3,4-二羟基苯甲醛通过OGT-PINK1途径对线粒体进行质量控制的机制研究  

Quality control mechanism of mitochondria by 3,4-dihydroxybenzaldehyde through OGT-PINK1 pathway

作　　者：罗苑 陈普 杨丽萍 段小花 LUO Yuan;CHEN Pu;YANG Li-ping;DUAN Xiao-hua(Yunnan Key Laboratory of Dai Medicine and Yi Medicines,Yunnan University of Chinese Medicine,Kunming 650500,China)

机构地区：[1]云南中医药大学云南省傣医药与彝医药重点实验室,云南昆明650500

出　　处：《中国中药杂志》2023年第12期3308-3316,共9页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(81960733);兴滇英才支持计划-青年人才专项(2022)。

摘　　要：基于O-连接N-乙酰氨基葡萄糖转移酶(O-GlcNAc transferase, OGT)-PTEN诱导假定激酶1(PTEN-induced putative kinase 1,PINK1)途径探讨3,4-二羟基苯甲醛(3,4-dihydroxybenzaldehyde, DBD)对线粒体进行质量控制的作用机制。通过建立大鼠局灶性大脑中动脉闭塞/再灌注(middle cerebral artery occlusion/reperfusion, MCAO/R)模型,将SD大鼠随机分为假手术组(sham),模型组(MCAO/R),DBD-L组(5 mg·kg^(-1))、DBD-H组(10 mg·kg^(-1)),每组12只,连续灌胃给药7 d后,除sham组外采用线栓法复制大鼠MCAO/R模型,再灌注24 h后检测大鼠神经功能、脑梗死面积百分率,以及HE和Nissl染色检测大鼠脑神经元的病理损伤。随后在电镜下观察线粒体超微结构,并用免疫荧光染色进一步检测自噬相关蛋白(light chain-3 protein, LC3)、选择性自噬接头蛋白(sequestosome-1,SQSTM1/P62)和自噬效应蛋白(beclin 1protein, Beclin1)的共定位。研究表明,通过诱导OGT-PINK1途径的线粒体自噬可确保线粒体质量,因此,该研究采用Western blot检测脑组织中OGT表达,线粒体自噬蛋白PINK1、E3泛素蛋白连接酶(Parkin),以及线粒体动力学蛋白动力相关蛋白1(dynamin-like protein 1,Drp1)和视神经萎缩蛋白1(optic atrophy 1,Opa1)的表达。结果显示,与sham组相比,MCAO/R组大鼠神经功能障碍,脑梗死面积明显增加(P<0.01),神经元形态结构的受损和尼氏体数量下降,伴有线粒体肿胀嵴消失;自噬的免疫荧光指标LC3及Beclin1的阳性细胞减少,而P62阳性细胞增加(P<0.01);Western blot检测显示,OGT、PINK1和Parkin的蛋白表达被抑制,线粒体动力学蛋白Drp1表达上调,相反Opa1表达下调(P<0.01),说明线粒体自噬水平的下降及线粒体动力学的失衡。然而DBD给药后对MCAO/R大鼠的行为缺陷和线粒体健康有积极作用,表现为神经元和线粒体形态结构的改善,以及尼氏体数量的增加;自噬相关指标LC3及Beclin1的阳性细胞增加,而P62阳性细胞减少(P<0.01)。此外,DBBased on the O-GlcNAc transferase(OGT)-PTEN-induced putative kinase 1(PINK1)pathway,the mechanism of 3,4-dihydroxybenzaldehyde(DBD)on mitochondrial quality control was investigated.Middle cerebral artery occlusion/reperfusion(MCAO/R)rats were established.SD rats were randomized into sham operation group(sham),model group(MCAO/R),DBD-L group(5 mg·kg^(-1)),and DBD-H group(10 mg·kg^(-1)).After 7 days of administration(ig),MCAO/R was induced in rats except the sham group with the suture method.Twenty-four h after reperfusion,the neurological function and the percentage of cerebral infarct area were measured.Based on hematoxylin and eosin(HE)staining and Nissl staining,the pathological damage of cerebral neurons was examined.Then the ultrastructure of mitochondria was observed under the electron microscope,and the co-localization of light chain-3(LC3),sequestosome-1(SQSTM1/P62),and Beclinl was further detected by immunofluorescence staining.It has been reported that the quality of mitochondria can be ensured by inducing mitochondrial autophagy through the OGT-PINK1 pathway.Therefore,Western blot was employed to detect the expression of OGT,mitophagy-related proteins PINK1 and E3 ubiquitin ligase(Parkin),and mitochondrial kinetic proteins dynamin-like protein 1(Drpl)and optic atrophy 1(Opal).The results showed that MCAO/R group had neurological dysfunction,large cerebral infarct area(P<0.01),damaged morphological structure of neurons,decreased number of Nissl bodies,mitochondrial swelling,disappearance of mitochondrial cristae,decrease of cells with LC3 and Beclinl,rise of cells with P62(P<0.01),inhibited expression of OGT,PINK1,and Parkin,up-regulated expression of Drpl,and down-regulated expression of Opal compared with the sham group(P<0.01).However,DBD improved the behavioral deficits and mitochondrial health of MCAO/R rats,as manifested by the improved morphology and structure of neurons and mitochondria and the increased Nissl bodies.Moreover,DBD increased cells with LC3 and Beclinl and decreased cells with P62

关 键 词：3 4-二羟基苯甲醛 脑缺血再灌注损伤 OGT-PINK1途径 线粒体质量控制 线粒体自噬 

分 类 号：R743.3[医药卫生—神经病学与精神病学]