Protective Effects And Mechanism Of Glycyrrhizin On Rat Myocardial Ischemia Reperfusion

Background:The definition of myocardial ischemia-reperfusion injury is that ischemic myocardium restore blood flow after reperfusion,with its structural damage, cell death, infarction extend and further damage of the heart function which can affect the prognosis of patients with myocardial infarction. Myocardial ischemia reperfusion injury has the pathogenesis of complex mechanism, including oxygen free radicals, calcium overload, inflammatory medium, in which inflammation is one of the important reasons to myocardial ischemia reperfusion injury. How to reveal the pathogenesis of key link and reasonable intervention to reduce ischemia-reperfusion injury is the focus of current research, and this thesis is the starting point.HMGB1is a kind of important " advanced " inflammatory cytokines, and HMGB1may also be pro-inflammatory cytokine regulatory networks in the segment of a center. At the late inflammatory response, HMGB1is released into the blood by endocrine and paracrine form leading to inflammatory cascade of further expansion. HMGB1,as an important " advanced " inflammatory cytokines, can not only make TNF-α, IL-1expression, and activate the ERK kinase, P38MAP K kinase, JNK kinase and prompt NF-κB nuclear translocation. These findings show that HMGB1may be a proinflammatory cytokine regulatory networks in the segment of a center, and can not only has a cascade of biological effects, but also regulate inflammatory cytokine secretion. And it is the " advanced " important medium to ischemia reperfusion injury. While searching for a drug to inhibit HMGB1become the important target of reducing myocardial ischemia reperfusion injury.Glycyrrhizin is a kind of active extract of licorice root, and has anti-inflammatory, immune function, anti allergic effect. A large amount of literature prove that glycyrrhizin can inhibit inflammation, reduce brain, spinal cord, intestine, liver and other organs of ischemia-reperfusion injury, but its protective effect in myocardial ischemia reperfusion injury has not yet been studied, which is one of the main starting point.Then, we design the glycyrrhizin on myocardial ischemia reperfusion injury experiment, in order to confirm whether it has myocardial protective effect, and the relationship with HMGB1, further elaboration of glycyrrhizin in ischemia reperfusion injury in the protection mechanism.Objective:1:To establish the rat model of ischemia reperfusion.2:To investigate the hemodynamic in ischemia reperfusion rats.3:To observe myocardial ischemia reperfusion and its pathological changes in rats.4:To study the expression of inflammatory factors and HMGB1expression in ischemia reperfusion rats.Methods:The experiment was divided into:sham operation group (Sham Group):In7days before operation, every rat tail vein was injected of saline1ml, constructed the model wearing only a line, not ligated; Ischemia reperfusion group (NS Group):In7days before operation, every rat tail vein was injected of normal saline lml, and the model of I/R injury was developed within30min of left coronary artery occlusion followed by a1-h reperfusion.; GLcyrrhizin intervention group (GL Group):In7days before operation, every rat tail intravenous was pretreated with GLcyrrhizin (10mg/kg). GLcyrrhizin plus recombinant group HMGB1(GL+Rhmgb1) group:In7days before operation, every rat tail intravenous was pretreated with GLcyrrhizin (10mg/kg). And Rat tail vein was immediately given recombinant HMGB1(100μg/rat) with ligation of coronary blood flow after30minutes. The model of I/R injury was developed within30min of left coronary artery occlusion followed by a1-h reperfusion. Serum HMGB1, inflammatory cytokine levels, and hemodynamic values were evaluated after I/R at different time points. In addition, the infarct size and pathological changes were investigated.Results:1:The experimental group showed no significant difference between hemodynamic (P>0.05)2:Myocardial infarction area was largest in NS group(P<0.05), GL may reduce the myocardial infarct size(P<0.05), and myocardial infarction