螺内酯对肢体缺血再灌注心肌损伤高迁移率族蛋白B1与Toll样受体4信号通路的影响

Effect of spironolactone on HMGB1-TLR4 signaling pathway following limbs ischemia/reperfusion-induced myocardial injury in rat

目的研究醛固酮受体拮抗剂螺内酯减轻肢体缺血再灌注致心肌损伤的信号传导机制。方法选择24只雄性Wistar大鼠,按随机数字法分为对照组、缺血再灌注组(缺血4 h再灌注4 h)和螺内酯组(建模前,螺内酯每日20 mg/kg灌胃,连续6 d),每组8只。观察各组大鼠血浆肌钙蛋白I(cTnI)水平;血浆和心肌白细胞介素6(IL-6)和高迁移率族蛋白B1(HMGB1)水平变化,用实时荧光定量PCR技术测大鼠心肌HMGB1、Toll受体4(TLR4)基因相对表达,免疫组织化学染色观察大鼠心肌HMGB1、TLR4蛋白表达。结果与对照组比较,缺血再灌注组大鼠血浆cTnI、血浆IL-6和心肌IL-6、血浆HMGB1和心肌HMGB1水平明显升高(P<0.05),心肌HMGB1 mRNA和TLR4 mRNA相对表达显著上调(P<0.05)。与缺血再灌注组比较,螺内酯组大鼠血浆cTnI、IL-6、HMGB1水平明显下降[(0.179±0.068)U/L vs(0.476±0.106)U/L,(102.05±15.17)ng/L vs(150.70±28.85)ng/L,(1.07±0.39)μg/L vs(2.48±0.68)μg/L,P<0.05],心肌IL-6、HMGB1水平明显降低[(66.26±20.44)pg/mg vs(117.02±21.44)pg/mg,(14.22±3.44)ng/mg vs(27.93±5.58)ng/mg,P<0.05],心肌HMGB1 mRNA、TLR4 mRNA表达显著下调(P<0.05)。对照组大鼠心肌细胞可见少量散在HMGB1和TLR4棕黄色阳性颗粒,缺血再灌注组大鼠心肌细胞内可见较多HMGB1、TLR4棕黄色阳性颗粒,螺内酯组心肌细胞HMGB1、TLR4棕黄色阳性颗粒较缺血再灌注组明显减少。结论醛固酮受体拮抗剂螺内酯能减轻肢体缺血再灌注后心肌损伤及炎性反应,机制可能与抑制HMGB1-TLR4信号通路有关。

Objective To study the mechanism of spironolactone(an aldosterone receptor antagonist)on alleviating limbs ischemia/reperfusion-induced myocardial injury in rats.Methods Twenty-four male Wistar rats were randomly divided into control group,ischemia/reperfusion group and spironolactone group(8 in each group).The plasma levels of troponin I(cTnI),interleukin-6(IL-6)and HMGB1 and the myocardial levels of IL-6 and HMGB1 were measured.The expressions of HMGB1 mRNA and TLR4 mRNA and protein in the myocardial tissue were detected by RT-PCR and immunohistochemical staining.Results The plasma level of cTnI,both plasma and myocardial levels of IL-6 and HMGB1 and the expression of HMGB1 mRNA and TLR4 mRNA were significantly higher in ischemia/reperfusion group than in control group(P<0.05).The plasma levels of cTnI,IL-6 and HMGB1 and myocardial levels of IL-6 and HMGB1 were significantly lower in spironolactone group than in ischemia/reperfusion group(0.179±0.068 U/L vs 0.476±0.106 U/L,102.05±15.17 ng/L vs 150.70±28.85 pg/mL,1.07±0.39μg/L vs 2.48±0.68μg/L,66.26±20.44 pg/mg vs 117.02±21.44 pg/mg,14.22±3.44 ng/mg vs 27.93±5.58 ng/mg,allP<0.05).The expressions of myocardial HMGB1 mRNA and TLR4 mRNA were significantly downregulated in spironolactone group than in ischemia/reperfusion group(P<0.05).Brown HMGB1 and TLR4 particles detected in myocardiocytes were much more in ischemia/reperfusion group than in control group while brown HMGB1 and TLR4 particles detected in myocardiocytes were much less in spironolactone group than in ischemia/reperfusion group(P<0.05).Conclusion Spironolactone can alleviate limbs ischemia/reperfusion-induced myocardial injury and inflammatory reactions by inhibiting the HMGB1-TLR4 signaling pathway.