Effect Of NLRP3 Inflammasomes Pathway Activation On Left Ventricular Remodeling In Uremia Mice Model

Background:Chronic kidney disease(CKD)is a common and frequent clinical disease with high incidence of cardiovascular complications and can seriously endangers human life.Ventricular remodeling is the major structural pathological change in patients with uremic cardiomyopathy.Histomorphological changes include cardiac hypertrophy and interstitial fibrosis.The mechanism of uremic cardiomyopathy is complex;Indoxyl sulfate(IS)causes micro-inflammation of the internal environment,activates inflammatory pathways,generates a large number of inflammatory factors and causes severe damage to the cardiovascular system.NLRP3 inflammasomes are involved in the CKD pathogenesis but there has been no report on the relationship between NLRP3 inflammasomes activation and uremic cardiomyopathyObjective:To investigate the relationship between NLRP3 inflammasomes pathway activation and left ventricular remodeling in uremic cardiomyopathy mice and its possible mechanismMethods:1?mice were randomly divided into sham and model groups.The model group was subjected to 5/6 nephrectomy to establish uremia mice model.8 weeks following mice model establishment,serum creatinine,blood urea nitrogen,plasma IS levels,cardiac HE staining,myocardial tissue ANP?BNP?NLRP3?Pro-caspase1?IL-1??IL-18 and p-P65 protein expression were measured2?Primary cardiomyocytes were extracted and divided into control group and control+IS(10?mol/dl)group and the expression of ANP and BNP proteins was detected by Western blot.CCK8 method was used to detect the optimal stimulation concentration and time of IS to stimulate mice mononuclear macrophages(RAW264.7).RAW264.7 were divided into control group and IS group and the expression of NLRP3?Pro-caspase1?IL-1??IL-18 and p-P65 proteins was measured.The contents of inflammatory factors IL-1? and IL-18 in the supernatant of both control and IS groups were determined by ELISA method.The macrophage medium supernatant was co-cultured with cardiomyocytes and divided into control group,macrophage medium group and macrophage medium+IS group.Western blot was used to detect the expression of NLRP3?Pro-caspase1?IL-1??IL-18?ANP?BNP proteins in each groupResults:4?Compared with the sham group,the renal function was decreased and serum creatinine,urea nitrogen and IS levels were significantly increased in the model group(P<0.05).Cardiac ultrasound revealed that LVAWd and LVPWd were significantly thickened,ejection fraction(EF)and fractional shortening(FS)were decreased(P<0.05).Left ventricular isovolumic relaxation time and the E/A were significantly increased(P<0.05)and the LVFS/MPI decreased significantly(P<0.05).The expression of ANP,BNP,NLRP3,Pro-caspase1,IL-1? and p-P65 protein was up-regulated in the myocardial tissue(P<0.05)and HE staining results showed myocardial hypertrophy in the model group.Immunofluorescence results showed that the expression of NLRP3?IL-1? in mice heart tissue was significantly increased in the model group.In vitro experiments showed that IS significantly induced up-regulation of ANP and BNP proteins expression in primary cardiomyocytes(P<0.05).The optimal concentration of IS stimulated macrophages measured by CCK8 method was 15?mol/dl and the optimal stimulation time was 3 hours.IS stimulated macrophages,NLRP3?Pro-caspase1?IL-1??IL-18 and p-P65 proteins expression were significantly up-regulated(P<0.05).ELISA revealed that the contents of inflammatory factors IL-1? and IL-18 in the supernatant of macrophage culture in IS group were significantly higher than those in the control group(P<0.05).5?In vitro experiments,the co-culture of IS+macrophage medium supernatant and cardiomyocytes compared with the control and macrophage medium groups showed the expression of ANP?BNP?NLRP3?Pro-caspase1?IL-1??IL-18 protein was significantly up-regulated(P<0.05)Conclusion:Left ventricular hypertrophy in uremia cardiomyopathy mice model is closely related to up-regulation of serum IS levels and the related mechanism may be highly associated with Nf-?B-mediated pathway activation of the NLRP3 inflammasomes.