bims-noxint 2020-01-05 papers

bims-noxint

Biomed News

on NADPH oxidases in tumorigenesis

Issue of 2020–01–05
twelve papers selected by
Laia Caja Puigsubira, Uppsala University

Angiotensin II and aldosterone activate retinal microglia.
Isoform-selective NADPH oxidase inhibitor panel for pharmacological target validation.
Alcohol promotes renal fibrosis by activating Nox2/4-mediated DNA methylation of Smad7.
Critical role of mitochondrial oxidative stress in acid aspiration induced ALI in mice.
Salusin-β mediates tubular cell apoptosis in acute kidney injury: Involvement of the PKC/ROS signaling pathway.
Mutant IDH1 Differently Affects Redox State and Metabolism in Glial Cells of Normal and Tumor Origin.
1α,25-dihydroxyvitamin D3 prevents renal oxidative damage via the PARP1/SIRT1/NOX4 pathway in Zucker diabetic fatty rats.
How the redox state regulates immunity.
Oxidative stress and cancer development: Are non-coding RNAs the missing links?
A novel rhynchophylline analog, Y396, inhibits endothelial dysfunction induced by oxidative stress in diabetes through EGF receptor.
NOX4 overexpression is a poor prognostic factor in patients undergoing curative esophagectomy for esophageal squamous cell carcinoma.
NADPH oxidase1 inhibition leads to regression of central sensitization during formalin induced acute nociception via attenuation of ERK1/2-NFκB signaling and glial activation.

Exp Eye Res. 2019 Dec 26. pii: S0014-4835(19)30415-4. [Epub ahead of print] 107902

Angiotensin II and aldosterone activate retinal microglia.

Indrajeetsinh Rana, Varaporn Suphapimol, Jack R Jerome, Dean M Talia, Devy Deliyanti, Jennifer L Wilkinson-Berka.

  Microglial cells are important contributors to the neuroinflammation and blood vessel damage that occurs in ischemic retinopathies. We hypothesized that key effectors of the renin-angiotensin aldosterone system, angiotensin II (Ang II) and aldosterone, increase the density of microglia in the retina and stimulate their production of reactive oxygen species (ROS) as well as pro-angiogenic and pro-inflammatory factors. Two animal models were studied that featured up-regulation of Ang II or aldosterone and included transgenic Ren-2 rats which overexpress renin and Ang II in tissues including the retina, and Sprague Dawley rats with ischemic retinopathy and infused with aldosterone. Complementary studies were performed in primary cultures of retinal microglia from neonatal Sprague Dawley rats exposed to hypoxia (0.5% O2) and inhibitors of the angiotensin type 1 receptor (valsartan), the mineralocorticoid receptor (spironolactone) or aldosterone synthase (FAD286). In both in vivo models, the density of ionized calcium-binding adaptor protein-1 labelled microglia/macrophages was increased in retina compared to genetic or vehicle controls. In primary cultures of retinal microglia, hypoxia increased ROS (superoxide) levels as well as the expression of the NADPH oxidase (NOX) isoforms, NOX1, NOX2 and NOX4. The elevated levels of ROS as well as NOX2 and NOX4 were reduced by all of the treatments, and valsartan and FAD286 also reduced NOX1 mRNA levels. A protein cytokine array of retinal microglia revealed that valsartan, spironolactone and FAD286 reduced the hypoxia-induced increase in the potent pro-angiogenic and pro-inflammatory agent, vascular endothelial growth factor as well as the inflammatory factors, CCL5 and interferon γ. Valsartan also reduced the hypoxia-induced increase in IL-6 and TIMP-1 as well as the chemoattractants, CXCL2, CXCL3, CXCL5 and CXCL10. Spironolactone and FAD286 reduced the levels of CXCL2 and CXCL10, respectively. In conclusion, our findings that both Ang II and aldosterone influence the activation of retinal microglia implicates the renin-angiotensin aldosterone system in the pathogenesis of ischemic retinopathies.

