摘要Nephropathy 1 Formula(N1F),a traditional Chinese medicine(TCM),has demonstrated promising clinical efficacy in diabetic nephropathy(DN).However,its underlying protective mechanisms remain insufficiently defined.In this study,a type 2 diabetes mellitus(T2DM)mouse model was established using a high-fat diet(HFD)and streptozotocin(STZ).Addition-ally,DN was simulated in vitro via exposure of mouse glomerular mesangial cells(MES-13)to high glucose(HG)and trimethylamine-N-oxide(TMAO).To elucidate the mechanistic basis of N1F's renoprotective effects,an integrative approach combining metabolomics,transcriptom-ics,and 16S ribosomal ribonucleic acid(rRNA)gene sequencing was employed.N1F treat-ment reduced the urinary albumin-to-creatinine ratio(UACR),preserved renal function,and attenuated histopathological damage and renal fibrosis in diabetic mice.Mechanistically,N1F modulated systemic TMAO levels and energy metabolism,altered gut microbiota composi-tion,and suppressed microbial production of TMAO-related metabolites.Under hyperglycem-ic conditions,TMAO induced excessive mitochondrial reactive oxygen species(mROS),im-paired mitochondrial dynamics,and disrupted cellular energy metabolism.In contrast,N1F normalized mROS levels,restored mitochondrial structure and function,enhanced oxidative phosphorylation(OXPHOS),increased ATP production,and reduced glycolytic dependency.Furthermore,N1F downregulated the expression of key pyroptosis-related proteins-includ-ing NOD-like receptor family pyrin domain-containing 3(NLRP3),N-terminal gasdermin D(GSDMD),cleaved-Casp1,interleukin-1β(IL-1β),and IL-18-in both in vivo and in vitro mod-els,indicating suppression of pyroptosis via inhibition of the TMAO-mROS-NLRP3 signaling axis.Collectively,these findings demonstrate that N1F exerts protective effects against DN by targeting mitochondrial dysfunction and pyroptotic injury,supporting its potential as a thera-peutic strategy for DN.