IL-1β下调Rho激酶活性介导脓毒症大鼠血管钙失敏的机制研究

Study on mechanism of IL-1β mediating vascular calcium desensitization in septic rats through down-regulating Rho kinase activity

目的探讨白细胞介素(IL)-1β下调Rho激酶活性介导脓毒症大鼠血管钙失敏的机制。方法 SD大鼠32只,按随机数字表完全随机分为假手术组、盲肠结扎穿孔(CLP)3h组、CLP 6h组、CLP 12h组,每组8只。经CLP复制脓毒症大鼠模型,检测不同时点血浆IL-1β浓度及肠系膜上动脉(SMAs)钙敏感性,分析二者之间的相关性。培养SMAs来源的血管平滑肌细胞(VSMCs)并与不同浓度重组人IL-1β孵育24h,观察IL-1β对其肌球蛋白轻链(MLC20)磷酸化水平、Rho激酶活性、G蛋白表达水平及RhoGEFs活性的影响。结果经CLP 3h后SMAs钙敏感性开始下降(P<0.05),而血浆IL-1β在CLP 6h后开始上升(P<0.05),SMAs钙敏感性变化趋势与血浆IL-1β浓度变化呈明显负相关(P<0.05)。IL-1β可降低VSMCs MLC_(20)磷酸化水平及Rho激酶活性(P<0.05),上调Gα11表达而下调Gα12表达(P<0.05),但对Gαq和Gα13表达无明显作用(P>0.05)。IL-1β可明显降低RhoGEF和PDZ-RhoGEF活性(P<0.05),升高p63RhoGEF活性(P<0.05)。结论 IL-1β通过下调Gα12表达,引起PDZ-RhoGEF和Rho激酶的活性下降,导致MLC_(20)磷酸化水平下降从而介导脓毒症大鼠血管钙失敏的发生;另外也能通过上调Gα11表达,引起p63RhoGEF活性增加而介导脓毒症大鼠血管钙敏感性增加,但总效应是使钙敏感性降低。

Objective To investigate the mechanism of interleukin-1β（IL-1β） mediating the vascular calcium desensitization of septic rats by down-regulating Rho kinase activity. Methods Thirty-two SD rats were randomly divided into the sham operation group,cecal ligation and puncture （CLP） 3 h group,CLP 6 h group and CLP 12 h group,8 cases in each group. The septic rat was duplicated by CLP. Then the plasma IL-1β level and calcium sensitivity of superior mesenteric arteries （SMAs） were detected at different time points. Their correlation was analyzed. VSMCc derived from SMAs were cultured and incubated with different concen- trations of human recombinant IL-1β for 24 h. Then the influences of IL-1β on the MLC50 phosphorylation level, Rho kinase activity, G protein expression level and RhoGEF activity were observed. Results The calcium sensitivity of SMAs after CLP 3 h began to decrease（P〈0.05）, while plasma IL-1β level began to increase after CLP 6 h （P〈0.05）,the change trend of SMAs calcium sensitivity was negatively correlated with plasma IL-1β level change（P〈0.05）. IL-1β could decrease the phosphorylation level of VSMCs myosin light chain（MLC20 ） and Rho kinase activity（P〈0.05）, up-regulate Gall expression and down-regulate Gα12 expression, but had no obvious effect on Gaq and Ga13 expression（P〉0.05）. IL-1β could significantly reduce RhoGEF and PDZ-RhoGEF activity（P〈0.05） but significantly increase p63 Rho GEF activity（P〈0.05）. Conclusion IL-113 induces the decrease of PDZ-Rho- GEF and Rho kinase activity by down-regulating Gal2 expression,causes the decrease of MLC20 phosphorylation level,thus mediates the occurrence of calcium desensitization in septic raticin addition,IL-1β may cause the increase of p63 RhoGEF activity by upregulating Gall expression,th.us mediates the increase of vascular calcium sensitivity in septic rats, but the total effect is the decrease of calcium sensitivity.