摘要
心肌能量代谢障碍在心力衰竭的发生、发展中的作用得到越来越多的关注。线粒体作为细胞内重要的细胞器,它不仅是心肌细胞氧化磷酸化产生能量的主要场所,在能量代谢方面有不可替代的作用,而且还与细胞中氧自由基的生成、细胞程序性死亡、细胞信号转导、细胞内多种离子的跨膜转运及电解质稳态平衡的调控等有关,在细胞的整个生命活动中充当着非常关键的角色。因此,在慢性心肌重构病理过程中,线粒体结构的变化和心肌细胞能量代谢障碍的规律,值得我们关注和研究。依托改善心肌重构为基础的治疗能够延缓心力衰竭进程的事实,我们假设：改善心肌重构会起到保护心肌细胞线粒体的作用,线粒体是与能量产生有关的亚细胞器,改善心肌细胞重构也会对线粒体能量代谢有益；川芎嗪、黄芪注射液及参附注射液能够改善心肌重构,也可能会对线粒体结构和心肌能量代谢产生有利影响。
目的：
1.通过大鼠原代心肌细胞培养,研究在心肌细胞肥大过程中,心肌细胞线粒体结构、功能和能量代谢情况。
2.探讨川芎嗪、黄芪注射液及参附注射液在抑制心肌细胞肥大过程中对线粒体的影响,能否保护线粒体、改善心肌细胞的能量代谢。
方法：
1.分离和培养大鼠原代心肌细胞,血管紧张素Ⅱ(KAngⅡ)共培养,通过BCA法去检测细胞总蛋白含量、倒置显微镜拍摄并测量细胞直径,反映心肌细胞肥大情况。
2.通过荧光显微镜测量线粒体膜电位(△Ψm)、酶标仪检测线粒体单胺氧化酶(MAO)活性、分光光度计检测线粒体细胞色素C氧化酶(COX)活性和线粒体外膜损伤率、SDS-聚丙烯酰胺凝胶电泳检测线粒体外膜Bcl-2、内膜ANT蛋白表达,反映肥大心肌细胞线粒结构和功能的损伤。
3.高效液相色谱法检测心肌细胞内ATP、ADP、AMP含量,并计算总腺苷酸池含量及能荷水平,反映肥大心肌细胞能量代谢情况。
4.以缬沙坦作为抑制心肌细胞肥大的阳性对照药,观察川芎嗪、黄芪注射液、参附注射液在抑制心肌细胞肥大过程中对线粒体结构、功能以及能量代谢的药理作用
5.观察能量代谢调节药物—曲美他嗪对肥大心肌细胞线粒体结构、功能以及能量代谢的影响。
结果：
1.在24 h和48 h两个时间点,模型组心肌细胞总蛋白含量、心肌细胞直径较同期空白组有所增多,但差异无统计学意义；在72 h和96 h两个时间点,模型组心肌细胞总蛋白含量、心肌细胞直径与同期空白组相比均明显增加。
2.与同期空白组相比,24 h时模型组心肌细胞MAO活性有所增加,但差异无统计学意义,48 h、72 h和96 h时模型组心肌细胞MAO活性均明显或显著性增加;在24h和48 h两个时间点,模型组心肌细胞线粒体COX活性较有所降低,但差异无统计学意义；72 h、96 h时模型组心肌细胞线粒体COX活性明显或显著性降低；在24 h、48 h、72 h、96 h四个时间点,模型组心肌细胞线粒体外膜损伤率与同期空白组相比均显著增加；在以上四个时间点,模型组心肌细胞线粒体△Ψm较同期空白组均显著下降；模型组心肌细胞线粒体外膜Bcl-2蛋白表达与同期空白组相比均有所下降,但差异均无统计学意义；24 h时心肌细胞线线粒体内膜ANT蛋白表达有所下降,但差异均无统计学意义,在48 h、72 h、96 h三个时间点模型组心肌细胞线线粒体内膜ANT蛋白表达均明显下降。
3.较同期空白组,在24 h、48 h、72 h、96 h四个时间点模型组心肌细胞ATP、ADP含量、心肌细胞总腺苷酸池含量、心肌细胞能荷水平均明显或显著性下降；24 h时模型组心肌细胞AMP含量有所增加,但差异无统计学意义,在48 h、72 h、96h三个时间点时模型组心肌细胞AMP含量较同期空白组明显或显著性上升
4.心肌细胞总蛋白含量,在72 h和96 h两个时间点与同期模型组相比,川芎嗪组明显减少,缬沙坦组显著减少,96 h时与同期模型组相比,黄芪注射液组和参附注射液组心肌细胞蛋白含量均明显减少；在以上两个时间点,黄芪注射液、川芎嗪和缬沙坦组心肌细胞直径均明显或显著性减少,72 h时较同期模型组,参附注射液组细胞直径亦明显缩小。
5.72 h和96 h时与同期模型组相比,黄芪注射液、川芎嗪和缬沙坦组MAO活性均明显或显著性降低；72 h时较同期模型组,参附注射液组亦能显著降低MAO活性；以上两个时间点较同期模型组,缬沙坦组、川芎嗪组、参附注射液组COX活性均明显升高,72 h时黄芪组注射液组线粒体COX活性明显升高,96 h时则升高显著；缬沙坦组和川芎嗪组线粒体外膜损害率在以上两个时间点较同期模型组均显著下降,黄芪注射液组和参附注射液组线粒体外膜损害率亦有明显降低；72 h和96 h时,与同期模型组相比,缬沙坦组和川芎嗪组的线粒体△Ψm均明显升高,72 h时黄芪注射液组和参附注射液组线粒体△Ψm明显升高,96h时则显著性升高；以上两个时间点,与同期模型组相比,缬沙坦组、川芎嗪组、黄芪注射液组和参附注射液组线粒体外膜Bcl-2和内膜ANT蛋白表达均有不同程度升高,但差异无统计学意义。
