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.
Zhongguo Zhong Yao Za Zhi. 2023 Jun;48(12):3308-3316. doi: 10.19540/j.cnki.cjcmm.20221212.401.
Based 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 Beclin1 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(Drp1) and optic atrophy 1(Opa1). 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 Beclin1, rise of cells with P62(P<0.01), inhibited expression of OGT, PINK1, and Parkin, up-regulated expression of Drp1, and down-regulated expression of Opa1 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 Beclin1 and decreased cells with P62(P<0.01). In addition, DBD promoted the expression of OGT, PINK1, Parkin, and Opa1 and inhibited the expression of Drp1, enhancing mitophagy(P<0.05, P<0.01). In conclusion, DBD can trigger PINK1/Parkin-mediated brain mitophagy through the OGT-PINK1 pathway, which plays a positive role in maintaining the health of the mitochondrial network. This may be a mitochondrial therapeutic mechanism to promote nerve cell survival and improve cerebral ischemia/reperfusion injury.
基于O-连接N-乙酰葡糖胺转移酶（OGT）-PTEN诱导的假定激酶1（PINK1）通路，研究了3,4-二羟基苯甲醛（DBD）对线粒体质量控制的作用机制。制备大脑中动脉闭塞/再灌注（MCAO/R）大鼠模型。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）和DBD-H组（10 mg·kg⁻¹）。给药7天（灌胃）后，除假手术组外，其余大鼠采用线栓法诱导MCAO/R。再灌注24小时后，测量神经功能和脑梗死面积百分比。基于苏木精-伊红（HE）染色和尼氏染色，检查脑神经元的病理损伤。然后在电子显微镜下观察线粒体的超微结构，并通过免疫荧光染色进一步检测轻链3（LC3）、聚集体蛋白1（SQSTM1/P62）和Beclin1的共定位。据报道，通过OGT-PINK1通路诱导线粒体自噬可以确保线粒体的质量。因此，采用蛋白质免疫印迹法检测OGT、线粒体自噬相关蛋白PINK1和E3泛素连接酶（Parkin）以及线粒体动力学蛋白动力相关蛋白1（Drp1）和视神经萎缩蛋白1（Opa1）的表达。结果显示，与假手术组相比，MCAO/R组存在神经功能障碍、脑梗死面积大（P<0.01）、神经元形态结构受损、尼氏体数量减少、线粒体肿胀、线粒体嵴消失、LC3和Beclin1阳性细胞减少、P62阳性细胞增加（P<0.01）、OGT、PINK1和Parkin表达受抑制、Drp1表达上调、Opa1表达下调（P<0.01）。然而，DBD改善了MCAO/R大鼠的行为缺陷和线粒体健康，表现为神经元和线粒体的形态结构改善以及尼氏体增加。此外，DBD增加了LC3和Beclin1阳性细胞，减少了P62阳性细胞（P<0.01）。另外，DBD促进了OGT、PINK1、Parkin和Opa1的表达，抑制了Drp1的表达，增强了线粒体自噬（P<0.05，P<0.01）。综上所述，DBD可通过OGT-PINK1通路触发PINK1/Parkin介导的脑线粒体自噬，这对维持线粒体网络的健康发挥积极作用。这可能是一种促进神经细胞存活和改善脑缺血/再灌注损伤的线粒体治疗机制。
