软脂酸致H9c2心肌细胞凋亡的机制研究
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摘要
在缺血性心脏病、心肌炎、慢性心衰等心脏疾病发生发展的病理过程中,心肌细胞数目减少(由心肌细胞死亡引起)或者残存的心肌细胞功能下降,是心功能下降的主要原因。一般认为心肌细胞是终末分化细胞,心肌细胞的损伤和死亡主要通过细胞坏死,但近年来的研究发现,心肌细胞凋亡亦存在于心肌的生理与病理过程中,是诸多心血管疾病发生的重要细胞学基础。资料表明,在许多心脏疾病中,饱和脂肪酸对心肌损伤起着关键作用,心肌细胞凋亡即为心肌损伤的主要后果之一。脂肪酸是成人心脏能量产生的主要来源,在多种疾病状态下,心肌都会出现脂肪酸代谢异常,如心肌急性缺血、心脏手术后脂肪酸水平可短期升高;而在糖尿病、肥胖、高脂血症等患者脂肪酸水平则长期升高。脂质升高可伴有心肌内脂质异常蓄积,导致心肌细胞糖脂代谢紊乱,引起心肌细胞损伤,凋亡及心肌收缩和舒张功能发生改变。心脏内脂质蓄积在糖尿病和肥胖病人心衰的发生发展过程中起着重要作用。
现有大量的动物和细胞培养实验也发现软脂酸(palmitate;PAL)可诱导多种细胞出现凋亡。但目前为止,脂肪酸引起细胞凋亡的具体分子细胞机制并不清楚。为探讨软脂酸损伤心肌细胞的机制,本文从以下三个部分进行了研究。
第一部分Pamitate对H9c2心肌细胞凋亡和NADPH氧化酶活性的影响
目的:明确palmitate致心肌细胞凋亡的最佳浓度与时间及其对心肌细胞内NADPH氧化酶亚基p47phox的影响。
方法:分组一:对照组与palmitate组(palmitate浓度分别为0.2,0.5,0.8,1.2mM)。培养24h后观察心肌细胞存活率、凋亡率,得到palmitate诱导心肌细胞凋亡的有效浓度。分组二:对照组与palmitate(0.5mM)组(处理时间分别为0、4、8、16、24、48h),观察心肌细胞存活率、凋亡率及caspase-3活性。分组三:对照组与palmitate(0.5mM)组,培养24h后检测p47phox mRNA和蛋白表达的变化。
结果:1.确定palmitate0.5mM为最佳干预浓度,且palmitate致心肌细胞凋亡损伤在一定范围内呈浓度依赖性。2.心肌细胞与palmitate0.5mM共孵育16h,存活率开始显著下降、凋亡率显著升高(P<0.01);且随着培养时间的延长,细胞凋亡数量逐渐增多,提示palmitate诱导心肌细胞凋亡呈显著时间依赖效应。3.与palmitate0.5mM共孵育24h后, NADPH氧化酶亚基P47phox mRNA及蛋白水平明显增加(P<0.05)。
结论:1.palmitate可诱导培养的H9c2心肌细胞发生凋亡,并对细胞凋亡率的影响呈显著的时间依赖性。2.palmitate可引起NADPH氧化酶亚基P47phox的基因及蛋白水平的明显上调。
第二部分Apocynin对palmitate处理H9c2心肌细胞的影响
目的:明确NADPH氧化酶是否参与palmitate诱导的H9c2心肌细胞凋亡。
方法:实验分为4组:对照组(Con),apocynin组(Apo,apocynin100uM),palmitate组(Pal,palmitate0.5mM),palmitate+apocynin组(Apo+Pal;palmitate0.5mM + apocynin100uM)。分别观察了Apocynin对palmitate处理心肌细胞的NADPH氧化酶亚基P47phox的mRNA水平及蛋白表达、荧光显微镜观察心肌细胞内ROS生成、酶标仪检测caspase-3活性,western-blotting检测心肌细胞的Bcl-2、Bax及caspase-3蛋白表达。
结果:
1. Apocynin对palmitate处理的心肌细胞内NADPH氧化酶亚基P47phox基因及蛋白水平的影响:Apocynin干预palmitate培养H9c2心肌细胞24h后,与对照组相比,单纯pal组H9c2心肌细胞内NADPH氧化酶亚基P47phox mRNA水平和蛋白表达均明显增加,具有统计学差异(P<0.05);pal+apo组与pal组相比,P47phox mRNA水平及蛋白表达明显下降,具有统计学差异(P<0.05)。
2. Apocynin对palmitate处理心肌细胞ROS产生的影响:与con组相比,pal组ROS生成明显增加;与pal组相比,pal+apo组ROS生成有所下降,阳性对照组强于pal组。
3. Apocynin对palmitate诱导H9C2心肌细胞caspase-3活性及蛋白表达的影响:与con组相比,caspase-3活性显著增加,具有统计学差异(P<0.05);与pal组相比,pal+apo组caspase-3活性明细下降,具有统计学差异(P<0.05)(见图2-4)。同时caspase-3蛋白表达的变化与其活性的变化相一致均具有统计学差异(P<0.05)。
4. Apocynin对palmitate诱导H9C2心肌细胞bcl-2,Bax蛋白表达的影响:与con组相比,pal组Bcl-2蛋白表达明显下调,具有统计学差异(P<0.05);与pal组相比,pal+apo组Bcl-2蛋白表达有显著改善,具有统计学差异(P<0.05)。而Bax蛋白表达变化则与Bcl-2的变化相反,且均具有统计学差异(P<0.05)。
