Gao Yuan, Guo Yiyang, Wu Mohan, Zheng Yanfei
（ 100029） School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 100029, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2025 Jan 20;56(1):74-82. doi: 10.12182/20250160504.
To investigate the molecular mechanism of Huangjing Zanyu Capsule (HJZY), a new class-Ⅲ traditional Chinese medicine for the treatment of male infertility developed by Wang Qi, an academician of the Chinese Academy of Engineering, based on AMPK-mediated mitophagy in the treatment of oligoasthenospermia.
Acrolein (ACR) was used to treat GC-2spd(ts) mouse spermatocytes to establish a cell model of oligoasthenospermia. The optimal ACR concentration and exposure time for subsequent modeling were determined by CCK8 cell viability assay. After successful modeling, the cells were cultured in complete medium containing different concentrations of HJZY. Then, cell viability was assessed by CCK8 assay after 24 hours, and the subsequent treatment concentration was determined based on the cell viability. After the GC-2spd cells adhered to the wall, they were divided into a normal control (NC) group, a modeling group, and an ACR + HJZY treatment group. The effect of HJZY on mitophagy was observed by confocal fluorescence microscopy. The three groups of cells were transfected with siRNA-NC and siRNA-, respectively, and divided into six groups, including siRNA-NC + control, siRNA-NC + ACR, siRNA-NC + ACR + HJZY, siRNA- + control, siRNA- + ACR, and siRNA- + ACR + HJZY groups. Western blot was performed to validate the regulatory effect of HJZY on mitophagy-related proteins, such as p-AMPK, LC3B, P62, PINK1, Parkin, TBK1, and ULK1, which were all proteins mediated by AMPK.
Through the cell viability assay, 34 μmol/L was selected as the the modeling concentration of ACR, and 20 minutes was selected as the modeling time The treatment concentration of HJZY was 160 μmol/L. Confocal fluorescence microscopy showed that HJZY had, to a certain degree, a positive regulatory effect on the mitochondrial membrane potential of damaged spermatogenic cells. The mitochondrial membrane potential of the model group decreased significantly compared with that of the NC group. After exposure to treatment, the cell membrane potential of the ACR + HJZY treatment group increased compared with that of the model group, and the difference was statistically significant ( < 0.05). Western blot results showed that the expression levels of p-AMPK/AMPK and PINK1 proteins in the siRNA-NC + ACR group were significantly lower than those in the siRNA-NC + control group ( < 0.001). The level of Parkin protein in the siRNA-NC + ACR group was lower than that in the siRNA-NC + control group, but the difference was not statistically significant. After the administration of HJZY, the levels of these 3 proteins increased, and those in the siRNA-NC + ACR + HJZY group were higher than those in the siRNA-NC + ACR group ( < 0.001). The expression levels of LC3B, P62, TBK1, and ULK1 proteins in the siRNA-NC + ACR group were higher than those in the siRNA-NC + control group ( < 0.01), and those in the siRNA-NC + ACR + HJZY group were lower than those in the siRNA-NC + ACR group ( < 0.05). After transfection with the gene-silencing siRNA-, the expression levels of p-AMPK/AMPK, PINK1, and Parkin proteins in the siRNA- + ACR group were lower than those in the siRNA- + control group ( < 0.01). After the administration of HJZY, there was no significant difference in the levels of these three proteins between the siRNA- + ACR + HJZY group and the siRNA- + ACR group. The expression level of LC3B protein in the siRNA- + ACR + HJZY group was still lower than that in the siRNA- + ACR group ( < 0.01). There was no significant difference in the levels of P62, TBK1, and ULK1 proteins between the siRNA- + ACR + HJZY group and the siRNA- + ACR group. Compared with the siRNA-NC + control group, the siRNA- + control group showed significantly decreased expression levels of p-AMPK/AMPK, ULK1, and TBK1 proteins ( < 0.001), decreased expression of PINK1 protein ( < 0.05), and increased expression of P62 protein ( < 0.001). Compared with the siRNA-NC + ACR group, the siRNA- + ACR group showed decreased expression of TBK1 protein ( < 0.001), decreased expression of LC3B protein ( < 0.01), and decreased expression of ULK1 protein ( < 0.05). The expression levels of PINK1 and Parkin proteins in the siRNA- + ACR group were lower than those in the siRNA-NC + ACR group, but the difference was not statistically significant. Compared with the siRNA-NC + ACR + HJZY group, the siRNA- + ACR + HJZY group showed decreased expression of p-AMPK/AMPK, PINK1, and Parkin proteins ( < 0.05), decreased expression of LC3B protein ( < 0.01), and increased expression of P62 protein ( < 0.001). There was no significant difference in the levels of TBK1 and ULK1 proteins between the siRNA- + ACR + HJZY group and the siRNA-NC + ACR + HJZY group.