6.72 h和96 h时较同期模型组,缬沙坦组、川芎嗪组以及黄芪注射液组均显著提高了心肌细胞内ATP、ADP含量,与同期模型组相比,72 h时参附注射液组心肌细胞ATP、ADP含量著提高,96h时亦明显提高；以上两个时间点较同期模型组,缬沙坦组显著降低了心肌细胞内AMP含量,黄芪注射液组和参附注射液组心肌细胞AMP含量亦明显降低,与同期模型组比较,72 h时川芎嗪组心肌细胞AMP含量著降低,96h时亦明显降低；72 h时与同期模型组相比,缬沙坦组明显提高了心肌细胞内总腺苷酸池含量,黄芪注射液组心肌细胞内总腺苷酸池含量显著提高,96 h时与同期模型组相比,参附注射液组心肌细胞内总腺苷酸池含量明显提高,缬沙坦组、川芎嗪组和黄芪注射液组则显著提高了心肌细胞内总腺苷酸池含量。以上两个时间点较同期模型组,缬沙坦组、川芎嗪组、黄芪注射液组以及参附注射液组均显著提高了心肌细胞的能荷水平。
7.72 h和96 h时较同期模型组,曲美他嗪组心肌细胞总蛋白含量和心肌细胞直径减少、心肌细胞MAO活性、线粒体外膜损伤率有所降低、线粒体外膜Bcl-2蛋白和内膜ANT蛋白表达有所提高,但以上差异均无统计学意义。较同期模型组,72 h时曲美他嗪组心肌细胞线粒体COX活性、线粒体△Ψm明显升高,心肌细胞内ATP、ADP含量明显上升,心肌细胞的能荷水平显著提高,96h时曲美他嗪组心肌细胞总腺苷酸含量明显升高。
结论：
1.在心肌细胞肥大过程中,存在线粒体的结构和功能的损害。
2.肥大心肌细胞存在能量代谢障碍,心肌细胞能量代谢障碍是心肌细胞肥大的早期敏感指标。
3.川芎嗪、黄芪注射液、参附注射液和缬沙坦在抑制血管紧张素Ⅱ所引起的心肌细胞肥大的过程中均具有一定的保护心肌细胞线粒体的作用。
4.川芎嗪、黄芪注射液、参附注射液和缬沙坦在抑制心肌细胞肥大、保护线粒体的过程中,能够改善心肌细胞能量代谢障碍。
5.曲美他嗪对肥大心肌细胞的能量代谢有一定的改善作用,但不能保护心肌细胞线粒体,不具有抑制心肌细胞肥大的作用。
In recent years, the cardiac energy metabolism obstacle in the occurrence and development of heart failure gets more and more attention. In cells, mitochondria as important organelles, are not only the main organelles that generate energy by oxidative phosphorylation, playing irreplaceable role in energy metabolism, but also involved in the cell oxygen free radical production, programmed cell death, signal transduction, various ions transshipment across cell membrane and the steady state of electrolyte. In the whole life of activity cells, mitochondria acts as a critical role. Therefore, regular pattern of mitochondrial structure change and cardiac energy metabolism obstacle in the process of chronic cardiac reconstruction is worth studying. Based on the fact that improving myocardial reconstruction treatment can delay heart failure progress, we assume that the treatment will protect mitochondrial function. Mitochondria are organelles producing energy. So improve the myocardial cell reconstruction will benefit mitochondrial energy metabolization. The traditional Chinese medicine of Benefiting Vital Energy, Activating Blood Circulation and Warming Yang are able to restrain myocardial reconstruction, and may also be benefit mitochondrial structure and cardiac energy metabolism.
