| Myocardial ischemia/reperfusion (MI/R) injury is being studied in recent years. Recoverying blood flow effectively and timely is the basic treatment of ischemic myocardium. But reperfusion can cause new injury, that is ischemia/reperfusion injury, which leads to malfunction of oxygen intake, arrhythmia, myocardial stunning, deformation in myocardial microstructure. Pathogenesis of MI/R injury includes energy metabolism obstacle, free radical injury, calcium overload and inflammatory cells infiltration and so on. All of them, intracellular calcium overload is one of the most important factors during MI/R injury. And the unbalance of the intracellular calcium is the common one among different mechanisms responsible for cell necrosis.According to traditional Chinese medicine, MI/R injury is referred to "Qi stagnation in the chest, palpitation and anxiety". Its pathogenesis is indicated by essence deficiency and superficiality excess. Deficiency refers to lackness of Heart-Qi, malnourishment of heart, excess refers to stagnation of Qi and bloodstasis.Shuang shen tong guan fang (SSTG), one formula composed by active fraction of Chinese medicine, was made of total saponins of panax Ginseng, total Salvianolic acids, total Corydalis yanhusuo alkaloids in proportion of 1 to 1 to 2.5. Ginseng root invigorate Qi. Radix Salviae Miltiorrhizae root activate blood circulation to dissipate blood stasis, restore menstrual flow and relieve pain. Corydalis yanhusuo activate blood circulation, invigorate Qi and relieve pain. These three kinds ofherbs were used together in term of simultaneous treatment of superficiality and origin of disease, to achieve the function of invigorating Qi, activating blood circulation, regulating Qi and relieving pain. The pathogenesis of MI/R injury corresponded to the indication of SSTG. Pharmacological experiments showed that SSTG had protective effects on ischemic myocardium while its mechanism was still unclear.Explorations into the mechanism of calcium overload during MI/R or hypoxia and reoxygenation will be helpful to clinical diagnose and treatment of MI/R injury. The study concerning with the protctive mechanisms of SSTG during MI/R injury is capable to provide the clinic with a modern traditional Chinese formula, whose mechanism has been illuminated and active components have been definite.As intracelluar calcium overload is one of the cardinal causes during MI/R injury, our study had been focused on calcium overload to explore the effects and mechanisms of SSTG on MI/R injury. Firstly, SSTG and its drug serum were prepared. The study protocols were listed as follows.(1) In the model of rat MI/R injury, the researchers observed pharmacological action and effects of SSTG on calcium and calcium-related signal molecules.(2) Using pharmacological and chemical research of drug serum concurrently, we aimed to search the best choice for SSTG drug serum preparation and to find the active components of SSTG against hypoxia/ reoxygenation injury.(3) With the model of hypoxia and reoxgyenization on cultured neonatal myocardiocytes, we observed the effects of SSTG drug serum on calcium and calcium-related signal molecule in cardiomyocytes.To elucidate the protective mechanism of SSTG on MI/R injury, we designedthe projects at different three levels: whole, cell and molecular.1 The effects of SSTG on myocardial ischemia and reperfusion injury in ratsMethods and Results1.1 Model of MI/R injuryLeft anterior descending branch of coronary artery was ligated and released to create myocardial ischemia reperfusion injury.1.2 Effects on myocardial ischemia infarctNitroblue tetrazolium (N-BT) staining. The infarction size and weight were detected by N-BT staining. Results: normal myocardium showed no infarct area. After MI/R, the size and weight of infarct area, percentage of infarct area/cardiac ventricle, percentage of infarct area/whole heart were (88.36 ±18.27) mm2. (0.208±0.046)g. ( 28.66±4.85 ) % , ( 23.58±4.43 ) % respectively. SSTG regressed the size and weight of infarct area, decreased the percentage of infarct area/cardiac ventricle and the percentage of infarct area/whole heart. The difference between SSTG and model group was statistically significant (P <0.05).1.3 Effects on Superoxide Dismutase (SOD) and Maleic Dialdehyde (MDA) of serumSpectrophotometric method. Results: The activity of SOD and concentration of MDA in normal group was (449.6+14.0)U/ml, (2.955+0.7)nmol/ml respectively. After MI/R, SOD was sharply reduced to (369.9+25. l)U/ml and MDA was elevated to (6.4+1.3)nmol/ml, which differ significantly from normal group (P <0.05). SSTG increased