{"title":"The role of METTL3-mediated CircStk4 modification in the treatment of chronic glomerulonephritis with Qi Teng Xiao Zhuo granule.","authors":"Xiujuan Qin, Huiyu Chen, Wenjia Zheng, Wenjie Hu, Xianjin Xu, Jiarong Gao","doi":"10.1016/j.phymed.2024.156183","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Qi Teng Xiao Zhuo granule (QTXZG), a compound preparation used in traditional Chinese medicine, is a highly effective treatment for chronic glomerulonephritis (CGN). Previously, the mechanism of circStk4 and the N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) modification of circStk4 in CGN was elucidated in vivo. Nevertheless, there hasn't been any research done on the connection between circStk4 and QTXZG's mechanism in CGN treatment.</p><p><strong>Purpose: </strong>The current study intended to clarify the molecular mechanism of QTXZG in CGN therapy by both in vitro and in vivo investigations.</p><p><strong>Methods: </strong>Mouse mesangial cells (MMCs) were used to measure the rate of proliferation and apoptosis using flow cytometry and the Cell Counting Kit-8 (CCK-8) assay. The expression of markers associated with proliferation, apoptosis, and autophagy was analysed using reverse transcription quantitative PCR (RT-qPCR), western blotting (WB), and immunofluorescence (IF), respectively. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) was utilized to analyse the m<sup>6</sup>A modification of circStk4, and METTL3 expression was assessed using RT-qPCR. Subsequently, miR-133a-3p and C1 expression was examined using RT-qPCR, WB, and IF. Adeno-associated virus 9 (AAV9)-circStk4 knockdown vector and a METTL3 inhibitor were used to explore the roles of METTL3 and circStk4 in CGN. Additionally, molecular docking and cellular thermal shift assays (CETSAs) were performed to assess the binding affinity between METTL3 and the active compounds in QTXZG.</p><p><strong>Results: </strong>Mechanistically, QTXZG reduced METTL3 expression and decreased circStk4 m<sup>6</sup>A levels while decreasing circStk4 levels and regulating the miR-133a-3p/C1 axis. Functionally, QTXZG inhibited MMCs and renal tissue proliferation, promoted apoptosis and autophagy, and reduced inflammation. In vivo experiments further confirmed that downregulated ircStk4 and METTL3 expression were accompanied by the therapeutic effects of QTXZG, resulting in a significant attenuation of renal injury, reduction in inflammation, inhibition of renal tissue proliferation and promotion of apoptosis and autophagy.</p><p><strong>Conclusion: </strong>The present study revealed that QTXZG reduced circStk4 m<sup>6</sup>A and METTL3 expression to regulate the circStk4/miR-133a-3p/C1 axis in the treatment of CGN and thus inhibited glomerular tissue/membrane cell proliferation and promoted autophagy and apoptosis; these results uncovered a new mechanism by which QTXZG reduced CGN and imply that METTL3 might be a target for innovative therapeutic approaches.</p>","PeriodicalId":20212,"journal":{"name":"Phytomedicine","volume":null,"pages":null},"PeriodicalIF":6.7000,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Phytomedicine","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1016/j.phymed.2024.156183","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"CHEMISTRY, MEDICINAL","Score":null,"Total":0}
引用次数: 0
Abstract
Background: Qi Teng Xiao Zhuo granule (QTXZG), a compound preparation used in traditional Chinese medicine, is a highly effective treatment for chronic glomerulonephritis (CGN). Previously, the mechanism of circStk4 and the N6-methyladenosine (m6A) modification of circStk4 in CGN was elucidated in vivo. Nevertheless, there hasn't been any research done on the connection between circStk4 and QTXZG's mechanism in CGN treatment.
Purpose: The current study intended to clarify the molecular mechanism of QTXZG in CGN therapy by both in vitro and in vivo investigations.
Methods: Mouse mesangial cells (MMCs) were used to measure the rate of proliferation and apoptosis using flow cytometry and the Cell Counting Kit-8 (CCK-8) assay. The expression of markers associated with proliferation, apoptosis, and autophagy was analysed using reverse transcription quantitative PCR (RT-qPCR), western blotting (WB), and immunofluorescence (IF), respectively. Methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) was utilized to analyse the m6A modification of circStk4, and METTL3 expression was assessed using RT-qPCR. Subsequently, miR-133a-3p and C1 expression was examined using RT-qPCR, WB, and IF. Adeno-associated virus 9 (AAV9)-circStk4 knockdown vector and a METTL3 inhibitor were used to explore the roles of METTL3 and circStk4 in CGN. Additionally, molecular docking and cellular thermal shift assays (CETSAs) were performed to assess the binding affinity between METTL3 and the active compounds in QTXZG.
Results: Mechanistically, QTXZG reduced METTL3 expression and decreased circStk4 m6A levels while decreasing circStk4 levels and regulating the miR-133a-3p/C1 axis. Functionally, QTXZG inhibited MMCs and renal tissue proliferation, promoted apoptosis and autophagy, and reduced inflammation. In vivo experiments further confirmed that downregulated ircStk4 and METTL3 expression were accompanied by the therapeutic effects of QTXZG, resulting in a significant attenuation of renal injury, reduction in inflammation, inhibition of renal tissue proliferation and promotion of apoptosis and autophagy.
Conclusion: The present study revealed that QTXZG reduced circStk4 m6A and METTL3 expression to regulate the circStk4/miR-133a-3p/C1 axis in the treatment of CGN and thus inhibited glomerular tissue/membrane cell proliferation and promoted autophagy and apoptosis; these results uncovered a new mechanism by which QTXZG reduced CGN and imply that METTL3 might be a target for innovative therapeutic approaches.
期刊介绍:
Phytomedicine is a therapy-oriented journal that publishes innovative studies on the efficacy, safety, quality, and mechanisms of action of specified plant extracts, phytopharmaceuticals, and their isolated constituents. This includes clinical, pharmacological, pharmacokinetic, and toxicological studies of herbal medicinal products, preparations, and purified compounds with defined and consistent quality, ensuring reproducible pharmacological activity. Founded in 1994, Phytomedicine aims to focus and stimulate research in this field and establish internationally accepted scientific standards for pharmacological studies, proof of clinical efficacy, and safety of phytomedicines.