Browsing by Author "Wu, Jinzi"
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Item Administration of 5-methoxyindole-2-carboxylic acid that potentially targets mitochondrial dihydrolipoamide dehydrogenase confers cerebral preconditioning against ischemic stroke injury(2018-03-14) Li, Rongrong; Li, Wenjun; Ren, Ming; Thangthaeng, Nopporn; Sumien, Nathalie; Liu, Ran; Yang, Shaohua; Simpkins, James; Forster, Michael J.; Yan, Liang-Jun; Wu, JinziPurpose: The purpose of this study was to investigate a possible role of mitochondrial dihydrolipoamide dehydrogenase (DLDH) as a chemical preconditioning target for neuroprotection against ischemic injury. Methods: We used 5-methoxyindole-2-carboxylic acid (MICA), a reportedly reversible DLDH inhibitor, as the preconditioning agent and administered MICA to rats mainly via dietary intake. Upon completion of 4 week's MICA treatment, rats underwent 1 h transient ischemia and 24 h reperfusion followed by tissue collection. Results: Our results show that MICA protected the brain against ischemic stroke injury as the infarction volume of the brain from the MICA-treated group was significantly smaller than that from the control group. Data were then collected without or with stroke surgery following MICA feeding. It was found that in the absence of stroke following MICA feeding, DLDH activity was lower in the MICA treated group than in the control group, and this decreased activity could be partly due to DLDH protein sulfenation. Moreover, DLDH inhibition by MICA was also found to upregulate the expression of NAD(P)H-ubiquinone oxidoreductase 1(NQO1) via the Nrf2 signaling pathway. In the presence of stroke following MICA feeding, decreased DLDH activity and increased Nrf2 signaling were also observed along with increased NQO1 activity, decreased oxidative stress, decreased cell death, and increased mitochondrial ATP output. We also found that MICA had a delayed preconditioning effect four weeks post MICA treatment. Conclusion: Our study indicates that administration of MICA confers chemical preconditioning and neuroprotection against ischemic stroke injury.Item Humanin Attenuates NMDA-Induced Excitotoxicity by Inhibiting ROS-dependent JNK/p38 MAPK Pathway(MDPI, 2018-09-29) Yang, Xiaorong; Zhang, Hongmei; Wu, Jinzi; Yin, Litian; Yan, Liang-Jun; Zhang, CeHumanin (HN) is a novel 24-amino acid peptide that protects neurons against N-methyl-d-aspartate (NMDA)-induced toxicity. However, the contribution of the different mitogen-activated protein kinases (MAPKs) signals to HN neuroprotection against NMDA neurotoxicity remains unclear. The present study was therefore aimed to investigate neuroprotective mechanisms of HN. We analyzed intracellular Ca(2+) levels, reactive oxygen species (ROS) production, and the MAPKs signal transduction cascade using an in vitro NMDA-mediated excitotoxicity of cortical neurons model. Results showed that: (1) HN attenuated NMDA-induced neuronal insults by increasing cell viability, decreasing lactate dehydrogenase (LDH) release, and increasing cell survival; (2) HN reversed NMDA-induced increase in intracellular calcium; (3) pretreatment by HN or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), an intracellular calcium chelator, decreased ROS generation after NMDA exposure; (4) administration of HN or N-Acetyl-l-cysteine (NAC), a ROS scavenger, inhibited NMDA-induced JNK and p38 MAPK activation. These results indicated that HN reduced intracellular elevation of Ca(2+) levels, which, in turn, inhibited ROS generation and subsequent JNK and p38 MAPK activation that are involved in promoting cell survival in NMDA-induced excitotoxicity. Therefore, the present study suggests that inhibition of ROS-dependent JNK/p38 MAPK signaling pathway serves an effective strategy for HN neuroprotection against certain neurological diseases.Item Pancreatic mitochondrial complex I exhibits aberrant hyperactivity in diabetes(Elsevier Inc., 2017-07-19) Wu, Jinzi; Luo, Xiaoting; Thangthaeng, Nopporn; Sumien, Nathalie; Chen, Zhenglan; Rutledge, Margaret A.; Jing, Siqun; Forster, Michael J.; Yan, Liang-JunIt is well established that NADH/NAD(+) redox balance is heavily perturbed in diabetes, and the NADH/NAD(+) redox imbalance is a major source of oxidative stress in diabetic tissues. In mitochondria, complex I is the only site for NADH oxidation and NAD(+) regeneration and is also a major site for production of mitochondrial reactive oxygen species (ROS). Yet how complex I responds to the NADH/NAD(+) redox imbalance and any potential consequences of such response in diabetic pancreas have not been investigated. We report here that pancreatic mitochondrial complex I showed aberrant hyperactivity in either type 1 or type 2 diabetes. Further studies focusing on