| BackgroundCoronary heart disease (CHD) has been seriously jeopardizing health and quality of life of human beings. There are about3800000men and3400000women die of the CHD each year around the world. The most ideal therapy for acute myocardial infarction myocardial measures is based in the coronary artery that has pass on full reperfusion, namely myocardial microcirculation reperfusion, it includes two aspects to epicardial reperfusion and myocardial tissue reperfusion. For the former, in thrombolysis, PCI and CABG developed today, to achieve this goal has become a reality. As for the latter, how to prevent from reperfusion injury and myocardial reperfusion is urgently needed to solve the problem. Studies in animal models of acute myocardial infarction tips, lethal reperfusion injury accounts for up to50%of the final size of myocardial infarction, therefore, the prevention of myocardial ischemia reperfusion injury can improve the acute myocardial infarction and clinical efficacy of reperfusion therapy, related research has become a hot spot, but so far have not been used in clinical medicine.Autophagy is a process of self degradation of organelles and proteins in cells, autophagy lysosome combined with, involved in the degradation process in cell survival, autophagy plays a crucial role in cell differentiation, and homeostasis, maintenance.Organelle proteins and damaged cells autophagy by degradation of cell is not stable, reduce the damage effect on cells, decomposed into amino acid metabolism to substrate, maintaining cell homeostasis.The overseas study confirmed that the rapid rise in ischemia reperfusion can induce the autophagy level. Domestic study indicated that autophagy found in many pathophysiological conditions plays an important role, such as tumor, infection, heart disease, neurodegenerative diseases. The regulation of autophagy is believed to be related to cardiovascular disease, including cardiomyopathy, cardiac hypertrophy, heart failure, ischemic heart disease and ischemic reperfusion injury. Autophagy in ischemia reperfusion injury may be associated with injury time. Macrophages in atherosclerotic plaques observed increased autophagosome. Autophagy by preventing apoptosis and plaque necrosis plays the role of the atherosclerotic plaque stability.Berberine is the main extracts of Rhizoma Coptidis, used for disease treatment of diarrhea, dysentery and other gastrointestinal infection, animal experiment and clinical research in other experiments that berberine has significant hypoglycemic, hypolipidemic effects, with in-depth study of berberine was found, except for the regulation of blood glucose and lipids in diabetes, berberine the anti platelet, anti atherosclerosis has remarkable curative effect. Pharmacological study showed that berberine has antioxidant properties, but study in autophagy in terms of berberine has not yet been reported, so this experiment using in vitro ischemia reperfusion model, at the cellular level simulation of myocardial ischemia reperfusion injury, to observe the mechanism of berberine and the damage to the protective effect and possible.ObjectivesResearch Report of autophagy is often used as a protective mechanism involved in cell protection effects, such as anti infection, inhibition of tumor cell proliferation inhibition of neural degeneration, anti-aging, anti reperfusion injury. Involved in a wide range of tumor pathology, cardiac ischemia, neurodegenerative diseases, metabolic diseases, respiratory diseases and other physiological processes and senescence physiology process.The above data, we discuss from the cell level:1. H9C2myocardial hypoxia reoxygenation model to simulate ischemia reperfusion injury, confirmed the effect of Berberine Preconditioning on H9C2myocardial cells during hypoxia reoxygenation injury at the cellular level.2. We explore the mechanism of protective effect of berberine on myocardial cells with hypoxia reoxygenation injury from the cellular level, provides a new way for the prevention of ischemia reperfusion injury.3. Berberine protects myocardial cells injured by hypoxia reoxygenation by the intervention of autophagy.MethodsThe first part of the experiment:we first develop myocardial H9C2rat myocardial cells, hypoxia reoxygenation model on myocardial ischemia reperfusion injury time points, the experiments were divided into5groups:control group, anoxia reoxygenation group3H4H4h, hypoxia reoxygenation hypoxia reoxygenation in6h