Ghrelin Inhibits Liver Fibrosis Mediated By Hepatic Ischemia-Reperfusion Injury

Objective: This study to observe the regulation of Ghrelin on mouse liver extra cellular matrix during hepatic ischemia reperfusion injury(HIRI)-mediated liver fibrosis.And under hypoxia / reoxygenation,observing Ghrelin’s inhibition of hepatic stellate cell activation,based on the PPAR-γ which is the crosstalk between TGF-β1 / Smad3 pathway and m TOR pathway,explore the mechanism of Ghrelin’s inhibition of HIRI-mediated liver fibrosis.Methods: C57 mice were divided into sham operation group(Sham group),HIRI group(H/R group)and Ghrelin intervention group(G-H/R group).70% of the blood flow into the liver was blocked for 90 minutes,and then the blood flow was restored at 0h,3h,6h,72 h,7d and 15 d to induce liver fibrosis in mice,and Ghrelin intervention was also given.Detection of alanine aminotransferase(ALT)level in plasma of mice;determination of hydroxyproline(Hyp)content in liver tissue;RT-PCR detection of liver tissue α-SMA,type Ⅰ collagen,typeⅢ collagen,MMP-13 and TIMP-2 m RNA expression level;HE staining to observe the morphological changes of liver tissue,Masson staining to observe the collagen deposition site in liver tissue.Resuscitate HSC-T6 cells and set them up three groups: normal control(Con),hypoxia / reoxygenation(H/R),and Ghrelin intervention(G-H/R).The apoptosis rate of each group and the expression levels of cytokines TGF-β1,α-SMA,type Ⅰ collagen,Smad3,p-Smad3,m TOR,p-m TOR,PPAR-γ protein in hepatic stellate cells were measured.Results: The vivo experimental result showed that compared with the Sham group,the plasma ALT level in the H/R group increased significantly(P <0.05),and the plasma ALT level in the G-H/R group decreased significantly compared with the H/R group at the same time point(P <0.05).RT-PCR results showed that compared with the Sham group,the expression level of α-SMA,type Ⅰ and Ⅲ collagen m RNA in the H/R group gradually increased with time(P <0.05).Compared with the H/R group at the same time point,the expression level of α-SMA,type Ⅰ and Ⅲ collagen m RNA in the G-H/R group significantly decreased(P <0.05).The content of Hyp in liver tissue of each group was consistent with theexpression trend of type Ⅰ and type Ⅲ collagen;the degree of liver fibrosis in pathological morphological response of liver tissue was consistent with the above change.Compared with the Sham group,the expression level of MMP-13 m RNA in the H/R group increased at H/R0 h and 3h,and then its expression level gradually decreased and decreased to a lower level on7 d and 15d(P <0.05).Compared with the Sham group,the expression level of TIMP-2m RNA in the H/R group was significantly increased at each time point,and the difference was statistically significant(P <0.05).Compared with the Sham group,the MMP-13 /TIMP-2 m RNA ratio in the H/R group increased at first and then gradually decreased.The G-H/R 0h,3h,6h and 72 h groups showed different lower levels of MMP-13 m RNA expression than the corresponding H/R group,and the G-H/R7 d and 15 d groups showed different degrees of higher than the corresponding H/R group(P <0.05),compared with H/R group,the expression level of TIMP-2 m RNA in G-H/R group decreased significantly at each time point,the difference was statistically significant(P <0.05).Compared with the H/R group,the MMP-13 / TIMP-2 m RNA ratio of the G-H/R group decreased at first and then increased.The vitro experimental result showed that the apoptosis rate of hepatic stellate cells in the H/R group was significantly increased compared with the Con group,and the apoptosis rate of the hepatic stellate cells in the G-H/R group was significantly reduced compared to the H/R group.Compared with the Con group,the expression levels of α-SMA and type Ⅰcollagen in the H/R group were significantly increased(P <0.05),and the protein expression levels of TGF-β1,Smad3,p-Smad3,m TOR and p-m TOR also increased significantly(P<0.05),while the expression level of PPAR-γ protein decreased significantly(P <0.05).Compared with the H/R group,the expression level of PPAR-γ protein in the G-H/R group was increased(P <0.05),while the expression level of TGF-β1,Smad3,p-Smad3,m TOR,p-m TOR protein was decreased(P <0.05),at the same time,the expression levels of α-SMA and type Ⅰ collagen in cells also decreased.Conclusion: Ghrelin can play a better inhibitory role in the process of HIRI-mediated liver fibrosis,to reduce or reverse liver fibrosis.The mechanism may be that Ghrelin can regulating the enzyme of extracellular matrix synthesis and degradation,putting theextracellular matrix production and degradation in a new balance so that inhibiting the excessive deposition of extracellular matrix;Ghrelin can inhibit the m TOR signaling pathway and TGF-β1 / Smad3 signaling pathway related proteins,so that the number of activated HSC is reduced;Ghrelin may also increase the cross-talk PPAR-γ of the two pathways to reduce the activation of HSC,or reverse the activated HSC and play an anti-fibrotic role.