摘要
目的:观察加味丹参饮(JDD)预处理对大鼠心肌细胞的延迟保护作用及其细胞信号转导机理。
方法:实验分三部分。第一部分:选用出生72h的大鼠,将培养72h的大鼠心肌细胞随机分6组。空白组正常培养;空白血清对照组加50%大鼠血清培养;含药血清对照组加含50%JDD的药物血清培养,缺氧/复氧(H/R)组予缺氧3h,再给氧1h;缺氧预处理(HPC)组、加味丹参饮预处理(JDDPC)组分别给予HPC、JDDPC,24h后再给予缺氧3h,再给氧1h。实验结束,以台盼蓝染色法观察各组细胞存活率,以比色法观察乳酸脱氢酶(LDH)、肌磷酸激酶(CK)的活性。
第二部分:将培养72h的大鼠心肌细胞随机分8组,空白组、空白血清对照组、含药血清对照组、H/R组、HPC组、JDDPC组处理同第一部分;HPC+多粘菌素B(PMB)组预处理前先加入PMB,余处理同HPC组;JDDPC+PMB组预处理前先加入PMB,余处理同JDDPC组。实验结束,以台盼蓝染色法观察各组细胞存活率,以比色法观察LDH、CK的活性,以PepTag非放射性蛋白激酶试剂盒检测蛋白激酶C(PKC)。
第三部分:将培养72h的大鼠心肌细胞随机分8组,空白组、空白血清对照组、含药血清对照组、H/R组、HPC组、JDDPC组处理同第一部分;HPC+格列本脲(GLI)组预处理前先加入GLI,余处理同HPC组;JDDPC+GLI组预处理前先加入GLI,余处理同JDDPC组。实验结束,以台盼蓝染色法观察各组细胞存活率,以比色法观察LDH、CK的活性,以Fura-2/AM为指示剂检测细胞内钙离子浓度,以RT-PCR检测热休克蛋白70mRNA(HSP70mRNA)。
结果:HPC组、JDDPC组细胞存活率明显高于H/R组(P<0.01),LDH、CK明显低于H/R组(P<0.01)。HPC+PMB组、HPC+GLI组细胞存活率、LDH及CK的活性与H/R组比较无显著性差异(P>0.05)。JDDPC+PMB组、JDDPC+GLI组细胞存活率高于H/R组(P<0.01),但低于HPC、JDDPC组(P<0.01);LDH、CK的活性低于H/R组(P<0.01),但高于HPC、JDDPC组(P<0.01)。
HPC组、JDDPC组PKC的活性、HSP70mRNA表达显著高于H/R组(P<0.01),细胞内钙离子浓度显著低于H/R组(P<0.01)。HPC+PMB组PKC活性、HPC+GLI组HSP70mRNA及细胞内钙离子浓度与H/R组比较无显著性差异(P>0.05)。JDDPC+PMB组PKC活性、JDDPC+GLI组HSP70mRNA表达高于H/R组(P<0.01或P<0.05),但低于HPC、JDDPC组(P<0.01);JDDPC+GLI组细胞内钙离子浓度低于H/R组(P<0.01),但高于HPC、JDDPC组(P<0.01)。
结论:①加味丹参饮预处理具有延迟保护作用。②PKC的抑制剂PMB、ATP敏感性钾通道(K_(ATP))的抑制剂GLI能完全阻断HPC的延迟保护作用,说明PKC是HPC的信号转导通路,K_(ATP)为HPC的终末效应物,能减少钙超载,同时诱导HSP70的高表达。③PKC的抑制剂PMB、K_(ATP)的抑制剂GLI不能完全阻断加味丹参饮预处理的延迟保护作用,说明加味丹参饮预处理不完全通过PKC信号转导途径及K_(ATP)通道发挥延迟保护作用,提示加味丹参饮预处理能从多种途径、多个靶点启动心肌内源性的调控保护。
Purpose: To observe the delayed protective function of the precondition of Jiawei Danshen decoction(JDD) to the rat's myocardial cell and its cell signal transduction mechanism.
