Protective Effect And Mechanism Of Dihydroquercetin On Myocardial Ischemia-Reperfusion Injury

ObjectiveMyocardial Ischemia-Reperfusion Injury(MIRI)refers to the peroxidative damage caused by the restoration of blood supply after myocardial ischemia,which promotes myocardial cell apoptosis and necrosis,aggravates myocardial dysfunction and structural damage,and causes arrhythmia,increasing the area of myocardial infarction,heart failure and other clinical phenomena.Studies have shown that Dihydroquercetin(DHQ)has anti-inflammatory,antioxidant,cardiovascular protection,antibacterial,antiviral biological activities.DHQ can resist ischemia-reperfusion injury,but its related molecular mechanism is not clear.Therefore,this topic intends to conduct in-depth research on the protective effect of DHQ against MIRI and its potential targets to reveal the possible molecules of DHQ against MIRI.The mechanism provides a theoretical basis for the clinical application of DHQ.Methods1.The protective effect and mechanism of DHQ on H/R-induced H9c2 cardiomyocytesFirst,a model of H/R-induced injury in H9c2 cardiomyoblasts was established.The optimal concentration of DHQ was determined colorimetrically by MTT assay,which was used in subsequent experiments.The supernates and cells were collected,respectively,after the different treatments for measuring the lactate dehydrogenase(LDH)levels,as well as glutathione peroxidase(GSH-Px)and superoxide dismutase in the cell supernatant(SOD)activity and malondialdehyde(MDA)content with the corresponding detection kit according to the manufacturers instructions;Flow cytometry was used to detect reactive oxygen species(ROS)levels;Mitochondrial membrane potential changes were observed and analyzed by JC-1 staining.Finally,western blot was used to detect p-PI3K and p-AKT in PI3K/AKT signaling pathway proteins in cardiomyocytes and the ratio of Bcl-2 and Bax and the endoplasmic reticulum stress signaling pathway related protein expression levels of PERK,p-eif2α,IRE1α,ATF6,GRP78,CHOP,p-JNK,and Caspase-12.2.The protective effect and mechanism of DHQ on MIRI in ratsThe Langendorff isolated heart perfusion technology was used to simulate myocardial ischemia-reperfusion injury model.Isolated heart contraction function parameters of heart rate,LVSP,+dP/dtmax and-dP/dtmax were recorded.The tissue myocardial enzyme CK,AST activity and antioxidant enzymes T-AOC,SOD,GSH-Px,CAT activity and ROS levels and MDA content were measured;HE staining method was used to observe the changes of myocardial histopathology in rats.Western blot was used to detect the above protein expression levels.Results1.The protective effect of DHQ on H/R-induced H9c2 cardiomyocytesBy MTT method,it was determined that different concentrations of DHQ had no significant effect on the proliferation of H9c2 cardiomyocytes;when the concentration of DHQ administered was 20 μM,H/R-induced cardiomyocyte damage was significantly inhibited,and the effect was the best.DHQ pretreatment could significantly inhibit H/R-induced release of LDH and increase of MDA levels in H9c2 cardiomyocytes and increase the activity of three antioxidant enzymesSOD,CAT and GSH-Px,reduce intracellular ROS levels,and inhibit H/R-induced damage to cardiomyocytes increases the red/green fluorescence ratio.Western blot results showed that DHQ pretreatment could up-regulate the expression of p-AKT and p-PI3K proteins,increase the ratio of Bcl-2/Bax,and down-regulate p-PERK,p-eif2α,IRE1α,ATF6,GRP78,CHOP,p-JNK,Caspase-12 expression level.2.The protective effect and mechanism of DHQ on myocardial ischemia-reperfusion injury in ratsOn the I/R model,the effect of DHQ on isolated cardiac contractile function parameters was analyzed.Compared with the I/R group,pre-administration of 20 μM DHQ could significantly increase LVSP,± dP/dtmax,and adjust heart rate.The PI3K inhibitor LY294002 could inhibit the improvement of DHQ on cardiac contractile function.Analysis of myocardial enzymes and antioxidant activity revealed that the DHQ pretreatment group could significantly inhibit the release of CK and AST,enhance the activity of T-AOC,SOD,CAT,GSH-Px,and also reduce the level of ROS and MDA,while LY2940 02 could significantly reverse the effect of DHQ.Histopathological results showed that DHQ pretreatment could significantly improved the degeneration of cardiomyocytes,reduce myocardial fiber rupture,widen the interstitial,reduced membrane rupture,and alleviated the cytoplasmic lysis,and other pathological features.The interstitium of cardiomyocytes was narrow,and the arrangement of cardiomyocytes was relatively neat and tight.After the administration of LY294002,the above-mentioned improvement was significantly inhibited.Western blot results showed that after DHQ pretreatment,the protein expression levels of p-AKT and p-PI3K could be increased,the ratio of Bcl-2/Bax could be increased,and endoplasmic reticulum stress signaling pathway related proteins GRP78,p-eif2α,p-PERK,IRE1α can be inhibited,ATF6,CHOP,p-JNK,and Caspase-12 expression levels,and after administration of LY294002,DHQ has no significant regulatory effect on the above proteins.ConclusionDHQ can inhibit oxidative stress and endoplasmic reticulum stress response by activating PI3K/AKT pathway,prevent the occurrence of apoptosis.and thus protect myocardial ischemia-reperfusion injury.This study provides a theoretical basis for the clinical application of DHQ.