结论:Palmitate可诱导H9c2心肌细胞p47phox基因及蛋白水平增加,心肌细胞内ROS生成增加,抗凋亡因子Bcl-2表达下降,促凋亡基因Bax表达上调,以及Caspase-3活性明显增加。NADPH氧化酶抑制剂apocynin可有效抑制上述改变,减少心肌内ROS生成和心肌细胞凋亡发生。
第三部分MAPK及NF-κB在Palmitate致H9c2心肌细胞凋亡中的作用
目的:明确MAPK及NF-κB在Palmitate致H9c2心肌细胞凋亡中的作用。
方法:分组一:对照组(Con),apocynin组(Apo,apocynin100uM),palmitate组(Pal,palmitate0.5mM),palmitate+apocynin组(Apo+Pal;palmitate0.5mM + apocynin100uM)。western-blotting检测Apocynin对palmitate处理心肌细胞NF-κB p65蛋白水平及p-p38,p-ERK,p-JNK蛋白表达。分组二:对照组(con),palmitate组(Pal,palmitate0.5mM),palmitate + SB203508组(pal+p38i),palmitate + PD98059组(pal+ERKi),palmitate + SP600125组(pal+JNKi)。western-blotting检测各组心肌细胞内Bcl-2,Bax蛋白表达以及caspase-3活性和细胞凋亡率的变化。
结果:
1.Apocynin对palmitate诱导H9c2心肌细胞P65表达的影响:与对照组相比,pal组胞核p65蛋白表达明细增强,有统计学差异(P<0.05);与pal组相比,pal+apo组p65蛋白表达明显下调,有统计学差异(P<0.05),而胞浆内p65蛋白表达,各组间比较无统计学差异(P>0.05)。
2.Apocynin对palmitate诱导H9c2心肌细胞p-p38,p-ERK,p-JNK蛋白表达的影响:与对照组相比,pal组p-p38、p-JNK蛋白表达明细增强,有统计学差异(P<0.05);与pal组相比,pal+apo组p-p38、p-JNK表达明显下调,有统计学差异(P<0.05)。各组间p-ERK蛋白表达,均无明显统计学差异(P>0.05)。
3. MAPK抑制剂对palmitate诱导心肌细胞Bcl-2,Bax蛋白表达的影响:与对照组相比,pal组抗凋亡蛋白Bcl-2表达显著下降,促凋亡蛋白Bax表达显著升高,有统计学差异(P<0.05);与pal组相比,pal+p38i组和pal+JNKi组Bcl-2蛋白表达上调,其中pal+JNKi组上调显著,有统计学意义(P<0.05),而pal+p38i上调不明显,无统计学差异(P>0.05);pal+p38i组、pal+JNKi组和pal+ERKi组Bax蛋白表达均有明显下调,有统计学差异(P<0.05)。
4. MAPK通路抑制剂对palmitate诱导H9c2心肌细胞caspase-3活性的影响:与对照组相比,pal组、pal+p38i组、pal+JNKi组caspase-3活性显著升高,有统计学差异(P<0.05);与pal组相比,pal+p38i组和pal+ERKi组caspase-3活性均有下降,但无统计学意义(P>0.05),pal+JNKi组caspase-3活性有明显下降(P<0.05)。
结论:p47phox mRNA及蛋白表达升高可导致NADPH氧化酶活性升高;NADPH氧化酶活性升高导致的氧化应激可能是palmitate致H9c2心肌细胞凋亡的上游机制之一;NF-κB和p38、JNK MAPK通路的激活可能参与了palmitate诱导的心肌细胞凋亡发生过程,从而启动抗凋亡因子Bcl-2下降和促凋亡因子Bax上调,caspase-3活性增强,最终实现细胞凋亡。
Recently, Lipid accumulation in the heart has been observed under conditions of elevated plasma free fatty acids, such as Type 2 diabetes mellitus and is linked with cardiomyocyte apoptosis, left ventricular contractile dysfunction. Fatty acids are a principal source of energy for the heart and the metabolism of fatty acids by the heart has been the subject of detailed reviews. Fatty acids, including palmitate, are present in high levels in the blood of patients following acute myocardial infarction and are responsible for increasing the extent and severity of myocardial injury.
