过氧化氢在肝硬化门静脉高压症大鼠肠系膜动脉RhoA/ROCK信号通路中的作用

The role of hydrogen peroxide on mesenteric artery RhoA/ROCK signal pathway in cirrhotic rats with portal hypertension

目的 探讨过氧化氢（H2O2）对胆总管结扎诱导的肝硬化门静脉高压症（PHT）大鼠肠系膜动脉收缩反应性的影响及其在RhoA/Rho激酶（ROCK）信号通路中的作用机制.方法 结扎胆总管建立肝硬化门静脉高压症大鼠模型.每日1次腹腔内注射生理盐水或聚乙二醇-过氧化氢酶（PEG-catalase,10 000 U/kg）共8d,相应处理正常大鼠.测定肠系膜动脉H2O2含量,利用血管灌流系统测定肠系膜微动脉对去甲肾上腺素的反应.检测大鼠肠系膜动脉α1肾上腺素能受体和β-arrestin2蛋白表达及其相互作用的变化,以及肠系膜动脉内ROCK-1蛋白量和活性的变化.结果 肝硬化PHT大鼠的离体肠系膜微动脉对去甲肾上腺素剂量反应曲线右移,EC50升高,PEG-catalase降低动脉过氧化氢含量后能逆转上述表现.各组肠系膜动脉的α1肾上腺素能受体含量不变.但是,PEG-cat-alase处理后肝硬化PHT大鼠肠系膜动脉内β-arrestin-2蛋白量降低,与α1肾上腺素能受体结合程度也降低;PHT大鼠肠系膜动脉内ROCK-1蛋白含量及其活性明显升高.结论 肝硬化PHT肠系膜动脉H2O2含量升高,引起抑制蛋白β-arrestin-2水平上升,与α1肾上腺素能受体结合能力增强,使得ROCK蛋白含量和活性均明显下降,造成肠系膜动脉对缩血管物质的收缩低反应性.

Objective To explore the role of hydrogen peroxide in mesenteric artery contraction of cirrhotic rats with portal hypertension,which was induced by bile duct ligation.Possible mechanism in RhoA/ROCK signal pathway was also part of the focus.Methods The bile duct ligation-induced cirrhotic rats and normal rats （control group） were treated equally with PEG-catalase（10 000 U/kg-1 · d-1,ip.） or by its vehicle for 8 days.Then the level of H2O2 in mesenteric arteries was detected.The contractile response to norepinephrine of arterioles was analyzed by vascular perfusion system.The protein expressions of the α1 adrenergic receptor,β-arrestin-2 and Rho kinase-1 （ROCK-1）,and the activity of ROCK-1 were measured by western blot.In addition,the interaction of α1-adrenergic receptor with β-arrestin-2 was assessed by co-immunoprecipitation.Results Compared to normal rats,the dose-response curve of the mesenteric arterioles in response to norepinephrine shifted to the right,and the EC 50 increased in the rats with portal hypertension.PEG-catalase treatment can decrease the hydrogen peroxide level in arteries,thus significantly lowered EC50 and improved the reactivity to norepinephrine of the mesenteric arterioles in portal hypertension rats.No significant difference in the α1-adrenergic receptor amounts was observed among groups.There was remarkable decreases in the protein expressions of β-arrestin-2 and its interaction with the α1-adrenergic receptor in cirrhotic rats with PEG-catalase treatment.PEG-catalase also increased the amount and activity of ROCK-1 in cirrhotic rats.Conclusions The level of hydrogen peroxide increases in the mesenteric arteries in bile duct ligation-induced cirrhotic rats.And it enhances the β-arrestin-2 expression and its interaction with the α1-adrenergic receptor,which subsequently decreases the amount and activity of ROCK as well as the contractility of mesenteric arteries in response to vasoconstrictors.