The Molecular Mechanisms Of The Dual Ningxin Capsule Intervention Myocardial Ischemia

Cardivascular disease caused by myocardial ischemia is one of the most deadly diseases. Currently many clinical medicaments are used for treatment of myocardial ischemia, but there are various problems. Therefore, it is important theoretically and pragmatically to the Chinese medicine modernization drive and new development of Chinese medicine drugs by studying the mechanism of Chinese medicines and new affected targets with our country prolific resources of traditional Chinese medicines and new research results of cardiovascular vital phenomena.Shuang Shen Ning Xin capsule (SSNX) is made up of Ginsenoside, salvianolic acid and corydalinesubstance. It has efficacy of nourishing qi and activating blood and regulating qi to alleviate pain. Currently, our lab has estalbished the assaying method of simplify chemical constituents in Vitro and/or blood and analyzed the HPLC fingerprints of samples obtained from rats after orally taking SSNX. Pharmacodynamics research indicates that SSNX could relieve myocardial damage of dog and/or acute myocardial ischemia(AMI) of rat, increase the activity of SOD in serum, decrease the content of MDA and CK, expand coronary artery, increases coronary flow and decrease myocardial oxygen consumption. It has protective effects for ischemia cardiac muscle.The essay aims at the material basis of the effects of SSNX on myocardial ischemia with sera pharmaceutical chemistry results. Besides, the essay establishes the model of cultured cells subjected to hypoxia and reoxygenation, and selects the safe components, ingredients of SSNX.On the above basis, we further studied the effects of SSNX and its effective components on myocardial cell calcium overload, observed the concentration of intracellular Ca²⁺ , calcium pump , L- type calcium channel electric current (I_{Ca l}) and the L-calcium channel subunits mRNA expression, moreover, illuminated the mechanism of SSNX's effect on myocardial ischemia from the point of calcium overload. Results were listed as follows:Part A: research on the material basis of SSNX's effect on myocardial ischemia 1 Effects of SSNX and its ingredients on proliferation of myocardial cell of ratResults: SSNX had no effects on myocardial cell proliferation in experimental dose range(l-1000mg/L)(P>0.05). And its three compositions in highdose(1000mg/ L)could inhibit the myocardial cell proliferation(P<0.001). But its ingredients: Dl-tetarhydropalmatine(dl-THP), salvianolic acid B(Sal B), ginsenosides Rgl(Rgl),ginsenosides Re(Re) and dehydeocorydaline (DHC) had no effects on myocardial cell proliferation(P>0.05). Conclusion: SSNX made by three compositions has less side effects than individual three compositions.2 Effects of SSNX and its ingredients on cultured cells subjected to hypoxia and reoxygenation.Results: Rgl, dl-THP, Sal B, and DHC could all reduce the leakage of lactic dehydrogenase(LDH) from cardiomyocytes subjected to hypoxia and reoxygenation(P<0.05). Sal B had the most evidents among the ingredients(P<0.01). Re had no effects on the leakage ofLDH(P>0.05).Ginsenoside, salvianolic acid and corydaline substance could all reduce the leakage of LDH from cardiomyocytes subjected to hypoxia and reoxygenation(P<0.05). Salvianolic acid had the most evidents among the ingredients(P<0.01).SSNX can remarkable reduce the leakage of LDH from cardiomyocytes subjected to hypoxia and reoxygenation(P<0.01).Conclusion: SSNX has no less effect than its individual ingredients. The results are consistent with the previous results of animal experiments in vivo. Dl-THP, Sal B, Rgl and DHC are the effective ingredients of SSNNX. Salvianolic acid and Sal B may play key roles on protecting cardiomyocytes.Part B: Effects of the SSNX's effective ingredients on Ca2+on myocardial cell1 Studies on mechanisms of the SSNX's effective ingredients on myocardial cellcalcium overload.1.1 Effects of the SSNX's effective ingredients on the concentration of intracellular Ca2+:We observed the effects of the SSNX's effective ingredients on the concentration of intracellular Ca2+ with spectrophotofluorometry. Results: Sal B, DHC and SSNX could reduce the concentration of intracellular Ca2+ caused by hypoxia and reoxygenation(P<0.05). Conclusion: Sal B, DHC and SSNX can relieve the calcium overload and have protective effects for cardiomyocytes.1.2 Effects of the SSNX's effective ingredients on the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase:We observed the effects of the SSNX's effective ingredients on the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase with colorimetry. Results: DHC and SSNX could increase the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase(P<0.05). Conclusion: DHC and SSNX can relieve the calcium overload by inhibiting exchange of Na+ /Ca2+ and improving the activity of calcium pump.1.3 Effects of the SSNX's effective ingredients on the L-calcium channel subunitsmRNA expression.We observed the effects of the SSNX's effective ingredients on the L-calciumchannel subunits mRNA expression in cultured ventricular myocytes with RT-PCR.Results: Sal B and SSNX could reduce the expression of the L-calcium channel a 1 torelieve the calcium overload(P<0.05). But DHC has no effects on the L-calcium channela !(P>0.05).1.4 Effects of the SSNX's effective ingredients on the contents of MDA in cultured ventricular myocytes.We observed the effects of the SSNX's effective ingredients on the contents of MDA in cultured ventricular myocytes. Results: Sal B, DHC and SSNX could reduce the contents of MDA in cultured ventricular myocytes(P<0.05). Conclusion: Sal B, DHC and SSNX can reduce oxygen free radical to relieve the calcium overload.1.5 Effects of the SSNX's effective ingredients on the activities of NOS and contents of NO.We observed the effects of the SSNX's effective ingredients on the activities of NOS and contents of NO in cultured ventricular myocytes supernatant. Results: Sal B, DHC and SSNX could reduce the activities of T-NOS and iNOS and the contents of NO(P<0.05).2 Effects of the SSNX's effective ingredients on L-type calcium channel currents in isolated rat ventricular myocytesThe single rat ventricular myocytes were obtained by enzymatic dissociation. The effects of the SSNX's effective ingredients on L-type calcium channel currents were Observed by whole cell patch clamp technique. Results: SSNX and Sal B could inhibit lea l in a concentration dependently manner without altering the shape of the current-voltage (I-V) curve, reversal potential and the steady-state activation curve of lea l(P<0.05). DHC has no effect on ICa L(P>0.05).Conclusion By improving activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase, inhibiting Ica-L, reducing the expression of the L-calcium channel, antioxidation, inhibiting NOS-NO, SSNX, Sal B and DHC can relive the calcium overload to protect cardiomyocytes.Dl-THP, Sal B, Rgl and DHC are the effective ingredients of SSNX. Among them Sal B may play a key role on protecting of cardiomyocytes.