Cardiomyocytes are terminally differentiated cells whose inability to reproduce underscores the seriousness of the loss of even a small amount of cardiac muscle cells in the situation of myocardial infarction, which is accompanied by high circulating concentrations of palmitate. The nature of palmitate-induced cardiomyocyte death, however, has not been well characterized. Recently, palmitate-induced apoptosis has been demonstrated in murine hematopoietic cell lines, embryonic chick astrocytes and neuronal cells, pancreatic beta cells and neonatal rat cardiomyocytes. Yet fatty acids have also been implicated in the induction of necrosis. Thus, it is unclear to what extent palmitate-induced cell death should be ascribed to the cascade of caspases, endonucleases and other factors that characterize the genetic program of apoptotic cell death.
The mechanisms underlying palmitate-induced cell death are incompletely understood. Palmitate is incorporated into de novo synthesis of ceramide, a lipid-signaling molecule implicated in the induction of apoptosis so that excess palmitate may induce cell death through increased intracellular ceramide concentration. An equally attractive hypothesis, that may be especially relevant for the heart, is that the adverse effect of excess fatty acids is due to other metabolic factors, so called toxic intermediates of fatty acid metabolism.
To more completely define the nature and mechanism underlying palmitate-induced cell death, cardiomyocytes were cultured from embryonic rat heart and were treated with palmitate.
Part I Effect of Pamitate on H9c2 myocardial cell apoptosis and activity of NADPH oxidase
Objective
To investigate the effect of palmitate on apoptosis concentration and time and the activity of NADPH oxidase in the H9c2 myocardial cell.
Methods
1. To find ideal concentration of palmitate the cardiomyocytes were divided into four groups: control group, 0.2mM palmitate group, 0.5mM palmitate group, 0.8mM palmitate group, and 1.2mM palmitate group. We examined the changes cell viability, cardiomyocyte apoptosis rate, and the activity of caspase-3 in the cardiomyocytes.
2. To observe time-effect of cell injury induced by palmitate, the cardiomyocytes were divided into five groups: 0h group, 4h group, 8h group, 16h group, 24h group and 48h. group. Cells were treated according to the instruction manual of AnnexinV-FITC KIT, then to perform FACS to measure the rate of apoptosis.
