Inhibition Role Of Y-27632 On Myosin Phosphatase In Rat Hepatic Stellate Cells

Hepatic fibrosis represents a reversible and dynamic process in response to a variety of chronic stimuli. Liver cirrhosis is the most advanced stage of fibrosis. The main pathological characteristic of hepatic fibrosis is the increased and irregular deposition of extracellular matrix (ECM) components. Activation of hepatic stellate cell (HSC), a perisinusoidal cell that resides in the liver in a quiescent stage, is responsible for the increased synthesis and deposition of ECM in the liver. Following a fibrogenic stimulus, the HSC undergoes a complex activation process in which the cell changes from a quiescent vitamin A-storing cell to an activated myofibroblast-like cell, which proliferates and becomes fibrogenic. An increase in DNA synthesis and cell proliferation occurs with HSC activation. Many signal transduction pathways are involved in the liver fibrogenesis in HSC. So we should look for a new signal transduction pathway and study the mechanism of liver fibrogenesis.Rho GTPase, also known as small G protein, regulates many cellular behaviors, such as cell migration, contraction, proliferation and differentiation through activating the downstream signal molecule-Rho associated coilel coil forming protein kinase (ROCK), also named Rho kinase. Central signal molecules in Rho/ROCK signal pathway are Rho GTPase, ROCK and myosin phosphatase (MP). Rho is activated by multiplicate stimulative signals, and Rho kinase is activated by activated small GTPase Rho. ROCK phosphorylates myosin phosphatase binding subunit at Thr697, and this phosphorylation inhibits MP, so myosin light chain (MLC) phosphorylation increases in cells. This leads to aggregation of the actin cytoskeleton and changes of cell behaviors.MP is composed of three subunits: the 38 kD catalytic subunit (PP1c), the 110 kD regulatory myosin phosphatase target subunit 1 (MYPT 1), also named myosin binding subunit (MBS) and the 20 kD regulatory subunit (M20). Activated ROCK phosphorylates MYPT 1 and so this phosphorylation lead to an inhibition of the MP activity. ROCK phosphorylates MYPT1 possibly at Thr695, Thr850, Ser854.Cell migration is an essential physiological progress involved in upgrowth, tissue damnification and inflammation responses. In the progress of liver fibrogenesis, that many HSC are recruited to injured sites is related to HSC migration. Rho GTPase and ROCKⅠare involved in regulating cell migration. Lysophosphatidic acid (LPA) enhanced the motility of hepatoma cells and facilitated their invasion, and the migration and motility of cancer cells can be inhibited by blocking Rho/ROCK pathway. So invasiveness of hepatocellular carcinoma is facilitated by the Rho/Rho-kinase pathway. Y-27632, a specific ROCK inhibitor, can inhibit many cell biological functions mediated by ROCK. Up to now, few studies have addressed the function of Rho/ROCK signal transduction pathway on the chronic inflammatory fibrosis of tissues.Objective: To investigate the inhibition role of Y-27632 on myosin phosphatase in rat HSC and effects of Rho/ROCK signal pathway on proliferation and migration in rat HSC.Methods: HSC were cultured in RPMI-1640 with 2% fetal bovine serum. After being stimulated by LPA, HSC were treated with Y27632 to block Rho/ROCK signal pathway. The protein levels of p-MYPT were analyzed by Western blotting. The proliferative inhibition of Y27632 on HSC was evaluated by MTT assay. Boyden chamber is given to detect the cell migration ability.Results:①The inhibition role of Y-27632 on the phosphorylation of myosin phosphatase in rat HSC activated by LPA: The level of p-MYPT protein expression by Western blotting in group of LPA (3.28±0.58) was significantly higher than that of control (1.18±0.29), P<0.01. In the group of Y27632 with LPA, the level of p-MYPT protein expression (0.63±0.10) was significantly lower than that of control, P<0.01. No difference was found between the protein expression of the control group and the DMSO group (1.10±0.23). The levels of p-MYPT protein expression had significant difference after HSC exposured to 30,50 μmol·L-1Y27632 with LPA for 12 h compared with that of LPA group, P<0.01, and the levels were 1.46±0.07 and 0.85±0.12, respectively. There was significantly difference between the protein expression of 30μmol·L-1 Y27632 group and 50μmol·L-1 Y27632 group (P<0.01). But there was no difference between that of 10μmol·L-1 Y27632 group (2.52±0.13) and LPA group (P>0.05). After HSC activated by LPA exposured to Y27632, the levels of p-MYPT protein expression respectively were 1.03±0.09, 0.64±0.08 for 24 h group and 48 h group compared with that of 0 h group (2.87±0.06), P<0.01. But there was no statistic difference between that of 12 h group (2.83±0.18) and 0 h group (P>0.05).②The inhinition effect of Y27632 on HSC migration: As Boyden chamber displayed, compared with LPA group, the migration ability had significant difference after HSC exposured to 30,50μmol·L-1 Y27632 with 50 ng·mL-1 LPA for 12 h, and the cells which brokethrough membrane were 21.10±1.87 vs 37.19±1.76,12.09±1.04 vs 37.19±1.76, respectively, P<0.01. There was statistic difference between the cells of 30μmol·L-1 Y27632 group and 50μmol·L-1 Y27632 group (P<0.01). But there was no difference between the cells of 10μmol·L-1 Y27632 group (34.07±0.93) and LPA group (P>0.05). After HSC activated by LPA exposured to Y27632, the cells which brokethrough membrane respectively were 18.04±0.70 and 17.09±0.63 for 24 h group and 48 h group compared with that of 0 h group (36.11±0.86), P<0.01. There was no difference between the cell migration of 24 h group and 48 h group (P>0.05). The cells was 34.07±0.93 for 12 h compared with that of 0 h group (P>0.05).③The inhibition effect of Y27632 on proliferation of HSC. Observed with inverted microscope, in the group of Y27632 with LPA, the cell shrunk and the cell spaces accreted. The caduceus cells obviously increased after 48 h. In the groups of control, DMSO and LPA, HSC revealed normol morphous. After exposure of HSC to LPA and Y27632, the proliferation of HSC was inhibited in the groups of 12 h, 24 h and 48 h compared with that of 0 h, and the inhibition rates were 20.11%, 42.48%, and 72.68%, respectively, in a time-dependent manner. But after HSC was exposed to LPA only, the proliferation rates at 12 h, 24 h and 48 h were 4.63%, 10.37% and 28.39% compared with that of 0 h. MTT results displayed that compared with control group, LPA stimulated the proliferation of HSC at 12 h, 24 h and 48 h. The proliferation rates were 46.81%, 53.26% and 80.59% respectively, P<0.01. In the group of Y27632 with LPA, the proliferation of HSC was inhibited compared with that of control group at 12 h, 24 h and 48 h. The proliferation inhibition rates respectively were 40.46%, 51.07% and 66.23%, P<0.01. But there was no statistic difference between the proliferation of control group and DMSO group at 12 h, 24 h and 48 h, P>0.05.Conclusions: Y27632 can inhibit the phosphorylation of myosin phosphatase in rat HSC activated by LPA in vitro, and blocking Rho/ROCK signaling pathway can inhibit migration and proliferation of HSC activated by LPA.