The role of METTL3-mediated CircStk4 modification in the treatment of chronic glomerulonephritis with Qi Teng Xiao Zhuo granule.

IF 6.7 1区 医学 Q1 CHEMISTRY, MEDICINAL
Xiujuan Qin, Huiyu Chen, Wenjia Zheng, Wenjie Hu, Xianjin Xu, Jiarong Gao
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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.

METTL3介导的CircStk4修饰在芪腾消渴颗粒治疗慢性肾小球肾炎中的作用
背景:芪腾消渴颗粒(QTXZG)是一种中药复方制剂,是治疗慢性肾小球肾炎(CGN)的高效药物。此前,已有研究阐明了circStk4和circStk4的N6-甲基腺苷(m6A)修饰在CGN中的体内作用机制。目的:本研究旨在通过体外和体内研究阐明 QTXZG 在 CGN 治疗中的分子机制:方法:采用流式细胞术和细胞计数试剂盒-8(CCK-8)检测小鼠间质细胞(MMCs)的增殖和凋亡率。分别使用逆转录定量 PCR(RT-qPCR)、免疫印迹(WB)和免疫荧光(IF)分析了与增殖、凋亡和自噬相关的标记物的表达。利用甲基化 RNA 免疫沉淀-qPCR(MeRIP-qPCR)分析 circStk4 的 m6A 修饰,并利用 RT-qPCR 评估 METTL3 的表达。随后,利用 RT-qPCR、WB 和 IF 检测了 miR-133a-3p 和 C1 的表达。研究人员使用腺相关病毒9(AAV9)-circStk4敲除载体和METTL3抑制剂来探讨METTL3和circStk4在CGN中的作用。此外,还进行了分子对接和细胞热转移试验(CETSAs),以评估METTL3与QTXZG中活性化合物的结合亲和力:结果:从机理上讲,QTXZG能降低METTL3的表达,降低circStk4 m6A的水平,同时降低circStk4的水平并调节miR-133a-3p/C1轴。在功能上,QTXZG 可抑制 MMCs 和肾组织增殖,促进细胞凋亡和自噬,减轻炎症反应。体内实验进一步证实,QTXZG的治疗作用伴随着ircStk4和METTL3表达的下调,从而显著减轻了肾损伤、减少了炎症反应、抑制了肾组织增殖、促进了细胞凋亡和自噬:本研究揭示了QTXZG在治疗CGN的过程中,通过降低circStk4 m6A和METTL3的表达来调节circStk4/miR-133a-3p/C1轴,从而抑制肾小球组织/膜细胞增殖,促进自噬和细胞凋亡;这些结果揭示了QTXZG减轻CGN的新机制,并意味着METTL3可能成为创新治疗方法的靶点。
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来源期刊
Phytomedicine
Phytomedicine 医学-药学
CiteScore
10.30
自引率
5.10%
发文量
670
审稿时长
91 days
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.
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