Management Of Gout-associated MSU Crystals-induced Nlrp3 Inflammasome Activation By Procyanidin B2: Targeting IL-1β And Cathepsin B In Macrophages

Objective: Procyanidin B2(Procyanidin B2,PCB2)is a naturally occurring phenolic compound and the main component of procyanidins in grape seeds.This research aimed to investigate whether procyanidin B2(PCB2),a natural dietary compound,can suppress the secretion of interleukin-1β(IL-1β)and Cathepsin B stimulated by MSU crystals.Thereby reducing gout inflammation.Methods: 1.In vivo models of gout-like inflammation are established by using air pouch model and mouse paw gout model respectively.(1)Mice air pouch model: C57BL/6 mice were subcutaneously injected with 5 ml of sterile air on the first day,and on the second day suppled 3 ml of sterile air to make air pouch.On the fourth day,pre-treated with procyanidin B2 by gavaged at the concentration of 30 mg/kg and 60mg/kg one hour in advance,following with MSU crystals for 6 hours,then collected back skin of air pouch,exudate and serum.(2)Mice paw gout model: C57BL/6 mice were used with procyanidin B2(60 mg/kg)by gavaged one hour in advance and MSU crystals were injected into the mouse paw to stimulate for 24 hours and then the mice paw tissues and serum were collected.Western blotting,ELISA,and histological analysis staining experiments were used to detect the expression of related inflammatory factors in the corresponding tissues,serum,and lavage fluid of mice.2.In vitro model of gout-like inflammation: 4 days after injection of thioglycolate,mouse peritoneal macrophages(MPM)were extracted and treated with PCB2 for 1 hour(4 μM,20 μM,100 μM),then stimulated with LPS 4 hours,with continuous incubation of MSU crystals(500 μg/ml)for 18 hours.The protein expression and release of IL-1βand Cathepsin B were detected by Western blotting,and the expression of NLRP3 was detected by Real-time PCR.Results:1.The experimental results of in vivo models showed that PCB2 could improve the symptom of swelling,and suppress the expression of IL-1β,Cathepsin B,F4/80,NE,NLRP3,HMGB1 and inflammatory immune cell infiltration in the skin and paw.2.The experimental results of in vitro model of mouse peritoneal macrophages showed that mouse peritoneal macrophages were stimulated with MSU crystals plus LPS occurred gouty inflammation,lead to release IL-1β and Cathepsin B,while PCB2 could improve the inflammatory response.Conclusions: Procyanidin B2 could improve the inflammation caused by MSU crystals,which might be related to NLRP3-related cytokines.It suggests that procyanidin B2 may be an effective candidate for gout prevention and treatment,and provide a new idea for the prevention and treatment of gout.