Study Of The Effect Of Qidan Tongmai Tablet On Myocardium Ischemia Reperfusion Injury And Its Mechanism Of Signaling Pathway

Following an acute myocardial infarction , rapidly initiating reperfusion strategies by re-establishing coronary blood flow with such as thrombolysis or percutaneous coronary angioplasty(primary PTCA) are essential to salvage viable myocardium. However, reperfusion of ischemic myocardium can itself result in exacerbating the cellular injury and dysfunction sustained during the ischemia period. Mounting evidences have bolstered the concept of reperfusion injury following ischemia. Ischemia/reperfusion injury (I/RI) becomes a key clinical problem. Cardiomyocyte apoptosis is boosted by reperfusion. Inhibition of cardiomyocyte apoptosis could reduce reperfusion injury and help the recovery of heart function. The phenomena of ischemia preconditioning(IPC) and ischemia postconditioning (IPostC) against apoptosis and infarct size provide a new strategy for inhibition of myocardium ischemia reperfusion injury. There is a common signaling pathway termed reperfusion injury salvage kinases (RISK) pathway about the mechanism of IPC and IPostC, which is a new direction for protecting the heart against ischemia reperfusion injury. Pharmacological agents and their mechanism of the signaling pathway were focused.Protecting the heart against ischemia reperfusion injury was one of the key points in the field of integrating the Traditional Chinese Medicine (TCM) with Western medicine. It has been investigated that the Chinese herbs could protection heart against ischemia reperfusion injury due to the mechanisms involved attenuation of Ca2+ overload, inflammation or oxygen radicals. However, the mechanism of signaling pathway is unknown .Our previous studies demonstrated the Qidan Tongmai tablet(QDTMT) could protect heart against ischemia in rat and dog,which is a compound of extractives of traditional Chinese herbs from dried roots of Astragalus membranaceus, Saliva miltiorrhiza (Labiate), Angelica sinensisi (Umbelliferae), the dried flower of Carthamus tinctorius(Compositae) and dried twig of Cinnamomum cassia(Lauraceae )and functions as invigorating qi and promoting blood circulation.In the present study, the effect of QDTMT against myocardium ischemia reperfusion injury was investigated, then the mechanisms of QDTMT against reperfusion-inducing cardiomyocyte apoptosis ,especially weather the protective effect was due to the modulation of RISK pathway, were to determined.1 Study of the effect of Qidan Tongmai tablet against myocardium ischemia reperfusion injury in ratsThe Sprague-Dawley(SD) male rats were divided randomly into four groups of sham, control, QDTMT-I (1.08g /kg ) and QDTMT- II(3.24g/kg). The model of acute myocardium ischemia reperfusion injury was performed by 40min occlusion of left descending coronary artery followed by 4h reperfusion in open-chest rats. The protective effect of in vivo pharmacological preconditioning of QDTMT administrating orally for 7d against myocardium ischemia reperfusion injury in rats was investigated by the area of Evans'Blue and triphenyltetrazolium chloride (TTC) staining and the levels of creatine kinase(CK) and cardiac troponinⅠ(cTnI) in serum. The results demonstrated pretreatment of QDTMT administrating orally at the dose of 1.08g/kg or 3.24g/kg for 7d could induce the protective effect against ischemia reperfusion injury, the ratio of infarct size and area at risk was significantly smaller than that in the control (36.7±8.9 % and 28.8±8.4% vs 49.1±10.3%; P﹤0.05;P﹤0.01).The levels of CK and cTnI in control were 12801.84±4102.70u/L and 64.89±13.03 ng/mL respectively, significantly higher than that in sham group(1552.83±451.07u/L and 1.40±0.64ng/ mL,P﹤0.01;P﹤0.01).The levels of CK in QDTMT groups (1.08g/kg or 3.24g/kg) were 10017.17±3345.46 u/L and 6973.50±1778.40u/L respectively and the levels of cTnI were 47.23±12.76 ng/mL and 35.13±14.42 ng/mL respectively,significantly lower than that in control( P﹤0.05;P﹤0.01).The result indicated pharmacological preconditioning of QDTMT administrating orally for 7d could protect the heart against ischemia reperfusion injury in rats.2 Study of the effect of Qidan Tongmai tablet against reperfusion- induced myocardial apoptosis and its mechanism in rats.The model and protocol were performed as previously described. The cardiomyocyte apoptosis was determined by terminal deoxynucleotidyl transfease-mediated dUTP nick-end labeling (TUNEL).The activity of NOS in heart tissue was assessed by chemical assay. The expressions of Bcl-2 and Bax in heart tissue were assessed by immunohistochemical stain. The