3. To observe the effect of palmitate on the NADPH oxidase subunit p47phox mRNA and protein level.
Results
1. A dose-dependent relation between the concentration of palmitate and H9c2 cardiomyocyte injury was observed. The H9c2 cardiomyocyte apoptosis rate was increased significantly, while the cellular viability was decreased in the 0.5mM group, this indicated that 0.5mmol/L palmitate was the ideal concentration to induc cardiomyocyte apoptosis.
2. A significant time-dependent relation between palmitate and cardiomyocyte injury was observed. H9c2 myocardial cell apoptosis rate was increased greatly after 16h treatment of palmitate .
3. p47phox mRNA and protein level was increased significantly campared with the control group after 24h treatment of palmitate.
Conclusions
1. H9c2 myocardial cell can be induced to injury and apoptosis by palmitate, and appears a significant time-dependent.
2. Palmitate induced significantly increase of p47phox mRNA and protein level in H9c2 myocardial cell.
Part II The effect of apocynin on the palmitate treated H9c2 cardiomyocytes
Objective
To investigate whether the NADPH oxidase involved in the process of the palmitate induced H9c2 myocardial cell apoptosis.
Methods
The experiment were divided into four groups: control group, apocynin group, palmitate group, and palmitate+apocynin group. We observed the ROS production using fluorescent probe, the P47phox mRNA was measured by RT-PCR. the Bcl-2、Bax and caspase-3 protein level were measured by western- blot.
Results
1. The effect of apocynin on the palmitate treated H9c2 cardiomyocytes NADPH oxidase P47phox mRNA and protein level: compared with the control, P47phox mRNA and protein level increased significantly in the palmitate group. (P<0.05). the apocynin can discrease the P47phox mRNA and protein level significantly(P<0.05).
2. The effect of apocynin on ROS production in the palmitate treated H9c2 cardiomyocytes: compared with the control,the ROS production significantly increased in palmitate group,apocynin can discrease the ROS production. The positive control is the best power in the ROS production.
3. Compared with the control,the caspase-3 activity, caspase-3 and Bax protein level were significantly increased in the palmitate group, and the Bcl-2 protein level was discrease in the palmitate group, apocynin can abatement the above change significantly(P<0.05).
Conclusions
Palmitate can increased the NADPH oxidase P47phox mRNA and protein level, and increased the ROS formation in the H9c2 myocardial cell, can led to the decrease of Bcl-2 and increase of Bax protein level, the caspase-3 activity also increased significantly. Apocynin can inhibit these changment.
Part III The effect of MAPK and NF-κB on the palmitate treated H9c2 cardiomyocytes apoptosis
Objective
To investigate the effect of MAPK and NF-κB on the palmitate induced H9c2 myocardial cell apoptosis.
Methods
First, the experiment were divided into 4 groups: control group, apocynin group, palmitate group and palmitate+apocynin group. The p65, p-p38,p-ERK,p-JNK were measured by western-blotting.
Then, the experiment were divided into 5 groups: control group, palmitate group, palmitate + SB203508 group(pal+p38i), palmitate + PD98059 group(pal+ERKi), palmitate + SP600125 group ( pal+JNKi ) . The Bcl-2, Bax were measured by western-blotting. The caspase-3 activity was detected by Enzyme Standardization instrument.
Results
1. Compared with the control, the P65 in nucleus protein was increased significantly in the palmitate group (P<0.05). the apocynin can discrease the P65 protein level significantly(P<0.05).
2. Palmitate can induce the p-p38、p-JNK protein level significantly(P<0.05). apocynin can discrease the p-p38、p-JNK protein level significantly(P<0.05).the p-ERK has no evident changement.
3. Palmitate decreased the Bcl-2 protein level and increased Bax significantly(P<0.05). the JNK inhibitor and p38 inhibitor inhibited the above changes. And only the JNK inhibitor had the statistical significanc(eP<0.05). The inhibitor of p38, JNK and ERK decreased the Bax protein level significantly compared with the palmitate group(P<0.05).