Keywords:  Aldosterone; Angiotensin II; Chemokines; Microglia; NADPH oxidase; Reactive oxygen species

DOI:  https://doi.org/10.1016/j.exer.2019.107902
Free Radic Biol Med. 2019 Dec 25. pii: S0891-5849(19)32560-2. [Epub ahead of print]

Isoform-selective NADPH oxidase inhibitor panel for pharmacological target validation.

V T Dao, Mahmoud H Elbatreek, S Altenhöfer, Ana I Casas, M P Pachado, C T Neullens, U Knaus, H H H W Schmidt.

  Dysfunctional reactive oxygen species (ROS) signaling is considered an important disease mechanism. Therapeutically, non-selective scavenging of ROS by antioxidants, however, has failed in multiple clinical trials to provide patient benefit. Instead, pharmacological modulation of disease-relevant, enzymatic sources of ROS appears to be an alternative, more promising and meanwhile successfully validated approach. With respect to targets, the family of NADPH oxidases (NOX) stands out as main and dedicated ROS sources. Validation of the different NOX isoforms has been mainly through genetically modified rodent models and is lagging behind in other species. It is unclear whether the different NOX isoforms are sufficiently distinct to allow selective pharmacological modulation. Here we show for five widely used NOX inhibitors that isoform selectivity can be achieved, although individual compound specificity is as yet insufficient. NOX1 was most potently (IC50) targeted by ML171 (0.1 μM); NOX2, by VAS2870 (0.7 μM); NOX4, by M13 (0.01 μM) and NOX5, by ML090 (0.01 μM). In addition, some non-specific antioxidant and assay artefacts may limit the interpretation of data, which included, surprisingly, the clinically advanced NOX inhibitor, GKT136901. In a human ischemic blood-brain barrier hyperpermeability model where genetic target validation is not an option, we provide proof-of-principle that pharmacological target validation for different NOX isoforms is possible by applying an inhibitor panel at IC50 concentrations. Moreover, our findings encourage further lead optimization and development efforts for isoform-selective NOX inhibitors in different indications.

Keywords:  NADPH oxidase; NOX inhibitors; Reactive oxygen species; Target validation

DOI:  https://doi.org/10.1016/j.freeradbiomed.2019.12.038
Clin Sci (Lond). 2020 Jan 03. pii: CS20191047. [Epub ahead of print]

Alcohol promotes renal fibrosis by activating Nox2/4-mediated DNA methylation of Smad7.

Qin Yang, Hai-Yong Chen, Jia-Nan Wang, Huai-Qin Han, Ling Jiang, Wei-Feng Wu, Biao Wei, Li Gao, Qiu-Ying Ma, Xue-Qi Liu, Qi Chen, Jia-Gen Wen, Juan Jin, Yan Huang, Wei-Jian Ni, Tao-Tao Ma, Jun Li, Xiao-Ming Meng.

  Alcohol consumption causes renal injury and compromises kidney function. The underlying mechanism of the alcoholic kidney disease remains largely unknown. In this study, an alcoholic renal fibrosis animal model was firstly employed which mice received liquid diet containing alcohol for 4-12 weeks. The Masson's Trichrome staining analysis showed that kidney fibrosis increased at week 8 and 12 in the animal model which was further confirmed by albumin assay, Western blot, immunostaining and real-time PCR of fibrotic indexes. In vitro analysis also confirmed that alcohol significantly induced fibrotic response in HK2 tubular epithelial cells. Importantly, both in vivo and in vitro studies showed alcohol treatments decreased Smad7 and activated Smad3. We further determined how the alcohol affected the balance of Smad7 and Smad3. Genome-wide methylation sequencing showed an increased DNA methylation of many genes and bisulfite sequencing analysis showed an increased DNA methylation of Smad7 after alcohol ingestion. We also found DNA methylation of Smad7 was mediated by DNMT1 in Ethyl alcohol (EtOH)-treated HK2 cells. Knockdown of Nox2 or Nox4 decreased DNMT1 and rebalanced Smad7/Smad3 axis, and thereby relieved EtOH-induced fibrotic response. The inhibition of reactive oxygen species by the intraperitoneal injection of apocynin attenuated renal fibrosis and restored renal function in the alcoholic mice. Collectively, we established novel in vivo and in vitro alcoholic kidney fibrosis models and found that alcohol induces renal fibrosis by activating oxidative stress-induced DNA methylation of Smad7. Suppression of Nox-mediated oxidative stress may be a potential therapy for long-term alcohol abuse-induced kidney fibrosis.