ERK1/2 (extracellular regulated protein kinases) and phosphorylation-ERK1/2(p-ERK1/2) in heart tissue were assessed by Western blot analysis. The results showed the apoptosis indexes(AI) in the groups of in vivo preconditioning of QDTMT at dosages of 1.08g/kg and 3.24g/kg for 7d were 14.3±3.8% and 10.6±2.0 % respectively, significantly smaller than the apoptosis index in control (21.3±4.2 % ;P﹤0.05;P﹤0.01).The activity of iNOS in heart tissue in control was higher significantly than that in sham (0.7484±0.1906U/mgprot vs 0.3454±0.1362 U/mgprot, P<0.01), the preconditioning of QDTMT could decrease the activity of iNOS, which was significantly smaller in the group of QDTMT-Ⅱ(0.4204±0.1351U/mgprot vs 0.7484±0.1906U/mgprot, P<0.01).The activities of cNOS in groups of the preconditioning of QDTMT (1.08g/kg or 3.24g/kg)were significantly higher than the activity in control (0.5827±0.1680U/mgprot and 0.7618±0.2077 U/mgprot respectively vs 0.3010±0.0617 U/mgprot, P<0.05;P<0.01 ).The activities of NOS were higher in groups of control, QDTMT-I and QDTMT-II than that in sham (P<0.05),but there was no significantly difference between groups of control and preconditioning of QDTMT (1.08g/kg or 3.24g/kg).The mean grey level of Bcl-2 or Bax decreased significantly in control compared with sham (P<0.05;P<0.01) .The mean grey levels of Bcl-2 in the group of in vivo preconditioning of QDTMT at doses of 1.08 g/kg and 3.24 g/kg orally for 7d were significantly lower than the level in control (147.82±19.51 and 118.65±16.31 vs 165.17±9.21 ;P<0.05;P<0.01, respectively).The mean grey levels of Bax in the groups of the preconditioning of QDTMT at dosages of 1.08 g/kg and 3.24 g/kg decreased slightly, which were no significantly lower than the level in control (151.67±16.69 and 160.71±19.82 respectively vs 139.43±11.70,P﹥0.05). The result from Western blot assessing ERK and p-ERK1/2 showed the level of ERK had not significantly difference between sham, control, and groups of QDTMT treatment (P﹥0.05). However, the preconditioning of QDTMT administrating orally at dose of 3.24g/kg for 7d resulted in 2.1 fold increase in the p-ERK1/2,which was significantly higher than control group (503.75±102.76 a.u vs 244.25±146.00a.u , P<0.01).The result indicated that the treatment of QDTMT could attenuate reperfusion-induced myocardial apoptosis in rat heart. Pharmacological preconditioning of QDTMT involved several targets of modulation of the activities of distinct NOS isoforms and the expressions of anti-apoptosis protein and pro-apoptosis protein, perhaps due to increasing the expression level of phosphorylation of the reperfusion salvage kinase of ERK1/2.3 Study of the correlation of the anti-apoptosis of Qidan Tongmai tablet and PI3K/Akt pathway in myocardial ischemial reperfusion injury in rat.To investigate the signaling pathway involved in the inhibition of apoptotic effect during myocardial ischemia reperfusion with QDTMT tablet, the SD male rats were divided randomly into four groups of sham, control, QDTMT and QDTMT with the inhibitor of PI3K/Akt pathway. After 7d of in vivo treatment with QDTMT or saline for 7d, the male SD rats were anesthetized and open-chest animals were subjected to 40 min of myocardium ischemia followed by 4h reperfusion. Cardiomyocyte apoptosis was determined both qualitatively and quantitatively by TUNEL methods with DAPI staining. The state of PI3K/Akt signaling cascade was determined by Western blot analysis. The result demonstrated apoptosis index (AI) was attenuated from 20.3±4.5% in control to 11.6±2.3% in QDTMT-treated animals (P<0.01). The state of p-Akt was induced a 2.5- fold increase by preconditioning of QDTMT. Pretreatment of LY294002 abrogated the anti-apoptosis effect of QDTMT significantly(17.0±5.5 % vs 11.6±2.3%,P﹤0.05;17.0±5.5% vs 20.3±4.5% in control, P﹥0.05). The results indicated that pharmacological preconditioning of QDTMT may able to inhibit the caidiomyocyte apoptosis against reperfusion-induced injury mediated by the increase of the expression of phosphorylating Akt .In conclusion, the result of the present study demonstrated that the pretreatment of QDTMT tablet has cardioprotective effect. The mechanisms of the preconditioning of QDTMT against ischemia reperfusion injury involved in modulation of the NOS isoform activity and increase in the expression of anti-apoptosis-related protein due to enhancing the phosphorlation of RISK. The anti-apoptosis effect could be abrogated by signaling pathway inhibitor. Based on these evidence, we conclude that the pharmacological preconditioning of QDTMT may protect heart against myocardium ischemia reperfusion injury via RISK pathway.