Experimental Study Of Tetrandrine On Regulation Proinflammatory Factors Attenuating Myocardial Ischemia/Reperfusion Injury

PARTⅠThe effect of tetrandrine on anoxia/reoxygenation-induced injury and the release of proinflammatory factors in cultured cardiomyocyte of neonate ratsExperimentⅠCulturing Neonate Rat Cardiocyte and Establishing Anoxia/Reoxygenation ModelObjective:To establish an anoxia/reoxygenation model of cultured cardiocyte using neonate rat and investigate the level of myocardial enzyme LDH,CK under anoxia/reoxygenation conditions.Methods:Using primary cultured SD neonate rat's myocardial cell establishes an anoxia/ reoxygenation model to simulate myocardial ischemia/reperfusion injury. Take this model as a platform to investigate the effect of tatrandrine on rat's myocardial ischemia/ reperfusion injury. Cardiomyocyte was identified by using immunohistochemistry method. Cultured cardiocytes were divided into 2 groups: control group (CON) and anoxia/ reoxygenation group (A/R). Each group was treated as follow: CON group-0.9% saline was added into culture fluid, and cardiocytes were cultured in normal circumstance 26h. A/R group-O2 and CO2 in the culture plate which carried cultured cardiocytes were blowed out by argon gas, and nutritive medium was replaced by non- saccharide non- serum culture medium, followed 95% argon gases were infused. The culture plated was sealed and cultured in incubator for 2h under anoxia, after that the seal was opened it was incubated in incubator for the next 24h reoxygenation. Lactate dehydrogenase (LDH) and creatine kinase (CK) were detected and compared during the process of cardiocyte anoxia/reoxygenation.Result:1. The result of cutured neonate rat in vitro: myocardial cell was separated and primary cultured 2h, most non-cardiocytes (mainly mechanocyte) were adherence and cardiocytes still floating in cultured flui. The shape of cultured cardiocyte is round or ellipse. At the day after cardiocyte was implanted 2d, some cardiocytes possessing beating ability were seen under inverted microscope. At the day after cardiocyte was implanted 3-4d, some poradic cardiocytes were connected each other to symplasm. These cultured cardioctes were stained by using antiα-Sacromeric immunohistochemistry method; more over 96% cardiocytes were present strong positive result. This result identified this model was established successful. 2. Anoxia lead LDH and CK increased lightly: there were no significant difference between CON group and A/R group before anoxia beginning (74.67±3.51 VS 73.08±2.08, p>0.05).This phenomenon indicates the two groups have comparability. The numerus of LDH and CK in A/R group were increased 3.5 (260.99±6.73 VS 74.67±3.51, p<0.01) and 2.68 (4.74±0.45 VS 1.77±0.40, p<0.01) times respectively after experienced 2h anoxia culture compared with their numerus before anoxia;There was no significant difference on the numerus of LDH and CK in CON group before and after cultured 2h (72.24±2.25 VS 73.08±2.08, p>0.05) (1.78±0.28 VS 1.73±0.12, p>0.05). 3. Reoxygenation lead to the LDH and CK in cultured cardiocyte increased higher: there were obviously increased on LDH and CK compared between anoxia 2h and reoxygenation 24h in A/R group, they were elevated 1.93 (503.72±6.26 VS 260.99±6.73, p<0.01) and 1.59 (7.54±0.66 VS 4.74±0.45, p<0.01) times respectively; but there were no significant difference found in CON group (74.32±2.78 VS 72.24±2.25, p>0.05) (1.93±0.21 VS 1.78±0.28, p>0.05). ExperimentⅡEffect of tetrandrine on anoxia/reoxygenation-induced release of proinflammatory factors in cultured cardiocyte of neonate ratsObjective:To investigate the effect of tetrandrine on anoxia/reoxygenation-induced the release of myocardial enzyme LDH, CK and proinflammatory factors: TNF-α, IL-1β, IL-6 in cultured cardiocytes of neonate rates.Mthods:After cardiocytes were cultured in vitro successfully, it were divided into 4 group: control group (CON), anoxia/reoxygenation group (A/R), tetrandrine