In order to explore the mechanism of kidney-tonifying Chinese medicine in improving autophagy of testicular mesenchymal cells in rats with kidney-essence deficiency, a rat model of kidney-essence deficiency was established by single-factor electrical stimulation. Immunohistochemistry and Western blot were used to detect the expression levels of autophagy marker proteins Beclin1, p62, LC3B, AMPK and mTOR in testicular cells of each group. Based on the close relationship between kidney essence deficiency and aging, Leydig cell aging model of testicular mesenchymal cells was established by free radical oxidative damage method, and AMPK inhibitor was combined to explore the molecular mechanism of Heshouwuyin activating autophagy. The results showed that Heshouwuyin could up-regulate the expression of Beclin1 protein and LC3B in testicular tissue, down-regulate the expression of p62 protein,- DOI:10.3969/j.issn.1000-1565.2024.06.009补肾中药通过AMPK-mTOR通路提高肾精亏虚大鼠睾丸间质细胞自噬能力李朝英1,李思睿2,张伟3,马昊飞2,牛双2,段豫磊2,牛嗣云2 (1.河北大学附属医院 新生儿科, 河北 保定 071000;2.河北大学 基础医学院, 河北 保定 071000;3.河北大学附属医院 医学工程中心, 河北 保定 071000)摘 要:为了探究补肾中药提高肾精亏虚大鼠睾丸间质细胞自噬能力的机制,采用单因素电刺激法建立肾精亏虚大鼠模型,免疫组化及Western blot检测各组睾丸细胞自噬标志蛋白Beclin1、p62、LC3B表达水平以及AMPK和mTOR表达变化.基于肾精亏虚与衰老的密切联系,通过自由基氧化损伤法建立睾丸间质细胞(Leydig细胞)衰老模型,联合AMPK抑制剂,探究何首乌饮激活细胞自噬的分子机制.结果显示:何首乌饮可上调睾丸组织Beclin1蛋白、LC3B表达,下调p62蛋白表达,促进AMPK磷酸化并抑制mTOR激活(P<0.05);细胞实验表明,何首乌饮可通过激活AMPK-mTOR信号通路提高Leydig细胞自噬能力(P<0.05),促进肾精亏虚大鼠睾丸Leydig细胞自噬进程.关键词:何首乌饮;肾精亏虚;睾丸组织;自噬;AMPK-mTOR中图分类号:Q291 文献标志码:A 文章编号:1000-1565(2024)06-0633-10Kidney-tonifying Chinese medicine improves autophagy of Leydig cells in rats with kidney essence deficiency through AMPK-mTOR pathwayLI Chaoying1, LI Sirui2, ZHANG Wei3, MA Haofei2, NIU Shuang2, DUAN Yulei2, NIU Siyun2(1. Department of Neonatology, Affiliated Hospital of Hebei University, Baoding 071000, China;2. School of Basic Medical Sciences, Hebei University, Baoding 071000, China;3. Medical Engineering Center, Affiliated Hospital of Hebei University, Baoding 071000, China)Abstract: In order to explore the mechanism of kidney-tonifying Chinese medicine in improving autophagy of testicular mesenchymal cells in rats with kidney-essence deficiency, a rat model of kidney-essence deficiency was established by single-factor electrical stimulation. Immunohistochemistry and Western blot were used to detect the expression levels of autophagy marker proteins Beclin1, p62, LC3B, AMPK and mTOR in testicular cells of each group. Based on the close relationship between kidney essence deficiency and aging, Leydig cell aging model of testicular mesenchymal cells was established by free radical oxidative damage method, and AMPK inhibitor was combined to explore the molecular mechanism of Heshouwuyin activating autophagy. The results showed that Heshouwuyin could up-regulate the expression of Beclin1 protein and LC3B in testicular tissue, down-regulate the expression of p62 protein,- 收稿日期:2024 -02-19;修回日期:2024-05-08 基金项目:国家自然科学基金资助项目(82374180);河北省中医药管理局科研项目(2020234);河北省重点研发计划项目-中医药创新专项(223777134D);河北大学医学学科培育项目(2023B10);河北省高等学校科学技术研究项目(ZD2022060) 第一作者: 李朝英(1980—),女,河北大学附属医院主治医师,主要从事早产儿营养评估.E-mail:lizhaoying725@126.com 通信作者:段豫磊(1994—),男,河北大学实验师,主要从事中药药理方向研究.E-mail:dyl15771@163.com牛嗣云(1967—),女,满族,河北大学教授,博士,博士生导师,主要从事生殖衰老方向研究.E-mail:nsy1688@163.com 第6期李朝英等:补肾中药通过AMPK-mTOR通路提高肾精亏虚大鼠睾丸间质细胞自噬能力河北大学学报(自然科学版) 第44卷promote AMPK phosphorylation and inhibit mTOR activation(P<0.05). Cell experiments showed that Heshouwuyin could affect the autophagy ability of Leydig cells by activating AMPK-mTOR signaling pathway(P<0.05).Therefore, Heshouwuyin may promote the autophagy process of Leydig cells in rats with kidney essence deficiency by activating AMPK-mTOR signaling pathway.
