异丙酚对糖尿病大鼠心肌缺血再灌注损伤的作用及其机制的研究
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摘要
近年来,糖尿病的发病率呈不断上升趋势,而心血管疾病是糖尿病最常见的并发症和死亡原因。心肌缺血再灌注(ischemic reperfusion, I/R)损伤是围术期面临的一种常见的病理生理变化,是冠脉溶栓术、经皮冠状动脉腔内成形术和冠状动脉旁路移植术后早期并发症的主要原因。因此,如何减轻或避免糖尿病病人的心肌缺血再灌注损伤已成为基础和临床研究的热点。异丙酚是一种非巴比妥类静脉麻醉药,具有快速、可靠的镇静催眠作用,临床广泛应用于麻醉诱导、麻醉维持和ICU镇静。据报道,异丙酚不仅化学结构类似于内源性抗氧化剂维生素E和外源性抗氧化剂丁羟基甲苯(BHT),具有清除自由基作用,而且具有钙通道阻滞作用。本实验室前期研究表明,异丙酚能够减轻心肌缺血再灌注损伤,但糖尿病心肌组织存在代谢紊乱,钙平衡调节异常,心肌肥大和纤维化等病理生理改变,有可能改变心肌对缺血再灌注损伤的反应性,一些对正常机体具有保护作用的措施在糖尿病机体上作用减弱或消失。异丙酚对糖尿病缺血再灌注心肌是否具有保护作用有待于探讨。因此,本研究通过建立糖尿病大鼠心肌缺血再灌注损伤模型,从氧化应激、炎性细胞因子、细胞凋亡、线粒体功能等方面,探讨异丙酚对糖尿病心肌缺血再灌注损伤的保护作用及机制,为其在临床的广泛应用提供理论基础和实验依据。
第一部分异丙酚对糖尿病大鼠缺血再灌注心肌组织氧化应激的影响
目的:观察异丙酚对糖尿病大鼠心肌缺血再灌注损伤的影响并从氧化应激方面探讨其作用机制。
方法:健康雄性SD大鼠(250-300g)100只,随机分为糖尿病组(diabetic group, D)76只和非糖尿病组(non-diabetic group, N)24只。腹腔注射链脲佐菌素(strepozotocin, STZ) 55 mg/ kg复制糖尿病大鼠模型,72h后剪尾尖采血测定血糖,以后每周测血糖,血糖持续≥16.7mmol/L为糖尿病大鼠(76只大鼠造模成功60只)。阻断大鼠左冠状动脉前降支30min,再灌注120min制备心肌缺血再灌注损伤模型。24只非糖尿病大鼠随机分为假手术组(non-diabetic sham, NS)和缺血再灌注组(NI/R);60只糖尿病大鼠随机分为假手术组(diabetic sham, DS)、缺血再灌注组(DI/R)和异丙酚低(DI/R+PL,3mg/kg/h)、中(DI/R+PM,6mg/kg/h)、高剂量组(DI/R+PH,12mg/kg/h),每组各12只。测定大鼠血糖和体重;Powerlab/30多导生理仪记录各组大鼠缺血再灌注120min时左室收缩峰压(LVSP)、左室舒张末压(LVDEP)、左室压力变化速率(±dp/dtmax),计算左室发展压(LVDP);测定血清中乳酸脱氢酶(LDH)、肌酸激酶同功酶(CK-MB)活性;测定心肌组织中超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,透射电镜观察心肌组织超微结构的变化。
结果:
1.腹腔注射STZ 3周时糖尿病各组大鼠血糖明显高于各自基础值和NS组(P<0.01),而体重明显低于各自基础值和NS组(P<0.05或P<0.01)。
2.再灌注120min时, NI/R组和DI/R组HR、LVDP、±dp/dtmax分别明显低于NS组和DS组,LVEDP分别明显高于NS组和DS组(P<0.05或P<0.01);DS组和DI/R组HR、LVDP、±dp/dtmax分别明显低于NS组和NI/R组,DI/R组LVEDP明显高于NI/R组(P<0.05或P<0.01);与DI/R组比较,DI/R+PM组和DI/R+PH组HR、LVDP、±dp/dtmax明显升高,LVEDP明显降低(P<0.05或P<0.01)。
3.再灌注120min时, NI/R组和DI/R组血清CK-MB、LDH活性、心肌组织MDA含量分别明显高于NS组和DS组,心肌组织SOD活性分别明显低于NS组和DS组(P<0.05或P<0.01);DI/R组上述指标变化较NI/R组更显著(P<0.05或P<0.01)。与DI/R组比较,DI/R+PM组和DI/R+PH组CK-MB、LDH活性、心肌组织MDA含量明显降低,心肌组织SOD活性明显升高(P<0.05或P<0.01)。
4.超微结构显示,DS组肌原纤维间间隙略增宽,线粒体稍增多,部分线粒体膜轻度肿胀;NI/R组和DI/R组心肌纤维排列紊乱,心肌细胞间及核周高度水肿,线粒体膜严重肿胀变形,嵴排列紊乱,断裂,溶解,空泡样变,DI/R组可见肌纤维收缩带和线粒体髓样变,呈同心圆排列,电子密度增高;与DI/R组比较,DI/R+PM组和DI/R+PH组心肌组织的超微结构特别是线粒体损伤明显减轻。
结论:糖尿病加重心肌缺血再灌注损伤,心肌超微结构的改变和氧化应激的增强可能是糖尿病加重心肌缺血再灌注损伤的原因,中、高剂量异丙酚通过减轻脂质过氧化反应,明显减轻了糖尿病大鼠心肌缺血再灌注损伤。
第二部分异丙酚对糖尿病大鼠缺血再灌注心肌组织炎性细胞因子的影响
目的:观察异丙酚对糖尿病缺血再灌注心肌组织NF-κB活性及其下游的炎性因子肿瘤坏死因子α(TNF-α)、白介素-1β(IL-1β)和白介素-6 (IL-6)的影响,探讨异丙酚对糖尿病心肌缺血再灌注损伤保护作用的机制。
