The Effect And Mechanisms Of Hypericum Perforatum Glycosides On H₂O₂Injury In Cardiomyocytes

Objective:1, To establish the culture methods of cardiomyocytes and researchthe cytotoxicity of hypericum perforatum glycosides(Hyp) on thecardiomyocytes for determining the concertration range of dose.2,To study the protective effect and mechanism of Hyp on oxidativestress injury in cardiomyocytes induced by H₂O₂.Methods:1,To study Cytotoxicity of Hypericum perforatum glycosides on thecardiomyocytes:The cardiomyocytes in primary culture were preparedfrom ventricular tissue of1-to3-day-old Sprague-Dawley rats,Morphocytology changes and Pulsation self-discipline were observedby microscopy, cardiomyocytes were detected by immunohistochemistry,the cells in good condition were randomly divided to eight groups:control group, DMSO solvent group, Hyp groups(doses of10,5,1,0.2,0.04,0.08g/L, respectively). the Cytotoxicity of Hyp on thecardiomyocytes was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)assay. The effect of beating frequenciesof cardiomyocytes were observed.2, To study Protective mechanism of Hypericum perforatumglycosides on oxidative stress injury in cardiomyocytes: Thecardiomyocytes in primary culture were prepared from ventricular tissueof1-to3-day-old Sprague-Dawley rats and the cells in good condition were randomly divided to seven groups: control group, H₂O₂Modelgroup, DMSO solvent group, Verapamil positive control group, Hyp low,medium and high dose group, all groups except the normal control wereestablished oxidative stress injury model in cardiomyocytes of neonatalrats induced by H₂O₂.The activity of the injured cardiomyocytes wasdetected by MTT. the activity of LDH, cNOS, iNOS, tNOS, cTnI,CK-MB,Na⁺-K⁺-ATP, Ca²⁺-Mg²⁺-ATP were measured by biochemistryapproaches. The expression levels of different subtypes of NOS mRNAwere determined by reverse transcription polymerase chain reaction.Results:1, Cardiomyocytes with high purity can be obtained bymorphocytology, beating and the result of immunohistochemistry. TheTC50and TC10of Hyp to the cardiomyocytes cultured in vitro was8.946g/L and0.028g/L respectively.Non–toxic concertrations of theadministration of Hyp had no effect on the pulse of the cardiomyocytes.2,After induced by H₂O₂, the cardiomyocytes activities of the Hypcontrol group were increased compared with that of the model group (P<0.01). when compared to the model group, the level of LDH, cNOS,iNOS, tNOS,Na⁺-K⁺-ATP, Ca²⁺-Mg²⁺-ATP in the Hyp groups (high andmedium doses) were significantly increased(P<0.01), The expressionlevels of nNOS mRNA in the Hyp each groups were increased(P<0.01),The expression levels of eNOS mRNA,iNOS mRNA in the Hyp groups(high and medium doses) were significantly increased(P<0.01).Conclusion:1,The methods of separation and culturing were simple,stable andreliable, Hyp was low toxicity to the cardiomyocytes cultured in vitro. 2, Hyp has protective effect on oxidative stress injury incardiomyocytes induced by H₂O₂. The mechanism may be related withinhancing the activity of ATP and blocking the Ca²⁺into the cell, affectingthe expression of different subtypes of NOS mRNA and protein.