The Role Of The Necroptosis Kinase RIP1K And RIP3K In Type 2 Diabetic Myocardial Fibrosis And Its Mechanism

Objectives:The role of necroptosis in the occurrence and development of visceral fibrosis has gradually attracted the attention of researchers,but its mechanism of necroptosis in diabetic myocardial fibrosis are still lacking.The objectives of this study includes:1.To observe the effects of gene knockdown of key upstream kinase receptor-related protein kinase 1 and 3(RIP1K,RIP3K)and their inhibitors on the injury and fibrosis of myocardial fibroblasts cultured in high glucose and high fat medium,and to clarify that the mechanism is related to restore the autophagic flux of myocardial fibroblasts.2.To study the interaction of RIP1K inhibitor Nec-1 and RIP3K inhibitor GSK on RIPlK-RIP3K necrosome,and to explore the feasibility of combination of low dose of Nec-1 and GSK to reduce the injury and fibrosis of myocardial fibroblasts induced by high glucose and high fat.3.To investigate the effect of Nec-1 on myocardial fibrosis in type 2 diabetes mellitus rats and its relationship with autophagic flux.Methods:In experiment,male SD rats were fed with high glucose and high fat and were treated with intraperitoneal injection of streptozotocin to establish the model of T2DM rats.In vitro experiment,primary neonatal rat cardiac fibroblasts were cultured in DMEM medium containing 25 mM glucose and 500 mM palmitate for 48 hours to establish the high glucose and high fat myocardial fibroblasts injury model.The expression of RIP1K and RIP3K vivo and in vitro was inhibited by pharmacological methods(Nec-1,GSK)or gene knockdown(RIP1K knockdown,RIP3K knockdown).ELISA was used to detect collagen I,collagen ?,LDH leakage rate and ATP content of cardiac fibroblasts.CCK-8 was used to detect cell proliferation,and flow cytometry was used to detect cell apoptosis and necrosis.RIP1K knockdown,RIP3K knockdown or Nec-1,GSK was used to observe the effects of the injury and fibrosis of myocardial fibroblasts induced by high glucose and high fat.Western blotting was used to detect the expression of LC3-II,P62,and active-Cathepsin D.mRFP-GFP-LC3 plasmid transfection and AO fluorescence staining were used to detect the changes of autophagosomes and lysosomes in the process of autophagic flux of cardiac fibroblasts.To examine the relationship between RIP1K-RIP3K necrosome and the restore of autophagic flux of cardiac fibroblasts,the autophagic flux blocker chloroquine(CQ,10 ?M)was used to inhibit the fusion process of autophagosomes and lysosomes.At the same time,the effects of Nec-1 combined with GSK on high glucose and high fat myocardial fibroblast injury and fibrosis were detected with Western blotting,flow cytometry and ELISA.In addition,the degree of myocardial fibrosis was measured with Masson's staining;cardiac function was measured with echocardiography;and the expression and distribution of RIP1K,RIP3K,collagen ?,collagen ?,a-SMA,LC3-?,P62 and active-Cathepsin D were measured with immunohistochemistry,Western blotting or ELISA assays.The effect of Nec-1 on myocardial fibrosis and its relationship with autophagic flux in T2DM rats were investigated.Results:1.The T2DM rats heart model and the high glucose and high fat myocardial fibroblast model were successfully established.In vivo experiment:the fasting blood glucose of diabetic rats(DM group)was significantly higher than that of control group(CON group).The body weight of rats in CON group and DM group increased gradually with time.From the 16th week of age,the body weight of rats in DM group was significant lower than that of rats in CON group.Compared with CON group,DM group showed the decrease in left ventricular end-diastolic volume,left ventricular end-systolic volume and heart weight;and the increase of negative insulin sensitivity index,heart/body mass ratio,collagen fiber area ratio between myocardium,collagen ?,collagen ? content;prolonged isovolumic diastolic time.There is no significant difference of insulin level between CON group and DM group.In vitro experiment:Compared with normal glucose control group(NG group,5 mM glucose+20 mM mannitol DMEM),the content of collagen ?,collagen ? and the expression of ?-SMA in myocardial fibroblasts were increased in high glucose and high fat group(HG group,25 mM glucose +500?M palmitate DMEM).The increased OD value indicated that the proliferation of myocardial fibroblasts was significant.2.In diabetic rats and high glucose and high fat myocardial fibroblasts,there is a higher levels of TNF-? and the expressions of RIP1K,RIP3K,p-RIP1K and p-RIP3K,the key proteins of necroptotic kinase were increased.3.RIP1K and RIP3K knockdown inhibited the expression of RIP1K and RIP3K in cardiac fibroblasts,increased ATP content,decreased LDH leakage,apoptosis and necrosis,collagen ?,collagen ? content,expression of a-SMA and cell proliferation in high glucose and high fat cultured myocardial fibroblasts,respectively.4.In high glucose and high fat cultured myocardial fibroblasts,RIP IK and RIP3K knockdown decreased the expression of LC3-?,P62 and active-Cathepsin D;mRFP-GFP-LC3 plasmid transfection and AO staining showed that RIP IK and RIP3K knockdown improved the autophagic flux process and enhanced the stability of lysosome membrane.5.RIP1K inhibitor Nec-1(100 ?M)could inhibit the expression of RIP1K and p-RIP1K,and RIP3K inhibitor GSK(10 ?M)inhibit the expression of RIP3K and p-RIP3K,reduced LDH leakage,increased ATP content,decreased collagen ?,collagen ?content and a-SMA expression in high glucose and high fat cultured myocardial fibroblasts,and also reduced the expression of autophagy and lysosome marker protein,improved the process of autophagic flux and increased the stability of lysosome membrane,respectively.Autophagic flux inhibitor CQ eliminated the above effects of Nec-1 and GSK.6.Interesting,knockdown of RIP1K or Nec-1 could inhibit the level of RIP3K and p-RIP3K,while knockdown of RIP3K or GSK could inhibit the level of RIP1K and p-RIP1K.Furthermore,low-dose of Nec-1(10 ?M)combined with low dose of GSK(1 ?M)significantly inhibited the expression of RIP IK and RIP3K,reduced LDH leakage,the expression of autophagy and lysosome marker proteins LC3-?,P62,active-Cathepsin D;increased ATP content,decreased collagen ?,collagen ? content and a-SMA expression,improved autophagic flux process and increased the stability of lysosome membrane.7.Comparison of 24 weeks old rats in Nec-1 treatment group and DM group,rats in Nec-1 treatment group showed that left ventricular end-diastolic volume and left ventricular end-systolic volume were increased;isovolumic diastolic time was shortened;negative insulin sensitivity index,heart/body mass ratio,collagen fiber area ratio,collagen I,collagen ? content and a-SMA expression were decreased,Simultaneously,autophagy and lysosome marker protein LC3-?,P62,active-Cathepsin D were decreased.Conclusion:Knockdown of RIP1K or RIP3K,or pharmacological inhibition of their phosphorylation can inhibit RIP1K and RIP3K activation and the interaction RIP1K and RIP3K,resulting in reducing the injury and fibrosis of high glucose and high fat cultured myocardial fibroblasts.The underlying mechanism may be related to the restoration of autophagic flux of myocardial fibroblasts by inhibition of RIP IK or RIP3K.Combination of low dose of Nec-1 and GSK can reduce the injury and fibrosis of high glucose and high fat myocardial fibroblasts,improve the autophagic flux of cardiac fibroblasts,and increase the stability of lysosomal membrane.Nec-1 intraperitoneal injection improves cardiac function and reduces myocardial fibrosis and autophagic lysosomal protein expression in T2DM rat hearts.