Keywords:  Alcohol; DNA methylation; NADPH Oxidases; Smad7; renal fibrosis

DOI:  https://doi.org/10.1042/CS20191047
Toxicol Mech Methods. 2020 Jan 03. 1-19

Critical role of mitochondrial oxidative stress in acid aspiration induced ALI in mice.

Gayatri Puri, Amarjit S Naura.

  Acute lung injury (ALI) is a pulmonary inflammatory disorder which causes significant mortality in critically ill patients. Intracellular oxidative stress has been considered to be the major component in the pathogenesis of ALI but exact source of intracellular ROS is not clearly known. The present study has been designed to elucidate the role of NADPH oxidase system and/or mitochondrial oxidative stress and its downstream pathway NLRP3 inflammasomes in mouse model of acid aspiration mediated ALI. Our data showed that acid aspiration induced lung inflammation was associated with enhanced oxidative stress as evident by data on MDA levels, nitrite levels and redox imbalance. Further acid aspiration resulted in elevation of expression of NADPH oxidase subunits (gp91 phox/p22 phox/p67 phox) as well as mitochondrial oxidative stress as reflected by aconitase activity, mitochondrial ROS levels. Interestingly, NADPH oxidase inhibitor, apocynin did not alter lung inflammation upon HCl instillation. Conversely, mitochondrial antioxidant mito-tempo resulted in significant amelioration of lung inflammation as indicated by suppression of pulmonary neutrophils and inflammatory cytokines namely IL-1β, TNF-α, IL-6 in BALF. Analysis of mitochondrial enzymes aconitase/mitochondrial ROS/Mn-SOD confirmed that reduction in lung inflammation by mito-tempo was associated with normalization of oxidative stress in mitochondria. Further, mito-tempo administration blunted phosphorylation of p65- NF-κB at Ser 536. Finally mito-tempo downregulated HCl-induced NF-κB dependent pro-inflammatory cytokines (IL-1β,TNF-α,IL-6) drastically at mRNA levels. Overall, our data supports that mitochondrial oxidative stress is crucial in modulating the HCl induced lung inflammation and identifies mitochondrial-targeted antioxidant as a potential therapeutic agent.

Keywords:  Acute lung injury; Inflammation; Mitochondria; NADPH oxidase; Oxidative stress

DOI:  https://doi.org/10.1080/15376516.2019.1710888
Redox Biol. 2019 Dec 20. pii: S2213-2317(19)31385-0. [Epub ahead of print]30 101411

Salusin-β mediates tubular cell apoptosis in acute kidney injury: Involvement of the PKC/ROS signaling pathway.

Qing-Bo Lu, Qiong Du, Hui-Ping Wang, Zi-Han Tang, Yuan-Ben Wang, Hai-Jian Sun.

  Salusin-β is abundantly expressed in many organs and tissues including heart, blood vessels, brain and kidneys. Recent studies have identified salusin-β as a bioactive peptide that contributes to various diseases, such as atherosclerosis, hypertension, diabetes and metabolic syndrome. However, the role of salusin-β in the pathogenesis of acute kidney injury (AKI) is largely unclear. In the present study, we investigated the roles of salusin-β in cisplatin or lipopolysaccharide (LPS)-induced renal injury. Herein, we found that salusin-β expression was upregulated in both renal tubular cells and kidney tissues induced by both cisplatin and LPS. In vitro, silencing of salusin-β diminished, whereas overexpression of salusin-β exaggerated the increased PKC phosphorylation, oxidative stress, histone γH2AX expression, p53 activation and apoptosis in either cisplatin or LPS-challenged renal tubular cells. More importantly, salusin-β overexpression-induced tubular cell apoptosis were abolished by using the PKC inhibitor Go 6976, reactive oxygen species (ROS) scavenger NAC, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (Apo) or p53 inhibitor Pifithrin-α. In animals, blockade of salusin-β alleviated PKC phosphorylation, ROS accumulation, DNA damage, and p53 activation as well as renal dysfunction in mice after administration of cisplatin or LPS. Taken together, these results suggest that overexpressed salusin-β is deleterious in AKI by activation of the PKC/ROS signaling pathway, thereby priming renal tubular cells for apoptosis and death.