the activity of SOD and decreased the content of MDA in serum, showing statistical difference compared with model group (P <0.05).1.4 Effects on Lactic Dehydrogenase (LDH), Creatin Phosphokinase (CK) in serumColorimetric method. Results: The concentration of LDH, CK in serum in normal group were (628.4±139.5 ) U/L and (536.4±151.7)U/L, which were elevated to ( 6734.3±2086.2 ) U/L, (3811.3±1496.0 ) U/L respectively. SSTG reduced the contents of LDH and CK. The difference between SSTG lower dose group and model group had statistical significance (P <0.05).1.5 Effects on Tumor Necrosis Factor-alpha (TNF-a), Intercellular adhesion molecule-1 (ICAM-1) in serumEnzyme-linked immunosorbent assay (ELISA). Results: The contents of TNF-cu ICAM-1 were (146.89±0.418)PG/ml, (62.79+1.890)PG/ml in normal group, which were increased to (70.019±3.088)PG/ml, (50.260+1.035)PG/ml after MI/R respectively. The difference between them had statistical significance (P <0.05). SSTG decreased the contents of TNF-a, ICAM-1 obviously (P <0.05 vs model group).1.6 Effects on Sodium ion (Na+), Calcium ion (Ca2+), Potassium ion (K+) contents of myocardiumAtomic absorption spectrophotometry. Results: The contents of Na+, Ca2+, K+ in normal group were (1090.56±141.22)mg/kg, (298.45±277.43)mg/kg, (3029±107.26)mg/kg. After MI/R injury, Na+ and Ca2+contents were elevated to (2012.67±280.10)mg/kg, (535.77±395.74)mg/kg, K+ contents was decreased to (1624.89±721.02)mg/kg. SSTG reduced Na+, Ca2+and elevated K+ contents of myocardium.1.7 Effects on pathology changes of myocardiumHematoxylin-eosin (HE) stain. Results: Normal myocardium fibers were in excellent alignment, and identified clearly. In addition, the cells were observed with undamaged membrane and without any indication of necrosis. After MI/R, myocardiocytes became disturbed and stained unequally, some of cardiomyocyteswere turbid and swelling, cardiac fibers were unclear or vanished. SSTG made these pathogenesis alterations less sever compared with model group.1.8 Effects on Nuclear factor-KB (NF-kB) of myocardiumImmuno-histochemical method. Results: The percentage of NF-kB positive expression , area of immuno-reactant, average absorbance, area density in normal group were ( 1.621 ±0.723 ) % , (0.916±1.248)um2, (0.081+0.056), (0.040±0.049) , which were increased to ( 16.444±7.519 % ) , (7.075±3.497)um2, (0.177±0.014), (0.112±0.021) after MI/R injury respectively. SSTG reduced the activity of NF-kB, decreased positive expression of nucleus, lowered the percentage of NF-kB positive expression, area of immuno-reactant, average absorbance and area density obviously (P <0.05 vs model group).1.9 Effects on Connexin 43(Cx43) of myocardiumImmunohistochemical method. Results: The percentage of Cx43 positive expression , the area of immuno-reactant, average grey level, area density in normal group were (20.86914.035 ) %, (10.562±3.357)um2, (148.442±12.741), (0.076±0.021), which were decreased to ( 16.444±7.519 % ) , (7.075±3.497)nm2, (0.177±0.014), (0.112+0.021) respectively after MI/R injury. SSTG suppressed the degradation of Cx43, elevated the percentage of Cx43 positive expression, the area of immuno-reactant, average greyness and area density obviously (P O.05 vs model group).Indications: SSTG protected myocardium against ischemia/reperfusion injury. And its mechanism was associated with its functions of calcium antagonism and regulation effects on calcium-related signal molecular. Then we established a further study on the calcium antagonism effects of SSTG drug serum at cell level.2 The study of pharmacology and pharmic chemistry of serum withSSTGPharmacological study of SSTG drug serum was carried on cultured rat cardiac myoctyes subjected to hypoxia and reoxygenation in order to confirm a regimen of SSTG drug serum preparation. By chemical research of SSTG drug serum simultaneously, we endeavored to search the pharmacologically active material of SSTG and SSTG drug serum against MI/R injury.Methods and Results2.1 Establishment of cardiomyocytes hypoxia/reoxygenation injuryThe cardiomyocytes were deprived of oxygen and glucose to mimic hypoxia/reoxygenation injury. After reoxygenation process finished, cardiomyocytes were used to study.2.2 Pharmalological research of drug serum2.2.1 Preparing of drug serumSD rats were treated orally with normal saline, SSTG 90mg/kg, SSTG45 mg/kg, SSTG22.5 mg/kg and Diltiazem Hydrochloride 16mg/kg respectively for five successive days. 30, 60, 90, 120 minutes after last drug administration, blood samples were withdrawn from abdominal aorta. Blood serum were separated and inactivated at 56°C for 30 minutes, then filtrated to remove bacterium and saved at -20 °C for a short-period.2.2.2 Determination of drug serum concentration (volume%) in reaction system3- (4,5-dimethylthiazol-2yl ) -2,5- dipenyl terazolium bromide(MTT) staining. Results: when drug concentrations were 5%, 10%, and 20%, OD volume of cardiomyocytes were concentration-dependently increased. The OD difference between in 20%drug serum treated group and normal group has statistically significance ( P <0.05 ) , which indicated that high concentration of drug serumwas a healthy hazard to cardiomyocytes. 