Abstract: BACKGROUND: The activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome has been found to be an important factor in the pathogenesis of gouty arthritis, and the activation of NLPR3 inflammasome can be effectively inhibited by regulating the PTEN-induced kinas 1 (PINK1)/Parkin signaling pathway.
OBJECTIVE: To investigate the effect of Duhuo Jisheng Decoction on the PINK1/Parkin/NLRP3 signaling pathway.
METHODS: (1) Animal experiment: Male Sprague-Dawley rats were randomly divided into control group, model group, positive control group (Qiushuixian tablets 0.3 mg/kg), and low-, medium-, and high-dose Duhuo Jisheng Decoction groups (6.9, 13.8, and 27.6 g/kg, respectively). A rat gouty arthritis model was constructed by injecting monosodium urate crystal suspension into the right hind ankle joint cavity of rats in all the groups except for the control group. (2) Cell experiment: Human monocytes (THP-1) were divided into blank control group, model group, Duhuo Jisheng Decoction-containing serum group, and PINK1 siRNA+Duhuo Jisheng Decoction-containing serum group. The cells in each group except the blank control group were stimulated with sodium urate to establish an in vitro gouty arthritis model. (3) The swelling degree, joint circumference, gait score, joint inflammation index and degree of pathological grading in the ankle joints of rats were observed. Enzyme-linked immunosorbent assay was used to detect the levels of uric acid, C-reactive protein, NLRP3, and interleukin 1β. Immunoblotting assay was used to detect the levels of proteins related to the PINK1/Parkin/NLRP3 pathway. Tetramethyl azolidine blue assay was used to detect cell activity. Immunofluorescent double staining was performed to detect the level of mitophagy. Immunofluorescence was used to detect the expression of NLRP3 and interleukin 1β. 
RESULTS AND CONCLUSION: (1) Compared with the control group, rats in the model group showed elevated ankle swelling and ankle circumference at 6-48 hours (P < 0.05), elevated gait scores and joint inflammation indexes at 24 and 48 hours (P < 0.05), elevated serum uric acid and C-reactive protein levels, degree of pathological classification, expression of PINK1, Parkin, and NLRP3 proteins, and interleukin 1β level in synovial tissues (P < 0.05). Compared with the model group, the protein expression levels of PINK1 and Parkin in all the Duhuo Jisheng Decoction groups were elevated, while the levels of the other indexes were decreased (P < 0.05). (2) Compared with the model group, NLRP3 activity, interleukin 1β level and mitochondrial outer membrane translocator protein 20 protein level in the Duhuo Jisheng Decoction-containing serum group were decreased (P < 0.05), while the proliferation inhibition rate, PINK1, Parkin and LC3B protein level were increased (P < 0.05). Compared with the model group, the mitochondrial autophagy level of cells in the Duhuo Jisheng Decoction-containing serum group was elevated (P < 0.05), while NLRP3 and interleukin 1β protein levels were decreased (P < 0.05). (3) Compared with the Duhuo Jisheng Decoction-containing serum group, the mitochondrial autophagy level of cells in the PINK1 siRNA+Duhuo Jisheng Decoction-containing serum group was decreased (P < 0.05), and the expression levels of NLRP3 and interleukin 1β were elevated (P < 0.05). To conclude, Duhuo Jisheng Decoction inhibits the activation of NLRP3 inflammasome by regulating the PINK1/Parkin signaling pathway, thereby exerting a therapeutic effect on gouty arthritis.   

Key words: Duhuo Jisheng Decoction, gouty arthritis, PTEN-inducible kinase 1, Parkin, NLRP3