| Part ?: Expressions of NLRP3 and autophagy in liver of NASH patients and miceOBJECTIVE: We collected liver tissue samples from NASH patients and established NASH mouse model to investigate the expression and activation of NLRP3 inflammasome and autophagy in the liver of NASH patients and mice.Methods:(1)Liver specimens of NASH patients and patients with normal liver were collected.HE staining and NAFLD activity score were done to divide the patients into Ctrl group and NASH group.Levels of serum triglyceride,FFA and IL-1? as well as NLRP3 inflammasome and autophagy-related proteins were detected.(2)NASH mice were established by HFD,and then,short-term starvation was given.Pathological changes were observed by HE oil red staining.The activation of autophagosomes was observed by transmission electron microscopy.WB was used to detect the expression of NLRP3 inflammsome and autophagy-related protein in the liver and KCs.Results:(1)The levels of P62,NLRP3,ASC and caspase-1 in the liver of patients with NASH were significantly higher than those of normal liver tissue(P<0.01).The conversion level of LC3 I to LC3 II and the expression level of Beclin-1 were lower than that of normal group(P<0.05).(2)The levels of P62,NLRP3,ASC and caspase-1 in the liver of NASH mice were significantly higher than those in normal liver tissues(P <0.01).Electron microscopy and WB suggested that the level of autophagy in the liver of NASH mice were decreased.WB results showed that the expression of autophagy-related protein in the short-term starvation group was more stable than that in the HFD group and reduced the activation of NLRP3.The levels of serum IL-1? in NASH patients and NASH mice were significantly increased(P<0.01).Conclusion: The expression of NLRP3 inflammasome in the liver of NASH patients and NASH mice were higher than that of normal liver,and the expression of autophagy was decreased.The levels of serum IL-1? in NASH patients and NASH mice were increased.Autophagy has been restored after short-term treatment of starvation,and inflammation has been relieved.Part ?: Effect of autophagy on NLRP3 inflammasome in KCsOBJECTIVE: To investigate the effect of autophagy on the activation of NLRP3 inflammasome and the secretion of IL-1? in KCs stimulated by PA.Methods: The isolated KCs were randomly divided into 6 groups: Ctrl group: common medium;DMSO group: DMSO medium;PA group: 0.5m M PA stimulation for 12h;PA + rapamycin group(PA + R): treated with 50?M Rapamycin for 4h followed by PA for 12h;PA + Wortmannin group(PA + W): treated with 50 ?M Wortmannin for 4h followed by PA for 12h;F+S: starvation for 4h and treated with PA for 12 h.WB and q PCR were used to detect the expressions of proteins and corresponding genes of LC3,P62,Beclin-1,NLRP3,ASC and Caspase-1.Autophagy was detected by transmission electron microscope and confocal microscopy.The level of IL-1? was detected by ELISA.Results:(1)The bilayer membrane structure of autophagosome in the cytoplasm of KCs in PA group was observed by transmission electron microscopy.Lipid droplets in PA + W group were larger and less autophagic bodies.In PA + R group the number of autophagosomes increased,and lipid droplets smaller.(2)m RFP-EGFP-LC3 double-labeled cells showed increased autophagy in PA group,and autophagy increased in PA + R group.PA + W group was significantly weakened.