group (Tet), simvastatin (Sim) in random. Each group was treated as follow: CON group-not treated anoxia/reoxygenation, continuous incubated 26h under normal circumstance. A/R group- first anoxia incubate carried, cells were incubated on the non- saccharide non- serum culture medium, which saturate by 95% argon gases 2h, reoxygenation incubate followed, cells were incubated in normal circumstance 24h.0.9% saline were added into culture fluid before the beginning of reoxygenation. Tet group and Sim group–the procedure of anoxia/reoxygenation was same to A/R group, the difference of these two groups was they added Tet (30μmol/L) or Sim (10μmol/L) respectively into culture fluid and incubated 60min before anoxia beginning. LDH, CK, TNF-α, IL-1β, IL-6 were detected after reoxygenation 24h.Conclusion:The LDH and CK were increased significantly in A/R, Tet, and Sim groups compared with CON group (p<0.01). The LDH and CK in Tet and Sim group were lower significant than A/R group (p<0.01). 2. The proinflammatory factors TNF-α, IL-1βand IL-6 were increased significantly in A/R, Tet, and Sim groups compared with CON group (p<0.01). And it were lower significant than A/R group (p<0.01). 3. The level of LDH, CK, TNF-α, IL-1β, IL-6 were no significant difference between Tet group and Sim group (p>0.05). PARTⅡTetrandrine regulate proinflammatory factors to attenuate rat myocardial ischemia/reperfusion injuryObjective:To investigate how tetrandrine through regulate the pro-inflammation factors TNF-α,IL-1β,IL-6 to attenuate rat ischemic/reperfusion injury.Methods:80 male Sprague-Dawley(SD) rats were randomly divided into 4 group: Sham group, ischemia/reperfusion (I/R) group, Tetrandrine group (Tet) and simvastatin group (Sim).The SD rat underwent 30 min of left anterior descending (LAD) coronary occlusion and 24h reperfusion to make ischemia/reperfusion (I/R) injury model in vivo. Sham group were not subjected to occlusion of artery. Tet group were injected tetrandrine to abdominal cavity 20min before ischemia starting. The rat in Sim group was administrated simvastatin 2mgkg/L intragastricly every day, administrating drugs lasted 14 days. The other procedures were same to the I/R group. Samples were collected after 24h reperfusion. The expression level of TNF-α,IL-1β,IL-6 protein in serum and myocardial tissue was detected by ELISA. LDH and CK were detected too. The neutrophil infiltration degree in myocardium was determined by using measuring the activity of myeloperoxidase (MPO) method. Cardiac function which includes FS%, EF and E/A was measured by using ultrasound. EB/TTC (Azovan Blue/2,3,5-Tripheny-2H-Tetrazoliam Chloride) dyeing method was used to measure the infraction size.Result:1. The effect of Tet on the release of myocardial enzyme during ischema/reperfusion: the LDH and CK were significantly higher in I/R, Tet and Sim groups compared with Sham group (p<0.01), but it were much lower in Tet and Sim groups compared with I/R group. 2. The effect of Tet on the cardiac function and infraction size: the cardiac function of systolic and dilator in experimental group was decreased significantly compared with normal heart's function. In Tet and Sim group, which was experienced pharmacological preconditioning their cardiac function were significant higher than I./Rgroup(p<0.01),but no significant difference between Tet and Sim on EF and E/A. intense There were intense liner correlation between myocardium(CK,LDH) and tumor necrosis factor alpha(TNF-α) (r=0.922,0.975, p<0.01). 