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

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.