| Background:Ischemic heart disease（IHD）is one of the most highly regarded cardiovascular diseases in the world.Timely and effective reperfusion strategies remain the current standard therapy for IHD.However,in the process of blood flow recovery,the damage of tissues and organs is aggravated and even life-threatening.Therefore,this injury is called Ischemia/reperfusion injury（I/RI）.During reperfusion,damaged cardiomyocytes and endothelial cells can release a series of inflammatory cytokines such as IL-1β,IL-6 and TNF-α,and then cause local inflammatory reactions,lead to increasing myocardial infarction size,and impairing heart function,myofibrosis and finally further aggravate ischemic heart disease.Toll-like receptor 4（TLR4）is a transmembrane protein that activates the downstream nuclear transcription factor NF-кB and causes a large number of inflammatory factors（such as IL-6,TNF-α,IL-1β）,those further aggravate myocardial damage.Many studies have shown that overexpression of autophagy during reperfusion accelerated the loss of cardiomyocytes.The relationship between inflammation and autophagy has a lot of speculation,but the TLR4/NF-кB signaling activation is the most important pathway which promotes the secretion of inflammatory factors,and in turn mediates the occurrence of autophagy.Therefore,it is especially important that we look for a new substance to balance the relationship between them.A number of epidemiological studies have reported that diets occurring extract of herbs,fruits and spices can reduce the risk of cardiovascular disease.Hesperetin（HES）is derived from the citrus fruits of the Rutaceae family.Its main medicinal ingredient is bioflavanone,which has various biological activities,such as improving blood lipids,anti-oxidation.antitumor and so on.Studies in recent years have shown that Hesperetin has a protective effect on ischemia/reperfusion injury of the retina,brain and other organs.But how it affects cardiac ischemia/reperfusion is unclear.Our previous studies have found that Hesperetin alleviated H2O2-induced oxidative damage in H9c2 cells.Therefore,this study aimed to investigate the effect and mechanism of Hesperetin on myocardial ischemia/reperfusion in rats,and provide new ideas for clinical treatment of ischemic heart disease.The study was divided into two parts:in vivo experiment,Hesperetin was pretreatment for 6 weeks to observe the effect on MI/R and the mechanism on the inflammation and autophagy;in vitro,hypoxia/reoxygenation（H/R）model is established,the effect of Hesperetin on hypoxia/reoxygenation-induced apoptosis,inflammation and autophagy in H9c2 cells will be observed.Objective:1.To observe the effect of Hesperetin on myocardial ischemia/reperfusion injury in rats;2.Explore the specific mechanism of Hesperetin exerting a protective effect on MI/R.Methods:1.Expermental animals and grouping:Male Sprague-Dawley rats（4 weeks old,weighing 90-100 g,n=50）were selected,and Hesperetin was dissolved in 0.5%sodium carboxymethyl-cellulose（CMC-Na）,the solution was administered orally to rats by gavage for a total of 6 weeks.They were randomly divided into sham group（Sham）,myocardial ischemia/reperfusion group（I/R）,Hesperetin pretreatment+myocardial I/R group（HES+I/R）,Hesperetinpretreatment+shamoperationgroup（HES）;TAK-242+myocardial I/R group（TAK-242）.Myocardial ischemia was performed for 45min,followed by reperfusion for 3 h.（1）Electrocardiogram was used to detect the effect of Hesperetin on arrhythmia induced by I/R in rats;（2）Echocardiographic tested the effect of Hesperetin on I/R cardiac function in rats before and after 24 hours operation;（3）LDH and CK-MB were used to detect the effect of Hesperetin on myocardial injury induced by I/R in rats;（4）ELISA method was measured by the MPO activity and serum levels of IL-6,TNF-α,IL-1β;（5）HE staining was used to observe the inflammatory infiltration of myocardial tissue;（6）TUNEL staining and Caspase3 activity were used to detect the effect of Hesperetin on myocardial apoptosis induced by I/R;（7）.IHC/IF was used to observe the expression of CD45,Beclin1,LC3,p62 in myocardial tissue;（8）Western blot was performed to detect the expression of IL-6,TNF-α,IL-1β,TLR4,p65,Beclin1,LC3,p62;（9）RT-PCR tested the effect of Hesperetin on the mRNA levels of IL-6,TNF-α,IL-1β,TLR4,p65,Beclin1,LC3.2.Experimental cells and grouping:H9c2 cells were divided into normal control.group（Control）,.hypoxia/reoxygenationgroup（H/R）,Hesperetin+hypoxia/reoxygenation group（HES+H/R）,TAK-242+hypoxia/reoxygenation group（TAK-242+H/R）,Hesperetin group+normal group（HES）.