The Study On The Role Of Syk Induced JNK And NLRP3 Activation In Diabetic Cardiomyopathy

Objective Diabetes mellitus(DM)is a metabolic disorder characterized by elevated blood glucose and lipid levels,Diabetic cardiomyopathy(DCM),as a common complication of DM,is the leading cause of heart failure and death in patients with DM.Decreased myocardial diastolic function was occured in early stage of DCM,and decreased myocardial systolic function was occured in late stage of DCM.The pathogenesis of DCM have not been fully elucidated.Spleen tyrosine kinase(Syk)or c-Jun N-terminal kinase(JNK)can regulate the immune response,studies have proved that inhibition of either Syk or JNK decreased the activity of NLRP3 in macrophages.Furthermore,expressions of p-JNK and NLRP3 were significantly increased in H9c2 cardiomyocytes treated with high glucose,and expression of p-Syk was significantly increased in HK-2 cells treated with high glucose,these results suggested that p-Syk,p-JNK and NLRP3 were associated with pathogenic mechanism of DM.However,the role of Syk in DCM inflammation and relationship among Syk,JNK and NLRP3 have not been reported.Therefore,we determined the expressions of Syk,JNK and NLRP3 in heart tissues of type 1 DCM rats and normal control rats in vivo,we also investigated the relationship among Syk,JNK and NLRP3 inflammasome activation in high glucose-induced neonatal rat cardiomyocytes and H9c2 cardiomyocytes in vitro.We expect to provide new evidences for the treatment of DCM.Methods1.We established the animal model of DM.SD rats were randomly divided into Ctrl group and DCM group.The DCM rats were injected intraperitoneally with65 mg/kg STZ solution,while the Ctrl rats were injected the same amount of citric acid solution(PH 4.2)in the same way.The blood glucose and body weight of the rats were measured and recorded after 3 days,if the blood glucose > 16.7mmol/L was considered as diabetic model.Echocardiography and hemodynamics were used to detect cardiac function changes of Ctrl and DCM rats.Then,the heart tissues were performed by HE and Masson staining,and the changes of heart tissues structure were observed under light microscope.2.Western Blot was used to detect the protein levels of p-Syk,p-JNK and NLRP3 in heart tissues of DCM and Ctrl rats at 16 week.3.H9c2 cardiomyocytes were randomly divided into Ctrl group(normal glucose of5.5 mmol/L treatment),HG group(high glucose of 25 mmol/L treatment),inhibitor control group(Syk inhibitor BAY61-3606,1 ?mol/L 2 h,or JNK inhibitor SP,20 ?mol/L 1 h,and then normal glucose of 5.5 mmol/L treatment),inhibitor treatment group(Syk inhibitor BAY61-3606,1 ?mol/L 2 h,or JNK inhibitor SP,20 ?mol/L 1 h,and then high glucose of 25 mmol/L treatment).RT-PCR was used to detect the m RNA levels of NLRP3,Caspase-1 and IL-1?after treatment with inhibitor.Western Blot was used to detect the protein level of NLRP3 after treatment with inhibitor.4.Newborn neonatal rat cardiomyocytes were randomly divided into Ctrl group(5.5 mmol/L glucose treatment),HG group(25 mmol/L glucose treatment),inhibitor control group(Syk inhibitor BAY61-3606,1 ?mol/L 2 h,or JNK inhibitor SP,20 ?mol/L 1 h,and then 5.5 mmol/L glucose treatment),inhibitor treatment group(Syk inhibitor BAY61-3606,1 ?mol/L 2 h,or JNK inhibitor SP,20 ?mol/L 1 h,and then 25 mmol/L glucose treatment).RT-PCR was used to detect the m RNA levels of NLRP3,Caspase-1 and IL-1? after treatment with inhibitor.Western Blot was used to detect the protein level of NLRP3 after treatment with inhibitor.5.Small interfering RNA was used to knock down Syk gene of H9c2 cardiomyocytes,and Syk silencing efficiency was determined by RT-PCR and Real-Time PCR.Western Blot was used to detect the protein level of NLRP3 after Syk-si RNA transfection.Real-Time PCR was used to detect the m RNA levels of NLRP3,Caspase-1 and IL-1? after treatment with Syk-si RNA.6.Western Blot was used to detect the protein level of p-JNK in H9c2 cardiomyocytes treated with Syk-si RNA or Syk inhibitor BAY61-3606.7.Western Blot was used to detect the protein level of p-JNK in neonatal rat cardiomyocytes treated with Syk inhibitor BAY61-3606.Results1.After a single intraperitoneal injection of STZ,DCM group rats showed polydipsia,polyphagia,polyuria typical symptoms,and lethargy,hair dry.While Ctrl group rats showed good spirit,shiny hair and quick movement.The random blood glucose level in DCM group was higher than that in Ctrl group(P < 0.01),the body weight was significantly decreased in DCM group(P < 0.01).The left ventricular function of two groups was obviously different at 12 week after STZ injection.The ratio of E/A,FS and EF in DCM group were markedly decreased(P < 0.05)compared to Ctrl group rats.HE staining showed that the size of myocardial cells in DCM group ratswas larger than that in Ctrl group,myocardial fibers were arranged irregularly and the inflammatory cells were infiltrated significantly.Masson staining showed that the collagen was accumulated in myocardial interstitial and perivascular of DCM group rats when compared with Ctrl group.These results indicate that we ssuccessfully established the animal model of type 1 DCM SD rats.2.Western Blot showed that the protein levels of cardiac p-Syk,p-JNK and NLRP3 in DCM rats were higher than Ctrl.3.RT-PCR showed that BAY61-3606 or SP inhibitor treatment significantly inhibited the high glucose-stimulated NLRP3,Caspase-1 and IL-1? m RNA expressions in H9c2 cardiomyocytes(P < 0.05).Western Blot showed that BAY61-3606 or SP inhibitor treatment significantly inhibited NLRP3 protein expression,the difference was statistically significantly(P < 0.05).4.RT-PCR showed that BAY61-3606 or SP inhibitor treatment significantly inhibited the high glucose-induced NLRP3,Caspase-1 and IL-1? m RNA expressions in neonatal rat cardiomyocytes(P < 0.05).Western Blot showed that BAY61-3606 or SP inhibitor treatment significantly inhibited NLRP3 protein expression,the difference was statistically significantly(P < 0.05).5.RT-PCR and Real-Time PCR showed that Syk-si RNA significantly silenced Syk protein expression(P < 0.05).Western Blot showed that NLRP3 protein expression was significantly suppressed after Syk-si RNA treatment,the differences was statistically significantly(P < 0.05).Real-Time PCR also showed that Syk-si RNA treatment significantly inhibited the NLRP3,Caspase-1 and IL-1? m RNA expressions,the difference was statistically significantly(P<0.05).6.Western Blot showed that Syk-si RNA pretreatment significantly abolished high glucose-induced p-JNK protein expression compared to NC-si RNA group(P<0.05).7.Western Blot showed that BAY61-3606 significantly inhibited p-JNK protein expression in H9c2 cardiomyocytes,and similar results were obtained in neonatal rat cardiomyocytes,the difference was statistically significantly(P<0.05).Conclusions In summary,we ssuccessfully established the animal model of SD rats type 1 diabetic cardiomyopathy,and the protein expressions of cardiac p-Syk,p-JNK and NLRP3 were elevated significantly in diabetic rats.p-Syk,p-JNK,NLRP3 and IL-1? were closely related to the pathogenesis of DCM.Syk induced JNK and NLRP3 activation was involved in the pathogenesis of DCM.