Wu Qian, Liu Jiao, Tian Li-Yu, Wang Ning
College of Pharmacy, Anhui University of Chinese Medicine Hefei 230012, China Anhui Province Key Laboratory of Research & Development of Chinese Medicine, Anhui University of Chinese Medicine Hefei 230012,China.
College of Pharmacy, Anhui University of Chinese Medicine Hefei 230012, China.
Zhongguo Zhong Yao Za Zhi. 2022 Apr;47(7):1897-1903. doi: 10.19540/j.cnki.cjcmm.20211029.404.
Mitochondrion, as the main energy-supply organelle, is the key target region that determines neuronal survival and death during ischemia. When an ischemic stroke occurs, timely removal of damaged mitochondria is very important for improving mitochondrial function and repairing nerve damage. This study investigated the effect of ligustilide(LIG), an active ingredient of Chinese medicine, on mitochondrial function and mitophagy based on the oxygen and glucose deprivation/reperfusion(OGD/R)-induced injury model in HT22 cells. By OGD/R-induced injury model was induced in vitro, HT22 cells were pre-treated with LIG for 3 h, and the cell viability was detected by the CCK-8 assay. Immunofluorescence and flow cytometry were used to detect indicators related to mitochondrial function, such as mitochondrial membrane potential, calcium overload, and reactive oxygen species(ROS). Western blot was used to detect the expression of dynamin-related protein 1(Drp1, mitochondrial fission protein) and cleaved caspase-3(apoptotic protein). Immunofluorescence was used to observe the co-localization of the translocase of outer mitochondrial membrane 20(TOMM20, mitochondrial marker) and lysosome-associated membrane protein 2(LAMP2, autophagy marker). The results showed that LIG increased the cell viability of HT22 cells as compared with the conditions in the model group. Furthermore, LIG also inhibited the ROS release, calcium overload, and the decrease in mitochondrial membrane potential in HT22 cells after OGD/R-induced injury, facilitated Drp1 expression, and promoted the co-localization of TOMM20 and LAMP2. The findings indicate that LIG can improve the mitochondrial function after OGD/R-induced injury and promote mitophagy. When mitophagy inhibitor mdivi-1 was administered, the expression of apoptotic protein increased, suggesting that the neuroprotective effect of LIG may be related to the promotion of mitophagy.
线粒体作为主要的能量供应细胞器，是决定缺血期间神经元存活与死亡的关键靶区域。当发生缺血性中风时，及时清除受损线粒体对于改善线粒体功能和修复神经损伤非常重要。本研究基于氧糖剥夺/复氧（OGD/R）诱导的HT22细胞损伤模型，探讨中药有效成分川芎嗪（LIG）对线粒体功能和线粒体自噬的影响。通过体外诱导OGD/R损伤模型，用LIG预处理HT22细胞3小时，采用CCK-8法检测细胞活力。利用免疫荧光和流式细胞术检测与线粒体功能相关的指标，如线粒体膜电位、钙超载和活性氧（ROS）。采用蛋白质免疫印迹法检测动力相关蛋白1（Drp1，线粒体分裂蛋白）和裂解的半胱天冬酶-3（凋亡蛋白）的表达。利用免疫荧光观察线粒体外膜转位酶20（TOMM20，线粒体标志物）和溶酶体相关膜蛋白2（LAMP2，自噬标志物）的共定位。结果表明，与模型组相比，LIG提高了HT22细胞的活力。此外，LIG还抑制了OGD/R诱导损伤后HT22细胞中的ROS释放、钙超载以及线粒体膜电位的降低，促进了Drp1表达，并促进了TOMM20和LAMP2的共定位。研究结果表明，LIG可改善OGD/R诱导损伤后的线粒体功能并促进线粒体自噬。当给予线粒体自噬抑制剂mdivi-1时，凋亡蛋白表达增加，提示LIG的神经保护作用可能与促进线粒体自噬有关。
