| Background:Cardiovascular disease is one of the most serious diseases that threaten human healthand coronary heart disease is the most common cardiovascular disease. The treatment ofcoronary heart disease is dominated by methods of positive thrombolysis, coronary stent,but when restores blood flow, it is found that functional metabolic disorder and structuralfailure of the cells worsen instead of being alleviated, i.e. resulting in myocardialischemia/reperfusion injury. Large amounts of active oxygen are produced duringreperfusion, which induces cells death. However, and despite a low tension in oxygen, asignificant reactive oxygen species generation during the ischemic phase was also reportedand associated with reperfusion-induced cell death. Heart is the biggest energyconsumption organ of human body. Once ischemia and hypoxia occur to myocardium,heart dysfunction will be more serious. At present, it is considered that the possiblemechanisms that cause myocardial damage includes ATP undersupply, calcium influx andsevere oxidative stress injury, all of which have increased the permeability ofmitochondrial membrane and have activated apoptosis pathway of mitochondria. Therefore, if some small biological molecule can cut the inner relations of the above oneor more links and prevent the activation of mitochondrial apoptosis pathway at the time ofmyocardial ischemia reperfusion, it can reduce the apoptosis of myocardial cells and playthe role of heart protection, that is, it can be used to prevent or treat insufficient bloodsupply of myocardium.Berberine (BBR), belonging to isoquinoline derivatives alkaloid, is a majorcomponent of Rhizoma coptidis(Huanglian), a herb used in traditional Chinese medicine.In traditional Chinese medicine, it has been used to treat diarrhea and digestive systemdisease for a very long time. At present, a growing number of studies reveal that BBR alsohas the functions of increasing myocardial contractility, improving myocardial blood flow,anti-arrhythmia, regulating pressure, reducing lipid and etc. BBR has good preventionfunction for various cardiovascular diseases, but its mechanism is still not clear. Theobjective of this experiment is to discuss the protective effect and its molecularmechanism of BBR on hypoxia/reoxygenation damage-induced apoptosis in H9C2myocardium cells.Objective:1. To observe the protective effect of BBR on hypoxia/reoxygenation-inducedapoptosis in H9C2myocardium cells2. To study the mechanisms of BBR on hypoxia/reoxygenation-induced apoptosis inH9C2myocardium cellsMethods:Experiment Ⅰ: Firstly, screen the effective time period of hypoxia/reoxygenation(H/R), and then establish the cardiac H/R injury model. Specific groups were as follows:the normal control group, the H9C2cardiomyocytes were treated according to thedifferent time periods of H/R groups (1h/23h;2h/22h;4h/20h;8h/16h;12h/12h;16h/8h), screening the final effective time period, and eventually we selected H/R4/20hgroup as the following experimental model group. Then, MTT method was used fordetecting effects of BBR on the viability of H9C2myocardial cells. Specific groups wereas follows: the normal control group, with different concentrations of BBR groups (10,20, 50,100,150,200μmol/L). Finally, observe the protective effects of BBR on cardiacmuscle, Specific groups were as follows: the normal control group (Con), BBR alonetreatment group (Ber), the H/R (4/20h) model group (H/R), the pretreatment groups(L-Ber; H-Ber) with different concentrations of BBR (10,100μmol/L+H/R). Hoechst33258marked with nuclear staining was used to observe changes of nuclear morphologyof H9C2apoptosis. Cell apoptosis ratio was detected by flow cytometry. LDH wasmeasured according to the instructions of lactate dehydrogenase (LDH) assay kit.ExperimentⅡ: To investigate the protective mechanism of BBR on H9C2cardiacH/R injury. According to the design of the experimentⅠ, set up the normal control group(Con), BBR alone treatment group (Ber), the H/R (4/20h) model group (H/R),pretreatment groups (L-Ber; H-Ber) with different concentrations of BBR (10,100μmol/L+H/R). The cell probe rhodamine123was used to observe changes of mitochondrialmembrane potential (MMP). Western blotting was used to detect the expression of Bcl-2,Bax, Cyt c, Smac, and Caspase-3,6,9proteins in cytoplasmic lysates.Results:ExperimentⅠ:1. MTT method was used to detect the viability of H9C2cells, compared with thecontrol group, the cell viability in the H/R model group was (48.6±1.3)%(P <0.01), butafter pretreated with10,100μmol/L BBR for24h, compared with the H/R group(48.6±1.3％), the cell viability in the pretreatment groups was increased to (67.6±2.2)%(P<0.01) and (82.6±1.5)%(P<0.01), respectively. However, BBR alone (100μmol/L) hadno obvious effect on H9C2cell viability (Fig.2).2. To assess the protective effect of BBR on the H/R-induced H9C2myocardial cellinjury, LDH release was considered as the observation index in this article. After cellswere treated with H/R (4/20h), compared with the control group, the LDH release wassignificantly increased,232.1±6.60%(Fig.2), but after pretreated with10,100μmol/LBBR for24h, compared with the control group, LDH release was significantly decreasedto173.9±5.6%(P <0.05),127.4±4.6%(P <0.01), respectively.3. Membrane permeable dye Hechst33258was used to observe the apoptosis of nucleus, nucleus was evenly diffused colored with smooth and neat edges under normalcircumstances. This experiment results also showed that the nucleus of normal controlgroup showed uniform diffused blue-stain, but the chromatin in the model group after H/R(4/20h) treatment was stained deeply and showed shrinkage and fragmentation, displayingbright blue fluorescence. But after pretreated with10,100μmol/L BBR, cell apoptosiswas significantly improved. Also BBR alone treatment had no significant effect on H9C2nucleus (Fig.3).4. FCM assay was used to detect the cell apoptotic ratio, this experiment resultsshowed that compared with the normal control group, the cell apoptotic ratios in the BBRalone treatment group and H/R (4/20h) model group were increased to46.2%from1.8%and1.6%, respectively. However, the cell apoptotic ratios pretreated with BBR (10,100μmol/L) were reduced to33.7%and8.4%, respectively (Fig.4).Experiment Ⅱ:1. A fluorescent dye rhodamine123was used to measure the mitochondrialmembrane potential. In order to clarify the impact of H/R (4/20h) and/or BBR on MMPchanges, we used a fluorescent probe rhodamine123to observe the integrity of cell MMP.Compared with the control (Fig.5), the fluorescence intensity of rhodamine123of H9C2cells after H/R (4/20hours) treatment was decreased significantly. And after BBRpretreatment, it dose-dependently attenuated changes of mitochondrial membranepotential caused by H/R (4/20hours) in H9C2cells. BBR (100μmol/L) alone had nosignificant effect on H9C2mitochondrial membrane potential.2. Western Blot results showed after H9C2cells were treated with H/R (4/20h), Bax,Cyt C, Smac and Caspase-3,6,9in the cytoplasm were significantly increased,respectively, but Bcl-2was significantly reduced. However, BBR (10,100μmol/L)pretreatment significantly inhibited their expression changes induced by H/R. In addition,BBR (100μmol/L) alone treatment did not produce significant effects on these proteinsexpressions in H9C2cells (Fig.6,7,8).Conclusion:1. BBR can attenuated H/R-induced apoptosis in H9C2cells. 2. Berberine decreases apoptosis by regulating Bcl-2family proteins and activatingcaspase pathway in Hypoxia/Reoxygenation-induced H9C2Cardiomyocytes... |
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