HJZY may exert its therapeutic effect on oligoasthenospermia by regulating AMPK-mediated mitophagy.
探讨中国工程院院士王琦研发的治疗男性不育的Ⅲ类新药黄精赞育胶囊（HJZY）基于AMPK介导的线粒体自噬治疗少弱精子症的分子机制。
采用丙烯醛（ACR）处理GC-2spd(ts)小鼠精母细胞建立少弱精子症细胞模型。通过CCK8细胞活力检测确定后续建模的最佳ACR浓度和作用时间。建模成功后，将细胞培养于含不同浓度HJZY的完全培养基中。24小时后通过CCK8检测评估细胞活力，并根据细胞活力确定后续处理浓度。待GC-2spd细胞贴壁后，分为正常对照组（NC）、模型组和ACR+HJZY处理组。通过共聚焦荧光显微镜观察HJZY对线粒体自噬的影响。分别用siRNA-NC和siRNA-转染三组细胞，分为六组，包括siRNA-NC+对照组、siRNA-NC+ACR组、siRNA-NC+ACR+HJZY组、siRNA-+对照组、siRNA-+ACR组和siRNA-+ACR+HJZY组。采用蛋白质免疫印迹法验证HJZY对p-AMPK、LC3B、P62、PINK1、Parkin、TBK1和ULK1等线粒体自噬相关蛋白的调控作用，这些蛋白均为AMPK介导的蛋白。
通过细胞活力检测，选择34μmol/L作为ACR建模浓度，20分钟作为建模时间，HJZY处理浓度为160μmol/L。共聚焦荧光显微镜显示，HJZY对受损生精细胞的线粒体膜电位有一定程度的正向调节作用。模型组线粒体膜电位较NC组显著降低。给予处理后，ACR+HJZY处理组细胞膜电位较模型组升高，差异有统计学意义（<0.05）。蛋白质免疫印迹结果显示，siRNA-NC+ACR组中p-AMPK/AMPK和PINK1蛋白表达水平显著低于siRNA-NC+对照组（<0.001）。siRNA-NC+ACR组中Parkin蛋白水平低于siRNA-NC+对照组，但差异无统计学意义。给予HJZY后，这三种蛋白水平升高，siRNA-NC+ACR+HJZY组高于siRNA-NC+ACR组（<0.001）。siRNA-NC+ACR组中LC3B、P62、TBK1和ULK1蛋白表达水平高于siRNA-NC+对照组（<0.01），siRNA-NC+ACR+HJZY组低于siRNA-NC+ACR组（<0.05）。用基因沉默siRNA-转染后, siRNA-+ACR组中p-AMPK/AMPK、PINK1和Parkin蛋白表达水平低于siRNA-+对照组（<0.01）。给予HJZY后，siRNA-+ACR+HJZY组与siRNA-+ACR组这三种蛋白水平无显著差异。siRNA-+ACR+HJZY组中LC3B蛋白表达水平仍低于siRNA-+ACR组（<0.01）。siRNA-+ACR+HJZY组与siRNA-+ACR组中P62、TBK１和ULK1蛋白水平无显著差异。与siRNA-NC+对照组相比，siRNA-+对照组中p-AMPK/AMPK、ULK1和TBK1蛋白表达水平显著降低（<0.001），PINK1蛋白表达降低（<0.05），P62蛋白表达升高（<0.001）。与siRNA-NC+ACR组相比，siRNA-+ACR组中TBK1蛋白表达降低（<0.001），LC3B蛋白表达降低（<0.01），ULK1蛋白表达降低（<0.05）。siRNA-+ACR组中PINK1和Parkin蛋白表达水平低于siRNA-NC+ACR组，但差异无统计学意义。与siRNA-NC+ACR+HJZY组相比，siRNA-+ACR+HJZY组中p-AMPK/AMPK、PINK1和Parkin蛋白表达降低（<0.05），LC3B蛋白表达降低（<0.01），P62蛋白表达升高（<0.001）。siRNA-+ACR+HJZY组与siRNA-NC+ACR+HJZY组中TBK1和ULK1蛋白水平无显著差异。
HJZY可能通过调节AMPK介导的线粒体自噬对少弱精子症发挥治疗作用。