Keywords:  Apoptosis; Cisplatin; DNA damage; LPS; Oxidative stress; Salusin

DOI:  https://doi.org/10.1016/j.redox.2019.101411
Cancers (Basel). 2019 Dec 16. pii: E2028. [Epub ahead of print]11(12):

Mutant IDH1 Differently Affects Redox State and Metabolism in Glial Cells of Normal and Tumor Origin.

Julia Biedermann, Matthias Preussler, Marina Conde, Mirko Peitzsch, Susan Richter, Ralf Wiedemuth, Khalil Abou-El-Ardat, Alexander Krüger, Matthias Meinhardt, Gabriele Schackert, William P Leenders, Christel Herold-Mende, Simone P Niclou, Rolf Bjerkvig, Graeme Eisenhofer, Achim Temme, Michael Seifert, Leoni A Kunz-Schughart, Evelin Schröck, Barbara Klink.

  IDH1R132H (isocitrate dehydrogenase 1) mutations play a key role in the development of low-grade gliomas. IDH1wt converts isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP+), whereas IDH1R132H uses α-ketoglutarate and NADPH to generate the oncometabolite 2-hydroxyglutarate (2-HG). While the effects of 2-HG have been the subject of intense research, the 2-HG independent effects of IDH1R132H are still ambiguous. The present study demonstrates that IDH1R132H expression but not 2-HG alone leads to significantly decreased tricarboxylic acid (TCA) cycle metabolites, reduced proliferation, and enhanced sensitivity to irradiation in both glioblastoma cells and astrocytes in vitro. Glioblastoma cells, but not astrocytes, showed decreased NADPH and NAD+ levels upon IDH1R132H transduction. However, in astrocytes IDH1R132H led to elevated expression of the NAD-synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT). These effects were not 2-HG mediated. This suggests that IDH1R132H cells utilize NAD+ to restore NADP pools, which only astrocytes could compensate via induction of NAMPT. We found that the expression of NAMPT is lower in patient-derived IDH1-mutant glioma cells and xenografts compared to IDH1-wildtype models. The Cancer Genome Atlas (TCGA) data analysis confirmed lower NAMPT expression in IDH1-mutant versus IDH1-wildtype gliomas. We show that the IDH1 mutation directly affects the energy homeostasis and redox state in a cell-type dependent manner. Targeting the impairments in metabolism and redox state might open up new avenues for treating IDH1-mutant gliomas.

Keywords:  IDH-mutation; IDH1; NAD-synthesis; glioma; metabolism; redox state

DOI:  https://doi.org/10.3390/cancers11122028
Am J Physiol Endocrinol Metab. 2019 Dec 31.

1α,25-dihydroxyvitamin D3 prevents renal oxidative damage via the PARP1/SIRT1/NOX4 pathway in Zucker diabetic fatty rats.

Dongxia Wang, Yanyan Li, Ning Wang, Gang Luo, Jun Wang, Can Luo, Wei Yu, Liping Hao.