4. Compared with the control, the caspase-3 activity increased significantly in the pal group, pal+p38i group and pal+JNKi group(sP<0.05). compared with the palmitate group, caspase-3 activity decreased in the pal+p38i group and pal+ERKi group, but the decrease has no statistical significance(P>0.05),and the caspase-3 activity significantly decreased in the pal+JNKi group(P<0.05).
Conclusions
The increase of p47phox mRNA and protein level can induce the NADPH oxidase activity, which can lead to oxidative stress, these maybe the upstream mechanism of palmitate induced H9c2 myocardial cell apoptosis. The NF-κB and p38, JNK MAPK activation may participate the palmitate induced H9c2 myocardial cell apoptosis, and lead to the decrease of Bcl-2 and increase of Bax protein level, and ultimately generate apoptosis.
现有大量的动物和细胞培养实验也发现软脂酸(palmitate;PAL)可诱导多种细胞出现凋亡。但目前为止,脂肪酸引起细胞凋亡的具体分子细胞机制并不清楚。为探讨软脂酸损伤心肌细胞的机制,本文从以下三个部分进行了研究。
第一部分Pamitate对H9c2心肌细胞凋亡和NADPH氧化酶活性的影响
目的:明确palmitate致心肌细胞凋亡的最佳浓度与时间及其对心肌细胞内NADPH氧化酶亚基p47phox的影响。
方法:分组一:对照组与palmitate组(palmitate浓度分别为0.2,0.5,0.8,1.2mM)。培养24h后观察心肌细胞存活率、凋亡率,得到palmitate诱导心肌细胞凋亡的有效浓度。分组二:对照组与palmitate(0.5mM)组(处理时间分别为0、4、8、16、24、48h),观察心肌细胞存活率、凋亡率及caspase-3活性。分组三:对照组与palmitate(0.5mM)组,培养24h后检测p47phox mRNA和蛋白表达的变化。
结果:1.确定palmitate0.5mM为最佳干预浓度,且palmitate致心肌细胞凋亡损伤在一定范围内呈浓度依赖性。2.心肌细胞与palmitate0.5mM共孵育16h,存活率开始显著下降、凋亡率显著升高(P<0.01);且随着培养时间的延长,细胞凋亡数量逐渐增多,提示palmitate诱导心肌细胞凋亡呈显著时间依赖效应。3.与palmitate0.5mM共孵育24h后, NADPH氧化酶亚基P47phox mRNA及蛋白水平明显增加(P<0.05)。
结论:1.palmitate可诱导培养的H9c2心肌细胞发生凋亡,并对细胞凋亡率的影响呈显著的时间依赖性。2.palmitate可引起NADPH氧化酶亚基P47phox的基因及蛋白水平的明显上调。
第二部分Apocynin对palmitate处理H9c2心肌细胞的影响
目的:明确NADPH氧化酶是否参与palmitate诱导的H9c2心肌细胞凋亡。
方法:实验分为4组:对照组(Con),apocynin组(Apo,apocynin100uM),palmitate组(Pal,palmitate0.5mM),palmitate+apocynin组(Apo+Pal;palmitate0.5mM + apocynin100uM)。分别观察了Apocynin对palmitate处理心肌细胞的NADPH氧化酶亚基P47phox的mRNA水平及蛋白表达、荧光显微镜观察心肌细胞内ROS生成、酶标仪检测caspase-3活性,western-blotting检测心肌细胞的Bcl-2、Bax及caspase-3蛋白表达。
结果:
1. Apocynin对palmitate处理的心肌细胞内NADPH氧化酶亚基P47phox基因及蛋白水平的影响:Apocynin干预palmitate培养H9c2心肌细胞24h后,与对照组相比,单纯pal组H9c2心肌细胞内NADPH氧化酶亚基P47phox mRNA水平和蛋白表达均明显增加,具有统计学差异(P<0.05);pal+apo组与pal组相比,P47phox mRNA水平及蛋白表达明显下降,具有统计学差异(P<0.05)。
2. Apocynin对palmitate处理心肌细胞ROS产生的影响:与con组相比,pal组ROS生成明显增加;与pal组相比,pal+apo组ROS生成有所下降,阳性对照组强于pal组。
3. Apocynin对palmitate诱导H9C2心肌细胞caspase-3活性及蛋白表达的影响:与con组相比,caspase-3活性显著增加,具有统计学差异(P<0.05);与pal组相比,pal+apo组caspase-3活性明细下降,具有统计学差异(P<0.05)(见图2-4)。同时caspase-3蛋白表达的变化与其活性的变化相一致均具有统计学差异(P<0.05)。
