基于AMPK介导的线粒体自噬研究黄精赞育胶囊治疗少弱精子症的分子机制

高源; 郭怡阳; 武默涵; 郑燕飞

doi:10.12182/20250160504

基于AMPK介导的线粒体自噬研究黄精赞育胶囊治疗少弱精子症的分子机制

Molecular Mechanisms of Huangjing Zanyu Capsule in Treating Oligoasthenospermia: A Study Based on AMPK-Mediated Mitophagy

摘要

摘要:
目的基于AMPK介导的线粒体自噬方面探究国医大师王琦院士研发的治疗男性不育的国家中药三类新药黄精赞育胶囊治疗少弱精子症细胞层面的分子机制。

方法选择丙烯醛（acrolein, ACR）作用于GC-2spd（ts）小鼠精母细胞制作少弱精子症细胞模型，通过CCK8细胞活性检测确定后续造模用ACR浓度和造模时间。造模成功后加入不同浓度含黄精赞育胶囊的完全培养基进行细胞培养，于24 h后CCK8细胞活性检测，根据细胞活力确定后续给药浓度。GC-2spd细胞贴壁后分为NC组、造模组和ACR+HJZY给药组，通过共聚焦荧光显微镜观测黄精赞育胶囊对线粒体自噬的影响；将上述3组细胞分别转染siRNA-NC和siRNA-AMPK，分为siRNA-NC+control、siRNA-NC+ACR、siRNA-NC+ACR+HJZY、siRNA-AMPK+control、siRNA-AMPK+ACR、siRNA-AMPK+ACR+HJZY 6组，通过Western blot验证黄精赞育胶囊对AMPK介导的p-AMPK、LC3B、P62、PINK1、Parkin、TBK1、ULK1等线粒体自噬相关蛋白的调控作用。

结果通过细胞活性检测实验确定ACR的造模浓度为34 μmol/L，造模时间为20 min，HJZY给药浓度为160 μmol/L。共聚焦荧光显微镜显示黄精赞育胶囊对受损生精细胞线粒体膜电位具有一定正向调节作用，造模组线粒体膜电位较NC组大幅降低，ACR+HJZY给药组给药后细胞膜电位较造模组有所升高，差异有统计学意义（P<0.05）；Western blot检测结果显示siRNA-NC+ACR组p-AMPK/AMPK和PINK1蛋白表达水平较siRNA-NC+control组下降（P<0.001），siRNA-NC+ACR组Parkin蛋白水平较siRNA-NC+control组有所下降，但差异无统计学意义，HJZY给药后这3种蛋白表达水平均较siRNA-NC+ACR组上升（P<0.001）；siRNA-NC+ACR组LC3B、P62、TBK1和ULK1蛋白表达水平均较siRNA-NC+control组上升（P <0.01），siRNA-NC+ACR+HJZY组上述蛋白表达水平均较siRNA-NC+ACR组有所下降（P<0.05）。转染敲减基因siRNA-AMPK后，siRNA-AMPK+ACR组p-AMPK/AMPK、PINK1和Parkin蛋白表达水平较siRNA-AMPK+control组有所下降（P<0.01）；HJZY给药后上述3种蛋白表达水平与siRNA-AMPK+ACR组比较差异均无统计学意义；siRNA-AMPK+ACR+HJZY组与siRNA-AMPK+ACR组相比，LC3B蛋白表达水平仍降低（P<0.01），但P62、TBK1和ULK1蛋白表达水平差异均无统计学意义；siRNA-AMPK+control组与siRNA-NC+control组相比，p-AMPK/AMPK、ULK1和TBK1蛋白表达水平下降（P<0.001），PINK1蛋白表达水平亦下降（P<0.05），P62蛋白表达水平上升（P<0.001）；siRNA-AMPK+ACR组与siRNA-NC+ACR组相比，TBK1蛋白表达水平下降（P<0.001），LC3B蛋白表达水平下降（P<0.01），ULK1蛋白表达水平亦下降（P<0.05），PINK1和Parkin蛋白表达水平有所下降但差异无统计学意义；siRNA-AMPK+ACR+HJZY组与siRNA-NC+ACR+HJZY组相比，p-AMPK/AMPK、PINK1和Parkin蛋白表达水平下降（P<0.05），LC3B蛋白表达水平亦下降（P<0.01），P62蛋白表达水平上升（P<0.001），TBK1和ULK1蛋白表达水平差异无统计学意义。

结论黄精赞育胶囊可能通过调节AMPK介导的线粒体自噬达到治疗男性少弱精子症的效果。

Abstract:
Objective 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.
Methods 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-AMPK, respectively, and divided into six groups, including siRNA-NC + control, siRNA-NC + ACR, siRNA-NC + ACR + HJZY, siRNA-AMPK + control, siRNA-AMPK + ACR, and siRNA-AMPK + 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.
Results 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 (P < 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 (P < 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 (P < 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 (P < 0.01), and those in the siRNA-NC + ACR + HJZY group were lower than those in the siRNA-NC + ACR group (P < 0.05). After transfection with the gene-silencing siRNA-AMPK, the expression levels of p-AMPK/AMPK, PINK1, and Parkin proteins in the siRNA-AMPK + ACR group were lower than those in the siRNA-AMPK + control group (P < 0.01). After the administration of HJZY, there was no significant difference in the levels of these three proteins between the siRNA-AMPK + ACR + HJZY group and the siRNA-AMPK + ACR group. The expression level of LC3B protein in the siRNA-AMPK + ACR + HJZY group was still lower than that in the siRNA-AMPK + ACR group (P < 0.01). There was no significant difference in the levels of P62, TBK1, and ULK1 proteins between the siRNA-AMPK + ACR + HJZY group and the siRNA-AMPK + ACR group. Compared with the siRNA-NC + control group, the siRNA-AMPK + control group showed significantly decreased expression levels of p-AMPK/AMPK, ULK1, and TBK1 proteins (P < 0.001), decreased expression of PINK1 protein (P < 0.05), and increased expression of P62 protein (P < 0.001). Compared with the siRNA-NC + ACR group, the siRNA-AMPK + ACR group showed decreased expression of TBK1 protein (P < 0.001), decreased expression of LC3B protein (P < 0.01), and decreased expression of ULK1 protein (P < 0.05). The expression levels of PINK1 and Parkin proteins in the siRNA-AMPK + 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-AMPK + ACR + HJZY group showed decreased expression of p-AMPK/AMPK, PINK1, and Parkin proteins (P < 0.05), decreased expression of LC3B protein (P < 0.01), and increased expression of P62 protein (P < 0.001). There was no significant difference in the levels of TBK1 and ULK1 proteins between the siRNA-AMPK + ACR + HJZY group and the siRNA-NC + ACR + HJZY group.
Conclusion HJZY may exert its therapeutic effect on oligoasthenospermia by regulating AMPK-mediated mitophagy.

HTML全文

参考文献(22)

施引文献

资源附件(0)