| Main components of Salivae Miltiorrhizae Liguspyragine Hydrochloride and Glucose Injection(SGI) are Salvia miltiorrhiza and ligustrazine hydrochloride. It is mainly used for the treatment of occlusive cerebrovascular diseases and other ischemic vascular diseases in clinical practice. At present, there are few reports about the protective effect of SGI on AMI and the risk assessment of SGI combined with commonly used injections.In order to obtain more comprehensive drug information, we carried out three parts studies of SGI from the perspectives of efficiency and safety.In Part 1,we studied the protective effects of SGI on AMI model induced by isoproterenol in rats. Purpose: This experiment used isoproterenol to establish a rat model of AMI. Then we evaluated preventive and therapeutic effects of SGI on AMI in rats and explored its possible mechanism through the observation of heart infarction size, electrocardiogram, cardiac function, myocardial enzyme detection and heart histomorphology. Method: 75 male SD rats were randomly divided into 6 groups: control group, model group, Panax Notoginsenosidum(PNS) group, SGI low-dose(L) group, SGI medium-dose(M) group and SGI high-dose(H) group. Animals were administered intravenously once a day, the control group and the model group were given an equal volume of 5% Glucose Injection, for 7 consecutive days. Each group was detected ECG before the model(0 min), and were administrated with 3mg/kg of isoproterenol(ISO) on 6th and 7th day after drugs treatment, once a day for 2 days, to establish the model of AMI. And indexes of cardiac function were evaluated after injection of isoprenaline on the 7th day. TTC staining was used to determine the area of myocardial infarction, H&E staining was used to observe the pathological changes of myocardial cell. We also determined activities of enzymes(BNP, CK, LDH and c Tn-I) in serum. Results: 1. Compared with the control group, the ECG ST segments in the model group elevated significantly and infarct size increased significantly(P<0.05). Indexes of cardic function, like cardiac output(CO), ejection fraction(EF), left axis shortening(LAS), left ventricular diastolic end diameter(LVDED), left ventricular end systolic diameter(LVESD), left ventricular diastolic end volume(LVDEV) and left ventricular end systolic volume(LVESV), in the model group were significantly different from the control group(P<0.01). Compared with the control group, the activities of CK, LDH, c Tn-I and BNP in model group were significantly different(P<0.01). These results suggested that the AMI model in rats could be successfully prepared by injecting ISO(3mg/kg) for 2 consecutive days. 2. Compared with model group, ECG ST segment of SGI groups declined. Among them, ECG ST segment of the SGI(H) group and SGI(M) group basically returned to normal level. 3. Infarct size of SGI groups and PNS group were significantly smaller than that of the model group(P<0.01 or P<0.05). 4. Activities of serum CK, LDH, c Tn-I and BNP in SGI(H) group were significantly lower than those in model group(P<0.01). Compared with model group, the serum levels of CK, c Tn-I and BNP in SGI(M) group were lower significantly and so as the serum levels of CK in SGI(L) group(P<0.05). 5. The H&E staining of myocardial tissue showed that the myocardial fibers of rats in the control group were in an orderly arrangement, and there were no atrophy, hypertrophy, necrotic foci, inflammatory cell infiltration or any other pathological changes. However, the myocardial damages were evident in the model group. The myocardial fibers were in a disorderly arrangement. Myocardial interstitial edema was found, and the tissue space was significantly widened. Neutrophil infiltration was also present. Many myocardial fibers were swollen, lysed, or ruptured, and the cross striation was vague or even disappeared. The myocardial cells underwent vacuolar degeneration. The myocardial damages in SGI and PNS groups were milder than those in the model group. Local mild swelling was found, mild interstitial edema was present, and the tissue space was slightly widened. A few inflammatory cells were infiltrated, and vacuolar degeneration could be observed sporadically. 6. Indexes of cardic function(CO, EF, LAS, LVDED, LVESD, LVDEV and LVESV) in SGI groups and PNS group were significantly different from those in the model group(P < 0.05 or P < 0.01).In Part 2, we studied the protective effect of SGI on doxorubicin induced myocardial injury in H9c2 cells. Purpose: To study the protective effect of SGI on doxorubicin(DOX) induced myocardial injury in H9c2 Cells and to explore its mechanism. Method: H9c2 cells were cultured in vitro. Cytotoxicity was detected by MTT method and intracellular calcium concentration was detected by fluorescence probe method. Results: 1. DOX can lead to the decrease of cell viability and the increase of intracellular calcium concentration. 