10% of concentration was decided to be used in experiments.2.2.3 The study of time-effect and dose-effect relationship of drug serumLDH determination in supernatant of cultured cardiomyocytes. Results: It was not the case that the higher the dose given to animal, the better the pharmacological effects. By study of time-effect, dose-effect relationship of drug serum, the best preparing regiment of SSTG drug serum was expected to reach. SSTG 90, 45, and 22.5 mg/kg were given to SD rats respectively for five successive days. Blood were taken at 30, 60, 90, 120 minutes after treatment.2.3 Chemical research of drug serumHigh performance liquid chromatography-electrochemical determination (HPLC-ECD), high performance liquid chromatography(HPLC). Results: Ginsenoside Rgl, Sahianolic acid B, Tetrahydropal matine could be traced and identified in SSTG drug serum. The peak time of these three representative components in serum was 60-120 minute.The results of pharmacological and chemical study of drug serum indicated that Ginsenoside Rgl, Salvianolic acid B, Tetrahydropal matine were representative components of pharmacologically active materials of SSTG drug serum to prevent cardiomyocytes from hypoxia/reoxgynation injury.3 Effects of SSTG drug serum on cardiomyocytes hypoxia/reoxygenation injuryMethods and Results3.1 Effects on cardiomyocytes intracellular calcium concentrationFluorescence spectrophotometrid. Results: Intracellular calcium concentration ofnormal cardiomyocytes was (242.974±19.131)nmol/L and increased to( 475.088+ 24.068 ) nmol/L after hypoxia/reoxygenation injury. SSTG drugserum suppressed the upwards of intracellular calcium concentration due to hypoxia/reoxygenation injury significantly (P <0.05 vs hypoxia/reoxygenation group).3.2 Effects on calcium inflow induced by K+Laster scanning confocal microscope. Results: With Ca2+ in extracellular fluid, Ca2+ inflowed when stimulated by K+, which leading to rapid elevation of basic intracellular calcium fluorescence intensity in singular cardiomyocyte. 100 and 200 seconds after non-drug serum treated, calcium fluorescence mostly still mount up. Suppressive rate of increasing fluorescence intensity at 100 seconds was 72.94%, and fluorescence intensity was returned almost to the non-stimulating situation after 200 seconds of SSTG drug serum treated. The effects of SSTG drug serum on the variations of Ca2+ fluorescence intensity in whole cardiomyocytes and nucleus induced by K+ stimulation were similar to those in plasm. Difference between non-drug serum and SSTG drug serum had statistical significance(/'<0.05).3.3 The changes of calcium fluorescence intensity induced by K+ in cells pretreated with drug serumLaster scanning confocal microscope. Results: Induced by K+, the increasing amplitude of calcium fluorescence intensity in myoctytoes plasma, pretreated with non-drug serum, was 886.4U. While 642U in cardiomyocytes plasma pretreated with SSTG drug serum. Then after calcium fluorescence intensity reached its peak, SSTG drug serum could suppresse the increasing of calcium fluorescence intensity in plasma at two successive phases: 0-100 and 100-200 second. The variations of calcium fluorescence intensity in whole cell and nucleus were similar to those in plasma induced by K+.3.4 Effects on increase of intracellular calcium stimulated by Thapsigargin, a kind of calcium pump inhibitorLaster scanning confocal microscope. Results: Without calcium in extracellular fliud, calcium fluorescence of plasma elevated quickly after Thapsigargin stimulation. Calcium fluorescence intensity was still increased 50 and 100 seconds after non-drug serum treated, while the calcium fluorescence intensity were inhibited by 5.64%, 76.91% at 50 and 100 second after SSTG drug serum treated respectively. The effects of SSTG drug serum on the variations of calcium fluorescence in whole cell and nucleus induced by Thapsigargin stimulation were similar to those in plasma. Difference between non-drug serum and SSTG drug serum had statistical significance (P<0.05).3.5. The variations of calcium