（1）.Cell viability and cell damage were measured by CCK-8 and LDH methods;（2）Hoechst33258 staining and Annexin V-FITC/PI double staining flow cytometry were detected cell apoptosis;（3）.Using the ELISA kit was detect the secretion of IL-6,TNF-α,IL-1βin the supernatant;（4）Western blot was used to detect the protein expression of TLR4,NF-кB,Beclin1,LC3 and p62;（5）JC-1 staining was used to detect the effect of mitochondrial membrane potential.Results:1.Hesperetin protects myocardial ischemia/reperfusion injury and regulates expression of the inflammation and autophagy by inhibiting TLR4/NF-κB signaling pathway1.1 Hesperetin improves cardiac dysfunction caused by I/RThe results of electrocardiogram showed that the frequency of arrhythmia in the I/R group increased significantly,and the frequency of the HES+I/R group was decreased.The results of the Echocardiography showed that no significant difference in LVEF and LVFS was observed before operation;24 h after operation:I/R group markedly reduced LVEF and LVFS compared to the sham group（P<0.01）,treatment with Hesperetin restored LVEF and LVFS to a similar level as the sham groups（P<0.05）.1.2 Hesperetin reduces I/R myocardial damageCompared with Sham group,CK-MB and LDH were significantly increased in I/R group（P<0.05）;Hesperetin pretreatment inhibited it（P<0.05）.HE staining results showed that the I/R group had myocardial fiber disorder,cell degeneration and necrosis,and a large number of inflammatory cell infiltration compared with the Sham group.The Hesperetin pretreatment,the myocardial arrangement was relatively neat,and a small amount of inflammatory cells infiltrated.1.3 Hesperetin can reduce myocardial tissue MPO activity,the levels of serum IL-1β,TNF-α,IL-6 induced by I/RThe results showed that compared with Sham group,MPO activity and the levels of IL-1β,TNF-αand IL-6 in I/R group were significantly increased（P<0.05）;Hesperetin was significantly reversed（P<0.05）.1.4 Hesperetin reduces the protein and mRNA levels of IL-1β,TNF-αand IL-6 in I/RWestern blot and RT-PCR results showed that compared with Sham group,I/R group increased the protein and mRNA levels of IL-1β,TNF-αand IL-6（P<0.01）;while HES+I/R group significantly reduced the expression level（P<0.05）.1.5 The effect of Hesperetin on the expression of CD45 induced by I/RThe results of immunohistochemistry showed that there were few positive staining particles in Sham group,and the expression of positive staining particles in CD45 was significantly increased in the I/R group at the area of inflammatory infiltration of myocardium damage.The treatment with Hesperetin was reduced positive staining particles compared with I/R group.1.6 Hesperetin inhibits TLR4/NF-κB signaling pathway induced by I/R.Western blot and RT-PCR showed that the proteins and mRNA expressions of TLR4and p65 were significantly up-regulated in I/R group compared with Sham group（P<0.01）.Hesperetin pretreatment was decreased（P<0.05）.1.7 Hesperetin reduces cardiomyocytes apoptosis subjected to I/RCompared with the Sham group,the Caspase3 activity and the number of TUNEL-positive cells in the I/R group increased（P<0.01）.After the treatment with Hesperetin,the Caspase3 activity and the number of TUNEL-positive cells decreased compared with the I/R group（P<0.05）.1.8 Hesperetin lessens the expression of I/R autophagy-related proteinsWestern blot and RT-PCR results showed that compared with Sham group,the expression levels of Beclin1 and LC3 in I/R group increased,and the protein level of p62decreased（P<0.01）.Compared with