The Role Of Rho A/ROCK Pathway In High Glucose-induced Proliferation And Transdifferentiation In Rat HSCs

Objectives:Diabetes mellitus could lead to non-alcoholic fatty liver disease, which can progress to liver fibrosis. While the pathogenesis that are involved in liver fibrosis caused by diabetes are not completely clear at present, the activation and transdifferentiation of hepatic stellate cells are remain considered to be the central event in liver fibrosis. By culturing HSCs in high glucose condition, the purpose of our study is to explore the role of Rho/ROCK pathway in high glucose-induced proliferation and transdifferenti- ation of HSCs, and to elaborate the regulatory effect of ROCK on MAPKs pathway, finally to provide new therapies for clinical liver fibrosis.Methods:Synchronized HSCs were divided into following groups: Normal glucose control group(NG:5.5 mmol/L glucose),High glucose group(HG:25 mmol/L glucose), Mannitol group(5.5 mmol/L glucose + 19.5 mmol/L mannitol),High glucose + fasudil group(HG + Fa, the concentrations of fasudil were 12.5, 25, 50 μmol/L, respectively). Proliferation was measured by MTS assay. The exent of α-SMA expression was presented by immunocytochemistry staining. The phosphorylation states of MYPT1 and MAP kinases(MAPKs), including extra cellular signal-regulated kinase 1/2(ERK1/2), c-jun kinase(JNK), and p38 MAPK were evaluated by Western blot analyses.Results:1 Effects of HG and fasudil on the proliferation in HSCs: As reflected in MTS assay, the proliferation extent was represented by OD value, compared with NG group, the proliferation of HSCs in HG group was significantly increased by 20.1%, P<0.01. In HG-stimulated cells, treatment with fasudil(12.5, 25, 50μM)significantly suppressed HG-induced increase in the proliferation of HSCs in a dose-dependent manner(decreased by 4.4%P<0.01], 7%[P<0.01]and 15.8% [P<0.01], respectively vs HG group). There was no significant difference in HSC proliferation between HOSM group and NG group(P>0.05). Thses results showed us that HG promoted the proliferation in HSCs and fasudil inhibited the high glucose-induced proliferation of HSCs in a dose-dependent manner. However, the high osmotic pressure has no effect on the HSC proliferation.2 Effects of HG and fasudil on the expression of α-SMA: We observed the contents of α-SMA in HSCs by using immunocytochemical staining methods. The cytoplasm of positive cells were staining for filemot and tan granulations around vacuolated nucleus. The NG group and HOSM group were all have slightly expression of α-SMA, and there was no statistically significant differences between them. The strongest positive staining for α-SMA was found in the cells of HG group. The contents of intracellular α-SMA in HG group were significantly higher than those in NG group(P<0.01). Fasudil(12.5, 25, 50μM) inhibited HG-stimulated increase in contents of α-SMA in a dose-dependent manner(P<0.01 vs HG group). The result revealed that the HSCs were in a quiescent state without stimulators, and HG could activated HSCs and promoted the synthesis of α-SMA, fasudil inhibited the high glucose-induced activation of HSCs in a dose-dependent manner. Same with previous study, high osmotic pressure has no effect on HSCs activation.3 Effects of HG and fasudil on Rho A/ROCK pathway: The myosin phosphatase target subunit 1(MYPT1) is the most important substrate of ROCK, when the ROCK pathway being activated, MYPT1 can be phosphorylated, so we measured the phosphorylation of MYPT1 as a maker of ROCK activity. From the Western blot,we found that there is no difference between NG and HOSM group. While exposure of HSCs to HG dramaticly increased the phosphorylation of MYPT1(P<0.01 vs NG group). All the three consentrations of fasudil we used inhibited HG-induced increase in phosphorylation of MYPT1(P<0.01 vs HG group). The results suggested that HG could active ROCK pathway while high osmotic pressure has no effect during the process. Application of fasudil could block the excessive activation 0f ROCK pathway stimulated by high glucose.4 Effects of HG andfasudil on MAPKs pathway: MAPKs mainly include ERK1/2, JNK, and p38 MAPK. The level of their phosphorylation indicates the degree of pathway activation.Compared with NG group, high osmotic pressure could not contribute to increasing the phosphorylation of ERK, JNK, and p38MAPK(P>0.05). In contrast, a marked increase of p-ERK, p-JNK, and p-p38 MAPK was observed in high glucose group(P<0.01 vs NG group), and fasudil inhibited their phosphorylation in a dose-dependent manner(P<0.01 vs HG group). The results pointed out high glucose could active MAPKs, they are signal molecules downstream ROCK pathway, and high osmotic pressure has no effect on the activation of MAPKs.Conclusions:1 High glucose promotes the proliferation and transdifferentiation in rat HSCs.2 MAPKs are signal molecules downstream ROCK, and high glucose promotes HSCs proliferation and transdifferentiation via ROCK cascade pathway.