[Effects and mechanisms of Supplemented Shenqi Dihuang Decoction Formula in improving podocyte pyroptosis in diabetic kidney disease by regulating dyslipidemia-mediated lipid droplet accumulation and TLR4/MyD88/NF-κB pathway].

This study aimed to investigate the effects and mechanisms of the Supplemented Shenqi Dihuang Decoction Formula(SSDDF) in improving podocyte pyroptosis in diabetic kidney disease(DKD) by regulating dyslipidemia-mediated lipid droplet accumulation and the Toll-like receptor 4(TLR4)/myeloid differentiation primary response gene 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. First, a modified DKD rat model was established. After successful modeling, the DKD rats were administered low-dose SSDDF(SSDDF-L), high-dose SSDDF(SSDDF-H), dapagliflozin(DAPA), or saline(vehicle) by gavage for six consecutive weeks. In the in vivo study, the general condition of the rats, blood glucose(BG), urinary albumin(UAlb), liver and renal function indexes, blood lipid profiles, and serum short-chain fatty acid(SCFA) levels were examined. Glomerular histopathological features, podocyte foot process morphology, and glomerular basement membrane(GBM) ultrastructure were compared. The protein expression levels of podocyte slit diaphragm structural molecules(nephrin and podocin) in renal tissue, key signaling molecules of the TLR4/MyD88/NF-κB pathway [TLR4, MyD88, phosphorylated inhibitor of NF-κBα(p-IκBα), and phosphorylated NF-κB p65(p-p65)],key molecules of the classical pyroptosis pathway [NOD-like receptor thermal protein domain-associated protein 3(NLRP3), apoptosis-associated speck-like protein containing a CARD(ASC), cleaved-caspase-1, and gasdermin D-N(GSDMD-N)], and effector inflammatory factors [interleukin-1β(IL-1β) and interleukin-18(IL-18)] were analyzed. Second, in the in vitro study, a lipotoxic podocyte injury model was constructed by treating MPC-5 cells with high glucose(HG) and palmitic acid(PA). The injured cells were then treated with either SSDDF-H-containing serum or the TLR4 inhibitor TAK-242. The lipid droplet characteristics and the expression levels of proteins associated with the TLR4/MyD88/NF-κB pathway and pyroptosis in podocytes were observed and analyzed. Additionally, in the network pharmacology study, potential targets of SSDDF and DKD were predicted and screened using the HERB and SwissTargetPrediction databases, and a "drug-component-target network" of SSDDF was constructed. DKD-related targets were integrated from the OMIM, GeneCards, and TTD databases. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses for the intersecting targets were performed using the DAVID database. Molecular docking was performed between the core components of SSDDF and key targets. The network pharmacology results suggested that the therapeutic effects of SSDDF on DKD were likely related to the regulation of lipid metabolism and inflammation-related signaling pathways. The in vivo results showed that SSDDF-L and SSDDF-H improved, to varying degrees, the general condition and the levels of BG, UAlb, serum creatinine(Scr), blood urea nitrogen(BUN), total cholesterol(TC), triglyceride(TG), the ratio of low-density lipoprotein cholesterol(LDL-C) to high-density lipoprotein cholesterol(HDL-C), and SCFA in DKD rats, ameliorated glomerular histopathological changes and podocyte ultrastructure, and improved renal protein expression levels of nephrin, podocin, TLR4, MyD88, p-IκBα, p-p65, NLRP3, ASC, cleaved caspase-1, GSDMD-N, IL-1β, and IL-18. Notably, regarding the improvement of proteinuria in the modified DKD rats, SSDDF was less effective than DAPA. However, for renal function and SCFA levels, SSDDF-H was superior to DAPA, and for blood lipid regulation, glomerulosclerosis, and podocyte injury, SSDDF showed similar efficacy to DAPA. The in vitro results showed that both SSDDF-H-containing serum and the TLR4 inhibitor reduced lipid droplet accumulation, inhibited TLR4/MyD88/NF-κB pathway activation, and alleviated podocyte pyroptosis, with comparable effects. In conclusion, SSDDF ameliorates podocyte pyroptosis in DKD by regulating dyslipidemia-mediated lipid droplet accumulation and the TLR4/MyD88/NF-κB signaling pathway. These findings provide pharmacological evidence for precisely elucidating the scientific connotation of the Jinling school's therapeutic approach to "Shenxiao disease".