4. Apocynin对palmitate诱导H9C2心肌细胞bcl-2,Bax蛋白表达的影响:与con组相比,pal组Bcl-2蛋白表达明显下调,具有统计学差异(P<0.05);与pal组相比,pal+apo组Bcl-2蛋白表达有显著改善,具有统计学差异(P<0.05)。而Bax蛋白表达变化则与Bcl-2的变化相反,且均具有统计学差异(P<0.05)。
结论:Palmitate可诱导H9c2心肌细胞p47phox基因及蛋白水平增加,心肌细胞内ROS生成增加,抗凋亡因子Bcl-2表达下降,促凋亡基因Bax表达上调,以及Caspase-3活性明显增加。NADPH氧化酶抑制剂apocynin可有效抑制上述改变,减少心肌内ROS生成和心肌细胞凋亡发生。
第三部分MAPK及NF-κB在Palmitate致H9c2心肌细胞凋亡中的作用
目的:明确MAPK及NF-κB在Palmitate致H9c2心肌细胞凋亡中的作用。
方法:分组一:对照组(Con),apocynin组(Apo,apocynin100uM),palmitate组(Pal,palmitate0.5mM),palmitate+apocynin组(Apo+Pal;palmitate0.5mM + apocynin100uM)。western-blotting检测Apocynin对palmitate处理心肌细胞NF-κB p65蛋白水平及p-p38,p-ERK,p-JNK蛋白表达。分组二:对照组(con),palmitate组(Pal,palmitate0.5mM),palmitate + SB203508组(pal+p38i),palmitate + PD98059组(pal+ERKi),palmitate + SP600125组(pal+JNKi)。western-blotting检测各组心肌细胞内Bcl-2,Bax蛋白表达以及caspase-3活性和细胞凋亡率的变化。
结果:
1.Apocynin对palmitate诱导H9c2心肌细胞P65表达的影响:与对照组相比,pal组胞核p65蛋白表达明细增强,有统计学差异(P<0.05);与pal组相比,pal+apo组p65蛋白表达明显下调,有统计学差异(P<0.05),而胞浆内p65蛋白表达,各组间比较无统计学差异(P>0.05)。
2.Apocynin对palmitate诱导H9c2心肌细胞p-p38,p-ERK,p-JNK蛋白表达的影响:与对照组相比,pal组p-p38、p-JNK蛋白表达明细增强,有统计学差异(P<0.05);与pal组相比,pal+apo组p-p38、p-JNK表达明显下调,有统计学差异(P<0.05)。各组间p-ERK蛋白表达,均无明显统计学差异(P>0.05)。
3. MAPK抑制剂对palmitate诱导心肌细胞Bcl-2,Bax蛋白表达的影响:与对照组相比,pal组抗凋亡蛋白Bcl-2表达显著下降,促凋亡蛋白Bax表达显著升高,有统计学差异(P<0.05);与pal组相比,pal+p38i组和pal+JNKi组Bcl-2蛋白表达上调,其中pal+JNKi组上调显著,有统计学意义(P<0.05),而pal+p38i上调不明显,无统计学差异(P>0.05);pal+p38i组、pal+JNKi组和pal+ERKi组Bax蛋白表达均有明显下调,有统计学差异(P<0.05)。
4. MAPK通路抑制剂对palmitate诱导H9c2心肌细胞caspase-3活性的影响:与对照组相比,pal组、pal+p38i组、pal+JNKi组caspase-3活性显著升高,有统计学差异(P<0.05);与pal组相比,pal+p38i组和pal+ERKi组caspase-3活性均有下降,但无统计学意义(P>0.05),pal+JNKi组caspase-3活性有明显下降(P<0.05)。
结论:p47phox mRNA及蛋白表达升高可导致NADPH氧化酶活性升高;NADPH氧化酶活性升高导致的氧化应激可能是palmitate致H9c2心肌细胞凋亡的上游机制之一;NF-κB和p38、JNK MAPK通路的激活可能参与了palmitate诱导的心肌细胞凋亡发生过程,从而启动抗凋亡因子Bcl-2下降和促凋亡因子Bax上调,caspase-3活性增强,最终实现细胞凋亡。
Recently, Lipid accumulation in the heart has been observed under conditions of elevated plasma free fatty acids, such as Type 2 diabetes mellitus and is linked with cardiomyocyte apoptosis, left ventricular contractile dysfunction. Fatty acids are a principal source of energy for the heart and the metabolism of fatty acids by the heart has been the subject of detailed reviews. Fatty acids, including palmitate, are present in high levels in the blood of patients following acute myocardial infarction and are responsible for increasing the extent and severity of myocardial injury.
