Paishi granule inhibits calcium oxalate-induced oxidative stress kidney injury after gut microbiota transformation: a multi-omics analysis combined in vivo and in vitro study

BACKGROUND: Renal stones are a common clinical problem with a high recurrence rate. In recent years, many Chinese herbs have been reported to effectively ameliorate calcium oxalate via different mechanisms. Gut microbiota plays an important role in the regulation of stone formation. In this study, we establish a novel molecular framework to illuminate that paishi granule (PSG) can treat calcium oxalate stone formation through gut microbiota transformation. METHODS: The UPLC-MS/MS of serum-PSG was analyzed. A rat model of calcium oxalate kidney stones and a calcium oxalate monohydrate cell model (COM) were established. Blood samples were measured for Scr and BUN level. The histological staining and immunohistochemistry were used for observing pathological morphology of the kidneys and the colon. 16S rRNA sequencing, fecal metabonomics analysis, RNA transcriptomics sequencing, enzyme-linked immunosorbent assays, RT-qPCR, and western blotting were used to identify the signal transduction pathway of PSG in treating renal calculi to systematically explain the function of PSG after transformation by the gut microbiota. RESULTS: PSG significantly alleviated renal injury induced by calcium oxalate stones, reduced BUN and Scr levels, and exerted antioxidant effects by enhancing T-SOD activity while decreasing LDH and MDA levels. Histological analysis of intestinal tissues demonstrated that PSG repaired intestinal barrier damage in EN rats. 16S rRNA sequencing revealed a decrease in the abundance of genera such as Romboutsia, Clostridium, and Enterobacter, and an increase in that of Blautia, Acetatifactor, and Kineothrix in EN group. SCFAs metabonomics analysis indicated significant reductions in acetate, propionate, and butyrate levels, which were improved after treatment with PSG, effectively ameliorating the disordered microbiota structure and metabolite profiles. The key differentially expressed genes, including β-arrestin2, TGF-β1, and NOX4, were identified using transcriptomics analysis, and enrichment analysis suggested the involvement of the MAPK signaling pathway. RT-qPCR and western blotting confirmed that PSG mitigated oxidative stress by suppressing the β-arrestin2/MAPK/NOX4 pathway. The renal protective effects of PSG were markedly attenuated in an antibiotic-induced model of gut-microbiota depletion. In vitro experiments further demonstrated that the PSG, after being transformed by the microbiota, could regulate the MAPK kinase cascade reaction through the mechanism mediated by β-arrestin2, inhibiting the process of NOX4 expression. CONCLUSION: PSG exerts its anti-lithic effects through a novel multi-target regulatory mechanism involving the gut microbiota-metabolite-oxidative stress axis, thereby establishing a new paradigm for mechanistic research in traditional Chinese medicine.