Guilu Erxian glue mitigates oxidative damage in mouse GC-1 spermatogonial cells by inhibiting autophagy via the Keap1/Nrf2 pathway

Q3 Medicine

Journal of Traditional Chinese Medical Sciences Pub Date : 2023-10-01 DOI:10.1016/j.jtcms.2023.09.002

Jin Ding , Wen Sheng , Wei Fu , Meixin Lin , Bonan Li , Xing Zhou , Qinghu He

{"title":"Guilu Erxian glue mitigates oxidative damage in mouse GC-1 spermatogonial cells by inhibiting autophagy via the Keap1/Nrf2 pathway","authors":"Jin Ding , Wen Sheng , Wei Fu , Meixin Lin , Bonan Li , Xing Zhou , Qinghu He","doi":"10.1016/j.jtcms.2023.09.002","DOIUrl":null,"url":null,"abstract":"<div><h3>Objective</h3><p>To explore the effects and underlying mechanisms of Guilu Erxian glue (GLEXG) on oxidative damage in a mouse GC-1 spermatogonial (MGS) cell model.</p></div><div><h3>Methods</h3><p>A cellular model for oxidative damage was created using MGS cells exposed to hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). Cell viability was assessed using the cell counting kit-8 assay, while reactive oxygen species (ROS) and malondialdehyde (MDA) levels were measured via flow cytometry and enzyme-linked immunosorbent assay, respectively. Western blotting and immunofluorescence techniques were employed to quantify the relative expression levels of sequestosome-1 (p62), nuclear factor erythroid 2-related factor 2 (Nrf2), microtubule-associated protein light chain 3β (LC3B), and Kelch-like ECH-associated protein 1 (Keap1). Quantitative real-time PCR was used to evaluate Keap1 mRNA expression. Transmission electron microscopy (TEM) was conducted to observe structural changes in autophagy-related vesicles.</p></div><div><h3>Results</h3><p>The cellular model of oxidative damage induced by H<sub>2</sub>O<sub>2</sub> showed reduced cell viability along with elevated levels of ROS and MDA. Treatment with 10% GLEXG-enriched serum significantly enhanced cell viability (<em>P</em> = .0002) while decreasing ROS and MDA levels (<em>P</em> = .0105 and <em>P</em> = .0033, respectively). In rapamycin-treated MGS cells, GLEXG treatment substantially upregulated the relative protein expression of p-mTOR, Nrf2, and p62 (all <em>P</em> < .01), and downregulated the expression of Keap1 and the LC3B-II/LC3B-I ratio (<em>P</em> = .002 and <em>P</em> = .0043, respectively). It also lowered ROS and MDA levels. TEM analysis revealed that GLEXG treatment considerably reduced the number of abnormally enlarged autolysosomes in rapamycin-treated MGS cells. In Keap1-siRNA-transfected MGS cells, the siRNA-Keap1-2311 knockdown site demonstrated higher efficiency. Furthermore, GLEXG treatment in these Keap1-siRNA-transfected cells notably upregulated the relative protein expression of Nrf2 and p62, decreased Keap1 expression and the LC3B-II/LC3B-I ratio, and reduced ROS and MDA levels.</p></div><div><h3>Conclusion</h3><p>GLEXG effectively mitigated oxidative damage in the MGS cell model by inhibiting autophagy through the Keap1/Nrf2 pathway.</p></div>","PeriodicalId":36624,"journal":{"name":"Journal of Traditional Chinese Medical Sciences","volume":"10 4","pages":"Pages 484-492"},"PeriodicalIF":0.0000,"publicationDate":"2023-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Traditional Chinese Medical Sciences","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2095754823000510","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"Medicine","Score":null,"Total":0}

引用次数: 0

Abstract

Objective

To explore the effects and underlying mechanisms of Guilu Erxian glue (GLEXG) on oxidative damage in a mouse GC-1 spermatogonial (MGS) cell model.

Methods

A cellular model for oxidative damage was created using MGS cells exposed to hydrogen peroxide (H₂O₂). Cell viability was assessed using the cell counting kit-8 assay, while reactive oxygen species (ROS) and malondialdehyde (MDA) levels were measured via flow cytometry and enzyme-linked immunosorbent assay, respectively. Western blotting and immunofluorescence techniques were employed to quantify the relative expression levels of sequestosome-1 (p62), nuclear factor erythroid 2-related factor 2 (Nrf2), microtubule-associated protein light chain 3β (LC3B), and Kelch-like ECH-associated protein 1 (Keap1). Quantitative real-time PCR was used to evaluate Keap1 mRNA expression. Transmission electron microscopy (TEM) was conducted to observe structural changes in autophagy-related vesicles.