  Diabetic nephropathy (DN) is one of the most important renal complications associated with diabetes and the mechanisms are yet to be fully understood. To date, few studies have shown the anti-oxidant effects of 1α,25-dihydroxyvitamin-D3 (1,25(OH)2D3) on hyperglycemia-induced renal injury. The aim of the present study was to explore the potential mechanism by which 1,25(OH)2D3 reduced oxidative stress in diabetic rat kidneys. In this study, we established a vitamin D-deficient spontaneous diabetes model: 5-6 weeks of age Zucker diabetic fatty (ZDF) rats were treated with or without 1,25(OH)2D3 for 7 weeks, age-matched Zucker lean (ZL) rats served as control. Results showed that ZDF rats treated with 1,25(OH)2D3 had decreased body mass, food intake, water intake and urine volume. 1,25(OH)2D3 ameliorated urine glucose, blood glucose and abnormal glucose tolerance. Additionally, 1,25(OH)2D3 significantly lowered microalbuminuria (MALB), decreased the glomerular basement membrane (GBM) thickness, and in some degree inhibited glomerular hypertrophy, mesangial expansion and tubular dilatation. Furthermore, 1,25(OH)2D3 attenuated renal oxidative damage, as reflected by the levels of MDA, GSH, 4-HNE, 8-OHdG and ROS production, and notably inhibited PARP1, activated SIRT1 and decreased the expression of NOX4. Of interest, the abovementioned proteins could be involved in the anti-oxidant mechanism of 1,25(OH)2D3 in diabetic rat kidneys. Our study showed that oxidative stress might be a major contributor to DN pathogenesis and uncovered the antioxidant role of 1,25(OH)2D3 in diabetic nephropathy which was associated with the PARP1/SIRT1/NOX4 pathway.

Keywords:  1,25-dihydroxyvitamin-D3; Diabetic nephropathy; Oxidative stress; PARP1/SIRT1/NOX4 axis

DOI:  https://doi.org/10.1152/ajpendo.00270.2019
Free Radic Biol Med. 2019 Dec 30. pii: S0891-5849(19)31607-7. [Epub ahead of print]

How the redox state regulates immunity.

Lisa Mullen, Manuela Mengozzi, Eva-Maria Hanschmann, Ben Alberts, Pietro Ghezzi.

Oxidative stress is defined as an imbalance between the levels of reactive oxygen species (ROS) and antioxidant defences. The view of oxidative stress as a cause of cell damage has evolved over the past few decades to a much more nuanced view of the role of oxidative changes in cell physiology. This is no more evident than in the field of immunity, where oxidative changes are now known to regulate many aspects of the immune response, and inflammatory pathways in particular. Our understanding of redox regulation of immunity now encompasses not only increases in reactive oxygen and nitrogen species, but also changes in the activities of oxidoreductase enzymes. These enzymes are important regulators of immune pathways both via changes in their redox activity, but also via other more recently identified cytokine-like functions. The emerging picture of redox regulation of immune pathways is one of increasing complexity and while therapeutic targeting of the redox environment to treat inflammatory disease is a possibility, any such strategy is likely to be more nuanced than simply inhibiting ROS production.

DOI: https://doi.org/10.1016/j.freeradbiomed.2019.12.022
Antioxid Redox Signal. 2019 Dec 31.

Oxidative stress and cancer development: Are non-coding RNAs the missing links?

Leonard Clinton D'Souza, Shruti Mishra, Anirban Chakraborty, Anusmita Shekher, Anurag Sharma, Subash Chandra Gupta.

