Hydrogen Sulfide Up-regulates WDR26 And Reduces Myocardial No-reflow

Acute myocardial infarction（AMI）is a high incidence of ischemic cardiomyopathy,which is more common in the middle-aged and elderly people.In recent years,AMI has an increasing trend among young people.Although percutaneous coronary intervention（PCI）can significantly reduce the mortality of patients,it may also lead to insufficient myocardial perfusion due to no-reflow（NR）or slow-reflow,which can partially offset or even completely offset the benefits of PCI.It’s an independent predictor of sudden cardiac death and adverse cardiac events.Its development is related to ischemic injury,reperfusion injury,microvascular dysfunction and distal microthrombotic embolism,in which the structure damage and dysfunction of microvascular endothelial cells play an important role.Hydrogen sulfide（H₂S）has been found to be the third gas signal molecule besides nitric oxide and carbon monoxide.Previous studies have shown that H₂S can relax blood vessels,inhibit vascular remodeling,reduce blood pressure,and then alleviate myocardial ischemia,atherosclerosis,pulmonary hypertension and other diseases.WD40 protein is composed of about 40 amino acid residues with conserved GH and WD sequences.WD40 family exists in many proteins with regulatory functions,mediates protein-protein interactions,and plays an important role in signal transduction,protein transport,chromosome modification,transcription or RNA processing.As a new member of WD40 protein family,the function of WDR26 remains to be further studied.At present,studies have found that WDR26 protein in involved in hematopoietic disorders,cancer,ischemia-reperfusion injury and other diseases or pathological processes,but whether it is involved in the inhibition of myocardial NR is unclear.Therefore,the purpose of this experiment is to study whether H₂S participates in myocardial NR,and further explore whether its beneficial effect is related to the expression of WDR26 in endothelial cells,so as to provide new methods and new ideas for clinical prevention and treatment of myocardial NR.Objective:To observe the effect of H₂S on myocardial NR in rats and whether the mechanism is related to WDR26.Methods:Forty male SD rats（200-300g）were randomly divided into 5groups:sham operation group（sham group）:threading under LAD without ligation;no reflow group（NR group）:ligating LAD for 1 hour and reperfusion for 3 hours;hydrogen sulfide+no-reflow group（NaHS+NR group）:intraperitoneal injection of NaHS（H₂S donor）when 45 minutes of ischemia and the rest treatment was the same as NR group.Propargylglycine（PPG）+no-reflow group（PPG+NR group）:at 45 minutes of ischemia,PPG（endogenous H₂S inhibitor）was injected intraperitoneally,and the rest treatment was the same as that of NR group.PPG+hydrogen sulfide+no-reflow group（PPG+NaHS+NR group）:at 45 minutes of ischemia,PPG and NaHS were injected intraperitoneally sequentially,and the rest treatment was the same as that of NR group.ECG was used to observe myocardial ischemia and reperfusion;Thioflavin-s staining was used to observe the changes of myocardial NR;hematoxylin eosin staining（HE）was used to observe the infiltration and injury of inflammatory cell in myocardial tissue;Western blot and immunofluorescence were used to detect the protein expression of WDR26.A stable H/R model of human vascular endothelial cells was established.To investigate the protective effect of exogenous H₂S on H/R injury of endothelial cells,endothelial cells were randomly divided into 7 groups:0μmol/L NaHS+H/R group,200μmol/L NaHS+H/R group,400μmol/L NaHS+H/R group,800μmol/L NaHS+H/R group,1600μmol/L NaHS+H/R group.Reactive oxygen species（ROS）were detected by DHE fluorescent probe reagent,cell survival rate was detected by cell counting kit-8（CCK-8）method,and cell damage was detected by malondialdehyde（MDA）method.Then,in order to explore whether H/R can induce the expression of WDR26 in endothelial cells,the cells were divided into two groups:control group and H/R group.The protein