The Effects Of HSP90 On The Complement-mediated Cardiac Immune Inflammatory Response In Myocardial Ischemic Postconditioning Regulated By MiR-499

Complement activation is an important characteristic of myocardial ischemia/reperfusion injury(IRI).In addition to cause direct damages to myocardial,complements can also induce cardiomyocyte apoptosis and necrosis by activating inflammatory response.Ischemic postconditioning(IPC)is a powerful endogenous protective mechanism for myocardial ischemia/reperfusion injury.Studies have shown that both HSP90 and miR-499 are involved in the cardiovascular protective effects of IPC.The aim of this study is to observe the effects of HSP90 and miR-499 on the complement system in myocardial ischemic postconditioning,and furtherly to reveal the mechanism of ischemic postconditioning.Firstly,a model of cardiomyocytes with hypoxia/ reoxygenation(H/R)injury and rats with IRI were established to test the effects of postconditioning on complement system and immune inflammatory response.Results showed that HPC and IPC significantly alleviated H/R(I/R)-induced expression of C3/C5 a,NF-?B p65,TNF-?,IL-1? and cardiomyocyte apoptosis.To further explore the mechanisms of the inhibition of postconditioning to complement expression and immune-inflammatory response,we tested the effects of geldanamycin,a specific inhibitor of HSP90,on the expression of C3/C5 a,NF-?B,TNF-?,IL-1?.Both cell and animal experiments showed that geldanamycin counteracted the inhibitory effects of postconditioning on C3/C5 a,NF-?B p65,IL-1?,TNF-?expression and cardiomyocyte apoptosis.At last,postconditioning was performed on cardiomyocytes and rats transfected with miR-499 overexpression or miR-499 inhibition vector.Results showed that miR-499 enhanced the inhibition of postconditioning to complement activation,inflammatory factors expression and cardiomyocyte apoptosis by up-regulating HSP90 expression.Part ? Effects of postconditioning on complement-mediated cardiac immune inflammatory response during myocardial ischemic postconditioningObjective To investigate the effects of postconditioning on the complement-mediated cardiac immune inflammatory response during myocardial ischemic postconditioning.Methods 1.The cardiomyocyte models of hypoxia/reoxygenation(H/R)injury were established and the cardiomyocytes were divided into 3 groups including control group(cardiomyocytes were cultured under normal conditions for 9 hours),H/R group(hypoxia 3h and reoxygenation 6h),hypoxic postconditioning(HPC)group(3 cycles of 5min H/R immediately after hypoxia 3h,then reoxygenation for 6h).Cardiomyocyte apoptosis was detected by using Hoechst 33258 staining.The expressions of C3,C5 a,NF-?B p65,TNF-?,IL-1? were detected by using Western blot.2.32 SD rats were randomly divided into 3 groups(n=8 in each group)including sham group,I/R group(LAD was occluded for 30 min and reperfused for 2h),isehemic post-conditioning(IPC)group(3 cycles of 30 s I/R immediately after occlussion 30 min,then reperfusion for 2h).Cardiomyocyte apoptosis was detected with TUNEL staining;Myocardial expressions of C3,C5 a,NF-?B p65,TNF-?,IL-1? were detected by using Western blot.Results 1.Compared with the control group,the expressions of C3,C5 a,NF-?B,TNF-?,IL-1? and cardiomyocyte apoptosis were significantly increased in H/R group.Compared with H/R group,the expressions of C3,C5 a,NF-?B p65,TNF-?,IL-1 and cardiomyocyte apoptosis were significantly reduced in HPC group.2.Compared with sham group,myocardial expressions of C3,C5 a,NF-?B p65,TNF-?,IL-1? and cardiomyocyte apoptosis were significantly increased in I/R group.Compared with I/R group,the expressions of C3,C5 a,NF-?B p65,TNF-?,IL-1?and cardiomyocyte apoptosis were significantly reduced in IPC group.Conclusions Ischemic(hypoxia)postconditioning inhibits complement activation induced by ischemia reperfusion(hypoxia/reoxygenation),alleviates complement-mediated immune inflammatory response and apoptosis.The mechanism may be related to the regulation of postconditioning on NF-?B signaling pathway.Part ? The effects of HSP90 and its mechanism on the complement-mediated cardiac immune inflammatory response during myocardial ischemic postconditioningObjective To investigate the effects of HSP90 and its mechanism on the complement mediated immune inflammatory response during myocardial ischemic postconditioning.Methods 1.The cardiomyocyte models of