The Effects Of NLRP3 Inflammasome On Damage Indeuced By Advanced Glycation End Products In Pancreatic β Cells

Objective: Advanced glycation end products(AGEs), the important derivative of glucose, could promote the occurrence and development of metabolic inflammation, but the mechanism is not clear. This study was designed to clarify the role of the NLRP3 inflammasome in the phenomenon of AGEs-induced pancreatic β cell damage.Method: In the first part of the experiment:(1) C57BL/6J(wild type, WT) and NLRP3 knock-out(NLRP3 KO) mice were injected intraperitoneally with glyceraldehyde derived AGEs(Gly-AGEs) or BSA(as a control) daily for 6 weeks. Intraperitoneal glucose tolerance test(GTT), insulin releasing test(IRT) and insulin tolerance test(ITT) were performed. Pancreatic tissue pathological changes were observed by HE staining. Islet cells apoptosis were detected using the TUNEL staining. Islet cells ultrastructure were observed by electron microscope. Immunofluorescent staining of islets insuin, glucagon, IL-1β, NLRP3 and F4/80 were performed. NLRP3 inflammasome and MCP-1 protein expression were assayed by western blotting. Activity of caspase 1 was measured using the colorimetric method. Level of MCP-1 was measured by ELISA.(2) C57BL/6J mice were injected intraperitoneally with Gly-AGEs for 6 weeks, clodronate liposome(COLD-lip) were used to deletion the peritoneal macrophage. Subsequently, the index and method were described previously.(3) MIN6 cells, mouse peritoneal macrophages and RAW264.7 cells were treated with Gly-AGEs and different interventions(RAGE and NLRP3 knockout and antioxidant NAC). Cell viability, ROS production, apoptosis and glucose stimulated insulin release were determined. Protein or gene expression of RAGE, NLRP3 inflammasome, ASC, caspase 1 and MCP-1 were measued by western blotting or real-time RT-PCR. Secretion of IL-1β was assayed by ELISA. In the second part of the experiment:(1) C57BL/6J mice were injected intraperitoneally with BSA, Gly-AGEs, AGEs and glucose derived AGEs(Glu-AGEs) for 6 weeks. The index and method were described previously.(2) After incubation of Gly-AGEs and Glu-AGEs, the changes of NLRP3 inflammasome and MCP-1 were detected. In the third part of experiment: the establishment of plasma in methyl ethyl two aldehyde(MG) method of HPLC-MS/MS concentration, and the determination of plasma MG levels in patients with newly diagnosed and evaluated T2 DM, MG and other parameters, such as the relationship between oxidative stress and metabolic indices. In the fourth part of the experiment:(1) WT and NLRP3 KO mice were injected intraperitoneally with methylglyoxal derived AGEs(MG-AGEs) or BSA daily for 6 weeks. The index and method were described previously.(2) After incubation of MG-AGEs and treatment with different interventions, the changes of RAGE expression, ROS level and NLRP3 inflammasome were detected.Result:(1) Impaired glucose tolerance and glucose-stimulated insulin secretion, increased pancreatic β cell apoptosis as well as damage of mitochondrial structure were present in Gly-AGEs-treated WT mice. These damages were ameliorated in NLRP3 KO mice.(2) Gly-AGEs actived NLRP3 inflammasome in WT mice pancreas, increased islet macrophage infiltration and enhance insulitis score. The caspase 1 activity, IL-1β secretion and macrophage infiltration were reduced in NLRP3 KO mice pancreas.(3) AGEs-evoked β-cell dysfunction and apoptosis in mice were improved by removal of macrophages through COLD-lip injection.(4) After incubation with Gly–AGEs, viability of MIN6 cells was decreased, ROS production and cell apoptosis was enhanced, and glucose-stimulated insulin release was impared. NLRP3 gene silencing could not improve these abnormal changes.(5) Gly-AGEs activated the NLRP3 inflammasome via the RAGE-ROS pathway in macrophage.(6) The MCP-1 protein expression and level was obviously reduced in pancreatic tissures of WT+AGEs mice, while NLRP3 KO partially reduced it.(7) Incubation of Gly-AGEs and IL-1β significantly upregulated MCP-1 m RNA expression in MIN6 cells. Treatment with IL-1ra decreased m RNA expression of MCP-1.(8) Gly-AGEs further activated the NLRP3 inflammasome and induced macrophage infiltration in mice islets campared with Glu-AGEs. The mice islet β-cell damage induced by Gly-AGEs injection was more obvious than Glu-AGEs.(9) Plasma MG level in patients with newly diagnosed T2 DM was significantly higher than that in control individuals. Furthermore, the increased plasma MG level is associated with the elevation of Hb A1 c and MDA.(10) MG-AGEs also activated the NLRP3 inflammasome in macrophages. NLRP3 KO could improve the MG-AGEs induced β cell dysfunction and apoptosis.Conclusion: AGEs activate NLRP3 inflammasome in macrophage through RAGE-ROS pathway, and promote the secretion of IL-1β. NLRP3 KO ameliorates the β cell structural and functional damage induced by AGEs. The in vivo toxicity of Gly-AGEs is stronger than that of Gly – AGEs. It is because that Gly-AGEs have the more activation of NLRP3 inflammasome and induction of macrophage infiltration in islets. MG, the precursor of AGEs, is enhanced in patients with newly diagnosed T2 DM.