The Effects Of Salvianolic Acid B On Metabolism Ca～(2+) During Myocardium Ischemia-Reperfusion And The Underlying Mechanism

The myocardial ischemia reperfusion injury （MI/R） is a main interference in thereperfusion therapy for acute myocardial infarction. The mechanism is generallyaccepted to be initially caused by the disturbance of energy metabolism, [Ca²⁺]ioverloading and oxygen free radicals. All of them, intracellular calcium overload isone of the most important factors during MI/R injury. Pharmacological experimentsshowed that Salvianolic acid B is the water-soluble effective components of Salviamiltiorrhiza Bunge, which had protective effects on ischemic myocardium. Ourfaculty working office had confirmed that Sal B had may offer myocardial protectiveeffect against MI/R injury through the isolated rat heart of myocardial MI/R study.OBJECTIVEInvestigate the protective effect of salvianolic acid B injection against themyocardial injury in isolated rabbit hearts caused by ischemia/reperfusion. And then,our study focused on calcium overload to explore the effects and mechanisms of Sal Bon MI/R injury. Studied the effects of Sal B on myocardial cell calcium overloadthrough established the model of cultured cells subjected to hypoxia andreoxygenation, observed the concentration of intracellular Ca²⁺, calcium pump, I-typecalcium channel electric current(I_Ca-L) and Sarcoplasmic reticulumCa²⁺-ATPasse（SERCA）, moreover, illuminated the mechanism of Sal B’s effect onmyocardial ischemia from the point of calcium overload.METHODS The experiment was performed in isolated rabbit hearts perfused on Langendorffapparatus. By observe the activities of creatine kinase（CK） and lactatedehydrogenase（LDH）, heart rate, coronary flow and cardiac histopathological change studythe effect of salvianolic acid B.The model of MI/R in cultured cardiac myocytes was set up by making the purecultured neonatal rat cardiac myocytes be lack of oxygen for 3 hours and then affordoxygen 1 hour. Cardiomyoctes were divided into different six groups: control group,H/R group, H/R+Sal B₁, H/R+Sal B₂, H/R+Sal B₃. Cell vitality of every group wasmeasured with MTT chromatometry. Concentration of cytoplasmic free calcium wasdetermined dual wavelength spectrofluorometer. The effects of Na⁺-K⁺-ATPase andCa²⁺-ATPase with colorimetry. The single rat ventricular myocytes were obtained byenzymatic dissociation. Iingredients on L-type calcium channel currents wereobserved by whole cell patch clamp technique. The activity of SERCA was alsoevaluated by determination of Ca²⁺-dependent p-nitrophenyl phosphatase （pNPPase）.RESULTS1. In contrast to the findings in the control group, the recovery of myocardialcontractility and heart rate was earlier in the test groups, the activities of creatinekinase（CK） and lactate dehydrogenase（LDH） were much lower.And in Sal B subsets,the morphological lesion was improved in different extent.2. The vitality of cardiomyocyte was raise in Sal B group.3. The concentration of cytoplasmic free calcium was distinctly raised in H/Rgroup. Sal B group could produce inhibition in concentration of eytoplasmie freecalcium.4. The activity of Na⁺-K⁺-ATPase, Ca²⁺-ATPase of normal myocytoes membranewere decreased after hypoxia and reoxygenation. Sal B group enhanced the activity ofNa⁺-K⁺-ATPase, Ca²⁺-ATPase.5. Cultured cardiomycytes and acute isolated cardiomycytes were used.Compared with normal neonatal rat cardiac myocytes, the single rat ventricularmyocytes were obtained by enzymatic dissociation and model ventricular myocytes, Sal B has no effect on L-type calcium current activating properties.5. The activity of SERCA was decreased significantly in H/R group, the activityof SERCA was increased in Sal B group significantly.CONCLUSIONSThe drug preconditioning with salvianolic acid B may offer myocardial protectiveeffect against ischemia/reperfusion injury. Results of experiments on cell andmolecule level indicated that the mechanism of Sal B protecting cardiomyocytesagainst hypoxial reoxgynation injury were associated with its effects of anti-calciumoverload during hypoxia/reoxygnation. The effect of calcium antagonism of Sal Bwere achieved by inhibiting calcium inflow, enhancing the activity of Na⁺-K⁺-ATPase,Ca²⁺-ATPase, promoting calcium re-absorption of calcium pump. But it had noeffected on cardiomyocytes I_Ca-L.