Role of Rho-Rock pathways induced by angiotensin II in hepatic stellate cell contraction

Abstract

To investigate the mechanism of Ca(2+)-independent pathways mediated by Rho-kinase in contraction of hepatic stellate cells (HSCs) induced by angiotonin II (Ang II). Human HSCs of the line HSC-T6 were cultured and randomly divided into 6 groups: negative control group, Ang II group treated by Ang II10 micromol/L for 15 min, Ang II + irbesartan (Ang II receptor inhibitor) group, exposed to irbesartan for 60 min prior to Ang II treatment, Ang II + Y27632 (Rho kinase specific inhibitor) exposed to Y27632 for 60 min prior to Ang II treatment, Ang II + ML-7 (myosin light chain kinase specific inhibitor) + saturo (protein kinase C specific inhibitor) group exposed to stauro for 60 min prior to Ang II treatment, and Ang II + Y27632 + ML-7 + stauro group, exposed to Y27632 and stauro for 60 min prior to Ang II treatment. The cell contraction was detected by silicone-rubber-membrane cultivation directly. The protein levels of MLC and phosphorylated MLC were detected by Western blotting 5, 15, 30, 60, and 120 min after Ang II treatment. RT-PCR was used to detect the expression of Rock2, RhoAGTP, and RhoGEF in Ca(2+)-independent pathways mediated by Rho-kinase. The silicone-rubber-membrane covered by Ang II treated HSCs showed obvious wrinkles indicating the contraction of HSCs. The ratios of phosphorylated MLC protein at the time pints 5, 15, 30, 60, and 120 min of the Ang II group to the control group (0 min) were 11.7 +/- 0.1, 26.9 +/- 0.1, 11.2 +/- 0.1, 4.1 +/- 0.1, and 1.0 +/- 0.1, showing that Ang II increased the phosphorylated MLC protein level time-dependently with the peak level at the time point of 15 minutes. The levels of phosphorylated MLC protein of the Ang II + irbesartan and Ang II + Y27632 groups were (1.12 +/- 0.09)and (1.22 +/- 0.10) respectively, both significantly lower than that of the Ang II group (1.33 +/- 0.06, both P < 0.01). The level of phosphorylated MLC protein of the Ang I + ML-7 + stauro group was (1.43 +/- 0.09), significantly higher than that of the Ang II + Y27632 group (0.64 +/- 0.04, P < 0. 01). The level of phosphorylated MLC protein of the Ang II + Y27632 + ML-7 + stauro group was (0.64 +/- 0.04), significantly lower than that of the Ang II group (P = 0. 003). The mRNA expression levels of Rock2, RhoAGTP, and RhoGEF of the Ang II group were (0.36 +/- 0.01), (0.80 +/- 0.01), and(0.65 +/- 0.11)respectively, all significantly higher than those of the control group [(0.12 +/- 0.01), (0.40 +/- 0.02), and (0.33 +/- 0.09) respectively, all P = 0. 000], and the mRNA expression levels of Rock2, RhoAGTP, and RhoGEF of the Ang II + irbesartan + group were (0.21 +/- 0.02), (0.62 +/- 0.02), and (0.41 +/- 0.10) respectively, all significantly lower than those of the control group. The mRNA expression levels of Rock2 (0.15 +/- 0.01) and RhoGEF (0.28 +/- 0.08) were lower, but The mRNA expression level of RhoAGTP (1.14 +/- 0.02) was higher in the Ang II + Y27632 group. The mRNA expression levels of Rock2, RhoAGTP, and RhoGEF of the Ang II + ML-7 + stauro group were (0.22 +/- 0.01), (0.55 +/- 0.03), and (0.44 +/- 0.10) respectively, all significantly higher than those of the control group. The mRNA expression levels of Rock2, RhoAGTP, and RhoGEF of the Ang II + Y27632 + ML-7 + stauro group were (0.23 +/- 0.01), (0.83 +/- 0.02), and (0.69 +/- 0.08) respectively, all significantly higher than those of the Ang II + ML-7 + stauro group. Ang II induces HSCs contraction in Ca(2+)-independent pathways mediated by Rho-kinase.