group,4H9h group,4H hypoxia reoxygenation the12h group were observed under inverted microscope, cell growth, cell activity was detected by MTT, was detected in the culture broth of LDH activity and myocardial cell MDA and the content of SOD in order to observe the activities of myocardial cell H9C2, cell apoptosis was detected by flow cytometry.The second part of the experiment:cultured myocardial H9C2rat myocardial cells hypoxia reoxygenation, determine the4H6h reoxygenation model of optimal time point for hypoxia, the experiments were divided into3groups:control group, hypoxia group, BBR+hypoxia reoxygenation group. Detection of MTT cell activity was detected in the culture broth, LDH activity and MDA in the myocardial cells and the content of SOD, the expression of Western Blotting detection of autophagy related protein Beclin-1and LC3, GFP-LC3gene expression was detected by detecting the autophagosome gene transfection, detection of MDC fluorescence microscopy stained particles and transmission electron microscopy, using Rh-123as a fluorescence probe by flow cytometry membrane mitochondrial H9C2myocardial cell detection potential (△ψ M). The changes were observed by transmission electron microscopy of mitochondrial membrane swelling.The third part of the experiment:we express by drug intervention autophagy, in order to observe the protective effect of H9C2mechanism in myocardial cells, were divided into5groups:control group, H/R group, Ra+H/R group,3-MA+BBR+H/R group, BBR+H/R, MTT cell activity detection, detection training activity in liquid LDH and MDA in the myocardial cells and the content of SOD, flow cytometry detection of TUNNEL and cell apoptosis, expression of Western Blotting detection of autophagy related protein Beclin-1and LC3, GFP-LC3gene expression was detected by detecting the autophagosome gene transfection, detection of MDC fluorescence microscopy stained particles and transmission electron microscopy, using Rh-123as a fluorescence probe by flow cytometry and H9C2to detect myocardial cell mitochondrial membrane potential (△ψ M). The changes were observed by transmission electron microscopy of mitochondrial membrane swelling.Results1. Culture H9C2cells, a cell model of hypoxia/reoxygenation, changes of MTT experimental detection of H9C2activity of myocardial cells. Compared with the control group, myocardial hypoxia reoxygenation in H9C24H6h,9h,12h, cell survival rate significantly decreased, there was significant (p<0.01). The in vitro cultured myocardial cell LDH activity and MDA and SOD content in myocardial cells, test results show that:compared with the control group, myocardial hypoxia reoxygenation in H9C24H6h,9h,12h, LDH and MDA were significantly increased, while the SOD value decreased, with significant difference (p<0.01),(see1-3,1-2). Flow cytometry assays were performed using Alexa Fluor488Annexin YFITe/P1double staining method. The results are shown in Figure1-4, the apoptosis of H9C2cells with hypoxia reoxygenation time closely related, after anoxia reoxygenation in, cell apoptosis rate increased significantly. Compared with the control group, myocardial hypoxia reoxygenation in H9C24H6h,9h,12h, cell apoptosis rate increased significantly, there was statistical significance (p<0.01, n=5).2. We use Western blot analysis of BBR protein expression in autophagy in each case. The results showed that, the enhanced H/R injury induced by H9c2in myocardial cells autophagy marker expression (Fig.2-4). Compared with the control group, the expression level of various autophagy marker compounds in H/R injury up-regulated (P<0.05). At the same time, we observed the expression level further enhanced in BBR group Beclin-1and LC3-Ⅱ (P<0.05,2-4). Then, we use the method of gene transfection into GFP-LC3carrier protein and to observe the changes of autophagy in H9C2cells (Figure2-4). In addition, we staining by MDC was confirmed by the changes of autophagy, increased during hypoxia reoxygenation, BBR group increased autophagy expression further (Figure2-5). Finally, we observe the change of autophagic bodies by means of transmission electron microscopy. The results showed that, compared with the control group, autophagosome in H9C2cells increased in H/R group (P<0.05,2-5). Compared with H/R group, the number of autophagosome BBR group significantly increased (P<0.05). These data again indicate the formation of BBR upregulation of H9C2myocardial autophagy lysosome. We use Rh-123as a fluorescence