[1] LI L H, YUAN Y, SHA J H. Potential clinical value of in vitro spermatogenesis[J]. Biol Reprod, 2022, 107(1): 95-100. DOI:10.1093/biolre/ioac076.
[2] WANG L, ZHU Y, WANG T, et al. Feasibility analysis of incorporating infertility into medical insurance in China[J]. Front Endocrinol, 2022, 13: 967739. DOI:10.3389/fendo.2022.967739.
[3] MIDZAK A S, CHEN H L, PAPADOPOULOS V, et al. Leydig cell aging and the mechanisms of reduced testosterone synthesis[J]. Mol Cell Endocrinol, 2009, 299(1):23-31. DOI: 10.1016/j.mce.2008.07.016.
[4] ERENPREISS J, FODINA V, POZARSKA R, et al. Prevalence of testosterone deficiency among aging men with and without morbidities[J]. Aging Male, 2020, 23(5):901-905. DOI: 10.1080/13685538.2019.1621832.
[5] PATAKY M W, YOUNG W F, NAIR K S. Hormonal and metabolic changes of aging and the influence of lifestyle modifications[J]. Mayo Clin Proc, 2021, 96(3):788-814. DOI: 10.1016/j.mayocp.2020.07.033.
[6] PI H F, LI M, ZOU L Y, et al. AKT inhibition-mediated dephosphorylation of TFE3 promotes overactive autophagy independent of MTORC1 in cadmium-exposed bone mesenchymal stem cells[J]. Autophagy, 2019, 15(4):565-582. DOI: 10.1080/15548627.2018.1531198.
[7] GAO F Y, LI G P, LIU C, et al. Autophagy regulates testosterone synthesis by facilitating cholesterol uptake in Leydig cells[J]. J Cell Biol, 2018, 217(6):2103-2119. DOI: 10.1083/jcb.201710078.
[8] WANG H J, SHAN B Y, DUAN Y L, et al. Effects of Heshouwuyin on gene expression of the insulin/IGF signalling pathway in rat testis and spermatogenic cells[J]. Pharm Biol, 2020, 58(1):1199-1210. DOI:10.1080/13880209.2020.1839511.
[9] 王建明,孙静,齐峰,等.何首乌饮对大鼠睾丸间质细胞类固醇激素合成急性调节蛋白和细胞色素P450胆固醇侧链裂解酶蛋白表达的影响[J].解剖学报,2017, 48(1):30-36. DOI:10.16098/j.issn.0529-1356.2017.01.006.
[10] NIU S Y, CHEN J B, DUAN F, et al. Possible mechanism underlying the effect of Heshouwuyin, a tonifying kidney herb, on sperm quality in aging rats[J]. BMC Complement Altern Med, 2014, 14:250. DOI: 10.1186/1472-6882-14-250.
[11] 王小杰,牛嗣云,张艳青,等.何首乌饮对雄性衰老大鼠生殖功能的保护作用[J].时珍国医国药, 2011, 22(3):532-535.
[12] 李少华,牛双,董子怡,等.齐墩果酸对衰老大鼠睾丸间质细胞胰岛素/IGF1通路关键基因表达的影响[J].河北大学学报(自然科学版),2022,42(4):403-410.DOI:10.3969/j.issn.1000-1565.2022.04.010.
[13] 李少华,姜丽萍,牛佳瑶,等.二苯乙烯苷调控胰岛素/IGF-1信号通路延缓大鼠睾丸间质细胞衰老的作用机制[J].河北大学学报(自然科学版),2021,41(3):297-303.DOI:10.3969/j.issn.1000-1565.2021.03.012.
