| Acute myocardial infarction(AMI) threatens human’s health seriously, its incidence and mortality increased year by year.The prevention and treatment are the most arduous task for medical staff and has become one of the most important public health problem in the face. At present, the main interventions of AMI are treated by percutaneous coronary intervention (PCI), drug thrombolysis and coronary artery bypass graft(CABG) surgery which rapidly opening the infarct-related coronary artery and thus reperfusion. However, we found in clinical practice that at the time of acute myocardial ischemia-reperfusion(MI/R), these treatments are accompanied by ischemia-reperfusion (I/R) injury which influence the therapeutic effect, while there may have serious adverse consequences, so the treatment strategies that reduce I/R injury are urgently needed. Effectively reduce I/R injury, preconditioning with drugs and drug intervention after reperfusion is the hotspot of cardiovascular research circles at home and abroad.Experts and scholars had invested a lot of energy to research which with little success, so whether our traditional Chinese medical which based on "righting" theory can play a more active role in inhibiting cell apoptosis in myocardial ischemia-reperfusion injury(MIRI). Salvianolic acid B(Sal-B) is a water soluble effective components of Salviamilti-orrhizaBge and has phenolic acid compounds and the strongest pharmacological activities in Salviamilti-orrhizaBge material composition.In recently years, Experiment research Sal-B can reduce the toxicity of oxygen free radicals in myocardial cells and inflammation. With protecting mitochondrial function, Sal-B maybe reduce or inhibit MIRI. At present, the mechanism of Sal-B on myocardial protection still has a lot of blind spots and need to be explored constantly.MIRI is outcome of multiple factors. Following the deep research in MIRI, it is found that PI3K-Akt signaling pathways play a important role in the process of MIRI, PI3K-Akt signaling pathway may play an important role with actively raise protection mechanisms in cells than other passive way. This topic observed effects of Sal-B on MI/R with many angles and different levels. At the molecular level to observe Sal-B on myocardial enzymology, oxygen free radical and so on. Observe effect on cardiac myocyte apoptosis at a cellular level. And from the level of organs to observe the influence of the heart whole, at the same time in functional genomics research Sal-B of apoptosis gene and genome level of protein expression. This study discusses the following aspects by experimental data. Sal-B has heart mitochondrial function on ischemia-reperfusion and the effect of myocardial cell apoptosis and inhibition of myocardial cell apoptosis through PI3K-Akt signaling pathways. The PI3K-Akt signaling pathway is possible targets of Sal-B. The mechanism of action of the drug may be expounded with cytokine signaling pathways. Traditional Chinese medicine maybe provide theoretical basis and new ideas for treatment of myocardial ischemia-reperfusion injury. Part1The mechanism and effects of Sal-B on myocardial ischemia-reperfusion injury in ratsObjective:Establishment of myocardial I/R injury models in rats, observe the effect of Sal-B on cardium of myocardial enzymology and myocardial infarction area in MI/R. To observe whether the mechanism of Sal-B to protection of myocardial cell is through PI3K-Akt signaling pathway, and the target of Sal-B in the PI3K-Akt signaling pathway.Methods:1. Construct models of myocardial I/R injury by ligation of the left circumflex coronary artery. With ST-segment elevation or depression, local tissue hyperemia and ST-segment resolution when reperfusion, to judge the success of ligation.2. Animal grouping and treatmentThe animals were randomly divided into five groups of10rats in each group.1) Sham operation group (sham group):the left circumflex coronary artery of experimental animals was only threaded about2mm under the roots but not ligated.2) Myocardial ischemia reperfusion group (MI/R):ligation after30min reperfusion for60min.3) Sal-B low dose group (Sal-B20mg/kg group):ligated for30min, under reperfusion for60min, at the same time gives Sal-B20mg/kg via the jugular vein.4) Sal-B high dose group (Sal-B60mg/kg group):ligated for30min, under reperfusion for60min, at the same time gives Sal-B60mg/kg via the jugular vein.5) Sal-B high dose group+PI3K/Akt inhibitors LY294002(Sal-B60mg/kg+LY group):ligated for30min, under reperfusion for60min, at the same time gives Sal-B60mg/kg and LY294002(0.3mg/kg) via the jugular vein.After the experiment, blood samples and heart tissues were collected for index detection.3. To analyzer the level of the plasma cTnI, CK-MB with automatic biochemical analyzer.4. Detection of myocardial