I/R group in HES+I/R group The expression protein and mRNA of Beclin1,LC3 decreased,and the protein level of p62 increased（P<0.05）.1.9.The administration of TAK-242 decreases I/R-induced expression of the autophagy-related proteinImmunofluorescence and Western blot showed that compared with Sham group,the the red fluorescent particles and expression protein of Beclin1 and LC3 increased,and the red fluorescent and protein level of p62 were reduced（P<0.05）.Compared with I/R group After TAK-242 was injected into the tail vein,the red fluorescent particles and protein levels of Beclin1 and LC3 were reduced,and the expression red fluorescent particles and protein of p62 was elevated（P<0.05）.2.Hesperetin regulates apoptosis,inflammation and autophagy induced by hypoxia/reoxygenation in the H9c2 cells by inhibiting TLR4/NF-κB pathway2.1 Establishment of hypoxia/reoxygenation model of the H9c2 cells and determination of optimal concentration of HesperetinCompared with the control group,the hypoxia 12 h/reoxygenation 3 h（H/R）cell survival rate decreased to about 50%（P<0.05）.Pretreatment of the H9c2 cells with different concentrations of Hesperetin（HES）at 0,6,12,25,50,100μM,compared with0μM,25μM HES significantly increased cell survival after H/R（P<0.05）;the level of LDH was significantly reduced（P<0.05）.The hypoxia was 12 h/reoxygenation for 3 h,and the optimal concentration of Hesperetin was 25μM for subsequent experiments.2.2 HES decreases H/R-induced apoptosis of the H9c2 cellsHoechst33258 staining and Annexin V-FITC/PI double staining flow cytometry showed that the number of apoptosis was increased compared with the Control group,while the number of cardiomyocyte apoptosis was decreased in the HES+H/R group compared with the H/R group.Similarly,Caspase3 activity was significantly elevated in the I/R group compared with the Control group（P<0.05）;Caspase3 activity was decreased after pretreatment with Hesperetin（P<0.05）.2.3 Hesperetin and TAK-242 reduce the secretion of inflammatory factor and expression of TLR4,p65 induced by H/R in H9c2 cellsThe ELISA results showed that the levels of TNF-α,IL-6 and IL-1βin the supernatant of Control group were lower;H/R induced the increase of the levels of TNF-α,IL-6 and IL-1βin the supernatant of the H9c2 cells（P<0.05）;the level of the HES+H/R group and TAK-242 group were decreased（P<0.05）.Western blot showed that compared with the Control group,the expression levels of TLR4 and p65 in the H/R group were significantly increased（P<0.05）;the trend was reversed in the HES+H/R group and TAK-242+H/R group（P<0.05）.2.4 Hesperetin and TAK-242 decrease expression of autophagy-related protein in H9c2cells subjected to H/RWestern blot showed that the expression protein of Beclin1 and LC3 in H/R group was up-regulated compared with Control group,and the expression of p62 protein was decreased（P<0.05）.After treatment with Hesperetin,the protein expression of Beclin1and LC3 was inhibited,and the protein level of p62 was increased（P<0.05）;similar results were obtained after TAK-242 administration（P<0.05）.2.5 Hesperetin and TAK-242 improve H/R-induced mitochondrial dysfunction in the H9c2 cellsThe results showed that compared with the Control group,the mitochondrial membrane potential of the H/R group decreased significantly.Compared with the H/R group,the mitochondrial membrane potential of the HES+H/R group and the TAK-242+H/R group increased（P<0.05）.The results of ATP showed that compared with the Control group,the level of ATP in the H9c2 cells of the H/R group was significantly decreased（P<0.05）;Compared with the H/R group,the HES+H/R group and TAK-242+H/R group were increased intracellular level of ATP（P<0.05）.Conclusion:1.Hesperetin has a protective effect on myocardial ischemia/reperfusion,and its mechanism is related to the reduction of apoptosis,inflammation and autophagy.2.Hesperetin regulates the relationship between inflammation,autophagy and apoptosis induced by MI/R via inhibiting the TLR4/NF-κB signaling pathway. |
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