probe by flow cytometry and H9C2to detect myocardial cell mitochondrial membrane potential (△ψ M). As shown in Figure2-6, compared with the control group, the mitochondrial membrane potential damage in H/R group myocardial cells induced by H9C2(P<0.05). Compared with group H/R, reduce the potential damage of mitochondrial BBR membrane group (P<0.05,2-6). We observed the changes of mitochondrial membrane swelling by transmission electron microscope. As shown in Figure2-6, transmission electron microscopy showed that, after H/R damage, mitochondrial H9C2myocardial cell swelling degree increased, the swelling area also increased (P<0.05, compared with the control group,2-6). The BBR group showed, the mitochondrial membrane area compared with the H/R group less, swelling degree is reduced, the results show that, H/R damage induced mitochondrial dysfunction.3. Then we through drug intervention autophagy to further explore the protective mechanism of berberine on hypoxia reoxygenation injury in oxygen, compared with the H/R group, berberine group LDH, MDA decreased, SOD increased significantly (P<0.01). Compared with3-MA group and berberine group, LDH, MDA increased, SOD decreased (P<0.01), and after treatment3-MA,3-MA group of LDH, MDA significantly increased, SOD decreased, with no significant differences between the H/R group (P>0.05);3-MA is the autophagy inhibitor, the inhibition of autophagy, cell protective effect of Berberine myocardial weakened. There is no difference between berberine group and control group LDH, MDA and SOD,(P>0.05). Then we use the MTT cell activity detection, results showed that, compared with control group, H/R cell activity in group decreased significantly (P<0.01). Compared with H/R group, the cell activity of berberine group increased significantly (P<0.01). The results showed that the protective effects of H9C2, berberine, myocardial H/R injury were increased significantly in the reflecting cell activity. After3-MA treatment, the activity of3-MA cells were significantly decreased, with no significant differences between the H/R group (P>0.05);3-MA is the autophagy inhibitor, the inhibition of autophagy, cell protective effect of berberine myocardial weakened. Cell activity of berberine group and control group was obviously increased (P>0.05). We use the flow and TUNNEL detection and comparison of H9C2myocardial oxygen complex, on apoptosis of oxygen injury results show, compared with the H/R group, apoptosis, berberine group were significantly reduced (P<0.01). The results showed that the protective effects of H9C2, berberine, myocardial H/R injury were reduced in cell apoptosis. After3-MA treatment, cell apoptosis in3-MA group increased obviously, with no significant differences between the H/R group (P>0.05);3-MA is the autophagy inhibitor, the inhibition of autophagy, on myocardial cell apoptosis effect of berberine was weakened. Cell activity of berberine group and control group was obviously increased (P>0.05). Compared with H/R group, the expression of autophagy protein Beclin-1and LC3berberine group increased significantly (P<0.01). The results showed that the induction of autophagy, berberine group. After3-MA treatment,3-MA group of autophagy significantly reduced. Berberine and rapamycin group autophagy protein increased significantly (P<0.01); there is no difference between berberine group and rapamycin group,(P>0.05).In addition, the expression of autophagy in each H9C2myocardial H/R injury in bodies of GFP-LC3, compared with H/R group, the expression of berberine group cell autophagic bodies in a significant increase in GFP-LC3(P<0.01). The results showed that the induction of autophagy, berberine group. After3-MA treatment,3-MA group of autophagy significantly reduced. Berberine and rapamycin treated cells autophagosome GFP-LC3increased significantly (P<0.01); there is no difference between berberine group and control group,(P>0.05), the level of berberine and rapamycin induced autophagy is consistent. We use the MDC staining and TEM comparison of detection effect on cell autophagic bodies H9C2H/R in myocardial cell injury.In the process of H/R, BBR increased autophagy expression further. The results showed that, compared with H/R group, the expression plasmid berberine group MDC staining was significantly increased (P<0.01). The results showed that berberine group, autophagic bodies. After3-MA treatment,3-MA group significantly decreased autophagosome. Berberine