SIGNIFICANCE: It is now clear that genetic changes underlie the basis of cancer and alterations in functions of multiple genes is responsible for the process of tumorigenesis. Besides the classical genes that are usually implicated in cancer, the role of non-coding RNAs (ncRNAs) and reactive oxygen species (ROS) as independent entitites has also been investigated. Recent Advances: The microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), two main classes of ncRNAs, are known to regulate many aspects of tumor development. ROS, which is generated during oxidative stress and pathological conditions,is known to regulate every step of tumor development. Conversely, oxidative stress and ROS producing agents can suppress tumor development. The malignant cells normally produce high level of ROS as compared to normal cells. The interaction between ROS and ncRNAs regulates the expression of multiple genes and pathways implicated in cancer suggesting a unique mechanistic relation among ncRNA-ROS-cancer. The mechanistic relation has been reported in hepatocellular carcinoma, glioma, and malignancies of blood, breast, colorectum, esophagus, kidney, lung, mouth, ovary, pancreas, prostate and stomach.The ncRNA-ROS regulate several cancer-related cell signaling pathways namely AKT, EGFR, FOXO3, Keap1, NF-B, Nrf2, p53, PTEN, and Wnt/GSK3β.
CRITICAL ISSUES: To date, most of the reports about ncRNAs-oxidative stress-carcinogenesis relation are based on cell lines. The mechanistic basis for this relation has not been completely elucidated.
FUTURE DIRECTIONS: Attempts should be made to explore the association of lncRNAs with ROS. The significance of ncRNAs-oxidative stress-carcinogenesis interplay should also beexplored through studies in animal models.

DOI: https://doi.org/10.1089/ars.2019.7987
Antioxid Redox Signal. 2019 Dec 31.

A novel rhynchophylline analog, Y396, inhibits endothelial dysfunction induced by oxidative stress in diabetes through EGF receptor.

Zhijun Wang, Lingling Chang, Jian Wu, Homh-Ming Pan, Qiuyan Zhang, Min-Jun Wang, Xiaoming Xin, Shanshan Luo, Ji-An Chen, Wei-Lu Wang, Xian Feng Gu, Wei Guo, Yizhun Zhu.

AIMS: Endothelial dysfunction appears in early diabetes mellitus partially because of epidermal growth factor receptor (EGFR) abnormal activation and downstream oxidative stress. The aim of this study was to determine whether Y396, a synthesized analog of rhynchophylline, could protect against endothelial dysfunction in diabetes and the underlying molecular mechanism.
RESULTS: Y396 could directly target the EGF receptor and inhibit its phosphorylation induced by high glucose and EGF, downstream translocation to the nuclear of E2F1, and its transcriptional activity and expression of Nox4. Diabetes-induced endothelium malfunction was ameliorated by Y396 treatment through EGF receptor inhibition. Downstream oxidative stress was decreased by Y396 in the aortas of T1DM mice and primary rat aorta endothelial cells (RAECs). Y396 could also ameliorate tunicamycin-induced oxidative stress in the aorta and RAECs. In addition, we also again determined the protective effects of Y396 on HFD/STZ-induced type 2 diabetes mellitus (T2DM). Innovations: This is the first study to demonstrate that Y396, a novel rhynchophylline analog, suppressed high-glucose-induced endothelial malfunction both in vivo and in vitro by inhibiting abnormal phosphorylation of EGFR. Our work uncovered EGF receptor as a novel therapeutic target, and Y396 as a potential therapy against diabetes-induced complication.
CONCLUSION: Y396 could directly bind with EGF receptor and inhibit its phosphorylation and downstream E2F1 transcriptional activity. It could also preserve tunicamycin-evoked endothelial dysfunction and oxidative stress. It could protect against diabetes-induced endothelium malfunction in vivo through EGF receptor inhibition and downstream oxidative stress.

DOI: https://doi.org/10.1089/ars.2018.7721
Surgery. 2019 Dec 27. pii: S0039-6060(19)30773-1. [Epub ahead of print]

NOX4 overexpression is a poor prognostic factor in patients undergoing curative esophagectomy for esophageal squamous cell carcinoma.

Yen-Hao Chen, Hung-I Lu, Chien-Ming Lo, Chang-Chun Hsiao, Shau-Hsuan Li.

