Qiteng Xiaozhuo granules activate mitophagy to alleviate chronic glomerulonephritis

Qiteng Xiaozhuo Granules (QTXZG), a traditional Chinese medicine formula, is widely recognized for its therapeutic effects. Previous studies have demonstrated that the Methyltransferase-like 3 (METTL3)/FOS Like Antigen 1 (FOSL1) N6-methyladenosine (m6A) axis plays a crucial role in the regulation of mitophagy in chronic glomerulonephritis (CGN) both in vitro and in vivo. However, it remains unclear whether QTXZG exerts its therapeutic effects in CGN through the METTL3/FOSL1 m6A axis to regulate mitophagy. This study aims to investigate the renal protective mechanisms of QTXZG by activating mitophagy in glomerular mesangial cells through the METTL3/FOSL1 m6A axis.

Adenine-induced CGN mice and lipopolysaccharide-stimulated mouse glomerular mesangial cells were employed as in vivo and in vitro models, respectively. The chemical characterization and quality control of QTXZG were performed using high-performance liquid chromatography fingerprinting, peak assignment, marker quantification, and analytical method validation. Renal function was evaluated via serum creatinine, blood urea nitrogen, immunoglobulin G, and immunoglobulin M quantification. Renal histopathology was analyzed using Hematoxylin and Eosin, Masson, and Periodic Acid-Schiff staining. QTXZG-containing serum concentration and treatment duration were optimized via cell counting kit-8 assays. Inflammation and oxidative stress were assessed by enzyme-linked immunosorbent assay and reactive oxygen species staining. Global m6A levels were quantified by colorimetric assays, and FOSL1 mRNA m6A modifications were validated via methylated RNA immunoprecipitation quantitative PCR. Actinomycin D experiments determined FOSL1 mRNA stability. METTL3/FOSL1 m6A axis and mitophagy were analyzed by western blot, transmission electron microscopy, MitoTracker Red staining, and microtubule-associated protein 1 light chain 3-mitochondria immunofluorescence co-localization.

High-performance liquid chromatography fingerprinting of ten QTXZG batches identified sixteen common peaks, assigned them to corresponding herbal sources, and quantified five representative marker compounds. QTXZG improved renal function and suppressed mesangial cell proliferation in CGN mice, while reducing pro-inflammatory cytokines and reactive oxygen species levels and enhancing superoxide dismutase activity. Importantly, QTXZG promoted autophagosome formation and mitigated mitochondrial damage, with enhanced microtubule-associated protein 1 light chain 3-mitochondria/voltage-dependent anion channel 1 co-localization confirming mitophagy induction. Mechanistically, QTXZG inhibited METTL3/FOSL1 expression, reduced global m6A levels and FOSL1 mRNA m6A enrichment, and destabilized FOSL1 transcripts. Critically, METTL3 overexpression reversed QTXZG's effects on inflammation, oxidative stress, mitophagy, and METTL3/FOSL1 m6A axis in lipopolysaccharide-stimulated mouse glomerular mesangial cells.

This study suggests that QTXZG induces mitophagy in glomerular mesangial cells in CGN by modulating the METTL3/FOSL1 m6A axis. Targeting FOSL1 m6A modification through the writer enzyme METTL3 may provide a potential therapeutic approach for the treatment of CGN.