Cardiomyocytes are terminally differentiated cells whose inability to reproduce underscores the seriousness of the loss of even a small amount of cardiac muscle cells in the situation of myocardial infarction, which is accompanied by high circulating concentrations of palmitate. The nature of palmitate-induced cardiomyocyte death, however, has not been well characterized. Recently, palmitate-induced apoptosis has been demonstrated in murine hematopoietic cell lines, embryonic chick astrocytes and neuronal cells, pancreatic beta cells and neonatal rat cardiomyocytes. Yet fatty acids have also been implicated in the induction of necrosis. Thus, it is unclear to what extent palmitate-induced cell death should be ascribed to the cascade of caspases, endonucleases and other factors that characterize the genetic program of apoptotic cell death.
The mechanisms underlying palmitate-induced cell death are incompletely understood. Palmitate is incorporated into de novo synthesis of ceramide, a lipid-signaling molecule implicated in the induction of apoptosis so that excess palmitate may induce cell death through increased intracellular ceramide concentration. An equally attractive hypothesis, that may be especially relevant for the heart, is that the adverse effect of excess fatty acids is due to other metabolic factors, so called toxic intermediates of fatty acid metabolism.
To more completely define the nature and mechanism underlying palmitate-induced cell death, cardiomyocytes were cultured from embryonic rat heart and were treated with palmitate.
Part I Effect of Pamitate on H9c2 myocardial cell apoptosis and activity of NADPH oxidase
Objective
To investigate the effect of palmitate on apoptosis concentration and time and the activity of NADPH oxidase in the H9c2 myocardial cell.
Methods
1. To find ideal concentration of palmitate the cardiomyocytes were divided into four groups: control group, 0.2mM palmitate group, 0.5mM palmitate group, 0.8mM palmitate group, and 1.2mM palmitate group. We examined the changes cell viability, cardiomyocyte apoptosis rate, and the activity of caspase-3 in the cardiomyocytes.
2. To observe time-effect of cell injury induced by palmitate, the cardiomyocytes were divided into five groups: 0h group, 4h group, 8h group, 16h group, 24h group and 48h. group. Cells were treated according to the instruction manual of AnnexinV-FITC KIT, then to perform FACS to measure the rate of apoptosis.
3. To observe the effect of palmitate on the NADPH oxidase subunit p47phox mRNA and protein level.
Results
1. A dose-dependent relation between the concentration of palmitate and H9c2 cardiomyocyte injury was observed. The H9c2 cardiomyocyte apoptosis rate was increased significantly, while the cellular viability was decreased in the 0.5mM group, this indicated that 0.5mmol/L palmitate was the ideal concentration to induc cardiomyocyte apoptosis.
2. A significant time-dependent relation between palmitate and cardiomyocyte injury was observed. H9c2 myocardial cell apoptosis rate was increased greatly after 16h treatment of palmitate .
3. p47phox mRNA and protein level was increased significantly campared with the control group after 24h treatment of palmitate.