infarction area.5. To observe the pathologic morphology in myocardial tissue by HE staining. Results:1. Effect on cardiac enzymes (cTnl, CK-MB) content in plasma The experimental results show that, compared with the Sham group, plasma cTnI and CK-MB content in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group, Sal B60mg/kg+LY group increased significantly (P<0.01). Sal-B20mg/kg group, Sal-B60mg/kg group compared with MI/R group, the plasma cTnI, concentration of CK-MB were lower (P<0.01). Sal-B60mg/kg group compared with Sal-B20mg/kg group, the plasma cTnI, CK-MB concentration decreased (P<0.01). Sal-B60mg/kg+LY group compared with Sal-B60mg/kg group, The plasma cTnl and CK-MB content were significantly lower(P<0.01).2. Comparison of myocardial infarction area in each group Compared with Sham group (15.2±3.3)%, myocardial infarction area in MI/R group (43.3±8.6)%was significantly increased (P<0.01). Compared with MI/R group, Sal-B20mg/kg group (33.5±7.1)%, Sal-B60mg/kg group (20.1±6.5)%myocardial infarction area were significantly lower (P<0.01). Compared with Sal-B20mg/kg group, myocardial infarction area in Sal-B60mg/kg group was reduced, compared with Sal-B60mg/kg group, the myocardial infarction area in Sal-B60mg/kg+LY group was increased (P<0.01).3. Pathological morphology of myocardial tissue in each group In Sham group, myocardial fibers was neat without inflammatory cell infiltration. Myocardial fibers was swelling and disorder, a large number of inflammatory cells infiltration appeared in myocardial interstitial in MI/R group, Sal-B20mg/kg and Sal-B60mg/kg+LY group. It was the most serious in MI/R group. Myocardial cell nuclei in Sal-B60mg/kg group was uniform and mild myocardial fibers swelling, occasional fibroblast cells and inflammatory cells infiltration, comparison with MI/R group, Sal-B20mg/kg group and Sal-B60mg/kg+LY group, myocardial inflammation and cardiac muscle fiber damage and focal myocardial necrosis were reduced significantly.Conclusions:1. Sal-B has myocardial the protection of cardiac function and can reduce the levels of CK-MB and cTNI and the myocardial infarction area in myocardial ischemia reperfusion.2. Ischemia-reperfusion myocardium protection of Sal-B was related to the dosage. Effect in high dose of Sal-B was more significant.3. The protective effect of Sal-B against myocardial ischemia-reperfusion may be through the PI3K-Akt signal pathway. Part2Studys of Sal-B intervention of cell apoptosis on myocardial ischemia-reperfusion in rats through PI3K-Akt signaling pathwaysObjective:To observe myocardial cell apoptosis of ischemia-reperfusion myocardium with Sal-B and the effect of PI3K-Akt signaling pathway in the experiment.Methods:1. Tunel was used to detect myocardial apoptosis.2. Immunohistochemical staining with Bax, Bcl-2protein expression.3. Western blot was used to determination of levels of caspase-3protein in myocardial tissue, expression of signaling proteins of total Akt (T-Akt) and phosphorylated Akt (p-Akt).Results:1. The change of myocardial apoptosis index in each group. Myocardial apoptosis index of each group:the Sham group (1.12±0.02), the MI/R group (36.12±4.37), Sal-B20mg/kg group (22.02±3.56), Sal-B60mg/kg (12.57±2.93), Sal-B60mg/kg+LY group (35.12±1.03). Compared with the Sham group, the apoptosis index in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group and Sal-B60mg/kg+LY group were increased (P<0.01). Compared with MI/R group, the myocardial cell apoptosis index in Sal-B20mg/kg group, Sal-B60mg/kg group were lower (P<0.01). Compared with Sal-B20mg/kg group and Sal-B60mg/kg+LY group, myocardial apoptosis in Sal-B60mg/kg group were significantly lower (P<0.01).2. The changes of Bcl-2and Bax protein expression in each group Compared with the Sham group, Bax protein expression levels in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group, Sal-B60mg/kg+LY group were increased significantly. Bax protein expression levels were lower (P<0.01). Compared with MI/R group, Bax protein expression levels in Sal-B20mg/kg group, Sal-B60mg/kg group were reduced, Bcl-2expression were significantly higher (P<0.01). Compared with Sal-B20mg/kg group, Bax protein expression level in Sal-B60mg/kg group was reduced, and Bcl-2expression was higher significantly (P<0.01). and Compared to Sal-B60mg/kg group, Bcl-2expression in Sal-B60mg/kg+LY was reduced, Bax protein expression was significantly higher (P<0.01).3. The changes of Caspase-3protein expression in each group Compared with the Sham group, Caspase-3protein expression levels in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group, Sal-B60mg/kg+LY group were increased significantly(P<0.01). Compared with MI/R group, caspase-3protein expression levels in Sal-B20mg/kg group, Sal-B60mg/kg group were reduced (P<0.01). Compared with Sal-B20mg/kg group, Caspase-3protein expression level in Sal-B60mg/kg group was reduced(P<0.01). Compared to Sal-B60mg/kg group, Caspase-3protein expression in Sal-B60mg/kg+LY was significantly higher (P<0.01).4. The changes of total Akt protein and p-Akt protein expression in each group According to the experimental results, the total Akt between groups has no significant difference. Compared with the Sham group, p-Akt protein expression levels in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group, Sal-B60mg/kg+LY group were reduced (P<0.01). Compared with MI/R group, p-Akt protein expression levels in Sal-B20mg/kg group, Sal-B60mg/kg group were increased (P<0.01). Compared with Sal-B20mg/kg group, p-Akt protein expression level in Sal-B60mg/kg group was increased (P<0.01). Compared to Sal-B60mg/kg group, p-Akt protein expression in Sal-B60mg/kg+LY was reduced significantly (P<0.01).Conclusions:1. Sal-B can inhibit myocardial apoptosis and protect myocardial function during myocardial ischemia reperfusion.2. Sal-B regulate the expression of apoptosis related gene, by increasing the Bal-2protein expression, reduce the levels of Bax protein and caspase-3protein through PI3K-Akt signaling pathways Part3Studys of Sal-B to protect cardiomyocyte mitochondrial function in ischemia reperfusion through PI3K-Akt signaling pathwayObjective:To observe the levels of the mitochondrial ROS and the levels of the plasma T-SOD and MDA with Sal-B and mitochondria change in myocardial ischemia-reperfusion injury through PI3K-Akt signaling pathway.Methods:1. The changes of myocardial cell mitochondrial ROS content by laser confocal detection.2. The changes of the plasma T-SOD and MDA concentration with enzyme-linked immunosorbent.3. To observe the changes of myocardial ultrastructure by Electron microscope.Results:1. The changes of myocardial cell mitochondrial ROS content in each group The ROS content in MI/R group was increased. Compared with MI/R group, ROS fluorescence in Sal-B20mg/kg group, Sal-B60mg/kg ere decreased(P<0.01). Sal-B can reduce ROS generation under the condition of MI/R. Compared with Sal-B20mg/kg group and Sal-B60mg/kg+LY group, ROS generation in Sal-B60mg/kg group was decreased (P<0.01).2. The changes of plasma T-SOD and MDA content in each group Compared with the Sham group,the plasma T-SOD content in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group, Sal-B60mg/kg+LY group were decreased and the levels of MDA were increased significantly (P<0.01). Compared with MI/R group, the plasma MDA contenin in Sal-B20mg/kg group, Sal-B60mg/kg group were reduced and the plasma T-SOD content was increased significantly (P<0.01). Compared with Sal-B20mg/kg group and Sal-B60mg/kg+LY group, the plasma MDA content in Sal-B60mg/kg group was decreased and the plasma T-SOD content was increased significantly (P<0.01).3. the changes of myocardial cell ultrastructure in each group Sham group:the myocardial cell morphology and the muscle membrane were integrity; Myocardial mitochondria structure was complete; Normal myocardial fibers arranged orderly, sarcomere periodic structure, light and shade shows clear, intercalated disc without expansion, Z line without deformation, muscle wire was complete.MI/R group:the myocardial cells were swelling significantly with muscle membrane rupture and defect, nucleus shrinkage, nuclear membrane fuzzy; myocardial mitochondrial damage evident vacuolization, and even dissolve; myocardial fiber disarray, the stromal cells in a large number of collagen fibers, the muscle fibers visible vacuoles of varying sizes, sarcoplasmic reticulum; sarcomere periodic structure was destroyed, with no clear shading, Z line deformation, diatortion, filaments break; local expansion intercalated disk, its sides into filamentous sarcoplasmic low electron density areas.Sal-B20mg/kg group:slight swelling, muscle fibers neatly arranged and dense collagen fibers were significantly higher, mainly in the stromal cells in myocardial mitochondrial swelling and damage was found more and accompanied by the phenomenon of convergence, part of the arrangement more chaos, fracture, compared with the control group, the situation improved slightly. Compared with MI/R group, there were improved.Sal-B60mg/kg group:most of the nuclei was complete and clear. Only a small portion of cell nuclear membrane was uncomplete, the structure of mitochondria basic were finded normal and orderly rows. The mitochondria had formed the part of fracture fragments and case. Myocardial fiber lined up, cells in the interstitial collagen fiber content was less. Comparison with Sal-B60mg/kg group, the change of ultrastructure in Sal-B60mg/kg+LY was bad.Conclusions:1. Sal-B can reduce oxygen free radicals, inhibit oxidative stress reaction, play to