[14] 辛华,郑洪新,谢晚晴,等.“异病”(原发性骨质疏松症和老年性痴呆)“同证”(肾精亏虚证)患者差异表达基因的同源性分析[J].辽宁中医药大学学报,2019,21(9):80-86.
[15] 孙洁,周安方,周艳艳,等.肾虚对雄性小鼠及其雄性仔鼠精子DNA损伤的影响[J].中华中医药学刊, 2010, 28(5):1044-1047. DOI: 10.13193/j.archtcm.2010.05.150.sunj.023.
[16] 孙洁,周安方,周艳艳,等.肾精亏虚对雄性小鼠及其雄性仔鼠生育力的影响[J].中华男科学杂志, 2007, 13(8): 754-757.
[17] 回陈红,刘昊坤,杜云帆,等.衰老大鼠睾丸组织受何首乌饮调控差异表达基因的筛选[J].时珍国医国药, 2017, 28(7):1583-1586.
[18] KE Y, ZHAN L H, LU T T, et al. Polysaccharides of dendrobium officinale kimura & migo leaves protect against ethanol-induced gastric mucosal injury via the AMPK/mTOR signaling pathway in vitro and vivo[J]. Front Pharmacol, 2020, 11:526349. DOI: 10.3389/fphar.2020.526349.
[19] GE Y C, ZHOU M, CHEN C, et al. Role of AMPK mediated pathways in autophagy and aging[J]. Biochimie, 2022, 195:100-113. DOI: 10.1016/j.biochi.2021.11.008.
[20] WANG M, ZENG L, SU P, et al. Autophagy: a multifaceted player in the fate of sperm[J]. Hum Reprod Update, 2022, 28(2):200-231. DOI: 10.1093/humupd/dmab043.
[21] VARGAS J N S, HAMASAKI M, KAWABATA T, et al. The mechanisms and roles of selective autophagy in mammals[J]. Nat Rev Mol Cell Biol, 2023, 24(3):167-185. DOI: 10.1038/s41580-022-00542-2.
[22] YAN Q, ZHANG Y, WANG Q, et al. Autophagy: a double-edged sword in male reproduction[J]. Int J Mol Sci, 2022, 23(23):15273. DOI: 10.3390/ijms232315273.
[23] ZHANG J L, YE R R, GRUNBERGER J W, et al. Activation of autophagy by low-dose silica nanoparticles enhances testosterone secretion in leydig Cells[J]. Int J Mol Sci, 2022, 23(6):3104. DOI: 10.3390/ijms23063104.
[24] XU R F, WANG F, ZHANG Z H, et al. Diabetes-induced autophagy dysregulation engenders testicular impairment via oxidative stress[J]. Oxid Med Cell Longev, 2023, 2023(1):4365895. DOI: 10.1155/2023/4365895.
[25] MIDZAK A S, CHEN H L, PAPADOPOULOS V, et al. Leydig cell aging and the mechanisms of reduced testosterone synthesis[J]. Mol Cell Endocrinol, 2009, 299(1):23-31. DOI: 10.1016/j.mce.2008.07.016.
[26] FU G Q, DAI J, LI Z, et al. The role of STAT3/p53 and PI3K-Akt-mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat[J]. Toxicol Appl Pharmacol, 2020, 404: 115151. DOI: 10.1016/j.taap.2020.115151.
[27] CAO W Y, LI J H, YANG K P, et al. An overview of autophagy: Mechanism, regulation and research progress[J]. Bull Cancer, 2021, 108(3):304-322. DOI: 10.1016/j.bulcan.2020.11.004.
[28] 郭凯华,高福禄,牛嗣云,等.何首乌饮对衰老大鼠睾丸组织细胞Rb/p53 信号转导通路的影响[J].解剖学报, 2010, 41(3):435-439.
[29] 李少华,姜丽萍,刘靓云,等.何首乌饮延缓大鼠睾丸间质细胞衰老与调控胰岛素/胰岛素样生长因子-1信号通路的关系[J].解剖学报,2020, 51(5):751-757. DOI: 10.16098/j.issn.0529-1356.2020.05.019.
[30] DASGUPTA B, SEIBEL W. Compound C/dorsomorphin: its use and misuse as an AMPK inhibitor[J]. Methods Mol Biol, 2018, 1732:195-202. DOI: 10.1007/978-1-4939-7598-3_12. (
