Studies On The Effect Of RIP3 Nitration On Crdio-Cerebral Ischemia Reperfusion Injury Of RAT And The Interven With Safflor Yellow

Interventional thrombolysis or drug reascularization is one of the important measures to protect the myocardium for ischemic heart disease, and greatly improved the prognosis of patients, but myocardial ischemia-reperfusion injury can be caused, which weakens the interventional thrombolysis in the clinical application effect of or drugs in some way. MIRI phenomenon forces people to start the related basic and clinical research of myocardial protection. Ischemia-reperfusion injury involves a lot of link including energy metabolic disorders, calcium overload, oxygen free radical injury, apoptosis an so on. Then scholars explore the molecular mechanism of cardio-cerebtal protection of drugs and to find new strategies, become research hot topic, such as how to inhibit apoptosis, in turn, reduce or avoid IRI, seeking to lessen IRI targets, and so on.Nitric oxide, is involved in many physiological and pathological processes of the disease, which is a kind of easily to spread the biological activity of small molecules which. It plays an important role in cellular function in regulating the REDOX signal. Besides the classic way is that NO’s activation relying on cyclic guanosine monophosphate (cGMP) sinaling pathyway to regulate the physiololgical function. NO can also occur through nucleophilic sulphur attached to protein modification after translation and generate nitrosothiols (RSNO) and then modify regulating cell function, therefore research protein thiol nitro and related signal transduction mechanisms will provide new means and methods to reduce IRI in clinical intervention. Under the condition of ischemia hypoxia and oxidative stress, the body will generate excessive NO and O2- and generates a large number of peroxynitrite anion ONOO" in a short period of time like waterfall. Then it makes intracellular tyrosine residues nitration to regulate protein function. There are many kinds of much more nitration in pathological damage process such as cardiovascular disease, inflammation, ischemic cerebral stroke and neural degenerative diseases. Protein nitration means NO free radicals react with O2- quickly, to generate a large number of ONOO - in a short period of time, the intracellular protein nitration of tyrosine residues into 3-nitrotyrosine(3-NT), which changes its structure and function. RIP3 plays an important role as cell stress sensing molecule in the regulation of cell apoptosis, cell death pathways. So What is right express RIP3 nitrification in IRI? What is the role in the whole process and what the position of the target is should be studied.The traditional Chinese medicine appeares a special advantage, although there are so many means and drug of reducing ischemia-reperfusion injury. Safflower yellow can scavenge free radicals, improve the cardio-cerebral cell energy metabolism, reduce calcium overload, inhibiting cell apoptosis to reduce IRI. But whether the effect of the molecular signaling pathways in play an important role in the inhibition of nitration? We perform relevant tests for applied molecular biology methods conducted a series of research through in vivo and in vitro experiments on animals,PART I:The study of the effect of RIP3 nitration in myocardium ischemia-reperfusion injury of rats in vivo with safflower yellow interventionBackgroundIschemia and reperfusion injury(IRJ) phenomenon refers to the organization because of ischemia injury occurred reversibility, can be survival after ischemia area gets reascularizate, but the dysfunction and structural damage is aggravating. Reperfusion arrhythmia, myocardial diastolic dysfunction, abnormal metabolism, and the changes of myocardial ultrastructure can happen after reperfusion of ischemic heart disease, overproduction of oxygen free radicals (OFR) is one of the important factors which cause MIRI. After ischemia hypoxia, it will generate excessive NO and O2- and quickly response to generate a large number of peroxynitrite anion (ONOO-), acting on the target protein tyrosine residues in the cell, make it nitration, then adjust the function of protein. Excessive nitration involved in mediating the various pathological damage process such as many kinds of ischemic cardio-cerebral injury, inflammation, and neural degenerative diseases.Receptor interacting protein 3 (RIP3) was identified as the earliest one of protein kinases, belongs to a member of the the RIPS family whose excessive expression leads to apoptosis. RIP3 takes part into various ischemia and necrotic disease, but there is few research in RIP3 covalent modification (nitration) during MIRI. Especially there is no series of related research of MIRI model with the application of molecular biology