Method: The experiment contains three steps.
Step 1:
Rat that were born 72 hours ago were chosen and rat myocardial cell, which had been cultured for 72 hours, was divided at random into 6 groups. The normal group was cultured in a usual way; the serum contrast group with 50% rat serum; and the medicine-serum group with 50% medicine serum containing JDD. The Hypoxic/reoxygenation(H/R) group was made to have three-hour's hypoxia and then reoxygenated for an hour. The Hypoxic preconditioning(HPC) group and the JDD preconditioning(JDDPC) group were given HPC and JDD precondition separately. After 24 hours, both of them were made to have three-hour's hypoxia and then reoxygenated for an hour. At the end of this step, the cell survival rate of each group was identified by TB straining while LDH and CK by colorimetry.
Step 2:
The rat myocardial cell, cultured for 72 hours, was divided into 8 groups at random. The normal group, the serum contrast group, the medicine-serum contrast group, the H/R group, the HPC group and the JDDPC group were treated the same as in Step 1. The HPC+PMB group was added PMB before precondition, and its following process was the same as the HPC group. The JDDPC + PMB group was added PMB before precondition, and the following process was the same as the JDDPC group . At the end of this step, the cell survival rate of each group was identified by TB straining, LDH and CK by colormetry, and PKC by PepTag(?) non-radioactive protein kinase reagent.
Step 3:
The rat myocardial cell, cultural for 72 hours, was divided into 8 groups at random. The normal group, the serum contrast group, the medicine-serum contrast group, the H/R group, the HPC group and the JDDPC group were treated the same as in Step 1. The HPC+GLI group was added GLI before precondition, and its following process was the same as the HPC group. The JDDPC + GLI group was added GLI before precondition, and the following process was the same as the JDDPC group . At the end of this step, the cell survival rate of each group was identified by TB straining, LDH and CK by colormetry. The concentration of calcium ion inside the cell was tested by Fura-2/AM, as the indicator, and the HSP 70m RNA by RT-PCR.
Result: The cell survival rate of HPC group and JDDPC group is obviously higher than the H/R group (P<0.01), while LDH and CK lower than H/R group (P<0.01). The cell survival rate, LDH and CK of HPC+PMB group and HPC+GLI group are similar to the H/R group (P>0.05). The cell survival rate of JDDPC + PMB group and JDD PC+ GLI group are higher than the H/R group (P<0.01), but lower than HPC and the JDDPC group (P<0.01). The LDH and CK of them are lower than the H/R group (P<0.01), but higher than HPC and JDDPC group.
The PKC activity and HSP70mRNA of the HPC group and JDDPC group are obviously higher than H/R group (P<0.01), but the concentration of calcium ion inside the cell is obviously lower than the H/R group (P<0.01). The PKC activity of HPC+PMB group and the HSP70mRNA and concentration of calcium ion inside the cell of HPC+GLI group are similar to H/R group (P>0.05). PKC activity of the JDDPC + PMB group and HSP 70mRNA of JDDPC + GLI group are higher than H/R group (P<0.01 or P<0.05), but lower than HPC and the JDDPC group. The concentration of calcium ion inside the cell of JDD PC+ GLI group is lower than the H/R group (P<0.01), but higher than HPC and the JDDPC group (P<0.01).
Conclusion: The precondition of JDD is of delayed protective function. PMB, the inhibitor of PKC, and GLI, of K_(ATP), can completely stop the delayed protective function of HPC, which means PKC is the signal transduction pathway of HPC and K_(ATP) is the final effector, K_(ATP) can reduce calcium overloan and induce a high convey of HSP70. PMB, the inhibitor of PKC, and GLI, of K_(ATP), can not completely stop the delayed protective function of the precondition of JDD, which means the precondition of JDD does not completely play its delayed protective function through PKC signal transduction pathway and K_(ATP) pathway, indicating that there are many pathways of the signal transduction in JDDPC.
引文
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