BACKGROUND: Nox4 has been associated with tumor progression in various types of malignancies. This study aimed to evaluate the importance of the expression of Nox4 in patients undergoing curative esophagectomy for esophageal squamous cell carcinoma.
METHODS: We reviewed retrospectively 121 patients with esophageal squamous cell carcinoma who had undergone a curative esophagectomy, including 67 patients with overexpression and 54 patients with low expression of Nox4 as evaluated by immunohistochemical analysis. In addition, 2 esophageal squamous cell carcinoma cell lines, TE11 and KYSE270, were treated with the Nox4 inhibitor GKT-137831 to explore the expression of Nox4, cell proliferative activity, and selected downstream pathways in these esophageal squamous cell carcinoma cell lines by Western blot analysis.
RESULTS: Univariate analysis showed that T1-2 status, absence of nodal metastasis, and low Nox4 expression were associated with greater disease-free survival (P = .001) and overall survival (P < .001), and Nox4 overexpression was an independent prognostic factor of worse disease-free survival and overall survival (P = .013 and P = .007, respectively). The esophageal squamous cell carcinoma cell lines treated with the Nox4 inhibitor GKT-137831 showed decreased cell proliferation in a dose-dependent manner (P < .01). Western blot analysis demonstrated that expression of AKT, phosphorylated AKT, mammalian target of rapamycin, and phosphorylated mammalian target of rapamycin were less in esophageal squamous cell carcinoma cells treated with the Nox4 inhibitor (P < .01).
CONCLUSION: This study suggests that Nox4 overexpression is a poor prognostic factor for patients with esophageal squamous cell carcinoma undergoing curative esophagectomy.

DOI: https://doi.org/10.1016/j.surg.2019.11.017
Neurochem Int. 2019 Dec 28. pii: S0197-0186(19)30539-X. [Epub ahead of print] 104652

NADPH oxidase1 inhibition leads to regression of central sensitization during formalin induced acute nociception via attenuation of ERK1/2-NFκB signaling and glial activation.

Sanjay Kumar, Manjula Vinayak.

  Role of NADPH oxidase1 in the development of inflammatory pain has been demonstrated by gene knockout studies. Nevertheless, pharmacological inhibition of NOX1 is a requisite approach for therapeutic utility. Recently, we have reported the anti-nociceptive effect of newly identified NOX1 specific inhibitor ML171 (2-acetylphenothiazine). Inhibition of NOX1 resulted in attenuation of nociceptive sensitization during acute inflammatory pain via inhibition of ROS generation and its downstream ERK1/2 activation. However, glial activation accompanying inflammation is closely related to the initiation and maintenance of pain. Peripheral nociceptive inputs activate the primary afferents via release of various chemical mediators which are potentially capable of mediating signals from neuron to glia in DRG and subsequently in spinal cord dorsal horn. The subsequent interactions between neuron and glia contribute to pain hypersensitivity. Thus, the present study was focused to investigate the effect of ML171 on ERK1/2 signaling, glial activation, and crosstalk between neuron and glia in a mouse model of formalin induced acute nociception. Thus, the present study was focused to investigate the effect of ML171 on ERK1/2 signaling, glial activation, and crosstalk between neuron and glia in DRG and dorsal horn of the spinal cord of lumbar region (L3-L5) in a mouse model of formalin induced acute nociception. Intraperitoneal administration of ML171 decreased nociceptive behavioral responses, i.e. the flinch and lick counts, in formalin induced nociceptive mice. Immunofluorescence and Western blot analysis demonstrated decreased levels of nociceptive mediators like p-ERK1/2, p-NFκB p65, Iba1 and GFAP in DRG as well as in spinal cord dorsal horn; supporting anti-nociceptive potential of ML171. Further, co-localization studies showed the neuron-glia crosstalk in tissue dependent manner. ERK1/2 was found to be activated in glia and NFκB in neurons in DRG; whereas in case of spinal cord ERK1/2 was activated in neurons and NFκB in astrocytes. Decrease in nociceptive behavioral response and activation of nociceptive mediators after intraperitoneal administration of ML171 strongly advocate anti-nociceptive potential of ML171. This is the first report demonstrating modulation of ERK1/2-NFκB signaling pathway, glial activation and regulation of neuron-glia crosstalk by NADPH oxidase1 inhibition towards its anti-nociceptive action.

Keywords:  Acute pain; ERK1/2; Glia; ML171; NFκB; NOX1

DOI:  https://doi.org/10.1016/j.neuint.2019.104652