参麦注射液及其活性成分对缺血再灌注后心肌细胞的影响
发布时间：2019-04-01 07:15
【摘要】：目的： 1、明确参麦注射液改善心肌缺血再灌注损伤是否通过调节钙调蛋白Phospholamban(PLB)的表达和磷酸化水平来改善细胞内钙浓度的机理；2、探讨人参皂苷对缺血心脏的心功能的改善作用及可能的钙相关的机理；3、比较参麦注射液复方制剂是否优于人参皂苷单纯制剂。 方法： 利用新生大鼠原代心肌细胞建立缺氧/复氧(I/R)模型,实验分为正常培养组(N)、正常培养+参麦组(N+SM)、正常培养+人参总皂苷组(N+TSPG)、正常培养+人参皂苷Rgl组(N+Rgl)、缺氧/复氧组(I/R)、缺氧/复氧组+参麦组(I/R+SM)、缺氧/复氧组+人参总皂苷组(I/R+TSPG)、缺氧/复氧+人参皂苷Rgl组(I/R+Rg1),采用流式细胞仪测定凋亡细胞百分率、细胞内钙的平均荧光值,实时荧光定量聚合酶式反应(RT-PCR)法测定心肌细胞肌浆网钙泵(SERCA)和PLB的mRNA表达量,Western Blot法测定心肌细胞SERCA和PLB、磷酸化PLB (p-PLB)的蛋白表达量。 结果： 心肌细胞缺氧／复氧后,PLBmRNA及蛋白表达无显著变化,p-PLB蛋白表达和SERCA的mRNA及蛋白表达均有不同程度的下降,而I/R+Rgl组较I/R组上述变化无明显差异(P0.05),I/R+SM组p-PLB蛋白表达、p-PLB/PLB、SERCA mRNA及蛋白表达均明显升高(P0.01),PLB/SERCA明显下降(P0.01),I/R+TSPG组SERCA mRNA及蛋白表达均明显升高(P0.01),PLB/SERCA明显下降(P0.05),p-PLB蛋白表达、p-PLB/PLB无明显差异(P0.05)。I/R+SM组较I/R+TSPG组SERCA mRNA及蛋白表达明显升高(P0.05)、PLB/SERCA明显降低(P0.05)。I/R+SM组较I／R组凋亡细胞百分率均有不同程度下降(P0.05),I/R+SM组较I/R组细胞内钙的平均荧光值均有不同程度的降低(P0.01),I/R+TSPG组、I/R+Rgl组细胞凋亡率变化无明显差异(P0.05),I/R+TSPG组细胞内钙离子浓度较I/R升高(P0.05),I/R+Rgl无明显变化。 结论： 1、参麦注射液能通过调节钙调蛋白PLB的磷酸化水平来改善细胞内钙浓度的机理来改善心肌缺血再灌注损伤；2、人参总皂苷可抑制缺血再灌注后心肌细胞内钙离子浓度,进而改善心功能,此过程可能跟促进SERCA表达有关,但不通过钙调蛋白PLB磷酸化途径；3、参麦注射液复方制剂可能优于人参皂苷单纯制剂。
[Abstract]:Objective: 1. To determine whether Shenmai injection can improve the intracellular calcium concentration by regulating the expression and phosphorylation of calmodulin Phospholamban (PLB) in myocardial ischemia-reperfusion injury. (2) to investigate the effects of ginsenoside on cardiac function of ischemic heart and the possible calcium-related mechanism; (3) to compare whether the compound preparation of Shenmai injection is superior to the simple preparation of ginsenoside. Methods: neonatal rat cardiomyocytes were used to establish hypoxia / reoxygenation (I / R) model. The model was divided into normal culture group (N), normal culture group (N SM), normal cultured ginseng total saponin group (N TSPG),). Normal cultured ginsenoside Rgl group (N Rgl), hypoxia / reoxygenation group, hypoxia / reoxygenation group ginseng wheat group (I / R SM), hypoxia / reoxygenation group Ginseng total saponin group (I / R TSPG),) In hypoxia / reoxygenation ginsenoside Rgl group (I / R Rg1), the percentage of apoptotic cells and the average fluorescent value of intracellular calcium were measured by flow cytometry. The mRNA expression of sarcoplasmic reticulum calcium pump (SERCA) and PLB in cardiomyocytes was measured by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). The protein expressions of SERCA and PLB, phosphorylated PLB (p-PLB) in cardiomyocytes were measured by, Western Blot method. Results: after hypoxia / reoxygenation, the expression of PLBmRNA and protein did not change significantly, but the expression of p-PLB protein and mRNA and protein of SERCA decreased to some extent. However, there was no significant difference between the two groups (P0.05). The expression of p-PLB protein, the expression of p-PLB, mRNA and protein in SM group were significantly increased (P0.01), and the expression of PLB/SERCA was significantly decreased (P0.01), but the expression of p-PLB protein in group I / R Rgl was significantly higher than that in group I / R (P < 0.01). The expression of SERCA mRNA and protein in TSPG group was significantly higher than that in control group (P0.01), while the expression of PLB/SERCA was significantly decreased (P0.05), and the expression of p-PLB protein was significantly decreased. There was no significant difference in p-PLB/PLB (P0.05). The expression of SERCA mRNA and protein in I / R SM group was significantly higher than that in I / R TSPG group (P 0.05). The percentage of apoptotic cells in PLB/SERCA SM group was significantly lower than that in I R group (P0.05), and the percentage of apoptotic cells was significantly decreased (P0.05). The average intracellular Ca ~ (2 +) fluorescence value in I / R SM group was significantly lower than that in I / R group (P0.01). There was no significant difference in apoptosis rate between I / R TSPG group and I / R Rgl group (P 0.05). The intracellular Ca ~ (2 +) concentration in I / R TSPG group was higher than that in I / R group (P 0.05), but there was no significant change in I / R Rgl. Conclusion: 1 Shenmai injection can improve the myocardial ischemia-reperfusion injury by regulating the phosphorylation level of calmodulin PLB to improve the intracellular calcium concentration. 2. Ginsenoside could inhibit intracellular calcium concentration and improve cardiac function after ischemia-reperfusion, which might be related to the promotion of SERCA expression, but not through calmodulin PLB phosphorylation pathway. 3. The compound preparation of Shenmai injection may be superior to the simple preparation of ginsenoside.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R542.2
