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目的观察白藜三醇(RES)对快速刺激离体兔右房自由基代谢的影响,探讨其心脏保护作用的可能机制。方法 32只家兔建立离体心脏Langendorff灌流模型,电极放置右心耳处,利用BL-420S生物机能系统以电压4.0 V,频率600次/分进行刺激,随机分为对照组(不起搏),快速起搏组(RAP组),还原型烟酰胺腺嘌呤二核苷酸氧化酶(NADPH)抑制剂夹竹桃麻素组(APO组),RES组,每组8只。对照组离体灌流70 min,不起搏;RAP组离体灌流30 min,快速起搏心房40 min;APO、RES组分别离体药物处理30 min,快速起搏心房40 min。灌流结束后,留存心房肌组织。比色法测定超氧化物岐化酶(SOD)、丙二醛(MDA)、体内活性氧(ROS)、NADPH活性,HE染色检测病理变化,免疫组化半定量分析calpain1的表达,用western blot方法检测calpain1、cTnT和gp91phox蛋白的表达,逆转录聚合酶链式反应方法检测calapin1、p22phox和gp91phox的mRNA表达。结果①与对照组比较,RAP、APO、RES组MDA、ROS和NADPH的表达均增加,SOD含量减少;与RAP组比较,APO、RES组MDA、ROS和NADPH表达均降低,SOD含量增加(P均<0.05);而APO组与RES组比较,相关氧化应激指标无差异;②对照组心肌细胞排列整齐;RAP组心肌细胞排列紊乱、稀疏,组织结构破坏;APO组心肌细胞排列较紧凑;RES组心肌细胞较APO组排列稍紊乱、稀疏;③与对照组比较,RAP、APO、RES组calpain1、gp91phox蛋白及mRNA表达和p22phox的mRNA表达均增加,cTnT蛋白表达降低(P<0.05);与RAP组比较,RES、APO组calpain1、gp91phox蛋白及mRNA表达和p22phox的mRNA表达均减低,cTnT蛋白表达增加(P<0.05);但与APO组比较,RES组相关蛋白及mRNA表达无差异。结论 RES可能通过部分抑制NADPH氧化酶活性,从而减少氧自由基的生成,进而减轻快速起搏右房导致的氧化应激损伤。
Objective To investigate the effects of resveratrol (TER) on the metabolism of right atrium (HR) in vitro in rabbits and to explore its possible mechanism of cardioprotection. Methods Langendorff perfusion model of isolated heart was established in 32 rabbits. The right atrial appendage was placed on the electrode. The BL-420S biofunctional system was used to stimulate at a voltage of 4.0 V and a frequency of 600 beats per minute. The rabbits were randomly divided into control group (no pacing) Fasting group (RAP group), reducing nicotinamide adenine dinucleotide oxidase (NADPH) inhibitor apocynin group (APO group) and RES group, 8 rats in each group. The rats in control group were perfused for 70 min without pacing. The rats in RAP group were perfused for 30 min in vitro and were rapidly atrial pacing for 40 min. The rats in APO group and RES group were given 30 mg / After perfusion, retained atrial muscle tissue. The contents of superoxide dismutase (SOD), malondialdehyde (MDA), reactive oxygen species (ROS) and NADPH were measured by colorimetric method. The pathological changes were detected by HE staining. The expression of calpain1 was detected by immunohistochemistry semiquantitative analysis. Methods The expressions of calpain1, cTnT and gp91phox proteins were detected by RT - PCR and mRNA expressions of calapin1, p22phox and gp91phox were detected by reverse transcriptase - polymerase chain reaction. Results Compared with the control group, the expression of MDA, ROS and NADPH increased and the content of SOD decreased in RAP, APO and RES groups. Compared with RAP group, the expression of MDA, ROS and NADPH decreased and the content of SOD increased (P <0.05). There was no difference in oxidative stress between the APO group and the RES group. (2) The cardiomyocytes in the control group were arranged neatly. The cardiomyocytes in the RAP group were disorganized, sparse and the tissue structure was destroyed. Compared with the control group, the mRNA and protein expressions of calpain1, gp91phox and p22phox in RAP, APO and RES groups were increased and the expression of cTnT protein was decreased in RES group (P <0.05). Compared with RAP group, the mRNA and protein expressions of calpain1, gp91phox and p22phox decreased and the expression of cTnT increased (P <0.05) in RES and APO groups, but there was no difference between APO group and APO group. Conclusions RES may partially inhibit the activity of NADPH oxidase and thus reduce the production of oxygen free radicals, thereby alleviating oxidative stress injury induced by rapid pacing of the right atrium.