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| Citation: |
GAO Yuan, GUO Yiyang, WU Mohan, et al. Molecular Mechanisms of Huangjing Zanyu Capsule in Treating Oligoasthenospermia: A Study Based on AMPK-Mediated Mitophagy[J]. Journal of Sichuan University (Medical Sciences), 2025, 56(1): 74-82. DOI: 10.12182/20250160504
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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-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.
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.
HJZY may exert its therapeutic effect on oligoasthenospermia by regulating AMPK-mediated mitophagy.
| [1] |
KATZ D J, TELOKEN P, SHOSHANY O. Male infertility-the other side of the equation. Aust Fam Physician, 2017, 46(9): 641-646.
|
| [2] |
TVRDA E, AGARWAL A, ALKUHAIMI N. Male reproductive cancers and infertility: a mutual relationship. Int J Mol Sci, 2015, 16(4): 7230-7260. doi: 10.3390/ijms16047230.
|
| [3] |
AJDUK A, ZERNICKA-GOETZ M. Quality control of embryo development. Mol Aspects Med, 2013, 34(5): 903-908. doi: 10.1016/j.mam.2013.03.001.
|
| [4] |
杨洪军, 李贤煜, 陈鹏, 等. 中药复方的现代基础研究进展述评. 中国科学基金, 2024, 38(3): 387-395. doi: 10.16262/j.cnki.1000-8217.20240416.001.
YANG H J, LI X Y, CHEN P, et al. Review on the progress of modern basic research on traditional Chinese medicine compound prescriptions. Bulletin of the National Natural Science Foundation of China, 2024, 38(3): 387-395. doi: 10.16262/j.cnki.1000-8217.20240416.001.
|
| [5] |
李博怿, 王琦, 朱斌, 等. 中医药治疗男性少弱精子症疗效的Meta分析. 北京中医药大学学报, 2018, 41(10): 863-872. doi: 10.3969/j.issn.1006-2157.2018.10.013.
LI B Y, WANG Q, ZHU B, et al. Meta-analysis of the efficacy of traditional Chinese medicine in the treatment of male oligoasthenospermia. Journal of Beijing University of Chinese Medicine, 2018, 41(10): 863-872. doi: 10.3969/j.issn.1006-2157.2018.10.013.
|
| [6] |
LI B Y, ZHENG C L, ZHOU K L, et al. Effects of Huang Jing Zan Yu Capsule on oligoasthenozoospermia rats sperm motility and genetic expression profiles. Oxidation Communications, 2016, 39: 3955-3964.
|
| [7] |
李向红, 哈灵侠, 陈庆, 等. 黄精赞育胶囊联合针挑疗法对弱精子症患者精液质量及性激素水平的影响. 现代中西医结合杂志, 2017, 26(27): 2965-2967, 2971.
LI X H, HA L X, CHEN Q, et al. Influence of Huangjing Zanyu Capsules combined with needle-pricking therapy on semen quality and sex hormone levels in patients with asthenospermia. Modern Journal of Integrated Traditional Chinese and Western Medicine, 2017, 26(27): 2965-2967.
|
| [8] |
瞿虎, 张靖, 江春强, 等. 黄精赞育胶囊治疗男性不育症患者的有效性和安全性分析. 现代诊断与治疗, 2015, 26(21): 4806-4809.
QU H, ZHANG J, JIANG C Q, et al. Analysis of the efficacy and safety of Huangjing Zanyu Capsule in the treatment of male infertility. Modern Diagnosis Treatment, 2015, 26(21): 4806-4809.
|
| [9] |
邓广鹏. 黄精赞育胶囊治疗少弱精子症的临床观察. 内蒙古中医药, 2017, 36(2): 14. doi: 10.3969/j.issn.1006-0979.2017.02.014.