Conclusions
1. H9c2 myocardial cell can be induced to injury and apoptosis by palmitate, and appears a significant time-dependent.
2. Palmitate induced significantly increase of p47phox mRNA and protein level in H9c2 myocardial cell.
Part II The effect of apocynin on the palmitate treated H9c2 cardiomyocytes
Objective
To investigate whether the NADPH oxidase involved in the process of the palmitate induced H9c2 myocardial cell apoptosis.
Methods
The experiment were divided into four groups: control group, apocynin group, palmitate group, and palmitate+apocynin group. We observed the ROS production using fluorescent probe, the P47phox mRNA was measured by RT-PCR. the Bcl-2、Bax and caspase-3 protein level were measured by western- blot.
Results
1. The effect of apocynin on the palmitate treated H9c2 cardiomyocytes NADPH oxidase P47phox mRNA and protein level: compared with the control, P47phox mRNA and protein level increased significantly in the palmitate group. (P<0.05). the apocynin can discrease the P47phox mRNA and protein level significantly(P<0.05).
2. The effect of apocynin on ROS production in the palmitate treated H9c2 cardiomyocytes: compared with the control,the ROS production significantly increased in palmitate group,apocynin can discrease the ROS production. The positive control is the best power in the ROS production.
3. Compared with the control,the caspase-3 activity, caspase-3 and Bax protein level were significantly increased in the palmitate group, and the Bcl-2 protein level was discrease in the palmitate group, apocynin can abatement the above change significantly(P<0.05).
Conclusions
Palmitate can increased the NADPH oxidase P47phox mRNA and protein level, and increased the ROS formation in the H9c2 myocardial cell, can led to the decrease of Bcl-2 and increase of Bax protein level, the caspase-3 activity also increased significantly. Apocynin can inhibit these changment.
Part III The effect of MAPK and NF-κB on the palmitate treated H9c2 cardiomyocytes apoptosis
Objective
To investigate the effect of MAPK and NF-κB on the palmitate induced H9c2 myocardial cell apoptosis.
Methods
First, the experiment were divided into 4 groups: control group, apocynin group, palmitate group and palmitate+apocynin group. The p65, p-p38,p-ERK,p-JNK were measured by western-blotting.
Then, the experiment were divided into 5 groups: control group, palmitate group, palmitate + SB203508 group(pal+p38i), palmitate + PD98059 group(pal+ERKi), palmitate + SP600125 group ( pal+JNKi ) . The Bcl-2, Bax were measured by western-blotting. The caspase-3 activity was detected by Enzyme Standardization instrument.
Results
1. Compared with the control, the P65 in nucleus protein was increased significantly in the palmitate group (P<0.05). the apocynin can discrease the P65 protein level significantly(P<0.05).
2. Palmitate can induce the p-p38、p-JNK protein level significantly(P<0.05). apocynin can discrease the p-p38、p-JNK protein level significantly(P<0.05).the p-ERK has no evident changement.
3. Palmitate decreased the Bcl-2 protein level and increased Bax significantly(P<0.05). the JNK inhibitor and p38 inhibitor inhibited the above changes. And only the JNK inhibitor had the statistical significanc(eP<0.05). The inhibitor of p38, JNK and ERK decreased the Bax protein level significantly compared with the palmitate group(P<0.05).
4. Compared with the control, the caspase-3 activity increased significantly in the pal group, pal+p38i group and pal+JNKi group(sP<0.05). compared with the palmitate group, caspase-3 activity decreased in the pal+p38i group and pal+ERKi group, but the decrease has no statistical significance(P>0.05),and the caspase-3 activity significantly decreased in the pal+JNKi group(P<0.05).
Conclusions
The increase of p47phox mRNA and protein level can induce the NADPH oxidase activity, which can lead to oxidative stress, these maybe the upstream mechanism of palmitate induced H9c2 myocardial cell apoptosis. The NF-κB and p38, JNK MAPK activation may participate the palmitate induced H9c2 myocardial cell apoptosis, and lead to the decrease of Bcl-2 and increase of Bax protein level, and ultimately generate apoptosis.
引文
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