方法:健康雄性SD大鼠(250-300g)100只,随机分为糖尿病组(D)76只和非糖尿病组(N)24只。腹腔注射STZ 55 mg/ kg复制糖尿病大鼠模型,72h后剪尾尖采血测定血糖,以后每周测血糖,血糖持续≥16.7mmol/L为糖尿病大鼠(76只大鼠造模成功60只)。阻断大鼠左冠状动脉前降支30min,再灌注120min制备心肌缺血再灌注损伤模型。24只非糖尿病大鼠随机分为假手术组(NS)和缺血再灌注组(NI/R);60只糖尿病大鼠随机分为假手术组(DS)、缺血再灌注组(DI/R)和异丙酚低(DI/R+PL,3mg/kg/h)、中(DI/R+PM,6mg/kg/h)、高剂量组(DI/R+PH,12mg/kg/h),每组各12只。分别于缺血前、缺血30min末、再灌注120min末记录心率(HR)、平均动脉压(MAP),并计算血压-心率指数(血压-心率指数=收缩压×心率)。ELISA法测定血清、心肌中肿瘤坏死因子α(TNF-α)、白介素6(IL-6)和白介素1β(IL-1β)的含量。半定量RT-PCR法检测心肌组织中TNF-α、IL-6和IL-1β的mRNA的表达。免疫组化染色分析心肌组织中NF-κB的核移位, Westernblotting检测心肌组织NF-κB的表达量。
结果:
1.缺血前糖尿病各组大鼠HR、MAP及血压-心率指数均低于非糖尿病大鼠(P﹤0.05或P﹤0.01),缺血再灌注后,各组大鼠上述指标呈进行性下降,DI/R组下降最显著(与NS组或DS组相比P﹤0.05或P﹤0.01),与DI/R组比较,DI/R+PM组、DI/R+PH组上述指标明显改善(P﹤0.05或P﹤0.01)。
2. DS组血清TNF-α、IL-6和IL-1β含量明显高于NS组(P﹤0.05);分别与NS组和DS组比较,NI/R组和DI/R组血清和心肌组织中TNF-α、IL-6和IL-1β含量明显升高,心肌组织中TNF-α、IL-6和IL-1βmRNA表达增强,NF-κB活化,明显从细胞浆移位于细胞核,表达量也显著增加,其中DI/R组变化显著(P﹤0.05或P﹤0.01)。与DI/R组比较,DI/R+PM组和DI/R+PH组血清、心肌组织中TNF-α、IL-1β含量明显降低,心肌组织中TNF-α、IL-6和IL-1βmRNA表达明显减弱,NF-κB从细胞浆向细胞核的移位被明显限制,NF-κB的表达量也明显降低(P﹤0.05或P﹤0.01)。
结论:NF-κB信号途径介导了糖尿病心肌I/R损伤。应用异丙酚后能明显抑制糖尿病心肌I/R后NF-κB的活化和TNF-α、IL-6和IL-1β的表达增加,心功能明显改善。
第三部分异丙酚对糖尿病大鼠缺血再灌注心肌组织细胞凋亡的影响
目的:采用糖尿病大鼠在体心肌缺血再灌注损伤模型,观察异丙酚对糖尿病大鼠缺血再灌注心肌组织细胞凋亡的影响,并探讨其机制。
方法:健康雄性SD大鼠(250-300g)100只,随机分为糖尿病组(D)76只和非糖尿病组(N)24只。腹腔注射STZ 55 mg/ kg复制糖尿病大鼠模型,72h后剪尾尖采血测定血糖,以后每周测血糖,血糖持续≥16.7mmol/L为糖尿病大鼠(76只大鼠造模成功60只)。阻断大鼠左冠状动脉前降支30min,再灌注120min制备心肌缺血再灌注损伤模型。24只非糖尿病大鼠随机分为假手术组(NS)和缺血再灌注组(NI/R);60只糖尿病大鼠随机分为假手术组(DS)、缺血再灌注组(DI/R)和异丙酚低(DI/R+PL,3mg/kg/h)、中(DI/R+PM,6mg/kg/h)、高剂量组(DI/R+PH,12mg/kg/h),每组各12只。光镜下观察心肌形态变化;流式细胞术(FCM)检测心肌细胞凋亡率;免疫组化法测定Bcl-2、Bax的蛋白表达;Westernblotting检测心肌组织天冬氨酸特异的半胱氨酸蛋白酶(Caspase-3)的蛋白表达量。
结果:
1.光镜下NI/R组和DI/R组大鼠部分区域心肌细胞浊肿,心肌纤维横纹不清或消失,核裂解、消失;DI/R组上述变化更加严重。DI/R+PM组和DI/R+PH组心肌细胞变性坏死程度较DI/R组轻。
2. NI/R组和DI/R组心肌细胞凋亡率、Bcl-2表达阳性细胞数百分率及Casepase-3的表达量分别明显高于NS组和DS组(P<0.05或P<0.01);与DI/R组相比,DI/R+PM组和DI/R+PH组Bcl-2表达阳性细胞数百分率明显升高(P<0.01);而心肌细胞凋亡率、Bax表达阳性细胞数百分率及Casepase-3表达量明显降低(P<0.05或P<0.01)。
结论:糖尿病加重缺血再灌注心肌组织的细胞凋亡,中、高剂量异丙酚对糖尿病大鼠心肌缺血再灌注损伤具有保护作用,减少Caspase-3、Bax的蛋白表达,增加Bcl-2的蛋白表达,抑制细胞凋亡可能是其作用机制之一。
第四部分异丙酚对糖尿病大鼠缺血再灌注心肌组织线粒体损伤的影响
目的:采用糖尿病大鼠在体心肌缺血再灌注损伤模型,观察异丙酚对糖尿病缺血再灌注心肌组织线粒体结构及功能的影响,进一步探讨异丙酚对糖尿病缺血再灌注心肌组织保护作用的可能机制。
方法:健康雄性SD大鼠(250-300g)100只,随机分为糖尿病组(D) 76只和非糖尿病组(N) 24只。腹腔注射STZ 55 mg/ kg复制糖尿病大鼠模型,72h后剪尾尖采血测定血糖,以后每周测血糖,血糖持续≥16.7mmol/L为糖尿病大鼠(76只大鼠造模成功60只)。阻断大鼠左冠状动脉前降支30min,再灌注120min制备心肌缺血再灌注损伤模型。24只非糖尿病大鼠随机分为假手术组(NS)和缺血再灌注组(NI/R);60只糖尿病大鼠随机分为假手术组(DS)、缺血再灌注组(DI/R)和异丙酚低(DI/R+PL,3mg/kg/h)、中(DI/R+PM,6mg/kg/h)、高剂量组(DI/R+PH,12mg/kg/h),每组各12只。透射电镜观察心肌组织超微结构的变化;差速离心法提取心肌线粒体,测定线粒体活力、膜肿胀度以及线粒体总ATP酶、超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)活性和丙二醛(MDA)含量。
结果:
1.超微结构显示,DS组线粒体稍增多,部分线粒体膜轻度肿胀;NI/R组和DI/R组心肌细胞核周高度水肿,线粒体膜严重肿胀变形,嵴排列紊乱,断裂,溶解,空泡样变,DI/R组可见线粒体髓样变,呈同心圆排列,电子密度增高;与DI/R组比较,DI/R+PM组和DI/R+PH组线粒体损伤明显减轻.
