Cardioprotection Mechanism Of Ischemic Postconditioning Mediated By TLR4/NF-κB Pathway Regulated By MiR-499/HSP90

Toll-like receptor 4（TLR4）-mediated cardiac immune inflammatory response is an important pathophysiological feature of myocardial ischemia/reperfusion（I/R）injury.Ischemic postconditioning（IPost C）means that after myocardial ischemia,multiple short-term myocardial reperfusion/stop perfusion treatments before resuming blood perfusion can significantly reduce myocardial I/R injury.The mechanism of IPost C’s cardioprotection is not fully understood.Our previous research showed that IPost C can up-regulate the expression level of heat shock protein 90（HSP90）,and reduce the apoptosis and damage of myocardial cells caused by I/R.How does HSP90 mediate the cardioprotective effect of postconditioning?Recent studies have shown that mi R-499 can regulate HSP90.Therefore,we hypothesized that mi R-499regulates the expression level of HSP90 in hypoxia postconditioning（HPC）and IPost C,thereby regulating the activation of TLR4 in the innate immune system,reducing cardiomyocyte apoptosis,myocardial infarction size and inflammatory factor IL-1β,IL-6 release,improve myocardial hypoxia/reoxygenation（H/R）and I/R injury.In this study,we established the model of H/R of isolated myocardial cells and I/R injury of in vivo rat heart,implemented hypoxia（ischemia）postconditioning,and used mi R-499 overexpression,inhibitory expression of viral vector and HSP90 inhibitor intervention.Subsequently,the expression levels of TLR4,IL-1β,IL-6 in the systemic circulation and local myocardial tissue were detected,and the internal relationship between HSP90 signaling pathway regulated by mi R-499 and immune inflammation in postconditioning was explored,and NF-κB signaling pathways was detected.Explore the signal transduction mechanism of mi R-499 regulating HSP90 in postconditioning from the cellular level and in vivo.This study is divided into two parts.Part I Effect of heat shock protein 90 on TLR4-mediated immune inflammation in hypoxia（ischemia）postconditioningObjective:To explore the mechanism by which HSP90 exerts cardioprotection against hypoxic（ischemic）during the period of hypoxia（ischemia）postconditioning by inhibiting TLR4-mediated immune inflammation response.Methods:1.Cell experiment part:After 3 hours of hypoxia in H₉C₂cardiomyocytes,the following treatments were performed:（1）reoxygenation for6 hours（H/R group）;（2）3 cycles of reoxygenation for 5 minutes/hypoxia for5 minutes before reoxygenation for 6 hours（HPC group）;（3）Apply HSP90specific inhibitor GA before performing（2）operation（HPC+GA group）;myocardial cells in the Control group were cultured under reoxygenation conditions for 9 hours.CCK-8 method was used to detect the survival rate of cardiomyocytes,and Western blot was used to detect the protein expression levels of HSP90,TLR4,NF-κB,IL-1βand IL-6.2.Animal experiment part:After 30 minutes of local myocardial ischemia in rats,the following treatments were performed:（1）reperfusion for 2 hours（I/R group）;（2）3 cycles of ischemia for 30s/reperfusion for 30s before reperfusion for 2 hours（IPost C group）;（3）Apply GA（1 mg/kg）and then perform the operation of（2）（IPost C+GA group）;rats in the Sham group were incubated for 2.5 hours under standard conditions.TUNEL staining was used to detect myocardial cell apoptosis,Evans blue+TTC staining was used to detect myocardial infarction size,fluorescent quantitative PCR and Western blot were used to detect HSP90,TLR4,NF-κB,IL-1βand IL-6 in local myocardial tissue of rats m RNA and protein expression levels,and ELISA method to detect circulating TLR4,NF-κB,IL-1βand IL-6 expression levels.Results:1.Compared with the HPC group,the survival rate of cardiomyocytes in the H/R group and the HPC+GA group were significantly reduced;but there was no statistically significant difference between the H/R group and the HPC+GA group（P>0.05）.Compared with the H/R group,the HPC group increased the protein expression level of HSP90,while simultaneously down-regulated the protein expression levels of TLR4,signaling factor NF-κB,and inflammatory cytokines IL-1βand IL-6;compared with the HPC group,the HPC+GA group down-regulated the protein expression level of HSP90,and at the same time up-regulated the protein expression levels of TLR4,signaling factor NF-κB,and inflammatory cytokines IL-1βand IL-6;there was no statistically