hypoxia/reoxygenation(H/R)injury were established and the cardiomyocytes were divided into 4 groups including control group(cardiomyocytes were cultured under normal conditions for 9h),H/R group(hypoxia 3h and reoxygenation 6h),hypoxic postconditioning(HPC)group(3 cycles of 5min H/R immediately after hypoxia 3h,then reoxygenation for 6h),HPC+GA group(Geldanamycin 1?M was added 30 min before HPC).Cardiomyocyte apoptosis was examined with Hoechst staining and flow cytometry.The expressions of HSP90,C3,C5 a,NF-?B p65,TNF-?,IL-1? and were detected by Western blot.2.32 SD rats were randomly divided into 4 groups(n=8 in each group)including sham group,I/R group(LAD was occluded for 30 min and reperfused for 2h),isehemic post-conditioning(IPC)group(3 cycles of 30 s I/R immediately after occlussion 30 min,then reperfusion for 2h),IPC+GA group(intraperitoneal injection of geldanamycin 0.6mg/kg 15 minutes before surgery).Cardiomyocyte apoptosis was detected with TUNEL staining;Serum and myocardial expressions of HSP90,C3,C5 a,NF-?B p65,TNF-?,IL-1? were detected by ELISA and Western blot.Results 1.Compared with H/R group,the expression of HSP90 was significantly increased while the expressions of C3/C5 a,NF-?B p65,TNF-?,IL-1? and cardiomyocyte apoptosis were significantly decreased in HPC group.Compared with HPC group,the expressions of C3,C5 a,NF-?B p65,TNF-?,IL-1? and cardiomyocyte apoptosis were significantly increased in HPC+GA group.2.Compared with I/R group,serum and myocardial expressions of HSP90 were significantly increased while the expressions of C3,C5 a,NF-?B p65,TNF-?,IL-1? and cardiomyocyte apoptosis were significantly reduced in IPC group.Compared with IPC group,the expressions of C3,C5 a,NF-?B p65,TNF-?,IL-1? and cardiomyocyte apoptosis were significantly increased in HPC+GA group.Conclusions HPC and IPC attenuate complement-mediated cardiac immune inflammatory response and cardiomyocyte apoptosis induced by H/R or I/R through up-regulating HSP90 expression.Part ? The effects of miR-499 on HSP90 and complement mediated immune inflammatory response during myocardial ischemic postconditioningObjective To investigate the effects of miR-499 on HSP90 and complement mediated immune inflammatory response during myocardial ischemia postconditioning.Methods 1.Mi R-499 overexpression,inhibition and negative control lentiviral vector were used to transfect H9C2 cardiomyocytes.The cells were divided into 4 groups including hypoxic postconditioning(HPC)group,miR-499 overexpression+hypoxic postconditioning(miR-499++HPC)group,miR-499 inhibition+hypoxic postconditioning(miR-499-+HPC)group and negative control+hypoxic postconditioning(NC+HPC)group.Cardiomyocyte apoptosis were detected with Hoechst 33258 staining and flow cytometry.The expressions of HSP90,C3,C5 a,NF-?B p65,TNF-?,IL-1 ? were detected by Western blot.2.Mi R-499 overexpression,inhibition and negative control adenovirus associated virus vector were used to transfect rats.Rats were divided into 4 groups(n=8 in each group)including ischemic postconditioning(IPC)group,miR-499 overexpression+ischemic postconditioning(miR-499++HPC)group,miR-499 inhibition+ischemic postconditioning(miR-499-+HPC)group and negative control+ischemic postconditioning(NC+HPC)group.Cardiomyocyte apoptosis was detected with TUNEL staining.Serum and myocardial expressions of HSP90,C3,C5 a,NF-?B p65,TNF-?,IL-1? were detected by ELISA and Western blot.Results 1.Compared with HPC group and NC + HPC group,the expression of HSP90 was significantly increased while cardiomyocyte apoptosis rate and expressions of C3,C5 a,TNF-?,IL-1?,NF-?B p65 were significantly decreased in miR-499++ HPC group.Conversely,the expressions of HSP90 were significantly decreased,apoptosis rate and expressions of C3,C5 a,NF-?B,TNF-?,IL-1 were significantly increased in miR-499-+HPC group.2.Compared with IPC group and NC+IPC group,the expression of HSP90 was significantly increased while cardiomyocyte apoptosis and expressions of C3,C5 a,NF-?B p65,TNF-?,IL-1? were significantly decreased in miR-499++IPC group.On the contrary,the expression of HSP90 was significantly decreased while cardiomyocyte apoptosis and expressions of C3,C5 a,TNF-?,IL-1? and NF-?B p65 were significantly increased in miR-499-+IPC group.Conclusions During the ischemic(hypoxia)postconditioning,miR-499 attenuates complement mediated immune inflammatory response by upregulating HSP90 expression.