抵当汤调节线粒体自噬对脑出血后神经损伤的保护作用及机制

Protective effect and mechanism of Didang Tang regulating mitochondrial autophagy on nerve damage after intracerebral hemorrhage

目的基于线粒体自噬研究抵当汤(DDT)对脑出血(ICH)后神经损伤的保护作用及机制。方法应用自体血注入法建立ICH大鼠模型,氯化钴(CoCl_(2))诱导PC12细胞建立神经细胞缺氧模型,并应用DDT进行治疗。随机分为假手术(Sham)组、ICH组、脑血康(NXK,0.64 mg/100 g)组、ICH+DDT低、中、高剂量(0.13、0.26、0.52 g/100 g)组、线粒体自噬诱导剂CCCP阳性对照(CCCP,4 mg/kg)组、CoCl_(2)(150μmol/L)组、CoCl_(2)+DDT低、中、高剂量(50、100、200μg/ml)组。Longa评分法观察DDT对ICH大鼠神经功能缺损的保护作用,通过检测超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性、谷胱甘肽过氧化物酶(GSH-Px)和丙二醛(MDA)含量探索DDT保护神经细胞氧化损伤的作用。利用Tunel染色和流式细胞术检测细胞凋亡,免疫荧光染色法观察线粒体膜电位变化,Western印迹和免疫组化法检测DDT对线粒体自噬的调控作用。结果DDT可以改善ICH神经功能评分,促进ICH后神经功能恢复;DDT能显著促进SOD、CAT及GSH-Px水平,显著降低MDA含量(P<0.05,P<0.01);DDT能显著减轻ICH病灶组织细胞凋亡,并能显著降低CoCl_(2)诱导PC12细胞的凋亡率(P<0.01,P<0.001);DDT能恢复CoCl_(2)诱导PC12细胞线粒体膜电位;DDT显著上调Parkin、PTEN诱导激酶(Pink)1和微管相关蛋白轻链(LC)3Ⅱ表达,显著抑制P62蛋白表达(P<0.05,P<0.01)。结论抵当汤能够调控线粒体自噬抑制神经细胞氧化损伤和细胞凋亡。

Objective Based on mitochondrial autophagy to study the protective effect and mechanism of Didang Tang(DDT)on nerve injury after intracerebral hemorrhage(ICH).Methods Autologous blood infusion was used to establish ICH rat models,CoCl_(2) induced PC12 cells to establish neuronal hypoxia models,and DDT was used for treatment.Rats were randomly divided into sham operation(Sham)group,ICH group,Naoxuekang(NXK,0.64 mg/100 g)group,ICH+DDT low,medium,and high dose(0.13,0.26,0.52 g/100 g)groups.Mitochondrial autophagy inducer CCCP positive control(CCCP,4 mg/kg)group,CoCl_(2)(150μmol/L)group,CoCl_(2)+DDT low,medium and high dose(50,100,200μg/ml)groups.The Longa scoring was used to observe the protective effect of DDT on neurological deficits in rats with ICH.Superoxide dismutase(SOD)and catalase enzymes(CAT)activity,glutathione peroxidase(GSH-Px)and malondialdehyde(MDA)content were detected to explore the protective effect of DDT on nerve cell oxidative damage.Tunel staining and flow cytometry were used to detect cell apoptosis.Fluorescence staining was used to observe the changes in mitochondrial membrane potential.Western blotting and immunohistochemistry were used to detect the regulatory effect of DDT on mitochondrial autophagy.Results DDT could improve the neurological function score and promote the recovery of neurological function of ICH.DDT could significantly promote SOD,CAT and GSH-Px levels,significantly reduce MDA content(P<0.05,P<0.01).DDT could significantly reduce cell apoptosis in focal tissues of ICH,and significantly reduce the percentage of apoptotic cells in PC12 cells induced by CoCl_(2)(P<0.01,P<0.001);DDT could restore the mitochondrial membrane potential in CoCl_(2)-induced PC12 cells.DDT could significantly increase the expressions of Parkin,PTEN induces kinase(Pink)1 and microtubule-associated protein light chain(LC)3Ⅱ(P<0.05,P<0.01),and significantly inhibit the expression of P62(P<0.05,P<0.01).Conclusions Didang Tang could inhibit nerve cell oxidative damage and apoptosis by regulating mitochondrial autophagy.