the role of protect the heart and reduce myocardial ischemia-reperfusion injury.2Sal-B inhibit oxidative stress and protect the function of mitochondrial function through the PI3K-Akt signaling pathways. Part4Effect and mechanism of Sal-B on the level of the plasma sICAM-1, sVCAM-1and NF-κBp65expression in myocardial ischemia-reperfusion Objective:To observe the plasma sVCAM-1and sICAM-1level and the NF-κB p65expression with Sal-B. To discusses the role of PI3K-Akt signaling pathways in myocardial ischemia-reperfusion.Methods:1. To detect the plasma sICAM-1concentration with ELISA.2. To detect the plasma sVCAM-1concentration with ELISA.3. western blot was used to detection of myocardial tissue content of NF-κB p65. Results:1. the change of the concentration of plasma sICAM-1in the each group The experimental results show that, compared with the Sham group, the concentration of plasma sICAM-1in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group, Sal-B60mg/kg+LY group were increased significantly (P<0.01). Sal-B20mg/kg group and Sal-B60mg/kg group compared with MI/R group, the concentration of plasma sICAM-1were lower (P<0.01). Sal-B60mg/kg group compared with Sal-B20mg/kg group, the concentration of plasma sICAM-1was decreased (P<0.01), Sal-B60mg/kg+LY group compared with Sal-B60mg/kg group, the concentration of plasma sICAM-1were significantly lower(P<0.01).2. the change of the concentration of plasma sVCAM-1in each group Compared with the Sham group, the concentration of plasma sVCAM-1in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group, Sal-B60mg/kg+LY group were increased significantly (P<0.01). Compared with MI/R group, the concentration of plasma sVCAM-1in Sal-B20mg/kg group and Sal-B60mg/kg group were lower (P<0.01). Sal-B60mg/kg group compared with Sal-B20mg/kg group, the concentration of plasma sVCAM-1was decreased (P<0.01), Sal-B60mg/kg+LY group compared with Sal-B60mg/kg group, the concentration of plasma sVCAM-1were significantly lower(P<0.01).3. the changes of myocardial tissue NF-κBp65content in each group Compared with the Sham group, NF-κBp65expression in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group, Sal-B60mg/kg+LY group were increased significantly (P<0.01). Compared with MI/R group, NF-KBp65expression in Sal-B20mg/kg, Sal-B60mg/kg group were lower (P<0.01). Compared with Sal-B20mg/kg and Sal-B60mg/kg+LY group, NF-κBp65expression Sal-B60mg/kg group was lower (P<0.01).Conclusions:1. Sal-B had reduced the levels of sICAM-1and sVCAM-1, improved myocardial microcirculation, play myocardial protection in ischemia-reperfusion through PI3K-Akt signaling pathways.2. Sal-B inhibit inflammation, improve the effect of myocardial microcirculation by the NF-kB signaling pathways. Part5Effect and mechanism of Sal-B on calcium sensitive receptor in myocardial ischemia reperfusionObjective:To observe the expression of calcium sensitive receptors with Sal-B in myocardial ischemia-reperfusion injury. To discuss the effects of PI3K-Akt signaling pathways on calcium sensitive receptor during myocardial ischemia reperfusion.Methods:1. RT-PCR was used to the detection of myocardial tissue CaSRmRNA transcription.2, CaSR protein expression with immunohistochemical methods.Results:1. The changes of CaSRmRNA expression on myocardial tissue in each group The experiment results show that compared with the Sham group, CaSRmRNA expression in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group, Sal-B60mg/kg+LY group were increased(P<0.01), CaSRmRNA expression was highest in MI/R group. Compared with MI/R group, CaSRmRNA expression in Sal-B20mg/kg group, Sal-B60mg/kg group were descreased (P<0.05). Compared with group Sal-B60mg/kg, the levels of CaSRmRNA expression in Sal-B60mg/kg+LY group was increased (P<0.05).2. The changes of CaSR protein expression with immunohistochemical methods in each group Compared with the Sham group, CaSR protein expression in MI/R group, Sal-B20mg/kg group, Sal-B60mg/kg group, Sal-B60mg/kg+LY group were increased(P<0.01), the levels of CaSR protein expression was highest in MI/R group. Compared with MI/R group, the levels of CaSR protein expression in Sal-B20mg/kg group and Sal-B60mg/kg group were descreased (P<0.05). Compared with group Sal-B60mg/kg, the level of CaSR protein expression in Sal-B60mg/kg+LY group was increased (P<0.05).Conclusions:1. The level of calcium sensitive receptor expression in Myocardial tissue was increased. Calcium sensitive receptors was involved in MIRI.3. Sal-B can reduce the expression of CaSR in myocardial cell in myocardial ischemia reperfusion injury4. The study for the traditional medicine was the first time to calcium sensitive receptor. It was more in-depth study of traditional medicine mechanism. |
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