Knockdown Of Nox4 Research Articles

Role of NOX1 and NOX5 in protein kinase C/reactive oxygen species‑mediated MMP‑9 activation and invasion in MCF‑7 breast cancer cells.

NADPH oxidases (NOXs) are a family of membrane proteins responsible for intracellular reactive oxygen species (ROS) generation by facilitating electron transfer across biological membranes. Despite the established activation of NOXs by protein kinase C (PKC), the precise mechanism through which PKC triggers NOX activation during breast cancer invasion remains unclear. The present study aimed to investigate the role of NOX1 and NOX5 in the invasion of MCF‑7 human breast cancer cells. The expression and activity of NOXs and matrix metalloprotease (MMP)‑9 were assessed by reverse transcription‑quantitative PCR and western blotting, and the activity of MMP‑9 was monitored using zymography. Cellular invasion was assessed using the Matrigel invasion assay, whereas ROS levels were quantified using a FACSCalibur flow cytometer. The findings suggested that NOX1 and NOX5 serve crucial roles in 12‑O‑tetradecanoylphorbol‑13‑acetate (TPA)‑induced MMP‑9 expression and invasion of MCF‑7 cells. Furthermore, a connection was established between PKC and the NOX1 and 5/ROS signaling pathways in mediating TPA‑induced MMP‑9 expression and cellular invasion. Notably, NOX inhibitors (diphenyleneiodonium chloride and apocynin) significantly attenuated TPA‑induced MMP‑9 expression and invasion in MCF‑7 cells. NOX1‑ and NOX5‑specific small interfering RNAs attenuated TPA‑induced MMP‑9 expression and cellular invasion. In addition, knockdown of NOX1 and NOX5 suppressed TPA‑induced ROS levels. Furthermore, a PKC inhibitor (GF109203X) suppressed TPA‑induced intracellular ROS levels, MMP‑9 expression and NOX activity in MCF‑7 cells. Therefore, NOX1 and NOX5 may serve crucial roles in TPA‑induced MMP‑9 expression and invasion of MCF‑7 breast cancer cells. Furthermore, the present study indicated that TPA‑induced MMP‑9 expression and cellular invasion were mediated through PKC, thus linking the NOX1 and 5/ROS signaling pathways. These findings offer novel insights into the potential mechanisms underlying their anti‑invasive effects in breast cancer.

Gut microbiota regulates the ALK5/NOX1 axis by altering glutamine metabolism to inhibit ferroptosis of intrahepatic cholangiocarcinoma cells

Intrahepatic cholangiocarcinoma (ICC) is a kind of hepatobiliary tumor that is increasing in incidence and mortality. The gut microbiota plays a role in the onset and progression of cancer, however, the specific mechanism by which the gut microbiota acts on ICC remains unclear. In this study, feces and plasma from healthy controls and ICC patients were collected for 16S rRNA sequencing or metabolomics analysis. Gut microbiota analysis showed that gut microbiota abundance and biodiversity were altered in ICC patients compared with controls. Plasma metabolism analysis showed that the metabolite glutamine content of the ICC patient was significantly higher than that of the controls. KEGG pathway analysis showed that glutamine plays a vital role in ICC. In addition, the use of antibiotics in ICC animals further confirmed that changes in gut microbiota affect changes in glutamine. Further experiments showed that supplementation with glutamine inhibited ferroptosis and downregulated ALK5 and NOX1 expression in HuCCT1 cells. ALK5 overexpression or NOX1 overexpression increased NOX1, p53, PTGS2, ACSL4, LPCAT3, ROS, MDA and Fe2+ and decreased FTH1, SLC7A11 and GSH. Knockdown of NOX1 suppressed FIN56-induced ferroptosis. In vivo, supplementation with glutamine promoted tumor growth. Overexpression of ALK5 repressed tumor growth and induced ferroptosis in nude mice, which could be reversed by the addition of glutamine. Our results suggested that the gut microbiota altered glutamine metabolism to inhibit ferroptosis in ICC by regulating the ALK5/NOX1 axis.

NADPH oxidase 4 facilitates progression of chondrosarcoma via generation of reactive oxygen species.

Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is an enzyme that regulates reactive oxygen species (ROS) generation, and its function in the development of chondrosarcoma remains unclear. In the present study, we studied NOX4 expression in chondrosarcoma by immunochemical examination, and analyzed the role of NOX4 in viability and apoptosis of human chondrosarcoma cell line SW1353 using NOX4 siRNA or NOX4 inhibitor GKT137831. NOX4 level significantly increased in tumor compared to that in para-carcinoma sample. The levels of NOX4 were positively correlated with histological grade and Musculoskeletal Tumor Society stage of the patients. NOX4 level was significantly increased in SW1353 compared with that in chondrocytes CHON-001. Knockdown of NOX4 or inhibition of NOX4 by GKT137831 both decreased generation of ROS, and induced growth inhibition and apoptosis in SW1353, accompanied with the activation of caspases (caspase-3, caspase-8 and caspses-9), upregulation of Bax, cytochrome C(cyt-c), cleaved-PARP and down-regulation of Bcl-2. Moreover, NOX4 siRNA and GKT137831 decreased the expression of p-Akt, p-ERK and p-p65 in SW1353 cells. In an in vivo study, NOX4 shRNA transfected SW1353 have shown impaired growth ability compared to the SW1353 when they were injected into the nude mice. Meanwhile, GKT137831 induced growth inhibition and apoptosis in SW1353 xenograft animals, together with increased expression of Bax, cyt-c, cleaved-PARP, and decreased expression of Bcl-2, p-Akt, p-ERK and p-p65. NOX4 plays a positive role in the development of chondrosarcoma and could serve as a promising target against chondrosarcoma clinically.

Identification of NOX4 as a New Biomarker in Hepatocellular Carcinoma and Its Effect on Sorafenib Therapy.

To improve the survival of patients with hepatocellular carcinoma (HCC), new biomarkers and therapeutic targets are urgently needed. In this study, the GEO and TCGA dataset were used to explore the differential co-expressed genes and their prognostic correlation between HCC and normal samples. The mRNA levels of these genes were validated by qRT-PCR in 20 paired fresh HCC samples. The results demonstrated that the eight-gene model was effective in predicting the prognosis of HCC patients in the validation cohorts. Based on qRT-PCR results, NOX4 was selected to further explore biological functions within the model and 150 cases of paraffin-embedded HCC tissues were scored for NOX4 immunohistochemical staining. We found that the NOX4 expression was significantly upregulated in HCC and was associated with poor survival. In terms of function, the knockdown of NOX4 markedly inhibited the progression of HCC in vivo and in vitro. Mechanistic studies suggested that NOX4 promotes HCC progression through the activation of the epithelial-mesenchymal transition. In addition, the sensitivity of HCC cells to sorafenib treatment was obviously decreased after NOX4 overexpression. Taken together, this study reveals NOX4 as a potential therapeutic target for HCC and a biomarker for predicting the sorafenib treatment response.

Nodakenin Induces ROS-Dependent Apoptotic Cell Death and ER Stress in Radioresistant Breast Cancer

Angelica gigas exerts powerful anti-tumor and anti-cancer effects in various cancer cell types. However, there have been few studies regarding the anti-cancer effect of nodakenin, a bioactive compound of Angelica gigas, in vivo and in vitro on breast cancers. I found that nodakenin, in a concentration-dependent manner, inhibits breast cancer cell viability and decreases the tumor volume in mice. Additionally, nodakenin induces caspase-3-dependent apoptosis in breast cancer cells; however, the combination of Z-VAD-FMK and nodakenin suppresses the caspase-3-dependent apoptotic cell death. Furthermore, nodakenin mediates apoptotic cell death via the PERK-mediated signaling pathway and calcium (Ca2+) release, and nodakenin combined with thapsigargin induces synergistic cell death by inhibiting sarco/endoplasmic reticulum (ER) Ca2+-ATPase. However, knockdown of PERK or CHOP inhibits Ca2+ generation and caspase-dependent apoptosis in nodakenin-treated breast cancer cells. Nodakenin induces ROS and Ca2+ generation, ER stress, and apoptotic cell death; however, the knockdown of Nox4 inhibits ROS generation and ER stress- and caspase-dependent apoptotic cell death. In addition, nodakenin combined with radiation overcomes radioresistance in radioresistant breast cancer cells by suppressing epithelial-mesenchymal transition phenotypes, including the decrease in E-cadherin and the increase in N-cadherin and vimentin. Therefore, these findings indicate that nodakenin may be a novel therapeutic strategy for breast cancers.

6-Shogaol Overcomes Gefitinib Resistance via ER Stress in Ovarian Cancer Cells.