Results

The cellular model of oxidative damage induced by H₂O₂ showed reduced cell viability along with elevated levels of ROS and MDA. Treatment with 10% GLEXG-enriched serum significantly enhanced cell viability (P = .0002) while decreasing ROS and MDA levels (P = .0105 and P = .0033, respectively). In rapamycin-treated MGS cells, GLEXG treatment substantially upregulated the relative protein expression of p-mTOR, Nrf2, and p62 (all P < .01), and downregulated the expression of Keap1 and the LC3B-II/LC3B-I ratio (P = .002 and P = .0043, respectively). It also lowered ROS and MDA levels. TEM analysis revealed that GLEXG treatment considerably reduced the number of abnormally enlarged autolysosomes in rapamycin-treated MGS cells. In Keap1-siRNA-transfected MGS cells, the siRNA-Keap1-2311 knockdown site demonstrated higher efficiency. Furthermore, GLEXG treatment in these Keap1-siRNA-transfected cells notably upregulated the relative protein expression of Nrf2 and p62, decreased Keap1 expression and the LC3B-II/LC3B-I ratio, and reduced ROS and MDA levels.

Conclusion

GLEXG effectively mitigated oxidative damage in the MGS cell model by inhibiting autophagy through the Keap1/Nrf2 pathway.

查看原文本刊更多论文

龟二仙胶通过Keap1/Nrf2通路抑制小鼠GC-1精原细胞的自噬，从而减轻小鼠GC-1精原细胞氧化损伤

目的探讨归绿二仙胶（GLEXG）对小鼠精原细胞（MGS）氧化损伤的影响及其可能机制。方法利用MGS细胞暴露于过氧化氢（H2O2），建立氧化损伤细胞模型。使用细胞计数试剂盒-8测定法评估细胞活力，同时分别通过流式细胞术和酶联免疫吸附测定法测量活性氧（ROS）和丙二醛（MDA）水平。采用蛋白质印迹和免疫荧光技术定量螯合体-1（p62）、核因子-红系2相关因子2（Nrf2）、微管相关蛋白轻链3β（LC3B）和Kelch样ECH相关蛋白1（Keap1）的相对表达水平。实时定量PCR用于评估Keap1mRNA的表达。透射电子显微镜（TEM）观察自噬相关囊泡的结构变化。结果H2O2诱导的细胞氧化损伤模型显示细胞活力降低，ROS和MDA水平升高。用富含10%GLEXG的血清处理显著提高了细胞活力（P=0.0002），同时降低了ROS和MDA水平（分别为P=0105和P=.0033）。在雷帕霉素处理的MGS细胞中，GLEXG处理显著上调p-mTOR、Nrf2和p62的相对蛋白表达（均为p<；.01），并下调Keap1的表达和LC3B-II/LC3B-I比率（分别为p=0.002和p=0.043）。它还降低了ROS和MDA水平。TEM分析显示，GLEXG处理显著减少了雷帕霉素处理的MGS细胞中异常增大的自溶体的数量。在Keap1 siRNA转染的MGS细胞中，siRNA-Keap1-2311敲低位点显示出更高的效率。此外，在这些Keap1 siRNA转染的细胞中，GLEXG处理显著上调了Nrf2和p62的相对蛋白表达，降低了Keap1的表达和LC3B-II/LC3B-I比率，并降低了ROS和MDA水平。结论GLEXG通过Keap1/Nrf2途径抑制自噬，有效减轻MGS细胞模型的氧化损伤。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Journal of Traditional Chinese Medical Sciences Medicine-Complementary and Alternative Medicine

CiteScore

1.90

自引率

0.00%

发文量

审稿时长

36 weeks

期刊介绍： Production and Hosting by Elsevier B.V. on behalf of Beijing University of Chinese Medicine Peer review under the responsibility of Beijing University of Chinese Medicine. Journal of Traditional Chinese Medical Sciences is an international, peer-reviewed publication featuring advanced scientific research in Traditional Chinese medicine (TCM). The journal is sponsored by Beijing University of Chinese Medicine and Tsinghua University Press, and supervised by the Ministry of Education of China. The goal of the journal is to serve as an authoritative platform to present state-of-the-art research results. The journal is published quarterly. We welcome submissions of original papers on experimental and clinical studies on TCM, herbs and acupuncture that apply modern scientific research methods. The journal also publishes case reports, reviews, and articles on TCM theory and policy.