technology in vivo. Safflor yellow can reduce MIRI with inhibiting the generation of free radicals or scavenge it, improving myocardial energy metabolism, reducing intracellular calcium overload and inhibitory effect of cell apoptosis and so_on. Howerver, the internal mechanism of SY protect MIRI, especially the studies on molecular signaling pathways, is the unsolved problem. So we interrogate:1. whether RIP3 nitration plays a major role in MIRI? 2. Whether traditional Chinese medicine (TCM) the cardio-protective effect of safflower yellow involve RIP3 nitration and RIP1 phosphorylation?Objective1. To observe the expression of RIP3 nitration and RIP1 phosphorylation, hemodynamics and tissue pathology after the SY post-conditioning ischemia-reperfusion myocardium of rats in vivo.2. Study molecular signaling pathway and the mechanism of the cardio-protection of rats with different doses S Y.Methods1. Male clean 108 SD rats were divided six groups randomly(n=18):Sham group, IR group, low dosage SY group (10 mg/kg), middle dosage group (30 mg/kg), high dosagegroup (90 mg/kg) and Edaravone group. The MIRI model in vivo was made through clip left anterior descending coronary artery (LAD). Sham group only wear suture and ligation. I/R group and drug intervention groups 1 min before reperfusion via femoral vein injection of normal saline(NS), different doses of SY and Eda, SY and Eda are diluted with amount of NS, Sham group was injected amount NS at corresponding time points.2. The index of heart function were recorded at different time point. The item includes heart rate(HR), left ventricular development pressure(LVDP), left ventricular end-diastolic pressure(LVEDP), maximum change rate of left ventricular pressure rise and fall. (±dp/dtmax). The time point is as follow:ischemia sharp(TO), ischemia 30min(T1), reperfusion 30 min (T2),60 min(T3),90 min(T4) and 120 min(T5).3. The right ventricle venous blood sampling was got at the end of 120 min reperfusion by puncture extraction. The malonaldehyde (MDA) content, activity of superoxide dismutase (SOD) and creatine kinase isoenzyme (CK-MB) were detected with the colorimetric method.4. Just at the end of the experiment, the animals were killed, myocardial infarction area (%) was measured by taking heart slice given 2,3,5-triphenyl tetrazolium chloride(TTC) dyeing. The express level of nitration of RIP3 and phosphorylation of RIP 1.Results1. There is no statistically significant difference in the heart function index (HR, LVDP, LVEDP, ±dp/dtmax) between groups before ischemia.2.Compared with sham group:The absolute value of HR, LVDP, LVEDP and ±dp/dtmax of IR group and different dosage group decreased at the end of ischemia 30min, reperfusion 90min and 120min, however, LVEDP increased. The content of MDA and the activity of CK-MB increased, the activity of SOD decreased, the express of N-RIP3、P-RIP1 increased, the proportion of myocardial apoptosis cells increased at the end of reperfusion 120min,3.Compared with IR group:The absolute value of LVDP,LVEDP, ±p/dtmax in high dosage SY group and Eda group increased at the end of reperfusion 90min and 120min. The content of MDA and the activity of CK-MB decreased, the activity of SOD increased, the express of N-RIP3、 P-RIP1 decreased, the myocardium infarction areas and proportion of myocardial apoptosis decreased. Myocardial pathologic change a little. There was no statistically significant difference of the index of low and middle dosage groups compared with IR group, little change in myocardial pathologic.Compared with low and middle dosage group:The absolute value of LVDP, ±dp/dtmax in high dosage SY group increased at the end of reperfusion 90min and 120min, LVEDP decreased instead. The content of MDA and the activity of CK-MB decreased, the activity of SOD increased, the express of N-RIP3、P-RIP1 decreased, the myocardium infarction areas and proportion of myocardial apoptosis significantly decreased.Conclusions1. In MIRI of rats in vivo, OFR generate excessive, RIP3 nitration increased and activated, which promoted the phosphorylation of the downstream signal molecule RIP1, aggravating MIRI.2. There is scavenge OFR effect with SY, thus reduce RIP3 nitration and phosphorylation of RIP1, to protect MIRI of rats, even there is concentration-response relationship.PART Ⅱ:The study of the effect of RIP3 nitration in isolated myocardium ischemia reperfusion injury of rats in vitro, with safflower yellow interventionBackgroundThe covalent modification of protein post-translated including phosphorylation, glycosylation, acetylation, nitration and so on. In cell signal transduction, protein phosphorylation was studied extensively and thoroughly, and more recently, protein nitration was taken seroiusly. Protein nitration means NO free radicals react with O2-quickly, generate