expression of WDR26 was detected by Western blot.In order to further explore the effect of H₂S on the expression of WDR26 in H/R endothelial cells,the cells were randomly divided into four groups:H/R group,NaHS+H/R group（400μmol/L）,PPG+H/R group,PPG+NaHS+H/R group.Finally,in order to determine whether H₂S can reduce the H/R injury of endothelial cells by regulating the expression of WDR26,lentiviral empty vector（LV-con）,LV-WDR26 and WDR26sh RNA were used to transfect the cells respectively,and stable transfection strains were obtained.Then the cells were divided into sixgroups:H/R+LV-Congroup,H/R+LV-WDR26group,H/R+WDR26sh RNAgroup,NaHS+H/R+LV-Congroup,NaHS+H/R+LV-WDR26 group,NaHS+H/R+WDR26sh RNA group.The secretion of ICAM-1 and ET-1 in supernatant was detected by Enzyme-Linked Immunosorbent Assays（ELISA）.Results:1.Compared with sham group,after LAD was ligated in other groups,ST-segment in lead II of ECG was significantly elevated,and ST-segment was decreased to varying degrees after removal of ligation.Compared with NR group,the ST-segment decline in NaHS+NR group was the most obvious,and the decline in PPG+NR group was smaller,while the NaHS+PPG+NR group had no significantly decline.The results of Thioflavin-s staining showed that compared with NR group,the NR area of NaHS+NR group was significantly reduced,while that of PPG+NR group was significantly increased.Western blot results showed that:compared with NR group,the expression of WDR26 in ischemic myocardium of rats in NaHS+NR group was increased（P<0.05）,while that in the PPG+NR group was the opposite.Immunofluorescence showed that the expression of WDR26 was more significant in H₂S+NR group than in NR group.2.The results of CCK-8showed that 200,400 and 800μmol/L NaHS could improve the survival rate and promote the proliferation of H/R cells,and reached the peak at 400μmol/L.The results of MDA content detection showed that 200,400 and 800μmol/L NaHS could reduce the release of MDA in H/R cells（P<0.05）,which reached the lowest level in 400μmol/L group.DHE fluorescence probe results showed that ROS fluorescence of H/R cells treated with 400,800 and 1600μmol/L NaHS was significantly weaker than 0μmol/L NaHS,and the fluorescence of H/R cells treated with 400μmol/L NaHS was the weakest,indicating that ROS release was the least,and then with the increase of NaHS concentration,ROS release began to increase.Western blot results showed that the expression level of WDR26 protein in H/R group was significantly higher than that in control group（P<0.05）,indicating that H/R could promote the expression of WDR26 in endothelial cells.Compared with H/R group,the expression level of WDR26 protein in NaHS+H/R group was higher than that in PPG+H/R group,but there was no significant difference in PPG+NaHS+H/R group,which indicated that NaHS could promote the expression of WDR26 protein in H/R endothelial cells.Compared with the control group and LV-Con group,the expression level of WDR26 protein in LV-WDR26 group was significantly increased,and the expression level of WDR26sh RNA group was significantly decreased,indicating that lentivirus transfection was successful.Compared with H/R+LV-Con group,the secretion of ET-1 and ICAM-1 in H/R+LV-WDR26 group was significantly decreased,indicating that overexpression of WDR26 could inhibit the secretion of ET-1 and ICAM-1 in H/R cells,while the secretion of ET-1 and ICAM-1 in H/R+WDR26sh RNA group was significantly increased,indicating that inhibition of WDR26 could promote the secretion of ET-1 and ICAM-1.Compared with H/R+LV-WDR26 group,the secretion of ET-1 and ICAM-1 decreased in NaHS+H/R+LV-WDR26 group,indicating that exogenous hydrogen sulfide can inhibit the secretion of ET-1 and ICAM-1 in H/R endothelial cells.Compared with H/R+LV-Control+NaHS group,the secretion of ET-1 and ICAM-1 increased in H/R+WDR26sh RNA+NaHS group,which indicated that inhibition of WDR26 can weaken the protective effect of H₂S on H/R cells.Conclusion:H₂S can up-regulate endothelial WDR26 and reduce myocardial no-reflow.