The Roles Of Rhoa/ ROCK And MAPKs Signaling Pathway In The Collagen Production Of Hepatic Stellate Cell In Rat Induced By Hyperglycemia

Objective: With the morbidity of diabetics is mounting up each year, Diabetes-induced chronic liver injury, is attracted to the attention of researchers, Nonalcoholic fatty liver disease(NAFLD),is the main form of expression at the early stage of Diabetes-induced chronic liver injury,which will develop into non-alcoholic steatohepatitis(NASH), fibrosis and Cirrhosis.Hepatic stellate cells(HSCs) activation is the central event during liver fibrogenesis. Activated HSCs undergo transdifferentiation to an activated myofibroblastic phenotype,and secrete a great deal of extracellular matrices.The collagens are the most important components of extracellular matrix,especially type-I collagen and type-III collagen.But the molecluar mechanisms of diabetes-induced hepatic fibrosis are not fully understood. The purpose of this study is to investigate the effect of high glucose on the hepatic stellate cells in vitro,and to observe whether the Rho A/ROCK and MAPKs pathway is involved in the collagen production of hepatic stellate cell induced by hyperglycemia,and clarify the mechanisms of liver fibrosis induced by diabetes, with the objective of providing a novel therapeutic basis for the treatment of diabetic hepatic fibrosis.Methods : Rat hepatic stellate cell-T6(HSC-T6) was cultured in vitro,The vitality of HSC-T6 was accessed by Trypan blue stain. Choose 2-5 generations of HSCs,Synchronized 2 hours by Serum free medium 1640. The cells were randomised into six groups: normal control group(NG: including 5.5 mmol/L glucose), the high glucose group(HG: including 25 mmol/L glucose), high osmotic pressure groups(OSM: for 5.5 mmol/ L glucose + 19.5 mmol/ L mannitol),the HG + fasudil group(12.5 umol/L, 25 umol/L, 50 umol/L).After 24 hours,The deposition and contents of collagen were evaluated by the hydroxyproline(HYP) determination.The m RNA expressions of type-I pro-collagen and type-III pro-collagen were assessed by Realtime fluorescence quantitative polymerase chain reaction.The phosphorylation of myosin phosphatase target subunit 1(p-MYPT1) of HSCs were measured by Western blot analysis, representing ROCK activity. And the phosphorylation level of Extracellular signal regulating kinase(ERK1/2), c- Jun amino terminal kinase(JNK) and p38 MAPK were also were measured by Western blot analysis,which represented the activity of MAPKs signaling pathway.Results:1 The effects of high glucose and fasudil on the contents of HYP in culture medium:Compared with the NG group, the contents of HYP in nutrition solution were significantly increased in the in the HG group(17.55±0.80 μg/ml vs. 26.24±0.91 μg/ml, P<0.01). The contents of HYP were markedly reduced in fasudil-treated(12.5 umol/L,25 umol/L,50 umol/L)compared with that in the HG group(23.22±0.62 μg/ml, 21.58±1.16 μg/ml, 19.31±0.37 μg/ml vs. 26.24±0.91 μg/ml, P < 0.01). Fasudil inhibited HG-induced increase in contents of HYP in a dose-dependent manner.There were no significant differences in the contents of HYP in nutrition solution between the NG group and the OSM group(17.24±0.67 μg/ml vs. 17.55±0.80 μg/ml, P>0.05).2 The effects of high glucose and fasudil on the gene expression of type-I and type-III procollagen:The m RNA expression of type-I and type-III procollagen m RNA in hepatic stellate cells in HG group were significantly up-regulated(P<0.01) than those in NG group.Compared with the HG group, Treatment HSCs with fasudil(12.5 umol/L,25 umol/L,50 umol/L) significantly suppressed the increase in accumulation of type-I and type-IIII procollagen induced by HG(P<0.01); There was no significant difference in m RNA expression of type-I and type-III procollagen between OSM group and NG group(P>0.05).These results suggested that High glucose induced increase of collagen synthesis, which was inhibited by fasudil. The change of osmolarity did not affect collagen synthesis.3 The effects of high glucose and fasudil on the activity of ROCK:The phosphorylation of myosin phosphatase target subunit 1(p-MYPT1) is a sign of ROCK signaling pathways activated, detecting MYPT1 can reflect the activity of ROCK. Compared with the NG group, exposure of HSCs to HG induced a significant up-regulation of the phosphorylation of MYPT1(P<0.01). In HG-stimulated cells,treatment it with fasudil(12.5 umol/L, 25 umol/L,50 umol/L) significantly suppressed the increase in accumulation of the phosphorylation of MYPT1 by HG in a dose-dependent manner(P<0.01).There were no significant differences in the phosphorylation of MYPT1 in HSCs between the OSM group and the NG group(P>0.05).These date suggested that high glucose stimulated the activity of ROCK,But the change of osmolarity did not affect the activity of ROCK, while fasudil could restrain the excessive activation of ROCK induced by high glucose.4 The effects of high glucose and fasudil on the MAPKs signaling pathway :MAPKs include Extracellular signal regulating kinase(ERK1/2), c- Jun amino terminal kinase(JNK) and p38 MAPK and its phosphorylation level on behalf of their activity.Compared with NG group, exposure of HSCs to high glucose led to a significant increase on the phosphorylation of ERK,JNK and p38MAPK(P<0.01), which was attenuated by fasudil. In HG-stimulated cells,treatment with fasudil(12.5 umol/L,25 umol/L,50 umol/L) significantly suppressed the increase in accumulation of the phosphorylation of ERK,JNK and p38MAPK(P<0.01).There were no significant differences in the phosphorylation of ERK,JNK and p38 MAPK in HSCs between the OSM group and the NG group(P>0.05).These results suggested that high glucose activated MAPKs pathway which was the downstream of ROCK,and the change of osmolarity was not responsible for MAPKs activation.Conclusions:1 High glucose induced increase of collagen synthesis in rat HSCs.2 MAPKs are the downstream signalling molecules of Rho A/ROCK. Rho A/ROCK activation is essential for the collagen synthesis of HSCs induced by High glucose,which might be associated with MAPKs pathway.