基于IRAK1/TRAF6/NF-κB通路探讨参附注射液治疗大鼠脓毒症心功能障碍的作用机制

Mechanism of Shenfu Injection in Treating Sepsis Induced Myocardial Dysfunction Through IRAK1/TRAF6/NF-κB Signaling Pathway

目的:基于白细胞介素-1受体相关激酶-1(interleukin-1 receptor-related kinase-1,IRAK1)/TNF受体相关因子6(TNF receptor associated factor 6,TRAF6)/核因子-κB(nuclear factor-κB,NF-κB)通路探讨参附注射液治疗大鼠脓毒症心功能障碍(sepsis induced myocardial dysfunction,SIMD)的作用机制。方法:将40只雄性SD大鼠随机分为对照组、假手术组、模型组和参附注射液组。对照组不采取任何干预措施,假手术组只寻找盲肠段但不进行结扎穿孔,其余大鼠采用盲肠结扎穿孔术制备脓毒症模型。造模后,大鼠尾静脉注射参附注射液或生理盐水(5 mL·kg^(-1)),12 h后重复注射1次。采用超声心动图检测大鼠心功能,包括左室收缩末内径(left ventricular end-systolic diameter,LVIDs)、左室舒张末内径(left ventricular end-diastolic diameter,LVIDd)、左室射血分数(left ventricular ejection fraction,LVEF)、短轴缩短率(fraction shortening,FS)。采用HE染色观察大鼠心肌组织病理形态;采用透射电镜观察大鼠心肌组织超微结构;采用ELISA检测大鼠血清白细胞介素-10(interleukin-10,IL-10)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、半胱氨酸天冬氨酸蛋白酶-3(cysteine aspartic acid specific protease-3,Caspase-3)含量;采用Western blot检测大鼠心肌组织IRAK1、TRAF6、NF-κB表达水平。结果:对照组与假手术组大鼠LVIDd、LVIDs、LVEF、FS的差异无统计学意义(P>0.05);与假手术组比较,模型组大鼠LVIDd、LVIDs增加(P<0.01)且FS、LVEF减少(P<0.001);与模型组比较,参附注射液组大鼠LVIDd、LVIDs减少(P<0.05)且FS、LVEF增加(P<0.001)。对照组与假手术组大鼠心肌组织结构正常;模型组大鼠心肌细胞明显水肿,存在心肌溶解及炎性细胞浸润;参附注射液组大鼠心肌细胞轻度水肿,少量炎性细胞浸润。对照组和假手术组大鼠心肌细胞线粒体结构正常;模型组大鼠心肌细胞内可见大量线粒体肿胀,嵴断裂,基质出现空白区;参附注射液组大鼠心肌细胞内大部分线粒体形态正常。对照组与假手术组大鼠血清IL-10、TNF-α、Bcl-2、Caspase-3含量的差异无统计学意义(P>0.05);与假手术组比较,模型组大鼠血清IL-10、TNF-α、Bcl-2、Caspase-3含量增加(P<0.05);与模型组比较,参附注射液组大鼠血清IL-10、TNF-α、Caspase-3含量减少(P<0.01)。对照组与假手术组大鼠心肌组织IRAK1、TRAF6、NF-κB表达水平的差异无统计学意义(P>0.05);与假手术组比较,模型组大鼠心肌组织IRAK1、NF-κB、TRAF6表达水平升高(P<0.05);与模型组比较,参附注射液组大鼠心肌组织IRAK1、NF-κB、TRAF6表达水平降低(P<0.05)。结论:参附注射液通过调节IRAK1/TRAF6/NF-κB通路,改善大鼠心功能,减轻心肌损伤和炎症反应,从而发挥治疗SIMD的作用。

Objective:To Explore the mechanism of Shenfu Injection in treating sepsis induced myocardial dysfunction(SIMD)based on interleukin-1 receptor associated kinase-1(IRAK1)/TNF receptor associated factor 6(TRAF6)/nuclear factor-κB(NF-κB)signaling pathways.Methods:Forty male SD rats were randomly divided into a control group,a sham operation group,a model group and a Shenfu Injection group.The control group were given no intervention,the sham operation group was only searched for the cecal segment but not performed perforation and ligation,and the remaining rats were used cecal ligation and perforation surgery to establish a sepsis model.After modeling,the rats were injected with Shenfu Injection or physiological saline(5 mL·kg^(-1))via the tail vein,and the injection was repeated 12 hours later.Use echocardiography to detect rat cardiac function,including left ventricular end-systolic diameter(LVIDs),left ventricular end-diastolic diameter(LVIDd),left ventricular ejection fraction(LVEF)and fraction shortening(FS).HE staining was used to observe the pathological morphology of rat myocardial tissue;Use transmission electron microscopy to observe the ultrastructure of rat myocardial tissue;ELISA was used to detect the serum levels of interleukin-10(IL-10),tumor necrosis factor-α(TNF-α),B-cell lymphoma-2(Bcl-2)and cysteine aspartic acid specific protease-3(caspase-3)in rats;The expression levels of IRAK1,TRAF6 and NF-κB in myocardial tissue were detected by Western blot.Results:There was no statistically significant difference in LVIDd,LVIDs,LVEF and FS between the control group and the sham operation group rats(P>0.05);Compared with the sham operation group,the LVIDd and LVIDs of the model group rats increased significantly(P<0.01),while FS and LVEF decreased significantly(P<0.001);Compared with the model group,the LVIDd and LVIDs of rats in the Shenfu Injection group decreased significantly(P<0.05),while FS and LVEF increased significantly(P<0.001).The myocardial tissue structure of rats in the control group and sham operation group was normal;The myocardial cells of the model group rats showed significant edema,with myocardial dissolution and inflammatory cell infiltration;The myocardial cells of rats in the Shenfu injection group showed mild edema and a small amount of inflammatory cell infiltration.The mitochondrial structure of myocardial cells in the control group and sham operation group rats was normal;A large number of mitochondria swelling,cristae rupture and blank areas in the matrix were observed in the myocardial cells of the model group rats;The morphology of most mitochondria in myocardial cells of rats treated with Shenfu Injection group was normal.There was no significant difference in the serum levels of IL-10,TNF-α,Bcl-2 and Caspase-3 between the control group and the sham operation group(P>0.05).Compared with the sham operation group,the serum levels of IL-10,TNF-α,Bcl-2 and Caspase-3 were significantly increased in the model group(P<0.05).Compared with the model group,the serum levels of IL-10,TNF-αand Caspase-3 in the Shenfu Injection group were significantly decreased(P<0.01).There were no significant differences in the expression levels of IRAK1,TRAF6 and NF-κB between the control group and the sham operation group(P>0.05).Compared with that of the sham operation group,the levels of IRAK1,NF-κB and TRAF6 in myocardial tissue of the model group were significantly increased(P<0.05).Compared with the model group,the Shenfu Injection group had significant decreases in the levels of IRAK1,NF-κB and TRAF6 in myocardial tissue(P<0.05).Conclusion:Shenfu Injection can improve cardiac function,reduce myocardial injury and inflammatory response by regulating IRAK1/TRAF6/NF-κB signaling pathway in rats,thus playing a role in the treatment of SIMD.