NLRP3 Inhibitor MCC950 Ameliorates Cartilage Degeneration By Restoring Impaired Autophagy And Activating Antioxidant Pathway

Objective1.To investigate inhibitory effect of MCC950 on expression of NLRP3,inflammatory factors and catabolic proteases in IL-1β stimulated chondrocytes in vitro experiments,and the regulatory effects of MCC950 on autophagy and oxidative stress.2.To explore the pathways related to inflammation,catabolism,autophagy and oxidative stress which might be regulated by MCC950.3.To investigate the protective effect of MCC950 on degeneration of OA cartilage in vivo experiment.Methods1.The OA related genes and pathways which MCC950 probably act on were selected and enriched through bioinformatics technology,and Degree Centrality,Closeness Centrality and Betweenness Centrality of target genes were analyzed and evaluated to selected core genes and pathways.2.The inflammatory chondrocyte model was established by stimulating chondrocytes and safe concentration range of MCC950 was screened by CCK-8 method.Then chondrocytes were stimulated by MCC950 and IL-1β respectively and collectively.Western blot was used to detect the inhibitory effect of MCC950 on the expression of NLRP3 protein,NLRP3,Caspase1,i NOS,Cox2,Caspse1,cartilage matrix protein Col2,matrix degrading enzyme MMP13 and ADAMTS5,autophagy related protein Atg5,Atg12,Beclin-1,LC3 I and LC3 II and age-related protein P16(INK4A).Expression of MAPK,PI3K/Akt/m TOR and Nrf2/HO-1/NQO1 pathways were also evaluated by Western blot.What’s more,3-MA was utilized to verify the regulation of inflammation and catabolism by autophagy.Production of ROS was labeled by DCFH-DA fluorescent probe and evaluated by flow cytometry and fluorescence microscope.In addition,the changes of autophagy flow in chondrocytes infected with m RFPGFP-LC3 double-labeled adenovirus were detected to further evaluate the autophagy regulation effect of MCC950.3.The OA mouse model was established by DMM surgery,and MCC950 was injected into articular cavity to evaluate its effect on degradation of cartilage.Saffron solid green staining was utilized to facilitated to observe cartilage degeneration in OA mouse,and the corresponding grade was evaluated by OARSI score.Results1.40 core target genes and 17 OA-related pathways which might be regulated by MCC950 were selected.Akt1 and MAPK1 were picked up as the TOP2 core target genes,and the weight of Akt1 was the highest according to the centrality evaluation.And PI3K/Akt and MAPK pathways are TOP2 core pathways.2.MCC950 had no toxic effect on chondrocytes at a concentration of 0.01-1 μM,and showed strong inhibitory effect on expression of NLRP3 protein at 1 μM.After stimulated by IL-1βfor 24 h,the expression of inflammatory factors i NOS,Cox2,Caspase1,cartilage matrix protein Col2,matrix degrading enzyme MMP13 and ADAMTS5,age-related protein P16(INK4A)were significantly up-regulated,and the protein expressions of autophagy markers Atg5,Atg12,Beclin-1,LC3 II were significantly down-regulated.MCC950 significantly inhibited the expression of inflammation,catabolism and senescence related proteins and up-regulated the expression of autophagy markers under inflammatory stimulation,it also increased the number of autophagosomes.The autophagy inhibitor 3-MA reversed the anti-inflammatory and catabolism effect of MCC950.In addition,MCC950 promoted the expression of Nrf2 nucleic protein and activation of Nrf2/HO-1/NQO1 pathway.Meanwhile,results from fluorescence microscope and flow cytometry confirmed the inhibitory effect of MCC950 on ROS.In addition,the activation of MAPK and PI3K/Akt/m TOR pathway under the stimulation of IL-1β was also inhibited.3.C57 mouse cartilage degeneration model could be successfully established by DMM surgery.The sanguine solid green staining showed that the mice in the DMM group had obvious wear and tear of cartilage,especially in the inner weight bearing area of articular cartilage.The wear of articular cartilage in mice injected with MCC950 in the articular cavity was significantly reduced compared with DMM group.Conclusion1.MCC950 showed better inhibitory effect on inflammation and catabolism related protein in IL-1β treated chondrocyte,and autophagy might play a crucial role in this process.Inhibition of inflammation and catabolism by MCC950 might be associate with inhibition of MAPK,PI3K/Akt/m TOR pathway which are well known to regulate autophagy.2.MCC950 may play an antioxidant role by promoting Nrf2 nuclear transcription and activating the Nrf2/HO-1/NQO1 pathway.3.MCC950 effectively alleviated the cartilage degeneration induced by DMM surgery in C57 mice.