins such as Collagen 1a1 and Collagen 3a1;3.Hic-5 regulation of hepatocyte proliferation can be accomplished via the MKK4/JNK signaling pathway and is primarily achieved by activated JNK1 phosphorylation. |
PDF Full Text Request