Objective:
1. Primary cultures of neonatal rat myocardial cells were performed to explore pathological problems about mitochondria changes in the process of myocardial hypertrophy.
2. Explore the influences of traditional Chinese drugs which benefit vital energy, activate blood circulation and warm yang on mitochondria and energy metabolism in the process of restraining myocardial cell hypertrophy caused by AngⅡ.
Methods:
1. Neonatal myocardial cells were dispersed and cultured with angiotensinⅡ(AngⅡ). We detected the total protein content with BCA method and measured cell diameter with inverted microscope, to show the situation of cardiomyocyte hypertrophy.
2. We detected mitochondrial membrane potential (A^m) with fluorescence microscope and mitochondrial single amine oxidase (MAO) activity with spectrophotometer and mitochondrial cytochrome oxidase (COX) activity and the damage percentage of mitochondrial outer membrane with microplate reader and protein expression of mitochondrial outer membrane Bcl-2, inner membrane ANT with western blot, to show the damage of mitochondrial construction and function in hypertrophic cardiomyocyte.
3. We detected the content of ATP、ADP、AMP with high performance liquid chromatography and calculated the total adenosine acid content and energy charge, to show the situation of energy metabolism in hypertrophic cardiomyocyte.
4. Valsartan as positive control, cells also were treated with ligustrazine、radix astragali injection and shenfu injection and then the pharmacological effects on mitochondrial structure, function and energy metabolism in the myocardial cells treated with AngⅡwere observed. 5. Observe the influences of energy metabolism drug(trimetazidine) on mitochondrial structure、function and energy metabolism in hypertrophic cardiomyocyte.
Results:
1. At 24 h and 48 h, compared with control, cells treated with AngⅡhad increased total protein content and enlarged diameter, but there were no difference between the two groups. At 72 h and 96 h, compared with control, total protein content and enlarged diameter were obviously increased.
2. At 24 h, compared with control, myocardial cells treated with Ang II had increased MAO activity,but there was no difference between them. At 48 h.72 h、96 h、myocardial cells MAO activity were obviously or significantly increased. At 24 h and 48 h, compared with control, myocardial cell treated with AngⅡCOX had decreased activity, but there was no difference between them. At 72 h and 96 h, compared with control, myocardial cell COX activity was obviously or significantly decreased. At 24 h、48 h.72 h and 96 h, compared with control, myocardial cells treated with AngⅡhad increased the damage percentage of mitochondrial outer membrane. At 24 h、48 h、72 h and 96 h, compared with control, myocardial cells treated with AngⅡhad significantly reduced mitochondrial△Ψ. At 24 h、48 h、72 h and 96 h, compared with control, myocardial cells treated with AngⅡhad declined expression of Bcl-2, but there was no difference between the two groups. At 24 h, compared with control, myocardial cells treated with Ang II had declined expression of ANT, but there was no difference between them. At 48 h、72 h and 96 h, compared with control, the expression of ANT were obviously declined.