本文编号：2451352
[Abstract]:Objective: 1. To determine whether Shenmai injection can improve the intracellular calcium concentration by regulating the expression and phosphorylation of calmodulin Phospholamban (PLB) in myocardial ischemia-reperfusion injury. (2) to investigate the effects of ginsenoside on cardiac function of ischemic heart and the possible calcium-related mechanism; (3) to compare whether the compound preparation of Shenmai injection is superior to the simple preparation of ginsenoside. Methods: neonatal rat cardiomyocytes were used to establish hypoxia / reoxygenation (I / R) model. The model was divided into normal culture group (N), normal culture group (N SM), normal cultured ginseng total saponin group (N TSPG),). Normal cultured ginsenoside Rgl group (N Rgl), hypoxia / reoxygenation group, hypoxia / reoxygenation group ginseng wheat group (I / R SM), hypoxia / reoxygenation group Ginseng total saponin group (I / R TSPG),) In hypoxia / reoxygenation ginsenoside Rgl group (I / R Rg1), the percentage of apoptotic cells and the average fluorescent value of intracellular calcium were measured by flow cytometry. The mRNA expression of sarcoplasmic reticulum calcium pump (SERCA) and PLB in cardiomyocytes was measured by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). The protein expressions of SERCA and PLB, phosphorylated PLB (p-PLB) in cardiomyocytes were measured by, Western Blot method. Results: after hypoxia / reoxygenation, the expression of PLBmRNA and protein did not change significantly, but the expression of p-PLB protein and mRNA and protein of SERCA decreased to some extent. However, there was no significant difference between the two groups (P0.05). The expression of p-PLB protein, the expression of p-PLB, mRNA and protein in SM group were significantly increased (P0.01), and the expression of PLB/SERCA was significantly decreased (P0.01), but the expression of p-PLB protein in group I / R Rgl was significantly higher than that in group I / R (P < 0.01). The expression of SERCA mRNA and protein in TSPG group was significantly higher than that in control group (P0.01), while the expression of PLB/SERCA was significantly decreased (P0.05), and the expression of p-PLB protein was significantly decreased. There was no significant difference in p-PLB/PLB (P0.05). The expression of SERCA mRNA and protein in I / R SM group was significantly higher than that in I / R TSPG group (P 0.05). The percentage of apoptotic cells in PLB/SERCA SM group was significantly lower than that in I R group (P0.05), and the percentage of apoptotic cells was significantly decreased (P0.05). The average intracellular Ca ~ (2 +) fluorescence value in I / R SM group was significantly lower than that in I / R group (P0.01). There was no significant difference in apoptosis rate between I / R TSPG group and I / R Rgl group (P 0.05). The intracellular Ca ~ (2 +) concentration in I / R TSPG group was higher than that in I / R group (P 0.05), but there was no significant change in I / R Rgl. Conclusion: 1 Shenmai injection can improve the myocardial ischemia-reperfusion injury by regulating the phosphorylation level of calmodulin PLB to improve the intracellular calcium concentration. 2. Ginsenoside could inhibit intracellular calcium concentration and improve cardiac function after ischemia-reperfusion, which might be related to the promotion of SERCA expression, but not through calmodulin PLB phosphorylation pathway. 3. The compound preparation of Shenmai injection may be superior to the simple preparation of ginsenoside.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R542.2
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