Abdominal sepsis is a common, life-threatening condition in the critically ill patients. The c-Jun N-terminal kinase (JNK) is known as a stress-activated protein kinase, in order to study the role of JNK on peritonitis-induced lung injury, the changes of plasma dihydrorhodamine 123 (DHR 123) oxidation level; the myeloperoxidase (MPO) and extravasations of Evans blue dye (EBD) of lung in wild-type (WT) mice with P. aeruginosa-induced peritonitis were determined first. Second, the specific JNK inhibitor, SP600125 or lefunomide, was given to WT mice immediately after P. aeruginosa injection and DHR oxidation, MPO activity, and EBD extravasations were examined. Third, JNK1-/- mice and JNK1+/- mice were subjected to peritonitis and assayed for DHR 123 oxidation, MPO activity, EBD extravasations, and reactive oxygen species (ROS). Fourth, chimeric mice (WT ¡÷ WT, JNK1-/- ¡÷ WT, WT¡÷JNK1-/-) were generated and used to determine the role of hematopoietic cells in peritonitis-induced lung damage. The results show that peritonitis induced DHR 123 oxidation; MPO activity and EBD extravasations in lungs and administration of specific JNK inhibitor decreased the peritonitis-induced DHR oxidation and lung damage. Also, both JNK1-/- and JNK1+/- mice showed a decreased DHR oxidation and lung damage after peritonitis. Finally, the decrease of DHR 123 oxidation, ROS, and lung damage in JNK1-/- ¡÷ WT chimeric mice suggests that that peritonitis-induced expression of iNOS and subsequent peroxynitrite production and lung damage depends on the JNK1 signaling of the hematopoietic cells.

Peroxynitrite (ONOO-), produced from the reaction of superoxide anion and nitric oxide (NO), plays important roles in many physiological and pathological processes as it is far more active than its precursors. However, the lack of specific and direct detection methods slow down the pace of its related research. To this end, five new peroxynitrite probes have been tested with different properties, including HKGreen-4A, 9-32, HKYellow-AM, 9-40 and MitoPN-1. Overwhelming evidence shows peroxynitrite is responsible for neurotoxicity in cerebral ischemia-reperfusion injury. Development of drugs for scavenging ONOO- becomes an important therapeutic strategy for ischemic stroke. Danshen, the dried root of salvia miltiorrhiza Bunge, is a traditional Chinese Medicine commonly used for cerebrovascular diseases. With the screening platform based on the new probe HKYellow-AM, several hydrophilic compounds from Danshen were identified to be peroxynitrite scavengers. Among them, Salvianolic acid B (SAB) possessed the highest peroxynitrite scavenging activity. Another compound, Sodium Danshensu (SDSS), the sodium salt of Danshensu, is not only a representative active compound of Danshen but also the degradation product of these hydrophilic compounds in vivo. Therefore, the potential neuroprotective effects of SAB and SDSS were studied. Both SAB and SDSS possessed strong ONOO- scavenging activity and consequently protected neuronal cell line SH-SY5Y and cortical neurons from both ONOO- and oxygen-glucose deprivation–reoxygenation induced cell death. SDSS also inhibited ONOO- formation partly by scavenging NO and superoxide. In vivo focal cerebral ischemia-reperfusion experiments revealed that both SAB and SDSS remarkably reduced protein nitration level, cell death and infarct volume in ischemia-reperfused rat brains. However, as “coin has its two sides”, whether peroxynitrite could also act as a signaling molecule has not been well known yet. The discovery of adult neurogenesis brings hope for brain repair after ischemic stroke. Recent studies indicate low concentration of peroxynitrite promotes endothelial cell growth for angiogenesis and contributes to hypoxia-induced muscle cell proliferation. To investigate the role of peroxynitrite in neurogenesis, several experiments on neural stem/progenitor cells (NSCs) were performed. The results indicated low concentrations of peroxynitrite promoted NSCs proliferation, self-renewal and neuronal differentiation. The increased peroxynitrite during hypoxia has been visualized by MitoPN-1 staining. Meanwhile, peroxynitrite decomposition catalysts (PDCs, FeTMPyP and FeTPPS) treatment reduced hypoxia-induced peroxynitrite formation, NSCs proliferation, self-renewal and neuronal differentiation. Moreover, effects of peroxynitrite on neurogenesis were partly through activating HIF-1α correlated with enhanced Wnt/β-catenin signaling pathway. In addition, the different effects of peroxynitrite on neurogenesis and neurotoxicity depended on its concentration. In summary, five new probes with different properties have been tested to be sensitive and specific peroxynitrite probes. With these probes, I found low concentration of peroxynitrite promoted neurogenesis, and endogenous peroxynitrite generation contributed to hypoxia-induced neurogenesis. On the other hand, high concentrations of peroxynitrite-induced neurotoxicity could be attenuated by SAB and SDSS, which partly contribute to their protective effects against ischemia-reperfusion injury. These findings extend the understanding on the biological function of peroxynitrite, drug discovery for targeting peroxynitrite, in both technological and experimental contexts. / published_or_final_version / Chinese Medicine / Doctoral / Doctor of Philosophy

