| BackgroundCardiovascular complications are the leading cause of death for patients with type 2diabetic mellitus,a disease affecting>94 million people in China.Inflammation in endothelial cells plays a critical role in the pathogenesis of vascular disease in obesity-related type 2 diabetes.It is therefore crucial to clarify the mechanism of inflammatory vascular injury in diabetic mellitus to search for novel therapeutic strategies.Adiponectin is an abundant adipocyte-derived plasma protein involved in myocardial protection and insulin sensitivity.Numerous epidemiological studies have shown that reduced adiponectin levels correlate with increased risk of cardiovascular disease in obesity and diabetes.Moreover,several clinical observations and basic studies have demonstratedthathypoadiponectinemiaisassociatedwithimpaired endothelium-dependent vasodilation.The anti-inflammatory and vascular protective effects of adiponectin have been well documented.Adiponectin decreases TNF-α-induced ICAM-1 expression and NF-κB activation in endothelial cells.However,the exact molecular mechanism by which adiponectin exerts its clinical effects has yet to be determined.Inflammasomes are large multiprotein complexes that consist of caspase-1,apoptosis-associated speck-like protein（ASC）,and NLRP（nucleotide-binding oligomerization domain-like receptor with a pyrin domain）.The ASC protein bridges the interaction between NLRP and caspase-1,making it essential for inflammasome activation and subsequent interleukin-1β（IL-1β）and IL-18 secretion.To date,four additional inflammasome proteins have been identified:NLRP1,NLRP3,NLRC4,and AIM2.The NLRP3 inflammasome is the most fully characterized and has been associated with a wide range of diseases,including infections,auto-inflammatory and autoimmune diseases,and metabolic disorders.The NLRP3 inflammasome,activated by endotoxins,K+channel openers,uric acid and reactive oxygen species（ROS）,plays a crucial role in insulin resistance and the pathogenesis of diabetes.Our laboratory has previously demonstrated that the NLRP3 inflammasome is excited in CMECs,but not in cardiomyocytes,playing a critical role in the pathophysiology of MI/R injury.However,the question of whether the NLRP3 inflammasome also plays an important role in diabetic vascular injury has not yet been addressed.It also remains unclear whether the NLRP3inflammasome is involved in adiponectin’s anti-inflammatory and vascular protective effects.Our previous data has indicated that adiponectin can reduce ROS production–an important activator of the NLRP3 inflammasome.Therefore,it is possible that the activation of the NLRP3 inflammasome may be linked with loss of adiponectin in diabetes mellitus.Aims1.To identify the role of NLRP3 inflammasome activation in diabetic vascular injury;2.To determine the role of NLRP3 inflammasome activation in adiponectin mediated anti-inflammatory and vascular protective signaling;3.To investigate the mechanism of adiponectin inhibition of NLRP3 inflammasome activation.MethodsPartⅠ:Role of NLRP3 inflammasome in diabetic vascular endothelial injury1.Western blot was used to determine NLRP3 inflammasome and caspase-1,IL-1βand IL-18 were tested;2.HUVECs were cultured with high glucose and high fat,the activation of NLRP3inflammasome was determined by Western blot and Elisa kit;3.Expression of NLRP3 inflammasome components were genetically inhibited by siRNA and inflammatory responses were determined by an Elisa kit.PartⅡ:Adiponectin decreases diabetic vascular endothelial injury by inhibiting activation of the NLRP3 inflammasome and the molecular mechanism1.Apoptotic cell death was determined by caspase-3 activity and inflammatory responses were determined by an Elisa kit;2.Activation of the NLRP3 inflammasome was determined by Western blot and an Elisa kit;3.HUVECs were cultured with high glucose and high fat,the activation of NLRP3inflammasome was determined by Western blot and Elisa kit;4.Feeding the mice with high-fat diet to establish diabetes mice by 8 weeks and intraperitoneally injected with 1400W（2 mg/kg,a selective iNOS inhibitor）and EUK134（5 mg/kg,a peroxynitrite scavenger）once a day for a total of 4 weeks;5.A commercial nitrotyrosine Elisa kit was used to assess nitrotyrosine in vascular tissues;6.Activation of the NLRP3 inflammasome was determined by Western blot and an Elisa kit.Results1.Abnormal glucose tolerance compared with control mice fed with chow diet.Adiponectin knockout mice exhibited a more severe abnormal glucose tolerance;2.Activation of the NLRP3 inflammasome was increased in diabetic vessels as determined by an increasing of NLRP3 expression,caspase-1 activity,IL-1βcontent and IL-18 content;3.MCC950,the NLRP3 specific inhibitor,can significantly reduce vascular injury in diabetic mice as evidenced by the improvement endothelium dependent vascular relaxation of and reduced apoptosis;4.Activation of NLRP3 was increased in HG/HG incubated endothelial cells and siRNA targeting NLRP3 significantly attenuated endothelial cell dysfunction,inflammatory responses and apoptosis;5.Adiponectin knockout mice show significantly increased NLRP3 inflammasome activation and vascular injury compared with WT mice in a diabetic mellitus model as evidenced by a more severe abnormal endothelium dependent vascular relaxation and inflammatory responses.MCC950 can significantly improve endothelium dependent vascular relaxation to comparable levels in APN-/-mice and WT diabetic mice.6.Activation of NLRP3 was increased in HG/HG incubated endothelial cells and adiponectin decreases NLRP3 inflammasome activation and attenuates endothelial cell injury as evidenced by improvement of endothelial function,reduced apoptosis and inflammatory response;7.NLRP3 inflammasome overexpression plasmid increased NLRP3 inflammasome activation upon HG/HF induction and adiponectin decreased HG/HF induced endothelial cell dysfunction and inflammatory response.Adiponectin’s effects on endothelial cell dysfunction and inflammatory response were abolished by the addition of the NLRP3transgene;8.Nitrotyrosine content and NLRP3 inflammasome activation were markedly higher in diabetic vascular tissue from APN-/-mice than tissue from WT mice,treatment with1400W or EUK134 reduced diabetic vascular nitrotyrosine formation and NLRP3inflammasome activation to comparable levels in APN-/-mice and WT mice.Conclusion1.NLRP3 inflammasome activation is partially responsible for diabetic vascular endothelial injury;2.Adiponectin decreases diabetic vascular endothelial injury by inhibiting the activation of NLRP3 inflammasome;3.Adiponectin inhibits activation of the NLRP3 inflammasome via anti-oxidative/nitrative stress in a model of diabetic mellitus. |