a large number of ONOO- in a short period of time, the intracellular protein nitration of tyrosine residues into 3-nitrotyrosine(3-NT). If the amount of 3-NT is more than the body’s normal removal ability, then a variety of important function of protein function is damaged or activity decline. It not only can damage mitochondria, DNA, but also can inhibit tyrosine phosphorylation, inducing cell apoptosis and senescence of casade process. The 3-NT can be detected in the pathological conditions, such as stress and inflammation, a variety of organizations, such as lung, liver, endothelial, etc whose generation and positioning can be reflected with the formation and distribution of tyrosine nitration.ONOO- shows the special biological activity. It is not only a strong oxidizer, but also a nitrating agent, Which can react with proteins, lipids, nucleic acids and other biological macromolecular. Under physiological conditions, there is many protein nitration in the normal tissue of the body. The regulation of signaling pathways is maybe associated with the low level of protein nitration reaction. However, more and more evidences suggest that excessive nitration is involved in mediating pathological damage process of many diseases such as the cardiovascular disease, inflammatory reaction, ischemic brain injury and neurodegenerative disorders. Previous studies in vivo have confirmed that the safflower yellow play a protective role by reducing RIP3 nitration and RIP1 phosphorylation. Can Safflower yellow still play a role of reliable myocardial protection except the in neuroendocrine factors? If so, whether it is also inhibits nitration to get it? We studied the effect and the internal mechanism of SY accordingly different doses of conditioning by the model of MIRI of rats in vitro with Langendorff apparutus.Objective1. To observe the myocardial hemodynamics, infarction areas, histopathological and apoptosis in isolated rat myocardium ischemia reperfusion.2. To try to illuminate the myocardial MIRI protection mechanism of molecular signaling pathways through expression of P-and N-RIP3, RIP1 with different doses SY in isolated rat myocardium,Methods1. Sixty isolated SD rat hearts were divided six groups randomly (n=10):Sham group(A), IR group(B), low dosage SY group (10 μmol/L, C), middle dosage group (30 μmol/L, D), high dosagegroup (90 μmol/L, E) and Edaravone group (F). After heparinization in rats, pentobarbital anesthesia, coring, cut large blood vessels, aortic keep 3-4 mm, suspension in Langendorff constant-voltage retrograde perfusion apparatus, cut off a small hole in left auricle, put into water sac connection transducer, the AD converter connected to the computer records the data.2. Cardiac function index monitoring is same as Part I. Recorded HR, LVDP and LVEDP and ±p/dtmax.Time points as the follow:immediately before ischemia (TO), at the end of ischemia 30 min(T1), at the end of the 30 min reperfusion (T2), at the end of the 60 min reperfusion(T3), at the end of the 90 min reperfusion (T4) and at the end of the 120 min reperfusion (T5).3. Methodology of measuring the myocardial infarction area, assessing myocardial cell injury and apoptosis are same as Part I.4.To detect and analyse RIP 1 phosphorylation and RIP3 nitration level with immunoprecipitation and Western Blotting.Results1. Compared with control group, the index of group IR decreased significantly. Compared with group IR, the index of left heart function in every intervention group significantly improved, and the middle dose group of SY is better.2. Compared with IR group, the infarction areas in group control, different dose SY group and edaravone group decreased, and the middle dose group of SY is the least. The activation of SOD increased and the content of MDA decreased in different dose SY group and edaravone group.3. Under light microscopy, myocardial fibers of control group rank neatly, boundaries seem clear, striation seem clearly rank. Myocardial fibers of IR group rank irregularly, part of them appeared fracture obviously, but striation is obscure. Myocardial fibers of different SY group rank neatly gradually, fracture decreased. Myocardial fibers of edaravone group rank neatly nearly, but it is sparse and the striation is visible. The TUNEL positive cells with tan nuclei are visible and vacuole formation. That of IR group is obvious. The myocardial cell apoptosis ratio decreased in different dose SY group and edaravone group.4. At the end of myocardial reperfusion 120min, the express level of RIP3 and RIP1 decreased in the drug intervention group. The nitration level of RIP3 decreased in middle, high dose SY group and edaravone group. The express and nitration level of RIP3 increased, however, the express level of RIP3 is low in high dose SY group and edaravone