Jin Ding , Wen Sheng , Wei Fu , Meixin Lin , Bonan Li , Xing Zhou , Qinghu He
{"title":"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","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":"{\"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}","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}
Guilu Erxian glue mitigates oxidative damage in mouse GC-1 spermatogonial cells by inhibiting autophagy via the Keap1/Nrf2 pathway
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.
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.
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