| At present,cardiovascular disease has become the main cause of death and disability worldwide.Myocardial ischemia/reperfusion(I/R)injury is the most common and dangerous.After ischemic reperfusion,damaged cardiomyocytes and endothelial cells can release a series of inflammatory cytokines such as IL-1?,IL-6 and TNF-?,and then cause local inflammatory reactions,leading to myocardial cell apoptosis and heart failure,Myofibrosis,increased myocardial infarct size,and impaired heart function.The study found that up-regulating the expression of hypoxia inducible factor-1?(HIF-1?)in rat myocardial I/R can reduce myocardial I/R injury.HIF-1? is an oxygen-sensitive transcription factor that is highly susceptible to degradation under normoxic conditions.Therefore,researchers used the proline hydroxylase(PHD)inhibitor dimethyloxalyl glycine(DMOG)to stabilize the expression of HIF-1? and study the protective effect of HIF-1? on the heart.It has been reported in the literature that HIF-1? can reduce the inflammatory damage in mammalian cells and Drosophila,and its effect is related to the inhibition of NF-?B and the expression of inflammatory factors.However,whether HIF-1? has a similar effect in rat myocardial I/R is still unclear.Therefore,the first part of the study was designed to investigate the effect of HIF-1? on myocardial inflammatory lesions induced by I/R in rats using DMOG to stabilize the expression of HIF-1?.Studies have reported that Toll-like receptor 4(TLR4)is involved in the regulation of myocardial I/R inflammatory lesions.During ischemia,a series of damage-associated proteins(DAMPs)released from damaged myocardial tissue activate TLR4.Subsequently,by responding to a series of signaling pathways,NF-?B expression is activated and released,resulting in increased release of pro-inflammatory cytokines IL-1,IL-6,and TNF-? and increased myocardial injury.If the TLR4/NF-?B signaling pathway is inhibited,cardiomyocyte loss and inflammatory damage caused by I/R are reduced.Our previous study found that HIF-1? can reduce myocardial mitochondrial damage in I/R rats,thereby protecting the structure and function of the heart.Whether the myocardial protection of HIF-1? is activated by activating TLR4/NF-?B signaling pathway has not been reported.Therefore,the second part of this study intends to clarify the relationship between HIF-1? and TLR4/NF-?B signaling pathway after clarifying the anti-inflammatory effect of HIF-1? in the first part,providing a new idea for elucidating the mechanism of HIF-1? cardioprotection.Objective:1.Observe the effect of HIF-1? on inflammatory injury induced by myocardial I/R in rats.2.To explore the possible mechanism of HIF-1? exerting anti-inflammatory effects.Methods:68 male SD rats(eight weeks old)with an average body weight of 230-250 g were selected and grouped into 4 groups at random:(1)Sham group;(2)Myocardial ischemia-reperfusion group(I/R));(3)HIF-1? stabilizer DMOG+ myocardial I/R group(DMOG+I/R);(4)HIF-1? inhibitor YC-1+ myocardial I/R group(YC-1+I/R).According to the classic left anterior descending coronary artery ligation model,myocardial ischemia was performed for 45 min,followed by reperfusion for 3 h,and anterior left ventricular tissue was obtained.HE staining was used to observe the inflammatory infiltration of myocardial tissue.Myocardial MPO activity was detected by kit.Western blot was used to detect the expression of HIF-1?,TLR4,NF-?B and I?B-? in myocardial tissue.The mRNA levels of IL-1?,IL-6,TNF-?,TLR4,NF-?B and I?B-? were detected by Real-time PCR..Results:1.DMOG pretreatment can increase HIF-1? protein level in rat myocardial tissue.Sham group had a small amount of HIF-1? protein expression,while I/R group HIF-1? protein content increased significantly,reaching 1.75 times(P<0.05)in Sham group;and after DMOG pretreatment,HIF-1? protein level It was further increased to2.82 fold(P<0.01)in the control group and also significantly higher than that in the I/R group(1.61-fold)(P<0.05).The use of HIF-1? inhibitor YC-1 showed that the protein level of HIF-1? in YC-1+I/R group was significantly lower than that in I/R group(P<0.05).It is suggested that DMOG can stabilize the expression of HIF-1?..2.HIF-1? can reduce the infiltration of inflammatory cells.The results of HE staining showed that the myocardial fibers of the Sham group were regular and no inflammatory cell infiltration was observed.Compared with the Sham group,a large number of inflammatory cells infiltrated in the model group,accompanied by significant cell degeneration and necrosis.Compared with the I/R model group,after the DMOG intervention,a small amount of inflammatory cell infiltration was observed,the myocardial arrangement was relatively neat,and the pathological changes were slight.After inhibiting HIF-1?,the swelling and necrosis of cardiomyocytes and the infiltration of inflammatory cells were more obvious.The results showed that HIF-1? could reduce the infiltration of inflammatory cells in the myocardium of I/R rats.3.HIF-1? can reduce the activity of MPO in myocardial tissue of rat I/R.Compared with Sham group,MPO activity in I/R group was significantly increased(1.33 ± 0.29 vs0.25 ± 0.80,P <0.01);while pre-administration of DMOG could significantly reduce the activity of MPO(0.730.14 vs 1.330.29,P < 0.05 vs I/R group);after HIF-1? inhibition,MPO activity increased again(2.200.43 vs 1.330.29,P<0.01 vs I/R group).It is suggested that HIF-1? can reduce the activity of MPO in myocardium.4.Effect of HIF-1? on expression of IL-1?,IL-6 and TNF-? induced by I/R in rat.Compared with the control group,the mRNA expression of IL-1?,IL-6 and TNF-?increased after myocardial ischemia and reperfusion in rats(P<0.01).The mRNAexpression of the three inflammatory factors decreased after administration of DMOG in advance(P<0.05).<0.01 vs I/R group);at the same time,it was found that after inhibiting the expression of HIF-1?,the mRNA level of inflammatory factors increased(P<0.01 vs.I/R group).It is suggested that HIF-1? can reduce the expression of inflammatory factors induced by I/R in rat myocardium.5.Effects of HIF-1? on expression of TLR4/NF-?B signaling pathway-associated proteins in myocardial I/R in rats.Myocardial TLR4 and NF-?B protein levels were significantly increased in the I/R group,which were approximately 1.90 fold and 2.23 fold higher than those in the Sham group(P<0.05).After administration of DMOG,the protein levels of TLR4 and NF-?B were reduced by 0.76 fold and 0.78 fold,respectively,compared with the I/R group(P<0.01).Real-time PCR results also showed that compared with I/R group,mRNA levels of TLR4 and NF-?B in DMOG+I/R group were significantly decreased(P<0.05).I?B-? is an inhibitor of NF-?B.Western blot results showed that I?B-? protein levels in the Sham group remained at a high level,while I?B-? protein levels in the I/R group decreased significantly(P<0.05).After administration of DMOG,the protein level of I?B-? increased again(P<0.01)compared with the I/R group.Real-time PCR results also showed that I?B-? mRNA levels in DMOG+I/R group were significantly higher than those in I/R group(P<0.05).These results suggest that HIF-1? has a certain inhibitory effect on TLR4/NF-?B signaling pathway.Conclusion:1.HIF-1? can reduce myocardial tissue inflammatory damage caused by rat I/R;2.The anti-I/R myocardial inflammation of HIF-1? is achieved by inhibiting the TLR4/NF-?B signaling pathway. |
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