2.再灌注120min时, DS组大鼠心肌线粒体总ATP酶和GSH-Px活性明显低于NS组(P﹤0.05);NI/R组和DI/R组心肌线粒体总ATP酶、SOD、GSH-Px活性分别明显低于NS组和DS组(P﹤0.05),MDA含量明显高于NS组和DS组(P﹤0.05),心肌线粒体膜肿胀、线粒体活力下降,表现为540nm和570nm处吸光度值下降(P﹤0.05),DI/R组上述指标变化较NI/R组更显著(P﹤0.05或P<0.01)。与DI/R组比较,异丙酚各组心肌线粒体总ATP酶、SOD活性升高,DI/R+PM组和DI/R+PH组GSH-Px活性、MDA含量、线粒体膜肿胀、线粒体活力明显改善(P﹤0.05或P<0.01)。
结论:糖尿病加重缺血再灌注心肌组织线粒体结构和功能的损伤,中、高剂量异丙酚对糖尿病大鼠心肌缺血再灌注损伤具有保护作用,增强线粒体ATP酶、SOD、GSH-Px的活性,降低线粒体脂质过氧化水平是其可能的作用机制。
In recent years, the prevalence of diabetes mellitus is rapidly increasing . Diabetes is one of the main causes for increased incidence of complication during perioperative period. Clinically, diabetes represents an important risk factor for cardiovascular accident and poor outcomes after coronary revascularization. Diabetes is thought as an independent predictor of low output syndrome after coronary artery bypass graft (CABG) surgery.
Myocardium ischemia/reperfusion (I/R) injury is not only a commonly occurred perioperative cardiac problem, but also a main early complication of coronary revascularization. So it has become a focus in basal and clinical study on how to relieve or avoid the myocardium ischemia-reperfusion injury in diabetic patients. Propofol, 2,6-diisopropylphenol, an intravenous anaesthetic with a rapid and short anesthesia, is frequently used during cardiac surgery and in postoperative sedation. There are reports that propofol can protect myocardium against the I/R injury in isolated rat hearts. It has been proposed that this action of propofol may be mediated by its ability to act as a free radical scavenger conferred by the phenolichydroxyl group in its structure or through inhibition of trans-membrane calcium channel, but the effects and the mechanisms of propofol against myocardial I/R injury in diabetes are not known or understood. In the present study, we investigated the effects of propofol on myocardium I/R injury in diabetic rats in vivo and explored the possible mechanisms at cellular and molecular level.
PartⅠEffects of propofol on oxidative stress induced by myocardium ischemia/reperfusion in diabetic rats
Objective: To investigate effects of propofol on myocardium ischemia/ reperfusion injury in diabetic rats and clarify the possible molecular mechanism from oxidative stress.
Methods: A total of 100 SD rats were randomly divided into two groups: non-diabetics group (n=24) and diabetics group (n=76). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin (STZ) 55 mg/kg, rats serving as controls were injected the same volume of sodium citrate. Finally, 60 diabetics rat models were successfully reproduced. The myocardium I/R model was reproduced by ligation of the left anterior descending coronary artery for 30 min and reperfusing for 120 min. The rats in non-diabetic group were randomly reassigned into sham-operated group(NS), I/R group(NI/R) , the rats in diabetic group were randomly reassigned into sham-operated group(DS), I/R group(DI/R) and propofol 3, 6, 12mg/kg/h(DI/R+PL, DI /R+PM, DI/R+PH) groups (n=12). Propofol(3, 6, 12mg/kg/h) was intravenously infused respectively at 10min before ischemia in rats of propofol groups. The blood glucose and weight were determined. The cardiac function indexes such as the left ventricular developed pressure (LVDP), the left ventricular end diastolic pressure (LVEDP), heart rate (HR),±dp/dtmax were recorded at the end of reperfusion. The lactate dehydrogenase (LDH), MB isoenzyme of creatine kinase (CK-MB) activities in the serum and the activities of SOD and the contents of malondialdehyde (MDA) in myocardium during the reperfusion period were measured. The pathological changes of myocardium mitochondria were obersved by electron microscope.
Results:
1. The blood glucose of rats in diabetic groups was much higher and the weight was much lower than that of non-diabetics group after 3 week injected STZ(P﹤0.05, P﹤0.01).
2. Compared respectively with those of NS or DS group, the cardiac function parameters (HR, LVDP,±dp/dtmax) were significantly decreased in NI/R group and DI/R group at the end of reperfusion, the LVEDP was markedly increased(P﹤0.05, P﹤0.01), the values of above parameters were more severe in diabetic rats than in non-diabetes rats(P﹤0.05, P﹤0.01). Compared with that of DI/R group, administration of propofol (6, 12mg/kg/h) could ameliorate the HR, LVDP, LVEDP and±dp/dtmax(P﹤0.05,P﹤0.01).
3. Compared respectively with those of NS or DS group, the activities of LDH, CK and content of malondialdehyde (MDA) in myocardium were increased, the activities of SOD in myocardium were decreased in NI/R group and DI/R group(P﹤0.05, P﹤0.01), the values of above parameters were more severe in diabetic rats than in non-diabetic rats(P﹤0.05, P﹤0.01), administration of propofol (6, 12mg/kg/h) could decrease the activities of LDH, CK in serum and content of MDA in myocardium respectively and significantly enhance the activities of SOD in myocardium(P﹤0.05, P﹤0.01).
4. In DI/R group, the pathological changes of mitochondria induced by I/R injury, such as membrane swelling, cristae disruption, dissolution or disappearance, glycogen granule reduction, were significantly worse than that of NI/R group, and could be alleviated by propofol (12 mg/kg/h).
Conclusion: Myocardium ischemia/reperfusion injury was aggravated by diabetes. Propofol has a beneficial myocardial protection against the ischemia/reperfusion injury in the diabetic rats. It could be concluded that the protection is related to diminishing oxidative stress, protecting mitochondria from peroxidative injury. Further studies will be needed to investigate the exact signal pathway.
PartⅡEffects of propofol on inflammatory response induced by myocardium ischemia/reperfusion in diabetic rats
Objective: To explore the molecular mechanisms of propofol in cardioprotection, the activation of NF-κB and its regulated inflammatory mediators expression were examined in the present experiment with the model of myocardial ischemia/reperfusion in diabetic rat .
