| Gout is the acute or chronic inflammation caused by the deposition of uric acid crystals in joints,tendons,surrounding tissues and it correlates closely to hyperuricemia.In compared with previous years,an obvious increasing in incidence of gout in China has occurred by recent years.In addition,gout often is concomitant with other kinds of metabolic diseases,such as hypertension and diabetes.Nonsteroidal antiinflammatory drugs(NSAIDs),glucocorticoids and colchicine are commonly used in therapies for acute gout.The currently available drugs,though effective,have some problems,such as serious side effects and limited effectiveness,thus,novel therapeutic agents are still needed.In the first chapter,we summarized the recent research progress of NLRP3 inflammasome,which is a part of innate immunity and closely related to the pathological processes in various inflammatory diseases.Its formation and activation process,relationships with several diseases are reviewed here.Moreover,some drugs aimed to treat related diseases via inhibiting the formation or activation of NLRP3 inflammasome has been reported.In the second chapter,the inhibition of NLRP3 inflammasome by AI-44 has been investigated in vitro and in vivo.To evaluate the effects of AI-44 on NLRP3 inflammasome,THP-1 cells stimulated by LPS and MSU are set as the in vitro model.We confirm its inhibition by determining the indicators that the activation of caspase-1 and Gasdermin D,the maturation and secretion of inflammatory cytokine IL-1β.Meanwhile,we study the probable mechanism of AI-44 to suppress the activation of MSU-induced NLRP3 inflammasome.We use AI-44-biotin conjugates as a probe for ligand fishing assay.After separation and identification the mixture of ligand fishing by SDS-PAGE and MS,we propose that cathepsin B(CTSB)is the probable target protein of AI-44.Subsequent experiments suggest that AI-44 can bind with CTSB and inhibit its activity.It reduces the hydrolysis of inhibitory domain on NLPR3.Furthermore,co-immunoprecipitation experiments indicate that the interaction between NLRP3 and CTSB is inhibited by the binding of AI-44 and CTSB.It also blocks the assembly of NLRP3 inflammasome complex and subsequent IL-1βproduction.In addition,we use the Autodock 4.2 program to predict the binding sites and mode of AI-44 and CTSB.A virtual screening on all AI serials compounds shows that AI-44 has the best affinity to CTSB.It’s interesting that AI-44 has showen a better anti-inflammation effect in low dosages in compared with CA-074Me,a commercial available inhibitor of CTSB.It means that development of a novel kind of CTSB inhibitor by setting AI-44 as a lead compound is potentially valuable.In the third chapter,we confirm the anti-inflammation effect of AI-44 in vivo by using MSU-induced mice acute gouty arthritis and peritonitis model.AI-44 has the similar inhibitory effects with in vitro model in mice foot paws,peritoneal lavage fluids and the recruitment of peritoneal exudate cells.More importantly,in histopathology,alleviation of edema,the infiltration of CD11b positive macrophages and Ly 6G positive neutrophils in foot paws from gouty arthritis mice by AI-44 has been observed.In conclusion,we confirm the inhibitory effect of AI-44 on NLRP3 inflammasome in vitro and in vivo and further research suggests that the anti-inflammation effect caused by its binding with cathepsin B.Besides,our study provides a lead compound for inflammatory diseases that are associated with NLRP3 activation. |
PDF Full Text Request