Abstract: Objective  To investigate the effect of Yiqi Qingre Xiaozheng Formula on pyroptotic injury in renal tubular epithelial cells induced by high glucose (HG) combined with lipopolysaccharide (LPS), and to elucidate the underlying mechanisms by which the formula ameliorates tubular cell pyroptosis in the context of diabetic kidney disease (DKD). Methods  Human renal proximal tubular epithelial cells (HK-2) were used for in vitro experiments. Pyroptosis was induced by stimulation with high glucose and lipopolysaccharide, and cell viability was assessed by using the CCK-8 assay to determine optimal modeling conditions. HK-2 cells were divided into control, model, and treatment groups (treated with Yiqi Qingre Xiaozheng Formula). Phalloidin staining was used to observe cytoskeletal morphology. Cell apoptosis was evaluated by Annexin V/PI double staining and flow cytometry. Immunofluorescence staining was performed to detect lysosomalc-associated membrane protein-1 (LAMP-1) and cathepsin B (CTSB). Western blotting was performed to assess the protein expression levels of key  NOD-like receptor family, pyrin domain containing 3（NLRP3) and apoptosis-associated speck-like protein containing a CARD（ASC). Lysosomal membrane permeabilizer LLOMe was used as a positive control. Levels of  interleukin-18 (IL-18) and IL-1β in the cell supernatant were quantified by ELISA to assess lysosomal membrane permeability and pyroptosis. Results  Stimulation with 30 mmol/L HG and 10 μg/mL LPS for 24 h significantly reduced HK-2 cell viability, while treatment with Yiqi Qingre Xiaozheng Formula restored cell viability. Compared with the control group, HG+LPS treatment led to disorganized cytoskeletal morphology, increased apoptosis, lysosomal membrane permeabilization, cytoplasmic release of CTSB, and upregulation of pyroptosis-related proteins NLRP3 and ASC, as well as increased pro-inflammatory cytokines of IL-1β and IL-18. In contrast, the formula stabilized cytoskeletal structure, inhibited apoptosis, protected lysosomal membrane integrity, and reduced CTSB release into the cytoplasm. Consequently, the expression of NLRP3 and ASC was downregulated, and the levels of IL-1β and IL-18 in the supernatant were significantly decreased. Conclusion  Yiqi Qingre Xiaozheng Formula mitigates high glucose-induced tubular cell injury in DKD. Its protective mechanism may be associated with inhibition of pyroptosis via modulation of the LAMP-1/CTSB/NLRP3 signaling axis.

Key words: diabetic kidney disease, lysosomes, pyroptosis, inflammatory cytokines, renal tubular epithelial cells