Methods: A total of 100 SD rats were randomly divided into two groups: non-diabetics group (n=24) and diabetics group (n=76). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin (STZ) 55 mg/kg, rats serving as controls were injected the same volume of sodium citrate. Finally, 60 diabetics rat models were successfully reproduced. The myocardium I/R model was reproduced by ligation of the left anterior descending coronary artery for 30 min and reperfusing for 120 min. The rats in non-diabetic group were randomly reassigned into sham-operated group(NS), I/R group(NI/R), the rats in diabetic group were randomly reassigned into sham-operated group(DS), I/R group(DI/R) and propofol 3, 6, 12mg/kg/h(DI/R+PL, DI/R+PM, DI/R+PH)groups (n=12). Propofol (3,6,12mg/kg/h) was intravenously infused respectively at 10min before ischemia in rats of propofol groups. Heart rate(HR), mean arterial blood pressure(MAP) and blood pressure-heart rate index (PRI) were recorded under basal conditions, during occlusion and at the end of reperfusion, respectively. The translocation of NF-κB in the cardiomyocytes was detected by immunohistochemistry and NF-κB expression were determined by Westernblotting. The concentrations of TNF-α, IL-6, IL-1βin serum and myocardium were evaluated by ELISA respectively. The cardiac amount of mRNA codifying for TNF-α, IL-6, IL-1βwere investigated by RT-PCR.
Results:
1. Compared with that of non-diabetic group, before ischemia the cardiac function parameters (HR, MAP, PRI) were decreased in diabetic group(P﹤0.05, P﹤0.01). At the end of reperfusion, the cardiac function parameters in NI/R group and DI/R group were lower than those of NS group or DS group and the value of the parameters were lower in DI/R group(P﹤0.05, P﹤0.01). Compared with those of DI/R group, administration of propofol (6, 12 mg/kg/h) resulted in improvement in HR, MAP and PRI, respectively(P﹤0.05, P﹤0.01).
2. Compared with that of NS group, the concentrations of TNF-α, IL-6, IL-1βin serum were increased in DS group(P﹤0.05). Compared with those of NS or DS group, the concentrations of TNF-α, IL-6, IL-1βin serum and myocardium, the expression of TNF-α, IL-6, IL-1βmRNA in myocardium were significantly increased in NIR and DI/R group respectively(P﹤0.05, P﹤0.01),In DI/R group, NF-κB was significantly translocated from the cytoplasm into the nucleus; however, NF-κB remained in the cytoplasm of cardiomyocyte in the sham group. And the expression of NF-κB in the nuclei in DI/R group was markedly higher than that of in the sham group(P﹤0.01).Compared with those of DI/R group, administration of propofol at 6, 12mg/kg/h significantly inhibited the NF-κB translocation into nucleus and attenuated the expression of NF-κB in the nuclei(P﹤0.05), decreased the concentrations of TNF-α, IL-6, IL-1βin serum and myocardium and the expression of TNF-α, IL-6, IL-1βmRNA respectively(P﹤0.05, P﹤0.05).
Conclusion: Propofol inhibits NF-κB activation, subsequent leads to down-regulation of NF-κB-dependent inflammatory gene expression and thus reduces the inflammatory response in myocardial ischemia reperfusion injury in the diabetic rats, which may be one of the molecular mechanisms of its cardioprotection.
PartⅢEffects of propofol on ischemia/reperfusion-induced cardiocyte apoptosis in diabetic rats
Objective: To investigate the effects of propofol on ischemia/reperfusion -induced cardiocyte apoptosis in diabetic rats and explore the possible mechanism.
Methods: A total of 100 SD rats were randomly divided into two groups: non-diabetics group (n=24) and diabetics group (n=76). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin (STZ) 55 mg/kg , rats serving as controls were injected the same volume of sodium citrate. Finally, 60 diabetics rat models were successfully reproduced. The myocardial I/R model was reproduced by ligation of the left anterior descending coronary artery for 30 min and reperfusing for 120 min. The rats in non-diabetic group were randomly reassigned into sham-operated group(NS), I /R group(NI/R) , the rats in diabetic group were randomly reassigned into sham-operated group(DS), I /R group(DI/R) and propofol 3, 6, 12mg/kg/h(DI /R+PL, DI/R+PM, DI/R+PH) groups (n=12). Propofol (3, 6, 12mg/kg/h) was intravenously infused respectively at 10min before ischemia in rats of propofol groups. The apoptotic rate of cardiomyocytes was evaluated by Flow Cytometry. The positive expressions of Bcl-2, Bax, caspase-3 in cardiomyocytes were respectively detected by immunohistochemistry and westernblotting.
Results: The apoptotic rates of cardiomyocytes in NI/R and DI/R group (18.18±2.12, 27.01±2.06) was significantly higher than that of NS or DS group (5.61±1.04,8.37±2.64)(P<0.01), and it is higher in DI/R group than in NI/R group(P<0.01). The apoptotic rates of cardiomyocytes in the three propofol groups were 24.03±5.05, 17.08±2.00, 13.67±3.07, respectively. The percentage of apoptosis cardiomyocytes in propofol 6, 12mg/kg/h group was markedly lower than that of DI/R group, respectively(P﹤0.01). Compared with those of DI/R group, the expression of Bax and casepase-3 were significantly decreased and the expression of Bcl-2 was markedly increased in propofol( 6,12mg/kg/h) group (P<0.05, P<0.01).
Conclusion: It could be concluded that diabetes exacerbates cardiocyte apoptosis induced by I/R , propofol has a beneficial myocardial protection against the I/R injury in diabetic rats by decreasing the expression of bax, caspase-3 and rate of cardiomyocytes apoptosis and increasing the expression of Bcl-2.
PartⅣEffect of propofol on mitochondria injury after myocardial ischemic/reperfusion in diabetic rats
Objective: To investigate effects of propofol on mitochondria structure and function after myocardial I/R injury in diabetic rats and explore the possible mechanism.
Methods: A total of 100 SD rats were randomly divided into two groups: non-diabetics group (n=24) and diabetics group (n=76). Diabetes mellitus was induced by intraperitoneal injection of STZ at 55 mg/kg, rats serving as controls were injected the same volume of sodium citrate. Finally, 60 diabetics rat models were successfully reproduced. The myocardium I/R model was reproduced by ligation of the left anterior descending coronary artery for 30 min and reperfusing for 120 min. The rats in non-diabetic group were randomly reassigned into sham-operated group(NS), I/R group(NI/R) , the rats in diabetic group were randomly reassigned into sham-operated group(DS), I /R group(DI/R) and propofol 3, 6, 12mg/kg/h(DI/R+PL, DI/R+PM, DI/R+PH) groups(n=12). Propofol(3, 6, 12mg/kg/h) was intravenously infused respectively at 10min before ischemia in rats of propofol groups. The pathological changes of myocardium mitochondria were obersved by electron microscope. The mitochondria were prepared by differential centrifugation. The activities of SOD, GSH-PX, ATPase and the contents of malondialdehyde (MDA) in myocardial mitochondria during the reperfusion period were measured. The swelling and activity of mitochondria were also determined.
Results:
1. The pathological changes of mitochondria induced by I/R injury, such as membrane swelling, cristae disruption, dissolution or disappearance, glycogen granule reduction, were significantly alleviated by propofol at 6, 12mg/kg/h.