area in GL+rHMGBl group was more serious than in GL group(P<0.05), but still lower than that of NS group(P<0.05);3:Myocardial fibers in Sham group were intact, while the nucleus and cytoplasm were integrity; myocardial cell in NS group were deranged, while myocardial cells were ruptured and dissoluted, with visible necrotic cells and inflammatory cell infiltration. Iinterstitial myocardial cells in G1group had edema part and a small amount of inflammatory cell infiltration, myocardial cells in GL+HMGB1group were arranged in disorder with edema and spotty necrosis, but the degree was lower than NS group (P<0.05).4:Cardiac troponin, LDH and AST were the highest level in NS group(P<0.05), and it were decreased in GL group(P<0.05). The infarct area was increased in GL+rHMGB1group than in GL group(P<0.05), but still lower than that of NS group(P<0.05).5:The levels of HMGB1and inflammatory cytokines were the highest level in NS group(P<0.05). The levels of HMGB1and inflammatory cytokines of TNF-a, IL-6levels were significantly decreased in GL group(P<0.05).Conclusions:1:HMGB1can increase the inflammatory cytokines TNF-a, IL-6expression, increasing the area of myocardial infarction.2:GL may be reduce myocardial ischemia reperfusion injury through inhibition of the expression of HMGB1. Background:GLcyrrhizin is a kind of licorice root extract active component, has effects of anti-inflammatory, anti-allergic, et al. it have been reported protective effect on ischemia reperfusion induced injury in experimental animal organs such as liver and brain and spine by foreign scholars. At the same time it has confirmed that glycyrrhizin on myocardial ischemia reperfusion injury has a protective effect in front of us research. But it have not been reported effects of glycyrrhizin intervention on apoptosis of myocardial cells, which is one of the starting points of the subject.In recent years, people have deepening understanding on apoptosis in myocardial ischemia reperfusion injury gradually. And the apoptosis is programmed cell death, which is one of the important mechanisms in myocardial ischemia reperfusion injury. and reducing myocardial apoptosis can significantly improve cardiac function. Bax and Bcl-2gene is a major gene to regulate apoptosis, and the protein level related directly to apoptosis. When Bax is increased, apoptosis is promoted. and while Bcl-2increased, apoptosis is inhibited. Cytochrome C, which is released from mitochondria, can induce Bax gene release from mitochondria into the cytosol, and causing apoptosis; and Bcl-2can prevent the release of cytochrome C, then apoptosis is inhibited. Caspases enzyme is the key enzyme in apoptotic cells, while Caspases-3is the key apoptotic protease in the Caspases cascade waterfall downstream. Bax protein can make cytochrome C through the mitochondrial membrane, then activate Caspases-9,and further activate Caspases-3, which leading to apoptosis. The Bax, Cyt C and Caspase-3protein plays an important role in apoptosis, so in this thesis, the glycyrrhizin intervention effects on Bax, Cyt C, is also studied.Recently, the mitogen-activated protein kinase (MAPK) pathway in myocardial cell apoptosis has been attracted more and more attention. MAPK has4major subfamilies:extracellular signal regulated kinase (ERK), extracellular signal-regulated kinase5(ERK5),c-Jun N-terminal kinase (JNK) and P38MAPK. The starting point of this issue is to confirm the effect of glycyrrhizin on apoptosis of myocardial cells in ischemia reperfusion, and its molecular mechanism related to protein kinase (MAPK) pathway, especially the JNK pathway. We are Aiming at the problem of this study, and further study the mechanism of the protection of myocardial ischemia-reperfusion injury.Objective:1:To clear the myocardial apoptosis after intervention by GLcyrrhizin.2:To investigate the expression of myocardial Bax, Cyt C and Caspase3activity by GLcyrrmhizin intervention.3:To study effect of GLcyrrhizin on phosphate kinase signal pathways, especially the role of JNK/Bax signal pathway, further elaborating on ischemia reperfusion myocardial apoptosis, and myocardial