3. At 24 h.48 h、72 h and 96 h, compared with control, myocardial cells treated with Ang II had obviously or significantly decreased the content of ATP、ADP、total adenosine acid pool and the level of energy charge. At 24 h, compared with control, myocardial cells treated with Ang II had increased the content of AMP, but there was no difference between the two groups. At 48 h.72 h and 96 h, compared with control, the content of AMP was obviously or significantly increased.
4. At 72 h and 96 h, compared with control, ligustrazine and valsartan had significantly reduced myocardial cell total protein content, At 96 h, compared with control, radix astragali injection and shenfu injection had obviously reduced myocardial cell total protein content. At 72 h and 96 h, compared with control, ligustrazine, radix astragali injection and valsartan had obviously or significantly reduced cell diameter. At 72 h, compared with control, shenfu injection had obviously reduced cell diameter.
5. At 72 h and 96 h. compared with control, ligustrazine. radix astragali injection and valsartan had obviously or significantly reduced myocardial cell MAO activity. At 72 h. compared with control, shenfu injection had obviously reduced myocardial cell MAO activity. At the two time points, compared with control, ligustrazine. shenju injection and valsartan had significantly increased mitochondrial COX activity. At 72 h, compared with control, radix astragali injection had obviously increased mitochondrial COX activity. At 96 h, mitochondrial COX activity was significantly increased. At the two time points, ligustrazine and valsartan had significantly reduced the damage percentage of mitochondrial outer membrane, radix astragali injection and shenju injection also had obviously reduced the damage percentage of mitochondrial outer membrane. At 72 h and 96 h, ligustrazine and valsartan had obviously improved mitochondrial AT compared with control. At 72 h, compared with control, radix astragali injection and shenju injection also had obviously improved mitochondrial△Ψ. At 96h, mitochondrial△Ψwas significantly improved. At the two time points, compared with control, ligustrazine、radix astragali injection、shenju injection and valsartan had increased expression of ANT. Bcl-2, but there was no differences between the two groups.
6. At 72 h and 96 h, compared with control, ligustrazine, radix astragali injection and valsartan had significantly increased content of ATP. ADP. At 72 h, shenfu injection had obviously increased contents of ATP、ADP compared with control. At 96 h, contents of ATP. ADP were significantly increased. At the two time points, valsartan had significantly decreased content of AMP, ligustrazine, radix astragali injection had obviously decreased content of AMP. At 72 h, compared with control, shenfu injection had obviously decreased content of AMP. At 96 h, the content of AMP were significantly decreased. At 72 h, compared with control, valsartan had obviously increased content of total adenosine acid pool, radix astragali injection had significantly increased content of total adenosine acid pool. At 96 h, compared with control, shenfu injection had obviously increased content of total adenosine acid pool, ligustrazine. radix astragali injection and valsartan had significantly increased content of total adenosine acid pool. At the two time points, compared with control, ligustrazine、radix astragali injection. shenfu injection and valsartan had significantly increased the level of energy charge.
7. At 72 h and 96 h, compared with control, trimetazidine had reduced myocardial cell total protein content, cell diameter, myocardial cell MAO activity, the damage percentage of mitochondrial outer membrane, increased expression of ANT, Bcl-2, but there were no differences between the two groups. At 72 h, compared with control, trimetazidine had obviously increased mitochondrial COX activity, mitochondrial△Ψ, contents of ATP. ADP. the level of energy charge. At 96 h, trimetazidine had obviously increased content of total adenosine acid pool compared with control.
Conclusions:
1. During the process of myocardial hypertrophy, damages of mitochondrial structure and function occurred.
2. Changes of myocardial cell energy metabolism occurred in hypertrophic cardiomyocyte. Energy metabolism obstacle is a sensitive indicator in the early stage of myocardial hypertrophy.
3. Ligustrazine. radix astragali injection、shenju injection and valsartan can restrain myocardial cell hypertrophy caused by AngⅡ, by protecting mitochondrial structure and function.
4. Ligustrazine. radix astragali injection、shenju injection and valsartan can improve energy metabolism of the myocardial cell by restraining myocardial cell hypertrophy and protecting mitochondria.
5. Trimetazidine had some good effects on energy metabolism in hypertrophic cardiomyocyte. Trimetazidine can't protect mitochondria and reverse myocardial cell hypertrophy.
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