In women, ovary cancer is already the fifth leading cause of mortality worldwide. The use of cancer therapies, such as surgery, radiotherapy, and chemotherapy, may be a powerful anti-cancer therapeutic strategy; however, these therapies still have many problems, including resistance, toxicity, and side effects. Therefore, natural herbal medicine has the potential to be used for cancer therapy because of its low toxicity, fewer side effects, and high success. This study aimed to investigate the anti-cancer effect of 6-shogaol in ovarian cancer cells. 6-shogaol induces ER stress and cell death via the reduction in cell viability, the increase in LDH cytotoxicity, caspase-3 activity, and Ca2+ release, and the upregulation of GRP78, p-PERK, p-eIF2α, ATF-4, CHOP, and DR5. Moreover, 6-shogaol treatment medicates endoplasmic reticulum (ER) stress and cell death by upregulating Nox4 and releasing ROS. The knockdown of Nox4 in ovarian cancer cells inhibits ER stress and cell death by blocking the reduction in cell viability and the enhancement of LDH cytotoxicity, caspase-3 activity, Ca2+, and ROS release. In gefitinib-resistant ovarian cancer cells, A2780R and OVCAR-3R, 6-shogaol/gefitinib overcomes gefitinib resistance by inhibiting EMT phenomena such as the reduction in E-cadherin, and the increase in N-cadherin, vimentin, Slug, and Snail. Therefore, our results suggest that 6-shogaol exerts a potential anti-cancer effect in ovarian cancer and combination treatment with 6-shogaol and gefitinib may provide a novel anti-tumor therapeutic strategy in gefitinib-resistant ovarian cancer.

Elevated Angiotensin-II Levels Contribute to the Pathogenesis of Open-Angle Glaucoma Via Inducing the Expression of Fibrosis-Related Genes in Trabecular Meshwork Cells Through a ROS/NOX4/SMAD3 Axis

Glaucoma including primary open-angle glaucoma (POAG) results from elevations in intraocular pressure (IOP). An eye-localized renin-angiotensin system (RAS) has been implicated in IOP regulation, although its mechanism of action and contribution to glaucoma is poorly understood. Here, we detected significant increases in the levels of angiotensin II (ANGII) in aqueous humor samples from POAG patients. Moreover, we determined that the concentrations of ANGII were positively correlated with IOP, suggesting a role for elevated ANGII levels in eye pathogenesis. Functional investigations demonstrated that ANGII induces the expression of fibrosis-related genes of transformed and primary human trabecular meshwork cells (HTMCs) through the transcriptional upregulation of key fibrotic genes. Parallel experiments using a murine periocular conjunctival fornix injection model confirmed that ANGII induces the expression of fibrosis-related genes in trabecular meshwork (TM) cells in vivo along with increasing IOP. ANGII was revealed to function through increasing the levels of reactive oxygen species (ROS) via selectively upregulating NOX4, with NOX4 knockdown or inhibition with GLX351322 alleviating fibrotic changes induced by ANGII. We further show that ANGII activates Smad3, with both GLX351322 and an inhibitor of Smad3 (SIS3) decreasing the phosphorylation of Smad3 and dampening the ANGII-induced increases in fibrotic proteins. Moreover, NOX4 and Smad3 inhibitors also partially rescued the elevated IOP levels induced by ANGII. Our collective results therefore highlight ANGII as a biomarker and treatment target in POAG together with establishing a causal relationship between ANGII and up-regulation of the expression of fibrosis-related genes of TM cells via a NOX4/ROS axis in cooperation with TGFβ/Smad3 signaling.

NOX4-derived ROS Regulates Aerobic Glycolysis of Breast Cancer through YAP Pathway.