protective mechanism.Methods:The experiment was divided into:sham operation group (Sham Group):In7days before operation, every rat tail vein was injected of saline1ml, constructed the model wearing only a line, not ligated; Ischemia reperfusion group (Ns Group):In7days before operation, every rat tail vein was injected of normal saline lml, and the model of I/R injury was developed within30min of left coronary artery occlusion followed by a1-h reperfusion.; GLcyrrhizin intervention group (GL Group):In7days before operation, every rat tail intravenous was pretreated with GLcyrrhizin (10mg/kg). GLcyrrhizin plus recombinant group HMGB1(GL+Rhmgb1) group:In7days before operation, every rat tail intravenous was pretreated with GLcyrrhizin (10mg/kg). And Rat tail vein was immediately given recombinant HMGB1(100μg/rat) with ligation of coronary blood flow after30minutes.The model of I/R injury was developed within30min of left coronary artery occlusion followed by a1-h reperfusion. Myocardial cell apoptosis were detected by Tunnel staining. Apoptosis-related events such as distribution of Bcl2, Bax cytochrome c expression between the mitochondrial and cytosolic fractions were evaluated by western blot analysis, and myocardial caspase-3activity was also measured. Furthermore, I/R-modulated activation of proteins belonging to the MAPK family, including phospho-ERK1/2, phospho-JNK and phospho-P38, was analyzed. Results:1、Tunnel staining showed that myocardial cell apoptosis in NS group was the most obvious (P<0.05), and myocardial cell apoptosis in GL group was decreased (P<0.05), and myocardial cell apoptosis in GL+rHMGB1group was more obvious than that in GL group, but still lower than that of group NS (P<0.05).2、Western blot showed that GL can change the Bax gene and cytochrome C intracellular distribution, and can inhibit Bax gene from the cytoplasm to the mitochondria transfer, and can reduce cytochrome C from mitochondria to the cytoplasmic transfer, though the effect of GLcyrrhizin plus HMGB1inhibition were attenuated. The overall change of Bax and cytochrome C in cell level was not exist (P>0.05).3:GL can inhibit INK cell pathway phosphorylation (P<0.05), while ERK1/2or P38phosphorylation had not been effected (P>0.05). And group GL+rHMGB1inhibition can attenuate that, but JNK phosphorylation level is still lower than that of NS group (P<0.05).Conclusions:1:GL can change the Bax gene and Cyt C distribution, and attenuate myocardial apoptosis after reperfusion so as to myocardial protection.2:The mechanism of Glycyrrhizin, which can reduce myocardial cell apoptosis on ischemia reperfusion rats, might be related to the inhibition of JNK phosphorylation. Background:Ischemia-reperfusion injury is aggravated when the functional metabolic disorders and tissue damage tissues and organs such as the heart, brain, kidney, after a certain time of ischemia, and after blood supply is restored, it not only cannot be restored to improve the function of tissues and organs, but deteriorated. The experimental table shows that the mitogen-activated protein kinase (MAPK) pathway mediates apoptosis in ischemia-reperfusion occurs. JNK plays an important role in the signal transduction pathways of apoptosis after myocardial ischemia, as an important way of cytokine signaling is widely involved in cell proliferation, differentiation, apoptosis and immune regulation, inflammation, cancer and other physiological process in ischemia-reperfusion, also has played an important role in cardiac arrest, the inflammatory response.Apoptosis is an active, procedural and physiological cell death, that under certain conditions, nuclear cells by starting the internal mechanism, through the endogenous DNA endonuclease activation occurs due to the natural process of cell death process, as a series of nuclear changes. C-Jun N-terminal kinase (JNK) is a mitogen activated protein kinase family (MAPK) member, is located in the cell cytoplasm, may be inflammatory mediators, cytokines, shock, trauma, ischemia reperfusion and other factors activate. A lot of experiments show that JNK cells is involved in the pathophysiological process