and rapamycin group autophagy protein increased significantly (P<0.01); there is no difference between berberine group and control group,(P>0.05), the level of berberine and rapamycin induced autophagy is consistent. Finally, we observe the change of autophagic bodies by means of transmission electron microscopy. The results showed that, compared with H/R group, the expression of autophagosome berberine group increased significantly (P<0.01). The results showed that berberine group, autophagic bodies. After3-MA treatment,3-MA group significantly decreased autophagosome. Berberine and rapamycin group autophagy protein increased significantly (P<0.01); there is no difference between berberine group and control group,(P>0.05), the level of berberine and rapamycin induced autophagy is consistent. We observe the change of autophagic bodies by means of transmission electron microscopy. The results showed that, compared with H/R group, the expression of autophagosome berberine group increased significantly (P<0.01). The results showed that berberine group, autophagic bodies. After3-MA treatment,3-MA group significantly decreased autophagosome. Berberine and rapamycin group autophagy protein increased significantly (P<0.01); there is no difference between berberine group and control group,(P>0.05), the level of berberine and rapamycin induced autophagy is consistent.. Finally, we use Rh-123as a fluorescence probe by flow cytometry and H9C2to detect myocardial cell mitochondrial membrane potential (△ψ M). The results showed that, compared with control group, M△ψ H/R group increased significantly (P<0.01). Compared with group H/R, M△ψberberine group decreased significantly (P<0.01). The results showed that the protective effects of H9C2, berberine myocardial H/R injury in decreasing cell△ψ M levels reflect. After3-MA treatment, M△ψ3-MA group significantly increased, with no significant differences between the H/R group (P>0.05); M△ψ berberine group and rapamycin group decreased significantly (P<0.01). There is no difference between berberine group and control group,(P>0.05). We observed the changes of mitochondrial membrane swelling by transmission electron microscope. The results show that, transmission electron microscopy showed that, after H/R damage, mitochondrial H9C2myocardial cell swelling degree increased, the swelling area also increased (P<0.05). After3-MA treatment,3-MA group of mitochondrial swelling area increased, with no significant differences between the H/R group (P>0.05); berberine group and rapamycin group significantly decreased mitochondrial swelling area (P<0.01). There is no difference between berberine group and rapamycin group,(P>0.05).Conclusions1. this study damage simulation of myocardial ischemia reperfusion injury induced by hypoxia reoxygenation myocardial cells of H9C2, after4h/of hypoxia reoxygenation after6h, can obviously cause the damage of myocardial cells, showed that myocardial hypoxia/reoxygenation injury model was constructed successfully, which provided a good platform for reoxygenation induced myocardial cell injury and autophagic mechanism in order to study the influence of berberine.2. This study damage simulation of myocardial ischemia reperfusion injury induced by hypoxia reoxygenation myocardial cells of H9C2, the use of Berberine Preconditioning myocardial cell injury, detection of autophagy and mitochondrial function, results showed that berberine inhibits mitochondrial dysfunction protect H9C2cells through the induction of autophagy.3. This study damage simulation of myocardial ischemia reperfusion injury induced by hypoxia reoxygenation myocardial cells of H9C2, the use of Berberine Preconditioning on myocardial cells injured by hypoxia reoxygenation, join the autophagy inducer rapamycin and autophagy inhibitor3-MA interference detection of autophagy and autophagy, mitochondrial function, the results show that:1.3-MA inhibits autophagy level, thereby increasing the cell mitochondrial dysfunction, lead to apoptosis, which weakens the protective effect of BBR on cell.2berberine inhibits mitochondrial dysfunction protect H9C2cells through the induction of autophagy. The level of autophagy induced3rapamycin, thereby reducing the mitochondrial dysfunction, reduce the incidence of apoptosis of cells, which may play a protective role.4berberine as autophagy inducer rapamycin induced autophagy, with basically the same. |
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