group, the express level of RIP 1 is low in low dose, high dose SY group and edaravone group. The nitration level of RIP3 in creased after ischmia reperfusion, but it decreased with the intervention with SY and edaravone. The express level of RIP1 increased and the result companied with different concentration of SY.Conclusions1. During the isolated myocardial ischemia reperfusion, the level of RIP3 nitration increased and promoted RIP1 phosphorylation level, both of them induced apoptosis, then increased the myocardial injury.2. SY protect the isolated ischemia reperfusion myocardium by decreasing the RIP3 nitration, RIP1 phosphorylation, reducing free radical damage, decreasing the ratio of myocardium apotosis. The protect effect of it changed with the dose of SY.PARTⅢ:The correlation and the active site study of PYK2 nitrosylation in ischemia reperfusion injuryBackgroundProline-rich tyrosine kinase 2 was non-receptor kinase family members of the focal adhesion kinase family. PYK2 participates in the signaling of G protein-coupled receptors, nicotinic acetylcholine receptors, membrane depolarization, and stress. PYK2 activation arose from its autophosphorylation on Tyr-402:PYK2 consists of a FERM, kinase, proline-rich, and focal adhesion targeting (FAT) domain. Tyr402 between the FERM and kinase domain autophosphorylated in PYK2 activation.Research shows that distribution of PYK2 tyrosine phosphorylation in cells have been detected by its tyrosine phosphorylation antibody. Its main tyrosine phosphorylation itself site is 402.Ischemia can lead to activation of PYK2, short induction of calcium ion in large quantities, caused by intracellular calcium overload.NO is a member of free radicals and an endogenous cell signaling molecule regulating many physiological and pathophysiological processes. NO and NO-related compounds exert both protective and cytotoxic effects depending on the cellular context and the nature of the NO group. Endogenous NO is synthesized from L-arginine by NO synthase (NOS) and is associated with S-nitrosylation. S-nitrosylation, the modification of the covalent attachment to the side chain of cysteine by NO group, is considered as an important post translational modification that has profound modulations on protein function. Intracellular Ca2+ targets protein phosphatases, protein kinases, and NOS. nNOS exists mainly in neurons and responds to calcium-calmodulin signaling constitutively. Interestingly, some studies show that PYK2 is a redox sensitive kinase activated by estrogen-induced ROS, but the mechanism of PYK2 activation is also not fully understood.As forementioned, we hypothesized that after cell OGD, Ca2+ influx via NMDAR leads to the formation of calcium-calmodulin signaling compounds. It help increase of endogenous NO synthesis, which leads to the PYK2 protein S-nitroso, and activate the PYK2, which finally induce cell death. In the present study, we initially examined that S-nitrosylated by reaction with GSNO. Secondly, we showed OGD induced S-nitrosylation and activation of PYK2 at different time points.Objective1. To detect the S-nitrosylation of P YK2 by exogenous nitric oxide.2. To detect S-nitrosylation and activation of PYK2 by the ischemia-reperfusion induced endogenous NO.3. To detect the location of mediating the S-nitrosylation of PYK2.Methods1. Use of PYK2-SPORT6 plasmid for template build PYK2 mutation physical grain.PYK2 enzyme activity area of each cysteine residues (C) mutation of glycine (G).2. Measurement of S-nitrosylated PYK2 with the biotin switch assay.3. Measurement of P-PYK2(activated PYK2) with Western blot.4. Detection of apoptosis-like cell death with DAPI staining.Results1. In cultured HEK-293 cells treated with GSNO, over-expression of PYK2 can form a significant nitroso protein, and present the dependence between concentration and time, and GSNO NEM can react with protein free thiol alkylation and closed thiol. S-nitrosylation of PYK2 can be inhibited by processed GNSO cell after being given NEM. S-nitrosylation of PYK2 can be inhibited too with the calcium carrier A23187 when being given 7NI which is an inhibitor of nNOS.2. Compared with wild type, the mutant decreased by about 86% with the measurement of nitrocellulose band density of S-nitrosylation PYK2 in the membrane.3. Compared with that of wild type, S-nitrosylation contributed to the functional activity of wild-type PYK2 with significantly elevated levels at 6 h, and 12 h of OGD. S-nitrosylation and phosphorylation of PYK2 decreased. The apoptosis cell significantly decreased in the SH-SY5Y cell of overexpressed C534G mutant PYK2.Conclusions1. PYK2 can be S-nitrosylated by exogenous NO(GSNO) or endogenous NO from nNOS.2. The cys-534 of PYK2 is the location of its S-nitrosylation3. PYK2 S-nitrosylation is involved in its activation.