of apoptosis.Studies had shown that JNK activation can be found in mitochondria and cytoplasmic after a few minutes ischemia-reperfusion,and it may play an important role in ischemia-reperfusion. Some researchers found that myocardial JNK activity increased significantly after ischemia-reperfusion, and the use of JNK inhibition SP600125can block Bax apoptosis pathway in myocardial ischemia-reperfusion injury. And using the JNK inhibitor AS601245can be significantly reduced ischemia-reperfusion induced neuronal apoptosis in cerebral ischemia-reperfusion model. Scholars also found that JNK activity was significantly increased after reperfusion, and JNK inhibitor can significantly alleviate liver cell apoptosis in hepatic ischemia reperfusion model.In this paper, the JNK inhibitor SP600125and recombinant HMGB1intervened, the role of JNK pathway and HMGB1in ischemia reperfusion injury were confirmed. So as to glycyrrhizin through inhibition of HMGB1and inhibition of JNK phosphorylation, it provide some evidence on ischemia-reperfusion myocardium protection. To solve these problems, we designed this experiment, the apoptosis of JNK pathway and HMGB1cells in myocardial ischemia and reperfusion in significance, further elaborating the mechanism of myocardial ischemia reperfusion injury.Objective:1To establish a rat myocardial ischemia reperfusion model, ischemia reperfusion in rat myocardial apoptosis, myocardial Bax, CytochromeC, Caspase3expression.2With the JNK inhibitor SP600125and recombinant HMGB1intervention, clear the apoptosis of JNK pathway and HMGB1cells in myocardial ischemia and reperfusion in significance, further confirmed that the mechanism of glycyrrhizin in myocardial ischemia reperfusion injury.Methods:The experiment was divided into:Ischemia reperfusion group (NS Group):In30minutes before the ligation of the coronary, every rat tail vein was injected of normal saline1ml, and the model of I/R injury was developed within30min of left coronary artery occlusion followed by a1-h reperfusion.; SP600125intervention group (NS+SP600125Group):In30minutes before the ligation of the coronary, every rat tail intravenous was pretreated with SP600125(0.5mg/kg). SP600125plus recombinant HMGB1(SP600125+rHMGB1) group:In30minutes before the ligation of the coronary, every rat tail intravenous was pretreated with SP600125(0.5mg/kg). And Rat tail vein was immediately given recombinant HMGB1(100μg/rat) when ligation of coronary blood flow. The model of I/R injury was developed within30min of left coronary artery occlusion followed by a1-h reperfusion. Myocardial cell apoptosis were detected by Tunnel staining. Apoptosis-related events such as distribution of BcI2, Bax cytochrome c expression between the mitochondrial and cytosolic fractions were evaluated by western blot analysis, and myocardial caspase-3activity was also measured.Results:1、The Western blot display that JNK activity in NS group is strongest (P<0.05), JNK activity in NS+SP600125is weakest (P<0.05).2、Tunnel staining results suggest that myocardial apoptosis in NS group is most obvious (P<0.05), and the degree of myocardial apoptosis in NS+SP600125group is decreased (P<0.05). myocardial apoptosis in SP600125+rHMGBl group is obvious than NS+SP600125group, but still lower than NS group (P<0.05);3:Western blot showed that SP600125can change the Bax gene and cytochrome C intracellular distribution, and can inhibit Bax gene from the cytoplasm to the mitochondria transfer, and can reduce cytochrome C from mitochondria to the cytoplasmic transfer, though the effect of SP600125plus HMGBl inhibition were attenuated. The overall change of Bax and cytochrome C in cell level was not exist (P>0.05).Conclusions:1:HMGB1intervention can aggravate the ischemia reperfusion injury, and provide an experimental basis for the glycyrrhizin inhibiting HMGB1protection mechanism.2:JNK cell signaling pathway play an important role in myocardial ischemia reperfusion injury. Intervention JNK approach can reduce reperfusion injury, as the glycyrrhizin inhibits the phosphorylation of JNK protect ischemia reperfusion myocardium provides theoretical basis.