Pachymaran Alleviates Acute Gouty Arthritis by Inhibiting NLRP3 Inflammasome Activation and Pyroptosis.

Abstract

Objective: Acute gouty arthritis is the most common rheumatic diseases, and leads to a heavy clinical burden, thereby to explore the treatment effects of pachymaran on acute gouty arthritis and elucidate its mechanism are meaningful.

Methods: Eighteen SPF C57BL/6 mice were randomly divided into three groups: the sham group, model group, and pachymaran group (200mg/kg), with 6 mice in each group. The acute gouty arthritis model of mice was established by injecting 0.025 mL sodium urate solution into the right ankle cavity of the mice. The pachymaran group was given 200mg/kg of pachymaran intragastrically, in the sham group and model group were given the same volume of normal saline, respectively, for 7 consecutive days. Blood samples were collected from the orbital venous plexus 1 h after the last administration, all mice were killed, and ankle tissue samples were collected. The pathological changes of mouse ankle synovial tissue were observed by HE staining. The expression levels of IL-1β and IL-18 inflammatory factors in the serum of mice were determined by ELISA. The ultrastructure of the synovial tissue of the mouse ankle joint was observed by transmission electron microscope. The protein expression levels of NLRP3, ASC, IL-1β, IL-18, GSDMD, and Caspase-1 in synovial tissue of mouse ankle were detected by Western blot assay. Mouse chondrocytes were cultured and divided into groups I, II, III, and IV. Group I was the control group without any drug intervention. The cells in groups II, III, and IV were stimulated with sodium urate solution (100μg/mL), and groups III and IV were intervened by pachymaran (200μg/mL), among which the NLRP3 agonist Nigericin sodium salt intervened group IV. The expression levels of NLRP3, IL-1β, GSDMD, and Caspase-1 proteins were detected by Western blot assay, and the apoptosis rate was detected by flow cytometry.

Results: Compared with the sham group, the pathological injury of mice ankle synovial tissue in the model group was significantly aggravated, as the membrane was incomplete, mitochondria were swollen, the ridge was unclear or even disappeared, and the pathological injury of mice ankle synovial tissue in the pachymaran group was significantly improved vs model group; the serum levels of IL-1β and IL-18 were increased in model group vs sham group, and pachymaran decreased these index vs model group; Compared with the sham group, protein expression levels of NLRP3, ASC, IL-1β, IL-18, GSDMD, and Caspase-1 were significant increased in model group, and pachymaran suppressed these proteins vs model group. The TEM results showed that in model group the wide swelling of mitochondria accompanied by disappearance of mitochondrial cristae vs sham group, and the mitochondrial ridge was slightly damaged, or the mitochondria were only swollen, and the ridge was still clearly visible in pachymaran group. In vitro experiments, Compared with group I, the protein expression levels of NLRP3, IL-1β, GSDMD, Caspase-1, and the apoptosis rate of chondrocytes in group II were significantly increased. Compared with group II, the protein expression levels of NLRP3, IL-1β, GSDMD, Caspase-1, and the apoptosis rate of chondrocytes in group III were significantly decreased. Compared with group III, the protein expression levels of NLRP3, IL-1β, GSDMD, Caspase-1, and the apoptosis rate of chondrocytes in group IV were significantly increased.

Conclusion: Pachymaran maintain the structural integrity of joints and alleviate the progression of acute gouty arthritis by inhibiting NLRP3 inflammasome activation and pyroptosis, pachymaran may be used and applied to clinical treatment.