2. SGI intervention could significantly alleviate the intracellular calcium overload(P < 0.01), and there is a certain improvement in cell viability. Antioxidant NAC intervention also has a similar effect.In Part 3, we studied combination risk of SGI combined with six injections based on isothermal titration calorimetry. Purpose: This article is to evaluate combination risk of SGI with six injections, which are used in combination with SGI in clinical practice by isothermal titration calorimetry(ITC). Method:Isothermal titration calorimetry(ITC) was used to evaluate combination risk of SGI and six clinical commonly used injections as follows: Levofloxacin and Sodium Chloride Injection(LSI), Cefoperazone Sodium and Sulbactam Sodium for Injection(CSI), Alprostadil injection(AI), Dextran 40 Glucose Injection(DI), Ossotide Injection(OI), Glycerol Fructose and Sodium Chloride Injection(GSI). Gibbs free energy(?G), enthalpy(?H) and entropy(?S) were used to decide reaction types of colliquefaction processes of different injections. We also carried out determinations of high performance liquid chromatography(HPLC), appearance, p H, insoluble particles and osmotic pressure, as supporting evidences.Results: 1. ITC results showed that in colliquefaction processes of SGI and LSI, CSI, AI, OI or GSI, ?G<0, so the reactions were spontaneous. Among them, the reactions of CSI, AI and SGI are enthalpy driven ones(│?H│<T│?S│), which means that the chemical reactions happened and the active ingredients might be changed. Besides, the reactions of LSI, OI, GSI and SGI are entropy driven ones(│?H│>T│?S│), which means physical reaction played a leading role and physical properties such as solubility of active ingredients might be changed. However, in colliquefaction process of SGI and DI, ?G>0, so the reaction was non-spontaneous and the mixture system was quite stable. 2. HPLC assay showed that the absorption peak area of GSI was changed after mixing with SGI. 3. Appearance and p H assay showed that no obvious changes had happened during 4h after combined injections were mixed with SGI. 4. Insoluble particle assay showed that the total number of insoluble particles of LSI and CSI and SGI has increased the trend during 4h after mixed SGI and LSI overran the limit of Ch.P. 5. Osmotic pressure assay showed that LSI, CSI, AI, DI, OI, GSI had no obvious change during 4h after mixed with SGI. Conclusions and prompts: 1. SGI could reduce myocardial infarct size, content of myocardial enzymes(CK, LDH, c Tn I and BNP and protect myocardial cells in AMI rats, reduce the, it could also inhibit myocardial cells injury and promote cardic function back to normal. Therefore, SGI has a good protective effect on isoproterenol induced AMI rats. 2. SGI can significantly alleviate the intracellular calcium overload induced by DOX, and medium-dose(0.28μmol/L) and high-dose(0.48μmol/L) had a better effect. It was suggested that the protective effect of SGI might be related to the decrease of intracellular calcium concentration. 3. Spontaneous reaction can occur in mixture of SGI and LSI, CSI, AI, OI, GSI, which meant that there exists combination risk and clinical use should be cautious. While spontaneous reaction can not occur in mixture of SGI and DI, which meant that combination risk is low.To sum up, this study conducted relevant experiments of SGI revaluation after being listed. The effectiveness researches found that SGI could reduce myocardial infarct size, content of myocardial enzymes(CK, LDH, c Tn I and BNP and protect myocardial cells in AMI rats, reduce the, it could also inhibit myocardial cells injury and promote cardic function back to normal. Therefore, SGI has a good protective effect on AMI. In addition, SGI can significantly reduce the intracellular calcium overload induced by DOX, indicating that the myocardial protective effect of SGI may be related to the decrease of calcium overload in cardiac muscle cells. Besides, the safety research found that spontaneous reaction can occur in mixture of SGI and LSI, CSI, AI, OI, GSI, which meant that there exists combination risk in clinical practice. But DI is just the opposite. And test results of p H value, insoluble particles and osmotic pressure also confirmed this point to varying degrees, suggesting that SGI used in combination with LSI, CSI, AI, OI, GSI should be cautious in clinical application. |
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