significant difference between the H/R group and the HPC+GA group（P>0.05）.2.IPost C significantly reduced the size of myocardial infarction,myocardial cell apoptosis,and the expression levels of TLR4,NF-κB,IL-1β,and IL-6 in circulating blood induced by I/R,and these beneficial effects were by up-regulating the expression of HSP90 and inhibiting the activation of TLR4 and its mediated cascade of inflammation;however,the application of GA to block the function of HSP90,IPost C’s cardioprotective effect was greatly reduced,indicating that IPost C has HSP90-dependent ischemic myocardium anti-inflammatory and anti-apoptotic protection.Conclusions:HSP90 may play a protective role of hypoxic（ischemic）myocardium during the period of hypoxia（ischemia）postconditioning by inhibiting the TLR4/NF-κB signal transduction pathway and the inflammatory response of the myocardial tissues local and systemic circulation.PartⅡ Effects of mi R-499 on HSP90 expression and TLR4-mediated immune inflammation during hypoxia（ischemia）postconditioningObjective: To explore the mechanism by which mi R-499 up-regulates the expression of HSP90 and inhibits TLR4-mediated immune inflammatory response during hypoxia（ischemia）postconditioning to exert the hypoxic（ischemic）cardioprotection.Methods:1.Cell experiment part: construction of mi R-499 overexpression,suppression expression and negative control lentiviral vectors,transfection of H9C2 cardiomyocytes;cardiomyocytes were divided into 4 groups: HPC group,HPC + negative control group（HPC + NC group）,HPC + mi R-499 inhibitory expression group（HPC + inhibitors group）and HPC + mi R-499 overexpression group（HPC + mimics group）.Fluorescence quantitative PCR was used to detect the m RNA expression level of mi R-499 in cardiomyocytes,CCK-8 method was used to detect the survival rate of cardiomyocytes,and Western blot was used to detect the protein expression levels of HSP90,TLR4,NF-κB,IL-1β and IL-6.2.Animal experiment part: construction of mi R-499 overexpression,suppression expression and negative control adeno-associated virus vectors,transfected SD rats;rats were divided into 4 groups（15 in each group）: IPost C group,IPost C + negative control group（IPost C + NC group）,IPost C + mi R-499 inhibitory expression group（IPost C + inhibitors group）and IPost C+mi R-499 overexpression group（IPost C + mimics group）.Fluorescent quantitative PCR was used to detect the m RNA expression level of mi R-499 in myocardial local tissue,TUNEL staining was used to detect myocardial cell apoptosis,Evans blue+ TTC method was used to detect myocardial infarction size,and fluorescent quantitative PCR and Western blot were used to detect HSP90,TLR4,NF-κB,IL-1β and IL-6 m RNA and protein expression levels in rat myocardial local tissue,and ELISA method to detect circulating TLR4,NF-κB,IL-1β and IL-6expression levels.Results: 1.Compared with the HPC group,the expression level of mi R-499 and HSP90 in cardiomyocytes in the HPC + inhibitors group was significantly reduced,the expression levels of TLR4,NF-κB,IL-1β and IL-6 were significantly increased,and the survival rate of cardiomyocytes was reduced;The expression levels of mi R-499 and HSP90 in cardiomyocytes in the HPC +mimics group was significantly increased,the expression levels of TLR4,NF-κB,IL-1β and IL-6 were significantly reduced,and the survival rate of cardiomyocytes was increased;There was no significant difference between the HPC group and the HPC + NC group（P> 0.05）.2.Compared with the IPost C group,the expression levels of mi R-499 and HSP90 in the myocardial local tissue of the IPost C + inhibitors group was significantly reduced,and the m RNA and protein expression levels of TLR4,NF-κB,IL-1β,IL-6,cardiomyocyte apoptosis and myocardial infarction size was significantly increased;the IPost C+ mimics group had the opposite result;the difference between the IPost C group and the IPost C + NC group was not statistically significant（P> 0.05）.These experimental results indicate that IPost C has anti-inflammatory and anti-apoptotic protection of mi R-499-dependent ischemic myocardium.Conclusions: mi R-499 is involved in the protection of hypoxia（ischemia）myocardium mediated by hypoxia（ischemia）postconditioning,which may be through the inhibition of TLR4,signaling factor NF-κB and subsequent activation of inflammatory cytokines in local myocardial tissue and systemic circulation.