2. In NI/R and DI/R group, the swelling of mitochondria was markedly increased and the activity of mitochondria was decreased (P﹤0.05), and the activities of SOD, GSH-PX, ATPase were decreased and the content of malondialdehyde (MDA) in myocardial mitochondria was increased(P﹤0.05) compared with those of NS and DS group, respectively. Compared with those of DI/R group, administration of propofol at the concentrations 6, 12mg/kg/h significantly enhanced the activities of SOD, GSH-Px and ATPase and the activity of myocardium mitochondria, ameliorated the mitochondria swelling and decreased the MDA contents, respectively(P﹤0.05).
Conclusion: Diabetes exacerbates the mitochondria damages induced by I/R. Propofol has a beneficial myocardial protection against the I/R injury in diabetic rats by increasing the activities of SOD, GSH-Px, ATPase and diminishing oxidative stress.
第一部分异丙酚对糖尿病大鼠缺血再灌注心肌组织氧化应激的影响
目的:观察异丙酚对糖尿病大鼠心肌缺血再灌注损伤的影响并从氧化应激方面探讨其作用机制。
方法:健康雄性SD大鼠(250-300g)100只,随机分为糖尿病组(diabetic group, D)76只和非糖尿病组(non-diabetic group, N)24只。腹腔注射链脲佐菌素(strepozotocin, STZ) 55 mg/ kg复制糖尿病大鼠模型,72h后剪尾尖采血测定血糖,以后每周测血糖,血糖持续≥16.7mmol/L为糖尿病大鼠(76只大鼠造模成功60只)。阻断大鼠左冠状动脉前降支30min,再灌注120min制备心肌缺血再灌注损伤模型。24只非糖尿病大鼠随机分为假手术组(non-diabetic sham, NS)和缺血再灌注组(NI/R);60只糖尿病大鼠随机分为假手术组(diabetic sham, DS)、缺血再灌注组(DI/R)和异丙酚低(DI/R+PL,3mg/kg/h)、中(DI/R+PM,6mg/kg/h)、高剂量组(DI/R+PH,12mg/kg/h),每组各12只。测定大鼠血糖和体重;Powerlab/30多导生理仪记录各组大鼠缺血再灌注120min时左室收缩峰压(LVSP)、左室舒张末压(LVDEP)、左室压力变化速率(±dp/dtmax),计算左室发展压(LVDP);测定血清中乳酸脱氢酶(LDH)、肌酸激酶同功酶(CK-MB)活性;测定心肌组织中超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量,透射电镜观察心肌组织超微结构的变化。
结果:
1.腹腔注射STZ 3周时糖尿病各组大鼠血糖明显高于各自基础值和NS组(P<0.01),而体重明显低于各自基础值和NS组(P<0.05或P<0.01)。
2.再灌注120min时, NI/R组和DI/R组HR、LVDP、±dp/dtmax分别明显低于NS组和DS组,LVEDP分别明显高于NS组和DS组(P<0.05或P<0.01);DS组和DI/R组HR、LVDP、±dp/dtmax分别明显低于NS组和NI/R组,DI/R组LVEDP明显高于NI/R组(P<0.05或P<0.01);与DI/R组比较,DI/R+PM组和DI/R+PH组HR、LVDP、±dp/dtmax明显升高,LVEDP明显降低(P<0.05或P<0.01)。
3.再灌注120min时, NI/R组和DI/R组血清CK-MB、LDH活性、心肌组织MDA含量分别明显高于NS组和DS组,心肌组织SOD活性分别明显低于NS组和DS组(P<0.05或P<0.01);DI/R组上述指标变化较NI/R组更显著(P<0.05或P<0.01)。与DI/R组比较,DI/R+PM组和DI/R+PH组CK-MB、LDH活性、心肌组织MDA含量明显降低,心肌组织SOD活性明显升高(P<0.05或P<0.01)。
4.超微结构显示,DS组肌原纤维间间隙略增宽,线粒体稍增多,部分线粒体膜轻度肿胀;NI/R组和DI/R组心肌纤维排列紊乱,心肌细胞间及核周高度水肿,线粒体膜严重肿胀变形,嵴排列紊乱,断裂,溶解,空泡样变,DI/R组可见肌纤维收缩带和线粒体髓样变,呈同心圆排列,电子密度增高;与DI/R组比较,DI/R+PM组和DI/R+PH组心肌组织的超微结构特别是线粒体损伤明显减轻。
结论:糖尿病加重心肌缺血再灌注损伤,心肌超微结构的改变和氧化应激的增强可能是糖尿病加重心肌缺血再灌注损伤的原因,中、高剂量异丙酚通过减轻脂质过氧化反应,明显减轻了糖尿病大鼠心肌缺血再灌注损伤。
第二部分异丙酚对糖尿病大鼠缺血再灌注心肌组织炎性细胞因子的影响
目的:观察异丙酚对糖尿病缺血再灌注心肌组织NF-κB活性及其下游的炎性因子肿瘤坏死因子α(TNF-α)、白介素-1β(IL-1β)和白介素-6 (IL-6)的影响,探讨异丙酚对糖尿病心肌缺血再灌注损伤保护作用的机制。
方法:健康雄性SD大鼠(250-300g)100只,随机分为糖尿病组(D)76只和非糖尿病组(N)24只。腹腔注射STZ 55 mg/ kg复制糖尿病大鼠模型,72h后剪尾尖采血测定血糖,以后每周测血糖,血糖持续≥16.7mmol/L为糖尿病大鼠(76只大鼠造模成功60只)。阻断大鼠左冠状动脉前降支30min,再灌注120min制备心肌缺血再灌注损伤模型。24只非糖尿病大鼠随机分为假手术组(NS)和缺血再灌注组(NI/R);60只糖尿病大鼠随机分为假手术组(DS)、缺血再灌注组(DI/R)和异丙酚低(DI/R+PL,3mg/kg/h)、中(DI/R+PM,6mg/kg/h)、高剂量组(DI/R+PH,12mg/kg/h),每组各12只。分别于缺血前、缺血30min末、再灌注120min末记录心率(HR)、平均动脉压(MAP),并计算血压-心率指数(血压-心率指数=收缩压×心率)。ELISA法测定血清、心肌中肿瘤坏死因子α(TNF-α)、白介素6(IL-6)和白介素1β(IL-1β)的含量。半定量RT-PCR法检测心肌组织中TNF-α、IL-6和IL-1β的mRNA的表达。免疫组化染色分析心肌组织中NF-κB的核移位, Westernblotting检测心肌组织NF-κB的表达量。