fluorescence intensity stimulated by Thapsigargin within cardiomyocytes pretreated with drug serumLaster scanning confocal microscope. Results: Stimulated by thapsigargin, the increasing amplitude of calcium fluorescence intensity in cardiomyoctytes plasma, pretreated with non-drug serum, was 1439U. While 1370U in cardiomyocytes plasma pretreated with SSTG drug serum. The decreasing rate of the increased calcium fluorescence of SSTG drug serum treated cardiomyocytes was twice as much as that of cardiomyocytes with non-drug serum treatment. The variations of calcium fluorescence intensity in whole cell and nucleus were similar to those in plasma stimulated by Thapsigargin.3.6 Effects on the activity of calcium-transfer-related enzyme Na+-K+-ATPase, Ca2+-Mg2+-ATPaseColorimetric method. Results: the activity of Na+-K+-ATPase, Ca2+-Mg2+-ATPase of normal myocytoes membrane were (52.032±10.517)nmolPi/mgprot/hour, (41.812±2.353 ) umolPi/mgprot/hour, which decreased to ( 23.864±4.800 ) umolPi/mgprot/hour, ( 25.240±2.555 ) umolPi/mgprot/hour respectively after hypoxia and reoxygenation. SSTG drug serum enhanced the activity of Na+-K+-ATPase and Ca2+-Mg2+-ATPase significantly (P<0.05 vs hypoxia andreoxygenation goup).3.7 Effects on calcium receptor calmodulin(CaM), calcium/CaM dependent kinase 118, ymRNAReverse transcription-polymerase chain reaction (RT-PCR) . Results: The expression of CaM and CaMPK II8 were weak in normal cardiomyocytes. CaM/GAPDH, CaMPK II5/GAPDH ratio were ( 0.54±0.24 ) , ( 0.35±0.34 ) respectively. CaM and CaMPK II5 expression were enhanced after hypoxia/reoxygenation and the ratio of CaM/GAPDH and CaMPK II 5/GAPDH were increased to (2.10±0.58) , ( 2.48±0.68 ) , which differ significantly from normal cardiomyocytes(P <0.05); SSTG drug serum depressed the expression of CaM and CaMPK II8 mRNA during hypoxia/reoxygenation injury, reduced CaM/GAPDH and CaMPK II8/GAPDH ratio, which showed statistical difference compared with non-drug serum treated cardiomyocytes^ <0.05). CaMPK II ymRNA expression could not be testified.3.8 Effects on total-Nitric Oxide Synthase(T-NOS), calcium-independent indusive Nitric Oxide Synthase (iNOS) and Nitric OxideColorimetric method. Results: the T-NOS, iNOS and NO in normal group were ( 0.178±0.518 ) U/ml, ( -2.192±0.373 ) U/ml, ( 301.882±19.165 ) umol/L. After hypoxia and reoxygenation, T-NOS, NOS and NO were increased to (18.13510.603 ) U/ml, (15.266±1.195 ) U/ml, ( 559.633115.635 ) umol/L respectively, which differed significantly relative to normal group(.P <0.05). SSTG drug serum inhibitws the activity of T-NOS, iNOS and decreased the contents of NO in supernatant of cultured cardiomyocytes during hypoxia/reoxygenation significantly (P<0.05 vs hypoxia and reoxygenation group)3.9 Effects on Intercellular adhesion molecule-l(ICAM-l) expression in cardiomyocytes induced by TNF-a, a calcium related signal molecule and A23187, a calcium carrierEnzyme immunoassay (EIA). Results: ICAM-1 expression was ( 0.43±0.02 ) in normal cardiomyocytes, which was increased to (0.5010.05 ) and (0.52±0.07)respectively after TNF-a and A23187 stimulation with non-drug serum treatment. SSTG drug serum suppressed the over-expression of ICAM-1 in cardiomyocytes induce by TNF-a and A23187. The difference between SSTG and non-drug serum treated group had statistical significance (PO.05 ) .3.10 The expression of ICAM-1 on cardiomyocytes induced by TNF-a and A23187 stimulation after SSTG drug serum pretreatmentEnzyme immunoassay (EIA). Results: ICAM-1 expression was(0.52±0.08 ) in normal cardiomyocytes and were increased to (0.75±0.06) and(0.84±0.19 ) on myoctyoes pretreatmed with non-drug serum. ICAM-1expression on cardiomyocytes pretreated with SSTG drug serum were lowed thanthose on cardiomyocytes pretreated with non-drug serum. The difference betweenSSTG and non-drug serum pretreated group has statistical significance(PO.05) .Indications: SSTG drug serum protected cardiomyocytes from hypoxia/reoxygenation injury, and the mechanisms were associated with its function of calcium antagonism and regulation effects on calcium-related signal molecules.4 ConclusionsWhole experiments results indicated that the mechanisms of SSTG protecting myocardium from ischemia/reperfusion injury were associated with its effects on calcium signal, calcium-related signal molecules and myocardial junction intercellular communication. SSTG was able to reduce the size and weight of infarct area, increase the activity of SOD, decrease the contents of MDA, suppress the release of CK and LDH, low Na+, Ca2+and increase K+...
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