S-Nitrosothiols have been found to undergo nucleophilic attack by the hydroperoxide anion to effect electrophilic nitrosation of the nucleophile. Peroxynitrite anion is formed in almost quantitative yields and the kinetics of the reaction examined, confirming attack through the deprotonated form of hydrogen peroxide. Conversely, under slightly acidic conditions peroxynitrous acid, the neutral form of peroxynitrite, has been shown to nitrosate an excess of thiol in an indirect pathway. Initially two moles of thiol are oxidised to the corresponding disulfide with the concomitant production of nitrite. Under mildly acidic conditions nitrous acid is formed which can then nitrosate excess thiol present. The reaction of a 2;1 excess of thiol over peroxynitrous acid has been shown to generate nitrous acid, which remains relatively stable, as there is no thiol remaining due to its oxidation to the disulfide. At higher acidities an additional source of nitrosation is uncovered which is explained in the terms of the formation of a protonated from of peroxynitrous acid with analogies toward the nitrous acidium ion. A comparison of the antioxidant potential of S-nitrosothiols versus thiols has also been examined using the powerful oxidant potassium bromate. Complex kinetic traces were generated but evidence was obtained which showed that S-nitrosothiols have enhanced antioxidant potential over thiols due to the nitroso moiety. The alkaline hydrolysis of S-nitrosothiols was also investigated with attack of the hydroxide ion postulated to proceed via nucleophilic attack on the sulfur atom of S-nitrosothiols. Also the reaction between S-nitrosothiols and phenolic compounds was found to proceed through different mechanisms depending on the ring substituents on the phenol.

Damage to cardiac contractile proteins such as myosin light chain 1 (MLC1), during oxidative stress is mediated by reactive oxygen species such as peroxynitrite (ONOO-), resulting in impairment of cardiac systolic function. The purpose of this study is to investigate the effects of the increased level of ONOO- on MLC1 degradation by the proteolytic enzyme matrix metalloproteinase-2 (MMP-2) during oxidative stress which ultimately decreases cardiac function.<p> In the present study two distinct models were utilized to demonstrate the mechanism by which MLC1 is modified by ONOO- and how these post-translational modifications lead to its increased degradation by MMP-2. In a model of newborn hypoxia-reoxygenation in piglets we demonstrated that ONOO--induced nitration and nitrosylation of tyrosine and cysteine residues of MLC1 increase its degradation by MMP-2. Furthermore, we found nitration of a tyrosine residue located adjacent to the cleavage site for MMP-2. We verified these results by using a model of isolated rat heart myocytes to determine that the same mechanism responsible for cardiac dysfunction in newborn piglets occurs in isolated myocytes and that the MMP-2 involved in degradation of MLC1 is located within the myocytes. Moreover, we were able to determine that this mechanism occurs during ischemia itself before the onset of reperfusion. Furthermore, we have found that pharmacological intervention aimed at inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO- scavenger FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), or inhition of MMP-2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility. The work presented here provides new evidence on the mechanisms of regulation of contractile proteins during the development of contractile dysfunction.

Damage to cardiac contractile proteins such as myosin light chain 1 (MLC1), during oxidative stress is mediated by reactive oxygen species such as peroxynitrite (ONOO-), resulting in impairment of cardiac systolic function. The purpose of this study is to investigate the effects of the increased level of ONOO- on MLC1 degradation by the proteolytic enzyme matrix metalloproteinase-2 (MMP-2) during oxidative stress which ultimately decreases cardiac function.<p> In the present study two distinct models were utilized to demonstrate the mechanism by which MLC1 is modified by ONOO- and how these post-translational modifications lead to its increased degradation by MMP-2. In a model of newborn hypoxia-reoxygenation in piglets we demonstrated that ONOO--induced nitration and nitrosylation of tyrosine and cysteine residues of MLC1 increase its degradation by MMP-2. Furthermore, we found nitration of a tyrosine residue located adjacent to the cleavage site for MMP-2. We verified these results by using a model of isolated rat heart myocytes to determine that the same mechanism responsible for cardiac dysfunction in newborn piglets occurs in isolated myocytes and that the MMP-2 involved in degradation of MLC1 is located within the myocytes. Moreover, we were able to determine that this mechanism occurs during ischemia itself before the onset of reperfusion. Furthermore, we have found that pharmacological intervention aimed at inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO- scavenger FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), or inhition of MMP-2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility. The work presented here provides new evidence on the mechanisms of regulation of contractile proteins during the development of contractile dysfunction.

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