A novel Chinese medicine formula against hyperuricemia-induced kidney inflammation involving pyroptosis inhibition via modulating AMPK-TLR4-NLRP3 pathway

Background: Hyperuricemia (HUA), characterized by elevated serum uric acid (SUA) level, serves as a precursor of gout. Gout formula (GF), an empirical formula, has been prescribed in Chinese medicine practice for many years and observed to be effective in relieving HUA and gout. This study aimed to investigate GF as an innovative therapeutic agent for HUA and to elucidate its underlying molecular mechanisms using in vivo and in vitro models of HUA.

Methods: A HUA mouse model was established by orally administering potassium oxonate (PO) and injecting with hypoxanthine (HX) to mice. Behavioral tests and histopathological analysis were performed to measure the efficacy of GF. Biochemical indicators such as SUA, urine uric acid (UA), Xanthine Oxidase (XOD), blood urea nitrogen (BUN), and creatinine (Cre), inflammatory markers, signaling pathway markers, uric acid transporters (UAT), and renal injury were assessed using enzyme-linked immunosorbent assay (ELISA), Western blot analysis, immunofluorescence staining, and Real-Time PCR analyses as appropriate. HK-2 cells stimulated with monosodium urate (MSU) were used to confirm the action mechanisms of GF against HUA.

Results: GF treatment significantly reduced the pain sensitivity and improved the tensile performance of the PO + HX-treated mice in the hot plate test and grip strength test, respectively. GF treatment also significantly reduced the levels of UA, BUN, and Cre, and alleviated the kidney injury induced by PO + HX in mice. In addition, GF suppressed inflammatory response by attenuating the protein expressions of CCR2, CCR5, CXCL10, TNF-α, IL-4, IL-6, and IL-1β, but upregulating AMPK phosphorylation. It also modulated the UAT by increasing OAT1 and OAT3, while decreasing URAT1 and GLUT9 protein expression. Moreover, increasing GF concentrations led to a decreased intracellular UA level, but increased extracellular UA in the MSU-stimulated HK-2 cells, consisting with the findings in the in vivo study. Furthermore, GF also mitigated the inflammation-induced Pyroptosis of HK-2 cells, and AICAR, an AMPK agonist, enhanced GF in suppressing the Pyroptosis via inhibiting the activation of AMPK-TLR4-NLRP3 pathway.

Conclusions: GF significantly alleviated kidney injury and inflammatory response induced by HUA in mice, and the action involved inhibiting the activation of the XOD and Pyroptosis, and modulating the expression of UAT through downregulating the TLR4-NF-κB p65-NLRP3 pathway. These findings amply indicate that GF is a promising therapeutic option for managing gout and kidney complications associated with HUA.

AMPK-TLR4-NLRP3 pathway; Gout formula; Hyperuricemia; Kidney inflammation; Pyroptosis.