结果:
1.缺血前糖尿病各组大鼠HR、MAP及血压-心率指数均低于非糖尿病大鼠(P﹤0.05或P﹤0.01),缺血再灌注后,各组大鼠上述指标呈进行性下降,DI/R组下降最显著(与NS组或DS组相比P﹤0.05或P﹤0.01),与DI/R组比较,DI/R+PM组、DI/R+PH组上述指标明显改善(P﹤0.05或P﹤0.01)。
2. DS组血清TNF-α、IL-6和IL-1β含量明显高于NS组(P﹤0.05);分别与NS组和DS组比较,NI/R组和DI/R组血清和心肌组织中TNF-α、IL-6和IL-1β含量明显升高,心肌组织中TNF-α、IL-6和IL-1βmRNA表达增强,NF-κB活化,明显从细胞浆移位于细胞核,表达量也显著增加,其中DI/R组变化显著(P﹤0.05或P﹤0.01)。与DI/R组比较,DI/R+PM组和DI/R+PH组血清、心肌组织中TNF-α、IL-1β含量明显降低,心肌组织中TNF-α、IL-6和IL-1βmRNA表达明显减弱,NF-κB从细胞浆向细胞核的移位被明显限制,NF-κB的表达量也明显降低(P﹤0.05或P﹤0.01)。
结论:NF-κB信号途径介导了糖尿病心肌I/R损伤。应用异丙酚后能明显抑制糖尿病心肌I/R后NF-κB的活化和TNF-α、IL-6和IL-1β的表达增加,心功能明显改善。
第三部分异丙酚对糖尿病大鼠缺血再灌注心肌组织细胞凋亡的影响
目的:采用糖尿病大鼠在体心肌缺血再灌注损伤模型,观察异丙酚对糖尿病大鼠缺血再灌注心肌组织细胞凋亡的影响,并探讨其机制。
方法:健康雄性SD大鼠(250-300g)100只,随机分为糖尿病组(D)76只和非糖尿病组(N)24只。腹腔注射STZ 55 mg/ kg复制糖尿病大鼠模型,72h后剪尾尖采血测定血糖,以后每周测血糖,血糖持续≥16.7mmol/L为糖尿病大鼠(76只大鼠造模成功60只)。阻断大鼠左冠状动脉前降支30min,再灌注120min制备心肌缺血再灌注损伤模型。24只非糖尿病大鼠随机分为假手术组(NS)和缺血再灌注组(NI/R);60只糖尿病大鼠随机分为假手术组(DS)、缺血再灌注组(DI/R)和异丙酚低(DI/R+PL,3mg/kg/h)、中(DI/R+PM,6mg/kg/h)、高剂量组(DI/R+PH,12mg/kg/h),每组各12只。光镜下观察心肌形态变化;流式细胞术(FCM)检测心肌细胞凋亡率;免疫组化法测定Bcl-2、Bax的蛋白表达;Westernblotting检测心肌组织天冬氨酸特异的半胱氨酸蛋白酶(Caspase-3)的蛋白表达量。
结果:
1.光镜下NI/R组和DI/R组大鼠部分区域心肌细胞浊肿,心肌纤维横纹不清或消失,核裂解、消失;DI/R组上述变化更加严重。DI/R+PM组和DI/R+PH组心肌细胞变性坏死程度较DI/R组轻。
2. NI/R组和DI/R组心肌细胞凋亡率、Bcl-2表达阳性细胞数百分率及Casepase-3的表达量分别明显高于NS组和DS组(P<0.05或P<0.01);与DI/R组相比,DI/R+PM组和DI/R+PH组Bcl-2表达阳性细胞数百分率明显升高(P<0.01);而心肌细胞凋亡率、Bax表达阳性细胞数百分率及Casepase-3表达量明显降低(P<0.05或P<0.01)。
结论:糖尿病加重缺血再灌注心肌组织的细胞凋亡,中、高剂量异丙酚对糖尿病大鼠心肌缺血再灌注损伤具有保护作用,减少Caspase-3、Bax的蛋白表达,增加Bcl-2的蛋白表达,抑制细胞凋亡可能是其作用机制之一。
第四部分异丙酚对糖尿病大鼠缺血再灌注心肌组织线粒体损伤的影响
目的:采用糖尿病大鼠在体心肌缺血再灌注损伤模型,观察异丙酚对糖尿病缺血再灌注心肌组织线粒体结构及功能的影响,进一步探讨异丙酚对糖尿病缺血再灌注心肌组织保护作用的可能机制。
方法:健康雄性SD大鼠(250-300g)100只,随机分为糖尿病组(D) 76只和非糖尿病组(N) 24只。腹腔注射STZ 55 mg/ kg复制糖尿病大鼠模型,72h后剪尾尖采血测定血糖,以后每周测血糖,血糖持续≥16.7mmol/L为糖尿病大鼠(76只大鼠造模成功60只)。阻断大鼠左冠状动脉前降支30min,再灌注120min制备心肌缺血再灌注损伤模型。24只非糖尿病大鼠随机分为假手术组(NS)和缺血再灌注组(NI/R);60只糖尿病大鼠随机分为假手术组(DS)、缺血再灌注组(DI/R)和异丙酚低(DI/R+PL,3mg/kg/h)、中(DI/R+PM,6mg/kg/h)、高剂量组(DI/R+PH,12mg/kg/h),每组各12只。透射电镜观察心肌组织超微结构的变化;差速离心法提取心肌线粒体,测定线粒体活力、膜肿胀度以及线粒体总ATP酶、超氧化物岐化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)活性和丙二醛(MDA)含量。
结果:
1.超微结构显示,DS组线粒体稍增多,部分线粒体膜轻度肿胀;NI/R组和DI/R组心肌细胞核周高度水肿,线粒体膜严重肿胀变形,嵴排列紊乱,断裂,溶解,空泡样变,DI/R组可见线粒体髓样变,呈同心圆排列,电子密度增高;与DI/R组比较,DI/R+PM组和DI/R+PH组线粒体损伤明显减轻.
2.再灌注120min时, DS组大鼠心肌线粒体总ATP酶和GSH-Px活性明显低于NS组(P﹤0.05);NI/R组和DI/R组心肌线粒体总ATP酶、SOD、GSH-Px活性分别明显低于NS组和DS组(P﹤0.05),MDA含量明显高于NS组和DS组(P﹤0.05),心肌线粒体膜肿胀、线粒体活力下降,表现为540nm和570nm处吸光度值下降(P﹤0.05),DI/R组上述指标变化较NI/R组更显著(P﹤0.05或P<0.01)。与DI/R组比较,异丙酚各组心肌线粒体总ATP酶、SOD活性升高,DI/R+PM组和DI/R+PH组GSH-Px活性、MDA含量、线粒体膜肿胀、线粒体活力明显改善(P﹤0.05或P<0.01)。
结论:糖尿病加重缺血再灌注心肌组织线粒体结构和功能的损伤,中、高剂量异丙酚对糖尿病大鼠心肌缺血再灌注损伤具有保护作用,增强线粒体ATP酶、SOD、GSH-Px的活性,降低线粒体脂质过氧化水平是其可能的作用机制。
In recent years, the prevalence of diabetes mellitus is rapidly increasing . Diabetes is one of the main causes for increased incidence of complication during perioperative period. Clinically, diabetes represents an important risk factor for cardiovascular accident and poor outcomes after coronary revascularization. Diabetes is thought as an independent predictor of low output syndrome after coronary artery bypass graft (CABG) surgery.