(P<0.01).The expressions of P62,NLRP3,ASC and caspase-1 in PA group were significantly higher than those in Ctrl group(P<0.01),and the expressions of LC3 II / LC3 I and Beclin-1 in PA group were significantly higher those that in Ctrl group(P <0.01).The expressions of NLRP3,ASC and caspase-1 in PA + W group were significantly higher than those in PA group(P<0.01),and the expression of P62 was significantly higher than that of PA group(P <0.01).The expressions of P62,ASC and caspase-1 in PA + R group were significantly lower those in PA group(P<0.01),and the expression of NLRP3 There was no significant difference between the two groups(P>0.05).The expressions of P62,NLRP3,ASC and caspase-1 in PA group were significantly higher than those in Ctrl group(P<0.01),and the expressions of ATG5,ATG7 and Beclin-1 in PA group were significantly higher than those in Ctrl group High(P<0.01).The expressions of ATC5,ATG7 and Beclin-1 in PA + W group were significantly lower than those in PA group(P<0.05).The expressions of P62,NLRP3,ASC and caspase-1 were increased compared with PA group(P <0.05).The expressions of LC3 II / LC3 I and Beclin-1 in PA + R group were significantly higher than that in PA group(P> 0.05).The expressions of P62,NLRP3,ASC and caspase-1 were decreased compared with PA group.(5)ELISA results showed that the level of IL-1? in PA group was higher than that in Ctrl group(P<0.01).The level of IL-1? in PA + W group was significantly higher than that in PA group(P<0.01).The level of IL-1? in PA + R group was significantly lower than that in PA group(P<0.01).CONCLUSION: PA stimulates KCs to increase NLRP3 inflammasome and autophagy.Activation of autophagy with rapamycin inhibits the activation of NLRP3 inflammasome and the secretion of IL-1?.In contrast,inhibition of autophagy with wortmannin promoted the activation of NLRP3 inflammasome and the secretion of IL-1?.autophagy and NLRP3 inflammasome under PA stimulation and the role of TRAF6 in it.Methods: The co-localization between autophagy and NLRP3 inflammasome was detected by laser scanning confocal microscopy.The relationship of ASC protein and LC3,Beclin-1 and P62 were detected by immunoprecipitation.The levels of ASC ubiquitination were detected by immunoprecipitation and WB with or without PA.The expression of ASC ubiquitination was detected by immunoprecipitation and WB in silencing of TRAF6.Results: The results of immunoprecipitation showed that PA enhanced ASC protein interaction with LC3,Beclin-1 and P62.The results of WB showed that total ubiquitination,K63 ubiquitination and K48 ubiquitination levels of ASC were enhanced after PA stimulation.After lentivirus silencing TRAF6,ASC total ubiquitination,K63 ubiquitination and K48 ubiquitination were found to be lower than those of PA group before silencing decline.CONCLUSION: PA-stimulated autophagy,P62 and NLRP3 inflammasome interact with each other,and K63 and K48 ubiquitination may be involved.TRAF6 may regulate the K63 ubiquitination of ASC to a certain extent to promote P62 to recognize ubiquitinated ASC,and then transport to autophagosome for degradation.Part ?: Role of TRAF6 on autophagy regulation of NLRP3 inflammasomeOBJECTIVE: To explore the mechanism of interaction between autophagy and NLRP3 inflammasome under PA stimulation and the role of TRAF6 in it.Methods: The co-localization between autophagy and NLRP3 inflammasome was detected by laser scanning confocal microscopy.The relationship of ASC protein and LC3,Beclin-1 and P62 were detected by immunoprecipitation.The levels of ASC ubiquitination were detected by immunoprecipitation and WB with or without PA.The expression of ASC ubiquitination was detected by immunoprecipitation and WB in silencing of TRAF6.Results: The results of immunoprecipitation showed that PA enhanced ASC protein interaction with LC3,Beclin-1 and P62.The results of WB showed that total ubiquitination,K63 ubiquitination and K48 ubiquitination levels of ASC were enhanced after PA stimulation.After lentivirus silencing TRAF6,ASC total ubiquitination,K63 ubiquitination and K48 ubiquitination were found to be lower than those of PA group before silencing decline.CONCLUSION: PA-stimulated autophagy,P62 and NLRP3 inflammasome interact with each other,and K63 and K48 ubiquitination may be involved.TRAF6 may regulate the K63 ubiquitination of ASC to a certain extent to promote P62 to recognize ubiquitinated ASC,and then transport to autophagosome for degradation. |
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