Qihuang Yishen formula attenuates renal tubular injury by modulating inflammation via the cGAS/STING pathway in diabetic kidney disease

BACKGROUND: The cGAS/STING-mediated inflammatory response plays a key role in diabetic kidney disease (DKD) pathogenesis. However, effective anti-inflammatory therapies to halt disease its progression remain unavailable. The Qihuang Yishen (QHYS) formula has been demonstrated to reduce proteinuria and halt the course of DKD. However, the underlying mechanism of QHYS remains unclear. PURPOSE: The purpose of this study was to determine whether QHYS had anti-inflammatory effects on DKD by targeting the cGAS/STING pathway both in vitro and in vivo. METHODS: In vivo, DKD models were established in KK-Ay mice using a high-fat diet. In vitro, human renal tubular epithelial cells (HK-2) were exposed to high glucose (HG) and palmitic acid (PA) for 24 h to simulate renal tubular injury. Both experimental settings received interventions with QHYS or canagliflozin (positive control drug). Bioactive components in QHYS-containing serum were characterized by HPLC-ESI/MS. Body weight, random blood glucose, renal function markers, urinary protein levels, renal tubular injury indicators, and renal histological lesions were evaluated. Mitochondrial ultrastructure was analyzed by transmission electron microscopy (TEM). Western blotting quantified mitochondrial proteins TFAM and TOM20. Cell viability was assessed via CCK-8 assay, while ELISA kits measured IL-6 and TNF-α levels in cell supernatant. Quantitative PCR assessed expression of mtDNA and inflammatory mediators (IL-6, TNF-α, IFN-γ and CXCL10 mRNA). For cGAS/STING pathway analysis, key proteins were evaluated through western blotting, immunofluorescence, and immunohistochemistry. Additionally, mtDNA transfection experiments in HK-2 cells elucidated QHYS's regulatory mechanism on this pathway. RESULTS: HPLC-ESI/MS identified 32 principal bioactive components in QHYS. In vivo experiments showed that QHYS significantly reduced Scr and UREA levels (p < 0.05), decreased UACR, 24 h UTP and renal tubular injury markers NAG and NGAL levels (p < 0.01), and alleviated renal histopathological damage in KK-Ay mice, including glomerular hypertrophy, mesangial matrix expansion, tubular vacuolar degeneration, and tubulointerstitial inflammation. Moreover, QHYS ameliorated mitochondrial structural damage, upregulated the expression of TFAM and TOM20 (p < 0.05), and suppressed cGAS/STING pathway activation (p < 0.01). In vitro experiments demonstrated that QHYS alleviated mitochondrial damage (p < 0.05) and mtDNA leakage (p < 0.01), and inhibited cGAS/STING signaling and downstream inflammation (p < 0.05). More importantly, transfecting mtDNA into the cytoplasm to mimic the leakage process demonstrated that QHYS-containing serum directly inhibits cGAS/STING pathway activation (p < 0.05), and this mechanism also alleviated renal tubular injury in DKD. CONCLUSION: QHYS attenuates renal tubular inflammatory damage via cGAS/STING pathway regulation. This pioneering study provides the first evidence that herbal medicine exerts renal tubule protection through multi-target regulation of innate immune-mediated inflammatory injury in metabolic nephropathy, offering novel scientific evidence for DKD intervention.