肝素对脓毒症小鼠急性肺损伤时FAK/RhoA/ROCK信号通路的影响

Effects of heparin on FAK/RhoA/ROCK signaling pathways during acute lung injury in septic mice

目的评价肝素对脓毒症小鼠急性肺损伤(ALI)时黏着斑激酶(FAK)/Ras同源家族成员A(RhoA)/Rho相关卷曲螺旋形成蛋白激酶(ROCK)信号通路的影响。方法SPF级健康成年雄性C57BL/6J小鼠30只,6~8周龄,体质量20~23 g,采用随机数字表法分为3组(n=10):对照组(C组)、ALI组和肝素组(H组)。采用腹腔注射LPS 15 mg/kg的方法制备脓毒症小鼠ALI模型,C组注射等量生理盐水。H组尾静脉注射肝素钠10 U,30 min后制备模型,C组和ALI组注射等量生理盐水。于注射LPS后24 h时深麻醉下采集眼球静脉血样,随后处死小鼠取肺组织。采用ELISA法测定血清IL-1β、IL-6和TNF-α浓度;HE染色法观察肺组织病理学结果并行肺损伤(LI)评分,确定肺湿质量/干质量(W/D)比值;免疫组化染色法观察血管内皮黏附因子1(VCAM-1)表达;Western blot法检测FAK、磷酸化FAK(p-FAK)、RhoA、结合GTP形式Rho蛋白A(RhoA-GTP)和ROCK表达。结果与C组相比,ALI组血清IL-1β、IL-6、TNF-α浓度、肺W/D比值、LI评分和VCAM-1表达水平升高,肺组织p-FAK、RhoA-GTP和ROCK表达上调(P<0.05);与ALI组相比,H组血清IL-1β、IL-6、TNF-α浓度、肺W/D比值、LI评分和VCAM-1表达水平降低,肺组织p-FAK、RhoA-GTP和ROCK表达下调(P<0.05)。结论肝素减轻脓毒症小鼠ALI的机制与抑制FAK/RhoA/ROCK信号通路有关。

Objective To evaluate the effects of heparin on focal adhesion kinase(FAK)/Ras homolog gene family member A(RhoA)/Rho-associated coiled-coil-containing protein kinase(ROCK)signaling pathways during acute lung injury(ALI)in septic mice.Methods Thirty SPF healthy adult male C57BL/6J mice,aged 6-8 weeks,weighing 20-23 g,were assigned into 3 groups(n=10 each)using a random number table method:control group(group C),ALI group,and heparin group(group H).Septic ALI model was prepared by intraperitoneal injection of lipopolysaccharide 15 mg/kg,while group C received the equal volume of normal saline.In group H,heparin sodium solution 10 U was injected via the tail vein at 30 min before developing the model.The equal volume of normal saline was injected in C and ALI groups.Venous blood samples were collected from the eyeballs under deep anesthesia at 24 h after lipopolysaccharide injection.The mice were subsequently sacrificed and lung tissues were obtained for determination of the serum concentrations of interleukin-1beta(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-alpha(TNF-α)(using enzyme-linked immunosorbent assay),wet/dry lung weight(W/D)ratio,expression of vascular endothelial adhesion factor 1(VCAM-1)(by immunohistochemical staining)and expression of FAK,phosphorylated FAK(p-FAK),RhoA,GTP-bound RhoA(RhoA-GTP)and ROCK(by Western blot)and for examination of the pathological changes.The lung injury was assessed and scored.Results Comparison with group C,the serum concentrations of IL-1β,IL-6 and TNF-α,W/D ratio and lung injury scores were significantly increased,and the expression of VCAM-1,p-FAK,RhoA-GTP and ROCK was up-regulated in ALI group(P<0.05).Compared with ALI group,the serum concentrations of IL-1β,IL-6 and TNF-α,W/D ratio and lung injury scores were significantly decreased,and the expression of VCAM-1,p-FAK,RhoA-GTP and ROCK was down-regulated in H group(P<0.05).Conclusions The mechanism through which heparin mitigates ALI is associated with the inhibition of the FAK/RhoA/ROCK signaling pathway in septic mice.