Myocardium ischemia/reperfusion (I/R) injury is not only a commonly occurred perioperative cardiac problem, but also a main early complication of coronary revascularization. So it has become a focus in basal and clinical study on how to relieve or avoid the myocardium ischemia-reperfusion injury in diabetic patients. Propofol, 2,6-diisopropylphenol, an intravenous anaesthetic with a rapid and short anesthesia, is frequently used during cardiac surgery and in postoperative sedation. There are reports that propofol can protect myocardium against the I/R injury in isolated rat hearts. It has been proposed that this action of propofol may be mediated by its ability to act as a free radical scavenger conferred by the phenolichydroxyl group in its structure or through inhibition of trans-membrane calcium channel, but the effects and the mechanisms of propofol against myocardial I/R injury in diabetes are not known or understood. In the present study, we investigated the effects of propofol on myocardium I/R injury in diabetic rats in vivo and explored the possible mechanisms at cellular and molecular level.
PartⅠEffects of propofol on oxidative stress induced by myocardium ischemia/reperfusion in diabetic rats
Objective: To investigate effects of propofol on myocardium ischemia/ reperfusion injury in diabetic rats and clarify the possible molecular mechanism from oxidative stress.
Methods: A total of 100 SD rats were randomly divided into two groups: non-diabetics group (n=24) and diabetics group (n=76). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin (STZ) 55 mg/kg, rats serving as controls were injected the same volume of sodium citrate. Finally, 60 diabetics rat models were successfully reproduced. The myocardium I/R model was reproduced by ligation of the left anterior descending coronary artery for 30 min and reperfusing for 120 min. The rats in non-diabetic group were randomly reassigned into sham-operated group(NS), I/R group(NI/R) , the rats in diabetic group were randomly reassigned into sham-operated group(DS), I/R group(DI/R) and propofol 3, 6, 12mg/kg/h(DI/R+PL, DI /R+PM, DI/R+PH) groups (n=12). Propofol(3, 6, 12mg/kg/h) was intravenously infused respectively at 10min before ischemia in rats of propofol groups. The blood glucose and weight were determined. The cardiac function indexes such as the left ventricular developed pressure (LVDP), the left ventricular end diastolic pressure (LVEDP), heart rate (HR),±dp/dtmax were recorded at the end of reperfusion. The lactate dehydrogenase (LDH), MB isoenzyme of creatine kinase (CK-MB) activities in the serum and the activities of SOD and the contents of malondialdehyde (MDA) in myocardium during the reperfusion period were measured. The pathological changes of myocardium mitochondria were obersved by electron microscope.
Results:
1. The blood glucose of rats in diabetic groups was much higher and the weight was much lower than that of non-diabetics group after 3 week injected STZ(P﹤0.05, P﹤0.01).
2. Compared respectively with those of NS or DS group, the cardiac function parameters (HR, LVDP,±dp/dtmax) were significantly decreased in NI/R group and DI/R group at the end of reperfusion, the LVEDP was markedly increased(P﹤0.05, P﹤0.01), the values of above parameters were more severe in diabetic rats than in non-diabetes rats(P﹤0.05, P﹤0.01). Compared with that of DI/R group, administration of propofol (6, 12mg/kg/h) could ameliorate the HR, LVDP, LVEDP and±dp/dtmax(P﹤0.05,P﹤0.01).
3. Compared respectively with those of NS or DS group, the activities of LDH, CK and content of malondialdehyde (MDA) in myocardium were increased, the activities of SOD in myocardium were decreased in NI/R group and DI/R group(P﹤0.05, P﹤0.01), the values of above parameters were more severe in diabetic rats than in non-diabetic rats(P﹤0.05, P﹤0.01), administration of propofol (6, 12mg/kg/h) could decrease the activities of LDH, CK in serum and content of MDA in myocardium respectively and significantly enhance the activities of SOD in myocardium(P﹤0.05, P﹤0.01).
4. In DI/R group, the pathological changes of mitochondria induced by I/R injury, such as membrane swelling, cristae disruption, dissolution or disappearance, glycogen granule reduction, were significantly worse than that of NI/R group, and could be alleviated by propofol (12 mg/kg/h).
Conclusion: Myocardium ischemia/reperfusion injury was aggravated by diabetes. Propofol has a beneficial myocardial protection against the ischemia/reperfusion injury in the diabetic rats. It could be concluded that the protection is related to diminishing oxidative stress, protecting mitochondria from peroxidative injury. Further studies will be needed to investigate the exact signal pathway.
PartⅡEffects of propofol on inflammatory response induced by myocardium ischemia/reperfusion in diabetic rats
Objective: To explore the molecular mechanisms of propofol in cardioprotection, the activation of NF-κB and its regulated inflammatory mediators expression were examined in the present experiment with the model of myocardial ischemia/reperfusion in diabetic rat .
Methods: A total of 100 SD rats were randomly divided into two groups: non-diabetics group (n=24) and diabetics group (n=76). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin (STZ) 55 mg/kg, rats serving as controls were injected the same volume of sodium citrate. Finally, 60 diabetics rat models were successfully reproduced. The myocardium I/R model was reproduced by ligation of the left anterior descending coronary artery for 30 min and reperfusing for 120 min. The rats in non-diabetic group were randomly reassigned into sham-operated group(NS), I/R group(NI/R), the rats in diabetic group were randomly reassigned into sham-operated group(DS), I/R group(DI/R) and propofol 3, 6, 12mg/kg/h(DI/R+PL, DI/R+PM, DI/R+PH)groups (n=12). Propofol (3,6,12mg/kg/h) was intravenously infused respectively at 10min before ischemia in rats of propofol groups. Heart rate(HR), mean arterial blood pressure(MAP) and blood pressure-heart rate index (PRI) were recorded under basal conditions, during occlusion and at the end of reperfusion, respectively. The translocation of NF-κB in the cardiomyocytes was detected by immunohistochemistry and NF-κB expression were determined by Westernblotting. The concentrations of TNF-α, IL-6, IL-1βin serum and myocardium were evaluated by ELISA respectively. The cardiac amount of mRNA codifying for TNF-α, IL-6, IL-1βwere investigated by RT-PCR.
