| Objective:The pharmacodynamics of total flavonoids of Jinhe Yangxin Prescription against myocardial ischemia-reperfusion injury was studied by establishing rat Langendroff's isolated myocardial ischemia-reperfusion injury model and in vivo myocardial ischemia-reperfusion injury model.On the basis of defining the anti-myocardial ischemia-reperfusion injury effect of total flavonoids of Jinhe Yangxin Prescription,the mechanism of action was studied by network pharmacology Line prediction and The mechanism of action was studied by Western blotting.Methods:1.Pharmacodynamic Study of Total Flavonoids of Jinhe Yangxin Prescription on Myocardial Ischemia Reperfusion InjurySeventy-two SD rats were randomly divided into six groups:normal group,model group,positive drug group?verapamil hydrochloride injection,dosage of 5×10-77 mol/L?,Jinhe Yangxin Prescription total flavone high,medium and low dose group?dosage of 16,8,4 mg/L,respectively?,with 12 rats in each group.The isolated rat hearts were prepared one week before the experiment.The isolated rat hearts were perfused with Langendorff and connected with ML870 8-channel physiological signal recording and analysis system.The isolated rat hearts were balanced for 20 minutes,stopped for 30 minutes,and reperfused for 60 minutes.The isolated rat models of myocardial ischemia-reperfusion injury were established.Three different concentrations of total flavonoids extract of Jinhe Yangxin Formula and verapamil hydrochloride injection were perfused in the drug group to observe the changes of hemodynamic indexes of HR?heart rate?,+dp/dt max?maximum rate of increase of left ventricular pressure?and-dp/dt max?maximum rate of decrease of left ventricular pressure?in rats at the equilibrium and end of reperfusion.After the bundle,the heart was removed from the perfusion device,and the left ventricle was cut off and fixed in 4%paraformaldehyde tissue fixation solution.The pathological changes of myocardium were observed by HE staining.Seventy-two SD rats were randomly divided into sham-operated group,model group,positive drug group(compound Danshen dropping pill,dosage of 0.081g·kg-1),Jinhe Yangxin Prescription total flavone high,medium and low dose group(dosage of 2,1,0.5g·kg-1,respectively),with 12 rats in each group.The rats in each group were fed adaptively for one week before the experiment,and were given orally for 14 days.The dosage volume was 2 mL/100g,once a day,and the model was made 2 hours after the last administration.The rat model of myocardial ischemia-reperfusion injury was established by ligating the left anterior descending coronary artery.The I/R period was 30 min/2h.After the experiment,abdominal aorta blood was collected and centrifuged(4°C,3000r·min-1,15 min),supernatant was put into refrigerator at-80?for freezing storage.Serological indexes such as lactate dehydrogenase?LDH?,creatine kinase isoenzyme?CK-MB?,malondialdehyde?MDA?,superoxide dismutase?SOD?,interleukin-6?IL-6?,tumor necrosis factor-alpha?TNF-alpha?were determined.After blood collection,the heart was taken and the infarct area was measured by TTC staining;the left ventricular tissue under ligation line was cut and the myocardial histopathological morphology was measured by HE staining.Part of the left ventricular tissue was taken and frozen in a refrigerator at-80°C.2.Study on the anti-myocardial ischemia-reperfusion injury mechanism of total flavonoids of Jinhe Yangxin Prescription based on network pharmacologyThe chemical constituents of the total flavonoids of Euphorbia,Herba Cranioides and Fructus Centralis in Jinhe Yangxin Prescription were collected by literature review.Potential active ingredients were screened according to the"five principles of medicine-like".Potential Targets of Active Components were predicted by reverse pharmacophore matching method of PharmMapper server.The phases of myocardial ischemia and reperfusion were also analyzed according to OMIM databases.To screen the target of Jinhe Yangxin Formula flavonoids against myocardial ischemia-reperfusion injury,through GO functional enrichment analysis and KEGG pathway analysis of the target,construct the network diagram of active ingredients,anti-myocardial ischemia-reperfusion injury targets and pathways,and explore the mechanism of Jinhe Yangxin Formula total flavonoids against myocardial ischemia-reperfusion injury.the expression levels of TLR-4,MyD88,p-NF-?B,NF-?B,p-p38,p38,p-JNK,JNK,p-ERK and ERK protein in left ventricular tissue were detected by Western blotting.Results:1.Pharmacodynamic Study of Total Flavonoids of Jinhe Yangxin Prescription on Myocardial Ischemia Reperfusion InjuryIschemia-reperfusion injury of isolated myocardium,The results of hemodynamics showed that the high,middle and low dose groups of total flavonoids of Jinhe Yangxin Prescription could increase the levels of HR,+dp/dt max,-dp/dt max at the end of ischemia-reperfusion in isolated hearts of rats;HE staining showed that each dose group of total flavonoids of Jinhe Yangxin Prescription could significantly improve the myocardial pathological injury caused by myocardial ischemia-reperfusion.Myocardial ischemia-reperfusion injury in vivo,The results of serological indexes showed that the total flavonoids of Jinhe Yangxin Formula could significantly improve the myocardial function injury induced by ischemia-reperfusion in vivo,reduce the production of LDH,CK-MB,MDA,IL-6,TNF-alpha,and increase the activity of SOD.TTC staining showed that the total flavonoids of Jinhe Yangxin Formula could significantly reduce the myocardial infarction area of rats.The HE staining showed that the pathological damage of myocardial cells could be significantly improved in each dose group of total flavonoid of Jinhe Yangxin Prescription.2.Study on the anti-myocardial ischemia-reperfusion injury mechanism of total flavonoids of Jinhe Yangxin Prescription based on network pharmacologyA total of 9 potential flavonoid active ingredients were obtained by network pharmacological analysis,and 95 predictive constituent targets were obtained.48 key targets related to anti-myocardial ischemia-reperfusion injury were screened.KEGG pathway enrichment analysis showed that the mechanism of anti-myocardial ischemia-reperfusion injury of total flavonoids of Jinhe Yangxin Prescription might be Toll-like receptor signaling pathway,NF-kappa B signaling pathway.Way and other signaling pathways are involved.Western blotting results showed that total flavonoid of Jinhe Yangxin Prescription could decrease the expression levels of TLR-4,MyD88,p-NF-kappa B,p-p38,p-JNK and p-ERK proteins in myocardial ischemia-reperfusion injury tissues to verify TLR4-NF-kappa B signaling pathway.Conclusion:The total flavone extract of Jinhe Yangxin Prescription has protective effect on myocardial ischemia-reperfusion injury in rats,and this effect is related to its regulation of TLR4-NF-kappa B signaling pathway. |
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