3.The effect of Tet on the neutrophil infiltration: the activity of MPO was significantly increased after reperfusion, its activity in experimental groups were much higher than Sham group(p<0.01), notwithstanding its activity in Tet nad Sim groups were significantly lower than I/R group(p<0.01). No significant difference was found between Tet and Sim group. There were intense liner correlation between MPO and TNF-α(r=0.936, p<0.01) 4. The effect of Tet on the release of proinflammatory factors(TNF-α,IL-1β,IL-6): The similar results were obtained after measuring the serum and tissue concentration of TNF-α,IL-1βand IL-6,that was in Tet and Sim group the expression of proinflammatory factors(TNF-α,IL-1β,IL-6) were significant lower compared with I/R group(p<0.01) and significant higher than shame group(P<0.01). The expression level of IL-1βand IL-6 has intense liner correlation with TNF-α(r=0.928, 0.948, p<0.01).PARTⅢThe molecule mechanism of tetrandrine attenuate myocardial ischemia/reperfusion injuryObjective:Observing the mRNA change of proinflammatory factors(TNF-α,IL-1β,IL-6) and modulating protein I-κBα,NF-κB during ischemia/reperfusion to reveal the molecular mechanism of tetrandrine attenuating myocardial ischemia/reperfusion injury.Methods:80 male SD rats were randomly divided into 4 groups: Sham group, ischemia/reperfusion (I/R) group, Tet group and Sim group. The SD rats underwent 30 min of left anterior descending (LAD) coronary occlusion and 24 hours reperfusion to make ischemia/ reperfusion (I/R) injury model in vivo. Sham group were not subjected to occlusion of artery. Tet group were injected tetrandrine to abdominal cavity 20 minute before ischemia starting. The rat in Sim group was administrated simvastatin 2mg/kg intragastricly every day, administrating drugs lasted 14 days. The other procedures were same to the I/R group. Samples were collected after 24h reperfusion. TNF-αmRNA, IL-1βmRNA, IL-6mRNA was detected by real time RT-PCR. Western-Blot and immunohistochemistry were used to detect the expression of phosphorylation I-κBα(P- I-κBα) in the kytoplasm of cardiocyte. Electrophoretic mobility shift assay (EMSA) was used to detect the expression NF-κB in the nuclear of cardiocyte.Recult:1. The result of real time of RT-PCR: after reperfusion the mRNA expression of proinflammatory factors increased significantly in experimental groups (p<0.01). The copies of mRNA in Tet and Sim group was decreased significantly compared with I/R group (p<0.01). 2. The result of Western-Blot and immunohistochemistry were proved that the expression of P- I-κBαincreased significantly in experimental groups (p<0.01) and the expression of P- I-κBαin Tet and Sim group was much lower than I/R group (p<0.01).3. There was the expression of NF-κB in I/R group and it was much higher compared with Sham, Tet, Sim group (p<0.01). Comparing the expression of NF-κB among Sham, Tet, Sim groups, there was no significant difference found (p>0.05). 3. The expression of P- I-κBαin cytoplasm had intense liner correlation with NF-κB(r=0.751, p<0.01).Conclusion:1.Neonate rat cardiocytes were separated and cultured in vitro, through the procduce of anoxia/reoxygenation establishing myocardial cell ischemia/reperfusion injury model.2. Ischemia/reperfusion can cause to cardiocyte damage.3. Inflammation raction of cardiocyte and cascade of cytokine induced by proinflammatory factors were emerge after ischemia/reperfusion.4. Proinflammatory factors participate ischemia/reperfusion injury.5. Tetrandrine can decrease the release of myocardial enzyme induced by ischemia/ reperfusion injuryr, diminish the infiliation of neutrophil; improve cardiac founction.6. Tetrandrine could rduce the production of proinflammatory factors.7. Tetrandrine can rduce the mRNA production of proinflammatory factors.8. Tetrandrine inhibite the phosphorylation of I-κBαin cytoplasm at local myocardium.9. Tetrandrine inhibite the activation of NF-κB, and inhibite its translocation into nuclear.10. The mechanism of tetrandrine inhibiting inflammation reaction, inturpting the cascade of cytokine, improving cardiac founction part releated to the founction of inhibiting the priduction of proinflammatory factors and blocking the key point of initiaiting agents inturping the vicious circule part releated to its multitarget founction such as: reducing the infilation of leukocytes, inhibiting the phosphorylation, blocking calcium channel.