Results:
1. Compared with that of non-diabetic group, before ischemia the cardiac function parameters (HR, MAP, PRI) were decreased in diabetic group(P﹤0.05, P﹤0.01). At the end of reperfusion, the cardiac function parameters in NI/R group and DI/R group were lower than those of NS group or DS group and the value of the parameters were lower in DI/R group(P﹤0.05, P﹤0.01). Compared with those of DI/R group, administration of propofol (6, 12 mg/kg/h) resulted in improvement in HR, MAP and PRI, respectively(P﹤0.05, P﹤0.01).
2. Compared with that of NS group, the concentrations of TNF-α, IL-6, IL-1βin serum were increased in DS group(P﹤0.05). Compared with those of NS or DS group, the concentrations of TNF-α, IL-6, IL-1βin serum and myocardium, the expression of TNF-α, IL-6, IL-1βmRNA in myocardium were significantly increased in NIR and DI/R group respectively(P﹤0.05, P﹤0.01),In DI/R group, NF-κB was significantly translocated from the cytoplasm into the nucleus; however, NF-κB remained in the cytoplasm of cardiomyocyte in the sham group. And the expression of NF-κB in the nuclei in DI/R group was markedly higher than that of in the sham group(P﹤0.01).Compared with those of DI/R group, administration of propofol at 6, 12mg/kg/h significantly inhibited the NF-κB translocation into nucleus and attenuated the expression of NF-κB in the nuclei(P﹤0.05), decreased the concentrations of TNF-α, IL-6, IL-1βin serum and myocardium and the expression of TNF-α, IL-6, IL-1βmRNA respectively(P﹤0.05, P﹤0.05).
Conclusion: Propofol inhibits NF-κB activation, subsequent leads to down-regulation of NF-κB-dependent inflammatory gene expression and thus reduces the inflammatory response in myocardial ischemia reperfusion injury in the diabetic rats, which may be one of the molecular mechanisms of its cardioprotection.
PartⅢEffects of propofol on ischemia/reperfusion-induced cardiocyte apoptosis in diabetic rats
Objective: To investigate the effects of propofol on ischemia/reperfusion -induced cardiocyte apoptosis in diabetic rats and explore the possible mechanism.
Methods: A total of 100 SD rats were randomly divided into two groups: non-diabetics group (n=24) and diabetics group (n=76). Diabetes mellitus was induced by intraperitoneal injection of streptozotocin (STZ) 55 mg/kg , rats serving as controls were injected the same volume of sodium citrate. Finally, 60 diabetics rat models were successfully reproduced. The myocardial I/R model was reproduced by ligation of the left anterior descending coronary artery for 30 min and reperfusing for 120 min. The rats in non-diabetic group were randomly reassigned into sham-operated group(NS), I /R group(NI/R) , the rats in diabetic group were randomly reassigned into sham-operated group(DS), I /R group(DI/R) and propofol 3, 6, 12mg/kg/h(DI /R+PL, DI/R+PM, DI/R+PH) groups (n=12). Propofol (3, 6, 12mg/kg/h) was intravenously infused respectively at 10min before ischemia in rats of propofol groups. The apoptotic rate of cardiomyocytes was evaluated by Flow Cytometry. The positive expressions of Bcl-2, Bax, caspase-3 in cardiomyocytes were respectively detected by immunohistochemistry and westernblotting.
Results: The apoptotic rates of cardiomyocytes in NI/R and DI/R group (18.18±2.12, 27.01±2.06) was significantly higher than that of NS or DS group (5.61±1.04,8.37±2.64)(P<0.01), and it is higher in DI/R group than in NI/R group(P<0.01). The apoptotic rates of cardiomyocytes in the three propofol groups were 24.03±5.05, 17.08±2.00, 13.67±3.07, respectively. The percentage of apoptosis cardiomyocytes in propofol 6, 12mg/kg/h group was markedly lower than that of DI/R group, respectively(P﹤0.01). Compared with those of DI/R group, the expression of Bax and casepase-3 were significantly decreased and the expression of Bcl-2 was markedly increased in propofol( 6,12mg/kg/h) group (P<0.05, P<0.01).
Conclusion: It could be concluded that diabetes exacerbates cardiocyte apoptosis induced by I/R , propofol has a beneficial myocardial protection against the I/R injury in diabetic rats by decreasing the expression of bax, caspase-3 and rate of cardiomyocytes apoptosis and increasing the expression of Bcl-2.
PartⅣEffect of propofol on mitochondria injury after myocardial ischemic/reperfusion in diabetic rats
Objective: To investigate effects of propofol on mitochondria structure and function after myocardial I/R injury in diabetic rats and explore the possible mechanism.
Methods: A total of 100 SD rats were randomly divided into two groups: non-diabetics group (n=24) and diabetics group (n=76). Diabetes mellitus was induced by intraperitoneal injection of STZ at 55 mg/kg, rats serving as controls were injected the same volume of sodium citrate. Finally, 60 diabetics rat models were successfully reproduced. The myocardium I/R model was reproduced by ligation of the left anterior descending coronary artery for 30 min and reperfusing for 120 min. The rats in non-diabetic group were randomly reassigned into sham-operated group(NS), I/R group(NI/R) , the rats in diabetic group were randomly reassigned into sham-operated group(DS), I /R group(DI/R) and propofol 3, 6, 12mg/kg/h(DI/R+PL, DI/R+PM, DI/R+PH) groups(n=12). Propofol(3, 6, 12mg/kg/h) was intravenously infused respectively at 10min before ischemia in rats of propofol groups. The pathological changes of myocardium mitochondria were obersved by electron microscope. The mitochondria were prepared by differential centrifugation. The activities of SOD, GSH-PX, ATPase and the contents of malondialdehyde (MDA) in myocardial mitochondria during the reperfusion period were measured. The swelling and activity of mitochondria were also determined.
Results:
1. The pathological changes of mitochondria induced by I/R injury, such as membrane swelling, cristae disruption, dissolution or disappearance, glycogen granule reduction, were significantly alleviated by propofol at 6, 12mg/kg/h.
2. In NI/R and DI/R group, the swelling of mitochondria was markedly increased and the activity of mitochondria was decreased (P﹤0.05), and the activities of SOD, GSH-PX, ATPase were decreased and the content of malondialdehyde (MDA) in myocardial mitochondria was increased(P﹤0.05) compared with those of NS and DS group, respectively. Compared with those of DI/R group, administration of propofol at the concentrations 6, 12mg/kg/h significantly enhanced the activities of SOD, GSH-Px and ATPase and the activity of myocardium mitochondria, ameliorated the mitochondria swelling and decreased the MDA contents, respectively(P﹤0.05).
Conclusion: Diabetes exacerbates the mitochondria damages induced by I/R. Propofol has a beneficial myocardial protection against the I/R injury in diabetic rats by increasing the activities of SOD, GSH-Px, ATPase and diminishing oxidative stress.
引文
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