白介素-1β对家兔内毒素休克血管钙敏感性的调节作用及与Rho激酶和PKC的关系

Regulatory role of interleukin-1β in vascular calcium sensitivity and its relationship with PKC and Rho kinase in rabbits following endotoxic shock

目的观察白介素-1β(IL-1β)对家兔内毒素休克及离体血管钙敏感性影响及与PKC、Rho激酶的关系。方法健康成年家兔35只,雌雄不拘,完全随机分为正常对照组,LPS30min组,LPS1h组,LPS2h组,LPS4h组,每组7只,经耳缘静脉注射1mg/kg LPS(O111∶B4)复制家兔内毒素休克模型,在给LPS后30min、1、2、4h取血清及肠系膜上动脉(superior mesenteric artery,SMA),用ELISA方法检测不同时相点血清中IL-1β的浓度;用离体血管环张力测定技术,测血管环钙敏感性的变化,分析其与血清IL-1β浓度的相关关系。另取家兔70只,采用随机数字表分为正常对照组,25ng/mlIL-1β组,50ng/ml IL-1β组,100ng/ml IL-1β组,150ng/ml IL-1β组,200ng/ml IL-1β组,200ng/ml IL-1β+PMA组,200ng/ml IL-1β+staurosporine组,200ng/ml IL-1β+AngⅡ组,200ng/ml IL-1β+Y27632组,每组7只。无菌条件下取正常家兔SMA,检测不同浓度重组人IL-1β对其钙敏感性的影响及与PKC、Rho激酶的关系。结果给LPS后30min至1h内成功复制出内毒素休克模型,测其平均动脉压(mean artery pressure,MAP)下降30%以上,血清中IL-1β在2h后即开始上升,至4h时显著升高(P<0.01);SMA钙敏感性在给LPS后30min、1h较正常对照组明显增高(P<0.01),而在给LPS后2、4h则明显降低(P<0.01),其变化趋势与血清IL-1β水平变化呈显著负相关(r=-0.675,P<0.01)。IL-1β可诱导离体SMA钙敏感性下降,其作用可被PKC和Rho激酶的激动剂拮抗。结论 IL-1β能够降低血管钙敏感性,且该作用与PKC、Rho激酶密切相关,IL-1β对内毒素休克后血管钙失敏可能有重要介导作用。

Objective To investigate the effect of interleukin-1β（IL-1β）on vascular calcium sensitivity in vitro and in rabbit after endotoxic shock,and to evaluate whether the effect is concerned with protein kinase C（PKC）and Rho kinase.MethodsA total of 35 adult rabbits were divided into 5 groups：normal control,LPS 30-minute,LPS 1-hour,LPS 2-hour,and LPS 4-hour groups（n=7）.Rabbits were given 1 mg/kg of LPS（O111∶B4）via ear-border vein to induce endotoxic shock.Then blood serum and superior mesenteric artery（SMA）were collected to measure IL-1β concentration and calcium sensitivity respectively at correspording time points.The vascular calcium sensitivity was measured with an isolated organ perfusion system.Then the correlation of calcium sensitivity of SMAs with IL-1β concentration in blood serum was analyzed.Effects of different concentrations of IL-1β（25,50,100,150 and 200 ng/ml）on vascular calcium sensitivity of SMAs and the relationship to PKC and Rho kinase were also observed with the aid of their agonists,inhibitors and antagonists（200 ng/ml IL-1β＋ 10^-7 mol/L PMA,＋10^-7/10^-6/10^-5 mol/L staurosporine,＋10-5 mol/L Y27632,and ＋10-9 mol/L AngⅡ）.ResultsEndotoxic shock model was successfully produced in 30 min to 1 h after LPS injection.The IL-1β concentration of blood serum started to increase at 2 h after injection,and it was then elevated very obviously at 4 h after injection（P〈0.01）.As compared to the control group,the calcium sensitivity of SMAs at early period（30 min and 1 h）after LPS injection was increased significantly（P〈0.01）,however,in 2 h and 4 h after LPS injection,it was decreased obviously（P〈0.01）.The changes of vascular calcium sensitivity was positively correlated with the IL-1β concentration in blood serum（r=-0.675,P〈0.05）.IL-1β reduced the calcium sensitivity of SMA in vitro,but the effect was reversed by PKC and Rho kinase agonists.ConclusionOur experiments reveal that IL-1β could decrease calcium sensitivity of rabbit in vitro,and its effect is closely related to PKC and Rho kinase.IL-1β may play a very important role in calcium desensitization following endotoxic shock in rabbits.