丹酚酸B对脓毒症大鼠心肌损伤的保护作用

Protective Effect of Salvianolic Acid B on Myocardial Injury in Septic Rats

目的:探讨丹酚酸B(salvianolic acid B,Sal-B)对脓毒症大鼠心肌损伤的作用及机制。方法:将50只雄性SD大鼠随机分为假手术组,模型组(行盲肠结扎穿孔术复制脓毒症动物模型),Sal-B组低、中、高剂量组(在模型基础上,经尾静脉注射Sal-B 6,12,24 mg·kg^-1)。采用酶联免疫吸附测定(ELISA)检测心肌坏死标志物和炎症因子,包括血清肌钙蛋白T(Tn T),肌酸激酶同工酶(CK-MB),心肌肌钙蛋白I(c Tn I)和肿瘤坏死因子-α(TNF-α),白细胞介素(IL)-1β,IL-6水平;比色法检测心肌中超氧化物歧化酶(SOD)的活性和丙二醛(MDA)的含量;采用苏木素-伊红(HE)染色观察各组心肌组织病理变化;采用原位末端标记法(TUNEL)检测心肌细胞凋亡情况;采用蛋白免疫印迹法(Western blot)检测心肌组织中凋亡相关蛋白半胱氨酸蛋白酶-3(Caspase-3),B淋巴细胞瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax)和自噬微管相关蛋白轻链3(LC3),Beclin-1表达水平。结果:与假手术组比较,模型组Tn T,CK-MB和c Tn I水平显著升高(P <0. 01);心肌组织TNF-α,IL-1β,IL-6水平显著升高(P <0. 01);心肌组织MDA含量显著升高(P <0. 01),SOD的活力显著降低(P <0. 01),TUNEL阳性心肌细胞比率显著升高(P <0. 01),Bax和Caspase-3蛋白表达水平升高,而Bcl-2表达显著降低(P <0. 01),自噬相关蛋白LC3,Beclin-1表达水平显著升高(P <0. 01);与模型组比较,Sal-B中、高剂量组Tn T,CK-MB和c Tn I水平均显著降低(P <0. 01),心肌组织TNF-α,IL-1β,IL-6水平显著降低(P <0. 01),心肌组织MDA含量明显降低(P <0. 05,P <0. 01),同时,心肌组织SOD的活性明显升高(P <0. 05,P <0. 01);TUNEL阳性心肌细胞比率明显降低(P <0. 05),Bax和Caspase-3蛋白表达下降,Bcl-2表达明显升高(P <0. 05,P <0. 01);同时增强自噬反应,LC3Ⅱ/LC3Ⅰ增加,Beclin-1表达水平明显上调(P <0. 05,P <0. 01)。结论:Sal-B通过影响自噬蛋白,降低脓毒症大鼠心肌炎症反应和氧化应激,抑制心肌细胞凋亡,从而减轻脓毒症大鼠心肌的损伤。

Objective: To explore the effect of salvianolic acid B ( Sal-B) on the myocardial apoptosis in rats with sepsis. Method: The 50 male SD rats were randomly divided into sham operation group,model group ( cecal ligation and perforation were performed to replicate the animal model of sepsis) and Sal-B low,medium and high-dose group ( 6,12,24 mg·kg ^- 1 ). The myocardial necrosis markers troponin T ( TnT),cardiac troponin I ( cTnI),creatine kinase-MB ( CK-MB) and the tumor necrosis factor-α( TNF-α),interleukin-1β( IL-1β) and interleukin-6 ( IL-6 ) were assayed by enzyme-linked immunosorbent assay ( ELISA). The antioxidant enzyme superoxide dismutase ( SOD) activities and malonaldialdehyde ( MDA) levels in myocardial homogenates were determined by spectrophotometrically. Haematoxylin-eosin ( HE) staining was used to evaluate the pathological change in rats. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay ( TUNEL) was used to detect apoptosis of myocardial cells in hearts,respectively. Western blot was used to detect the expression levels of apoptosis-related protein cysteine protease-3 ( Caspase-3),b-lymphocytoma-2 ( Bcl-2),Bcl-2-related X protein ( Bax),autophagy microtubule-related light chain 3 ( LC3) and Beclin-1 in myocardial tissue. Result: Compared with sham group,the levels of TnT,CK-MB and cTnI in model group were significantly increased ( P < 0.01). The levels of TNF-α,IL-1β and IL-6 in myocardial tissue were significantly increased ( P < 0.01). MDA content in myocardial tissue was significantly increased ( P < 0.01 ),SOD activity in myocardial tissue was significantly decreased ( P < 0.01),the ratio of TUNEL positive myocardial cells was significantly increased ( P < 0.01),Bax and Caspase-3 protein expression levels were significantly increased,while Bcl-2 expression was significantly decreased ( P < 0.01),autophagy related protein LC3 and Beclin-1 expression levels were significantly increased ( P < 0.01). Compared with model group,the levels of TnT,CK-MB and cTnI in Sal-B medium and high-dose groups were significantly decreased ( P < 0.01),the levels of TNF-α,IL-1 and IL-6 in the myocardial tissue were significantly decreased ( P < 0.01),MDA content in myocardial tissue was significantly decreased ( P < 0.05,P < 0.01),and the SOD activity in myocardial tissue was significantly increased ( P < 0.05,P < 0.01). The ratio of TUNEL positive cardiomyocytes was significantly decreased ( P < 0.05),Bax and Caspase-3 protein expressions were decreased,and Bcl-2 expression was significantly increased ( P < 0.05,P < 0.01). The autophagy response was enhanced, the LC3 Ⅱ/LC3 Ⅰ ratio was increased, and Beclin-1 expression was significantly up-regulated ( P < 0.05,P < 0.01). Conclusion: Sal-B showed a protective effect on the myocardial apoptosis in septic rats maybe by depressing inflammatory infiltration,anti-oxidative effects and improving the exceptional expression of the proteins such as Bax,Bcl-2,Caspases-3 by enhancing the level of autophagy.