氧化苦参碱通过TGF-β1/Smads信号通路对脓毒性休克大鼠心肌损伤的保护作用

Protective effect of oxymatrine on myocardial injury in septic shock rats through the TGF-β1/Smads signaling pathway

目的 探究氧化苦参碱(OMT)通过TGF-β1/Smads信号通路对脓毒性休克大鼠心肌损伤的保护作用机制。方法 雄性Sprague-Dawley大鼠随机分为7组(n=8):CON组、OMT组、CLP组、CLP+DEX组[CLP+地塞米松(DEX),10 mg/kg]和CLP+OM T-M组(CLP+OMT 52 mg/kg)、CLP+OMT-H组(CLP+OMT 26 mg/kg)、CLP+DEX组(CLP+OMT 13 mg/kg)。感染性休克模型由CLP诱导。由右侧颈总动脉测定大鼠心率(HR)、平均动脉压(MAP)、左心室内压变化率(LVdp/dtmax)、左心室收缩末期压(LVESP)和通过插入心导管术测量左心室舒张末压(LVEDP)。取出心脏进行组织学分析,苏木精-伊红染色后,光镜下观察心肌组织病理变化。RT-PCR分析TNF-α和IL-1β的相对表达。蛋白质印迹分析TGF-β1和Smad2蛋白表达。通过放射免疫测定法测定心肌组织中TNF-α和IL-1β的水平。结果 与CON相比,OMT组的心功能参数,包括HR、MAP、LVSP、LVEDP和±LVdp/dtmax指数未受影响。在CON和OMT组之间未发现心肌组织的差异。心内膜完整,无水肿及纤维结缔组织增生。心肌条纹清晰,细胞核居中,未见血管舒张和炎性细胞浸润。心外膜在基质中表现出完整性,没有炎性渗出物。与CON相比,OMT组的心肌组织未见明显变化,均具有正常的形状和清晰的结构。CLP组的超微组织心肌组织受到显著损伤。与CLP相比,CLP+OMT组超微组织心肌组织损伤明显减轻。CLP+OMT-L组表现出最高水平的损伤。CLP+DEX组的超微组织心肌组织几乎正常。蛋白质印迹分析显示OMT对照心肌组织中TGF-β1和Smad2蛋白水平与CON组相似,但在CLP组中观察到显著更高(P<0.05)。然而,与CLP相比,CLP+OMT和CLP+DEX组的TGF-β1和Smad2蛋白水平显著降低(P<0.05)。放射免疫分析显示,与CON和OMT组相比,CLP组TNF-α和IL-1β蛋白水平显著升高(P<0.05)。与CLP组相比,发现CLP+OMT和阳性对照中TNF-α和IL-1β蛋白水平显著降低(P<0.05)。OMT对照和CON心肌组织中的TNF-α和IL-1β mRNA水平正常,CLP组的水平显著增加(P<0.05)。与CLP组相比,CLP+OMT和阳性对照中TNF-α和IL-1β mRNA水平显著降低(P<0.05)。结论 OMT通过抑制TGF-β1/Smads信号通路,在治疗感染性休克诱导的心肌损伤方面表现出巨大的治疗潜力。

Objective To explore the protective mechanism of oxymatrine on myocardial injury in septic shock rats through the transforming growth factor-β1(TGF-β1)/Smads signaling pathway. Methods Male Sprague-Dawley rats were randomly divided into 7 groups(n=8): CON group, OMT group, CLP group, CLP+DEX group(CLP+dexamethasone 10mg/kg), CLP+OMT-M group(CLP+OMT 52mg/kg), CLP+OMT-H group(CLP+OMT 26mg/kg), and CLP+OMT-L group(CLP+OMT 13mg/kg). The septic shock model was induced by the cecal ligation and puncture(CLP). In the right common carotid artery, the heart rate(HR), mean arterial pressure(MAP), rate of change of left ventricular pressure(LVdp/dtmax), left ventricular end-systolic pressure(LVESP) and left ventricular end-diastolic pressure(LVEDP) were measured by the cardiac catheterization. Rat heart was removed for histological analysis. After hematoxylin and eosin staining, the pathological changes of myocardial tissue were observed under a light microscope. The mRNA and protein expressions of tumor necrosis factor-α(TNF-α) and interleukin 1β(IL-1β), and TGF-β1 and Smad2 were analyzed by real-time reverse transcription polymerase chain reaction(RT-PCR) and Western blot, respectively. The positive expressions in myocardial tissue were measured by radioimmunoassay.Results Cardiac function parameters, including HR, MAP, LVSP, LVEDP and ±LVdp/dtmax index, were comparable between OMT group and CON group. No differences in myocardial tissues were found between CON and OMT groups. The endocardium was intact without edema or fibrous connective tissue hyperplasia. Myocardial stripe was clear, nucleus was centered, and no vasodilation and inflammatory cell infiltration was observed. The epicardium was intact in the stroma without inflammatory exudates. Compared with CON group, the myocardium of OMT group did not have obvious changes, and all of them had normal shape and clear structure. The ultrastructure and myocardial tissues of CLP group were significantly damaged. Compared with CLP group, the injury of ultrastructure and myocardial tissue in CLP+OMT group was significantly alleviated. The CLP+OMT-L group showed the severest injury. The ultrastructure and myocardial tissue of CLP+DEX group were almost normal. Western blot showed that the protein levels of TGF-β1 and Smad2 in myocardium of OMT group were similar to those in CON group, but significantly higher in CLP group(both P<0.05). However, compared with CLP group, the protein levels of TGF-β1 and Smad2 in CLP+OMT and CLP+DEX groups were significantly downregulated(both P<0.05). Radioimmunoassay showed that compared with CON and OMT groups, protein levels of TNF-α and IL-1β in CLP group were significantly upregulated(P<0.05). Compared with CLP group, the protein levels in CLP+OMT group and the positive control group were significantly downregulated(P<0.05). The mRNA levels of TNF-α and IL-1β in OMT control and CON were normal, which in CLP group were significantly upregulated(P<0.05). Compared with CLP group, the mRNA levels of TNF-α and IL-1β in CLP+OMT and positive control were significantly downregulated(P<0.05). Conclusion OMT shows a great therapeutic potential in the treatment of septic shock-induced myocardial injury by inhibiting the TGF-β1/Smads signaling pathway.