DENG G P. Clinical observation of Huangjing Zanyu Capsules in the treatment of oligoasthenospermia. Inner Mongolia Journal of Traditional Chinese Medicine, 2017, 36(2): 14. doi: 10.3969/j.issn.1006-0979.2017.02.014.
|
| [10] |
ASHRAFI G, SCHWARZ T L. The pathways of mitophagy for quality control and clearance of mitochondria. Cell Death Differ, 2013, 20(1): 31-42. doi: 10.1038/cdd.2012.81.
|
| [11] |
PALIKARAS K, LIONAKI E, TAVERNARAKIS N. Mechanisms of mitophagy in cellular homeostasis, physiology and pathology. Nat Cell Biol, 2018, 20(9): 1013-1022. doi: 10.1038/s41556-018-0176-2.
|
| [12] |
TAMARGO-GÓMEZ I, MARIÑO G. AMPK: regulation of metabolic dynamics in the context of autophagy. Int J Mol Sci, 2018, 19(12): 3812. doi: 10.3390/ijms19123812.
|
| [13] |
APARICIO I M, ESPINO J, BEJARANO I, et al. Autophagy-related proteins are functionally active in human spermatozoa and may be involved in the regulation of cell survival and motility. Sci Rep, 2016, 6: 33647. doi: 10.1038/srep33647.
|
| [14] |
HERZIG S, SHAW R J. AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol, 2018, 19(2): 121-135. doi: 10.1038/nrm.2017.95.
|
| [15] |
WANG C C, CHEN H J, CHAN D C, et al. Low-dose acrolein, an endogenous and exogenous toxic molecule, inhibits glucose transport via an inhibition of Akt-regulated GLUT4 signaling in skeletal muscle cells. Int J Mol Sci, 2021, 22(13): 7228. doi: 10.3390/ijms22137228.
|
| [16] |
李博怿. 基于自噬-凋亡交互作用探讨黄精赞育胶囊治疗少、弱精子症的分子机制. 北京: 北京中医药大学, 2022. doi: 10.26973/d.cnki.gbjzu.2022.000115.
LI B Y. Exploration of the molecular mechanism of Huangjing Zanyu Capsules in the treatment of oligoasthenospermia based on the interaction between autophagy and apoptosis. Beijing: Beijing University of Chinese Medicine, 2022. doi: 10.26973/d.cnki.gbjzu.2022.000115.
|
| [17] |
李红林, 傅广波, 吕述彦. 精子线粒体功能检测与男性不育相关性的研究进展. 临床检验杂志, 2021, 39(4): 287-290. doi: 10.13602/j.cnki.jcls.2021.04.12.
LI H L, FU G B, LYU S Y. Research progress on the correlation between sperm mitochondrial function detection and male infertility. Chinese Journal of Clinical Laboratory Science, 2021, 39(4): 287-290. doi: 10.13602/j.cnki.jcls.2021.04.12.
|
| [18] |
HARDIE D G. AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes Dev, 2011, 25(18): 1895-908. doi: 10.1101/gad.17420111.
|
| [19] |
WU J, DANG Y, SU W, et al. Molecular cloning and characterization of rat LC3A and LC3B-two novel markers of autophagosome. Biochem Biophys Res Commun, 2006, 339(1): 437-442. doi: 10.1016/j.bbrc.2005.10.211.
|
| [20] |
MOSCAT J, DIAZ-MECO M T. p62 at the crossroads of autophagy, apoptosis, and cancer. Cell, 2009, 137(6): 1001-1004. doi: 10.1016/j.cell.2009.05.023.
|
| [21] |
王娜娜. 氧化应激通过调节ALS相关蛋白TBK1和OPTN影响线粒体自噬的机制. 苏州: 苏州大学, 2019. doi: 10.27351/d.cnki.gszhu.2019.002059.
WANG N N. Mechanism of oxidative stress affecting mitophagy by regulating ALS-related proteins TBK1 and OPTN. Suzhou: Soochow University, 2019. doi: 10.27351/d.cnki.gszhu.2019.002059.
|
| [22] |
SALIMI A, PIRHADI R, JAMALI Z, et al. Mitochondrial and lysosomal protective agents ameliorate cytotoxicity and oxidative stress induced by cyclophosphamide and methotrexate in human blood lymphocytes. Hum Exp Toxicol, 2019, 38(11): 1266-1274. doi: 10.1177/0960327119871096.
|
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