The Role Of JAK-STAT Signal Pathway In Ischemia/Reperfusion Injury And Mechanism Of Cardioprotection By Irbesartan

The Role of JAK-STAT Signal Pathway in Ischemia/ Reperfusion Injury and Mechanism of Cardioprotection by IrbesartanIntroductionWith the development of emergency thrombolysis, percutaneous tranluminal coronary angioplasty(PTCA) and coronary artery bypass graft(CABG), myocardial Ischemia/Reperfusion(I/R) injury has been becoming the major problem which hinders ischemic myocardium acquiring the best therapeutic effect by reperfusion therapy. Many basic and clinical studies showed that the mechanism of ischemia/reperfusion injury included three links of triggering substance, medial substance and effector, most of the studies aimed directly at trigging substance and effector, however there are not satisfactory therapeutic measure. This results, in part, from our limited tmderstanding of medial substance, which was cellular signal pathway. It is conduced to invest the pathogenesy of I/R injury at the level of molecular level to explore the relationship between cellular signal transduction pathway and I/R injury, then supplies new effective target for the treatment of I/R injury.The influence of I/R injury on the heart is mediated by a complex cellular signal network consisting of multiple cellular signal transduction pathway, and existes interaction in multistrata and polynucleation. Recent studies demonstrated that Janus kinase signal transducers and activators of transcdption(JAK-STAT) pathway related to I/R injury, howerver there were little study on it. The problem at present are as following: First, which members of STAT family are activated when I/R occur, and how is the time course of activating? The effect of different family members on heart is protective or harmful? Second, it was not clear that how the JAK-STAT signal pathway exerted its effect on heart. Third, If cardioprotection of the medicine preconditioning is involved in this signal pathway? All these questions are still to be further studied.Renin angiotensin systerm(RAS) plays an important role in myocardial I/R injury. Lots of animal experiments demonstrated that angiotoninⅡreleased largely during I/R, and caused I/R injury by AT1 receptor coupling with G protein. AT1 receptor antagonist had the role of cardioprotection angist I/R injury, but also existed adverse results. Especially, it was dispute about its effect on apoptosis. JAK-STAT pathway is activates mainly by G protein couple receper and cytokine receper. It was not very clear that if role of AT1 receptor antagonist in protection against I/R injury is related to the JAK-STAT pathway.Apoptosis has become increasingly recognized as one of the mechanisms of cell death during myocardial I/R injury. It had shown that RAS and the JAK-STAT pathway were involed tightly in myocardial apoptosis during I/R injury. Apoptosis is a complex process from ecto-signal to cyto- sigal pathway, then to apoptotic related gene and enzyme. Bcl-2, Bax and caspases-3 are apoptotic related gene and enzyme controlling apoptosis. RAS, the JAK-STAT pathway and Bcl-2, Bax, caspase-3 affect on apoptosis in three different decks respectively. It is not clear how change they occur during I/R, what relationship are among them, and what manner they play a part in.The study applied Langendorff isolated myocardial ischemia/reperfusion model, to explore the following questions. First, if the main number STAT1 and STAT3 of the JAK-STAT signal pathway is activated during I/R injury and the changing regularity of time course? Second, the role of the JAK-STAT pathway in the I/R injury; and if cardiprotection by AT1 receper antagonist. Final, the changes of apoptotic related gene and enzyme, and the relationship with RAS and the JAK-STAT pathway during I/R injury. These would supply experimental and theory basement for clinical therapy of I/R injuryMethods1. The study of the activating time course of the JAK-STAT signal pathway during myocardial I/R injury of rats The Langendorff isolated myocardial ischemia/reperfusion model was applied. 42 male Wistar rats were divided randomly into 7 groups, and 6 ones for every group. The groups were control group, ischemia30min group(R0), ischemia/reperfusion5min group, (R5) ischemia/reperfusionl 5min group(R15), ischemia/reperfusion30min group(R30), ischemia/reperfusion60min group(R60), and ischemia/reperfusion120min group(R120). In control group, isolated rats hearts were perfuse with modified Krebs-Henseleit(KH) buffer for 180 min, in ischemia/reperfusion group, hearts were perfusion with KH buffer for 30min, followed by 30minutes of globle ischemia and 0minute, 5minutes, 15minutes, 30minutes, 60minutes and 120 minutes of reperfusion respectively. The left ventricular developed pressure(LVDP) and Left ventficular first derivative of developed pressure(dp/dt_max) were monitored dynamicly; The extent of myocardial necrosis was observed by GCA bright green staining; the phosphorylated STAT1 and STAT3 proteins were located by immunohistochemical method; and the total as well as the phosphorylated STAT1 and STAT3 protein expression were detected by immunoblotting.2. The protective role of Irbesartan against myocardial I/R injury of rats through the JAK-STAT signal pathwayThe Langendorff isolated heart perfusing model was applied. 