streptozotocin (STZ)-induced diabetes indicate that complex I hyperactivity could be attenuated by metformin. Moreover, complex I hyperactivity was accompanied by increased activities of complexes II to IV, but not complex V, suggesting that overflow of NADH via complex I in diabetes could be diverted to ROS production. Indeed in diabetic pancreas, ROS production and oxidative stress increased and mitochondrial ATP production decreased, which can be attributed to impaired pancreatic mitochondrial membrane potential that is responsible for increased cell death. Additionally, cellular defense systems such as glucose 6-phosphate dehydrogenase, sirtuin 3, and NQO1 were found to be compromised in diabetic pancreas. Our findings point to the direction that complex I aberrant hyperactivity in pancreas could be a major source of oxidative stress and beta cell failure in diabetes. Therefore, inhibiting pancreatic complex I hyperactivity and attenuating its ROS production by various means in diabetes might serve as a promising approach for anti-diabetic therapies.Item Redox imbalance and aberrant mitochondrial enzymatic activities in diabetic lung(2016-03-23) Jin, Zhen; Yan, Liang-Jun; Wu, JinziThe lung is a known target of diabetic injury, but the underlying mechanisms of damage remains poorly understood. We hypothesized that pulmonary cellular redox imbalance and mitochondrial abnormalities contribute to diabetic lung injury. To test this hypothesis, we induced diabetes in rats by streptozotocin and measured redox imbalance parameters including aldose reductase activity, poly ADP ribose polymerase (PARP) activation, NAD+ and NADPH contents along with mitochondrial functional parameters represented by the enzymatic activities of complexes I to IV. Results indicate that aldose reductase activity was elevated and PARP was upregulated in diabetic lung, while the contents of both NAD+ and NADPH were decreased in diabetic lung, demonstrating an excess NADH-linked redox imbalance problem in diabetic lung. Consequently, the enzymatic activities of complexes I to IV were all elevated in diabetic lung mitochondria due to an NADH oversupply. We also found that the enzymatic activities of dihydrolipoamide dehydrogenase (DLDH) and mitochondrial sirtuin 3 (Sirt3), both of which are inducible enzymes and are NAD+-dependent, were impaired in diabetic lung, and such an impairment was due to a decreased level of protein expression for both DLDH and Sirt3. For DLDH functional impairment in diabetic lung, protein acetylation also appeared to play a role as DLDH acetylation was increased. Additionally, we found that an increased complex I activity in diabetic pulmonary mitochondria was partly due to hyperglycemia-induced upregulation of nicotinamide N-methyltransferase (NNMT) and a concomitant increase in the expression of NDUFS3, a complex I subunit that is responsible for complex I assembly. The overall outcome of this NADH-driven redox imbalance and aberrant mitochondrial enzyme functions were a decreased ATP content, an increased NAD(P)H dehydrogenase, quinone 1 (NQO1) activity, and an elevated hydrogen peroxide concentration that reflects an aggravated oxidative stress. These findings demonstrate that diabetic lung exhibits NADH/NAD+-linked redox imbalance and abnormal mitochondrial function that likely contribute to energy deficiency and oxidative damage involved in diabetic lung injury.Item Two dimensional blue native/SDS-PAGE to identify mitochondrial complex I subunits modified by 4-hydroxynonenal (HNE)(2015-03) Wu, Jinzi; Luo, Xiaoting; Yan, Liang-JunAbstract The lipid peroxidation product 4-hydroxynonenal (HNE) can form protein-linked HNE adducts, thereby impacting protein structure and function. Mitochondrial complex I (NADH-ubiquinone oxidoreductase), containing at least 45 subunits in mammalian cells, sits in a lipid-rich environment and is thus very susceptible to HNE modifications. In this paper, a procedure for the identification of HNE-modified complex I subunits is described. Complex I was isolated by first dimensional nongradient blue native polyacrylamide gel electrophoresis (BN-PAGE). The isolated complex I band, visualized by either Coomassie blue staining or silver staining, was further analyzed by second dimensional SDS-PAGE. HNE-modified proteins were visualized by Western blotting probed with anti-HNE antibodies. HNE-positive bands were then excised and the proteins contained in them were identified by mass spectrometric peptide sequencing. The method was successfully applied for the identification of two complex I subunits that showed enhanced HNE-modifications in diabetic kidney mitochondria. Keywords: blue native/SDS-PAGE, diabetes, 4-hydroxynonenal, mitochondria, reactive oxygen species, streptozotocin