Effect And Mechanism Of Ligustrazine On Myocardial Ischemia/Reperfusion Injury

Objective: Myocardial ischemia/reperfusion(I/R)injury is a pathological process of further aggravating tissue damage after restoring perfusion of ischemic myocardium,which severely limits the therapeutic effect of revascularization for ischemic heart disease,therefore,it is important to avoid the damage caused by reperfusion while open the blood vessels to restore the blood supply to the ischemic myocardium.Ligustrazine,also known as Tetramethylpyrazine(TMP),is a characteristic alkaloid extracted from the traditional Chinese medicine-Ligusticum wallichii.TMP has a wide range of pharmacological effects and has a certain protective effect on myocardial I/R injury.The specific mechanism of TMP protecting against myocardial I/R injury is rarely explored in depth with the direct drug target as the entry point.Therefore,the specific mechanism of TMP protecting against myocardial I/R injury remains to be elucidated.This study aims to explore the mechanism of TMP protecting against myocardial I/R injury based on the combination of drug affinity reaction target stability(DARTS)and liquid chromatography-mass spectrometry(LC-MS/MS)technology.Methods: Section one: In the in vivo experiment,36 male Sprague-Dawley(SD)rats were randomized into Sham,TMP(40mg/kg),I/R,and I/R+TMP(10mg/kg,20mg/kg and 40mg/kg,respectively)groups,with 6 rats in each group.30 min after TMP treatment,the left anterior descending coronary artery was ligated to establish the myocardial I/R injury model(ischemia for 30 min,reperfusion for 120min).TTC staining was used to assess the area of myocardial necrosis.Kits were used to detect the contents of CK-MB,c Tn T,and LDH in serum.The level of inflammatory infiltration was evaluated using HE staining.In the in vitro experiment,H9C2 cells were divided into Control,TMP (20μM),Hypoxia/Reoxygenation(H/R),and H/R+TMP(5,10,20μM,respectively)groups.H9C2 cells induced H/R(hypoxia for 8h,reoxygenation for 2h)injury and treat by TMP for 24 h.CCK8 and flow cytometry was used to detect the cell viability and apoptosis rate in each group,respectively.Section two: In the in vitro experiment,H9C2 cells were divided into two groups: the H/R and H/R+TMP(10u M)groups.Combination of DARTS and LC-MS/MS was used to identify the differentially expression protein between of the two groups;Western blot further verified the direct binding ability between TMP with the target differential protein and assess the expression levels of target differential proteins in samples of section one.Section three: In the in vivo experiment,24 male SD rats were randomly assigned to the Sham,I/R,I/R+TMP+si RNA-NC,and I/R+TMP+si RNA-Myl2 groups,of which I/R+TMP+si RNA-Myl2 group was treated with ligustrazine after si RNA-Myl2 was injected into the myocardium and the I/R model was constructed.The area of myocardial necrosis was observed by TTC staining.The contents of CK-MB,c Tn T and LDH were detected by kits.HE staining was applied to evaluated the inflammatory infiltration level.Network pharmacology was used to analyze the pathway enrichment of the targets of TMP in the treatment of myocardial I/R injury.Western blot was used to evaluate the expression levels of pyroptosis markers Cleaved-caspase-1,GSDMD,IL-18,and IL-1β.Results: Section one: TMP dose-dependently decreased the infarct size in I/R rats and the apoptosis rate of H9C2 cells following H/R,reduced the serum levels of CK-MB,c Tn T and LDH,and alleviated the level of inflammatory infiltration in I/R rats.Section two: DARTS combined with LC-MS/MS technology identified a total of 613 differential proteins between the H/R and H/R+TMP groups,and the 283 up-regulated and 335 downregulated differential proteins in the H/R+TMP group.Among them,the expression of Myl2 protein changed most significantly.Western blot results showed that TMP up-regulated the protein expression level of Myl2 in a concentration-dependent manner,the Myl2 expression was considerably decreased following I/R and H/R,and TMP treatment was able to reverse the I/R and H/R-induced decrease in Myl2 expression.Section three: Compared with the I/R group,the myocardial infarction area of the rats in the I/R+TMP group was significantly reduced,the contents of CK-MB,c Tn T and LDH were effectively reduced,and the level of inflammatory infiltration was reduced.After further inhibiting the expression of Myl2,therapeutic effect of TMP was partial reversed.The network pharmacology analysis showed that the target of TMP protects against myocardial I/R injury were enriched in TNF,NOD-like receptor and other signaling pathways,and the NOD-like receptor signaling pathway was related to pyroptosis.Western blot results showed that compared with the I/R group,TMP could significantly down-regulate the expressions of pyroptosis markers Cleaved-caspase-1,GSDMD,IL-18,and IL-1β,while inhibiting the expression of Myl2 reversed the expression of the above pyroptosis markers.Conclusion: TMP protects against myocardial I/R injury by upregulating Myl2 and inhibiting cardiomyocyte pyroptosis following myocardial IR injury.