65 male Wistar rats were randomly divided into 5 groups, and 13 ones for each group. The groups were control group, ischemia/reperfusion group, AG490 group, Irbesartan preconditioning group and Irbesartan postcinditioning group. The change of LVDP and dp/dt_max were monitored dynamicly; The myocardial infarct size was measured by TTC staining after 120 min of reperfusion; The total and phosphorylated STAT1 and STAT3 protein expression were detected by immunoblotting after 30min of reperfusion.3. The effect of Irbesartan on apoptosis and the related gene expression during myocardial I/Rinjury of ratsThe Langendorff isolated heart perfusing model was applied. 25 male Wistar rats were randomly divided into 5 groups, and 5 ones for each group. The groups were control group, ischemia/reperfusion group, AG490 group, Irbesartan preconditioning group and Irbesartan postcinditioning group respectively. The myocardial apoptotic index was detected by TUNNEL method; The mRNA expression of Bcl-2 and Bax was detected by RT-PCR method; The protein expression of Bcl-2, Bax and Caspase-3 was detected by immunoblotting method.Results1. The study of the activating time course of the JAK-STAT signal pathway during myocardial I/R injury of rats①With the prolongation of reperfusing time, The values of LVDP and dp/dt_max decreased gradually after KH buffer perfusion for 30min, and decreased to the lowest level after 120 min of reperfusion(p＜0.01).②extent of myocardial necrosis stained by GCA bright green increased gradually with the prolongation of reperfusing time, and the most obvious result appeared after reperfusion for 120 min.③The immunohistochemical localization demonstrated that in control group, protein of p-STAT1 and p-STAT3 appeared the light brown grana in the cytoplasm. Compared with control group, brown grana of p-STAT1 in the cytoplasm did not increase after 30min of ischemia, and it began to rise and translocated to nucleus after 5min of reperfusion, brown grana of p-STAT1 reached peak afer 30min of reperfusion. Howerver, brown grana of p-STAT3 in the cytoplasm increased remarkablely and translocated to nucleus after 30min of ischemia, and did not increased again during reperfusion.④The Westeron-blot demonstrated that the protein of p-STAT1 and p-STAT3 expressed slightly in control group. Compared with control group, the protein expression of p-STAT1 didn't increase obviously after 30min of ischemia, and it began to increase after 5min of reperfusion, then reached the peak after 30 min of reperfusion. While the protein expression of p-STAT3 increased significantly after 30min of ischemia, and it did not incrase again during reperfusion. The t-STAT1 and t-STAT3 had no change in the period of ischemia and reperfusion.⑤The proportionality of p-STAT1 and p-STAT3 was correlate negtively with LVDP and dp/dt_max(r=—0.894,—0.886, p＜0.001).2. The protective role of Irbesartan against myocardial I/R injury of rats through the JAK-STAT signal pathway①Compared with control group, myocardial infarct size was increased obviously in I/R group(p＜0.01), the values of LVDP and dp/dtmax were all decreased(p＜0.01), and the protein expression of p-STAT1 and p-STAT3 were all increased(p＜0.01), especially, the protein expression of p-STAT1 approached 4 times of that in control group, and the proportionality of p-STAT1/p-STAT3 upregulated. The total STAT1 and STAT3 had no change.②Compared with I/R group, AG490, Irbesartan preconditioning and postconditioning reduced myocardial infarct size (p＜0.01), and the values for both LVDP and dp/dt_max were elevated markedly(p＜0.01), At the same time, all the three could block the increasing of protein expression of p-STAT1 and p-STAT3(p＜0.05 or p＜0.01), and the proportionality of the two down-regulated; The three had no effect on the protein expression of total STAT1 and STAT3.3. The effect of Irbesartan on apoptosis and the related gene expression during myocardial I/Rinjury of rats①Compared with control group, the myocardial apoptotic index increased significantly in I/R group (p＜0.01); The mRNA and protein expression of Bax were all increased (p＜0.01), while the mRNA and protein expression of Bcl-2 had no change. the proportionality of Bax and Bcl-2 up-regulated; The protein expression of active Caspase-3 also increased about 4 times (p＜0.01).②Compared with I/R group, AG490, Irbesartan preconditioning and postconditioning caused a marked lowering of myocardial apoptotic index(p＜0.01); The mRNA and protein expression of Bax all decreased(p＜0.01), While the mRNA and protein expression of Bcl-2 all increased(p＜0.05). The proportionality of Bax and Bcl-2 down-regulated; the protein expression of active Caspase-3 decreased, too(p＜0.01). Conclusion(1) JAK-STAT signal pathway was activated during myocardial I/R.(2) The blockage of JAK-STAT signal pathway lightened myocardial I/R injury, which suggested that the activation of this pathway can promot myocardial I/R injury.(3) The role of cardioprotection was taken on by Irbesartan during myocardial I/R. Its mechanism was related to the blockage of JAK-STAT signal pathway, down-regulating of the expression of Bax, up-regulatating of the expression Bcl-2, and the activity decrease of Caspase-3.