Background: NOX4 is highly expressed in breast cancer and is closely associated with cell invasion and metastasis. The involvement of NOX4 in glycolysis in breast cancer remains unclear. The aim of this study was to investigate the role and mechanism of NOX4 in glycolysis in breast cancer. Methods: NOX4 expression in breast cancer cells was detected by qRT-PCR and western blotting. siRNAs and plasmids were used to silence or enhance the expression of NOX4. The mRNA and protein expression of HK2, GLUT1, PKM2, LDHA, and YAP was detected by qRT-PCR and western blotting, and the 18F-FDG uptake rate was detected by γ-radiometer. Detection of reactive oxygen species (ROS) in cells was performed using a commercial ROS kit. After transfection, CCK8, EDU and Transwell experiments were conducted to detect cell proliferation and migration ability. MicroPET imaging was used to detect the effects of NOX4 on tumor metabolism. Immunohistochemistry was used to detect the expression of NOX4, glycolytic enzymes HK2, GLUT1, PKM2, LDHA, the proliferation index KI67, and the activation of YAP pathway molecule. Results: In this study, the expression of NOX4 in MDA-MB-231 and MDA-MB-453 was higher than in MCF10A. qRT-PCR and western blotting experiments showed that NOX4 downregulation decreased the expression of glycolytic enzymes HK2, GLUT1, PKM2, LDHA, and 18F-FDG uptake. Conversely, the overexpression of NOX4 enhanced the expression of HK2, GLUT1, PKM2, LDHA, and 18F-FDG uptake. Proliferation and migration experiments showed that after down-regulation of NOX4, cell proliferation and migration ability decreased, while NOX4 overexpression promoted cell proliferation and migration ability. Additionally, ROS content and YAP expression decreased after NOX4 down-regulation, while ROS content and YAP expression increased following NOX4 overexpression, which was reversed by N-acetyl cysteine (NAC), a ROS inhibitor. Furthermore, exposure to NAC and Peptide17, a YAP inhibitor, blocked the increase in glycolytic enzyme expression, and the enhancement of proliferation and migration caused by NOX4 overexpression. In addition, in animal experiments, the results of the MicroPET imaging showed that the glucose metabolism rate of the NOX4 inhibitor group was significantly lower than that of the control group. ROS levels in the NOX4 inhibitor group was lower than that in the control group. Immunohistochemistry showed that the expression of HK2, GLUT1, PKM2, LDHA, KI67, and YAP in the NOX4 knock-down group were decreased. Conclusions: NOX4 affects breast cancer glycolysis through ROS-induced activation of the YAP pathway, further promoting the proliferation and migration of breast cancer cells.

NADPH Oxidase Isoforms in COPD Patients and Acute Cigarette Smoke-Exposed Mice: Induction of Oxidative Stress and Lung Inflammation.

Cigarette smoke (CS) is a major risk factor for chronic obstructive pulmonary disease (COPD), which represents the third leading cause of death worldwide. CS induces reactive oxygen species (ROS) production, leading to pulmonary inflammation and remodeling. NADPH oxidases (NOXs) represent essential sources of ROS production in the cardiovascular system. Whether and how NOX isoforms are activated in COPD patients and in response to acute cigarette smoke (ACS) remains incompletely understood. In the present study, the expression of NOX isoforms was examined in the lungs of end-stage COPD patients. In addition, mice silenced of NOX1 or NOX4 expression using in vivo RNA interference (RNAi), and NOX2-deficient (NOX2−/y) mice, were exposed to ACS for 1 h using a standard TE-10B smoking machine. In lung sections isolated from COPD patients undergoing lung transplantation, protein expression of NOX1, NOX2, NOX4, or NOX5 was markedly upregulated compared to non-smoking donor controls. Likewise, ACS upregulated protein expression of NOX1, NOX2, and NOX4, production of ROS, inflammatory cell infiltration, and mRNA expression of proinflammatory cytokines TNF-α and KC in the mouse lung. In vivo RNAi knockdown of NOX1 or NOX4 decreased ACS induced ROS production, inflammatory cell influx, and the expression of TNF-α and KC, which were accompanied by inhibition of the NF-κB-COX-2 axis. Although ACS induced ROS production was reduced in the lungs of NOX2−/y mice, inflammatory cell influx and expression of NF-κB/COX-2 were increased. Taken together, our results demonstrate for the first time that NOX isoforms 1, 2, 4 and 5 all remain activated in end-stage COPD patients, while NOX1 and NOX4 mediate oxidative stress and inflammatory responses in response to acute cigarette smoke. Therefore, targeting different isoforms of NOX might be necessary to treat COPD at different stages of the disease, which represents novel mechanistic insights enabling improved management of the devastating disease.

A novobiocin derivative, XN4, triggers ferroptosis in gastric cancer cells via the activation of NOX4

Context A novobiocin derivative, XN4, has been shown to promote cell apoptosis in chronic myeloid leukaemia. Objective This study explores the mechanism by which XN4 promotes ferroptosis of gastric cancer (GC) cells. Materials and methods Human GC SGC-7901 and BGC-823 cells were treated with different XN4 concentrations (0, 0.1, 0.5, 1.0, 5.0, and 10.0 μmol/L) to evaluate effects of XN4. Additionally, cells were pre-treated for 24 h with si-NOX4, for 1 h with the iron chelator deferoxamine mesylate (DFO) or for 1 h with the lipid peroxidation inhibitor liproxstatin-1 before being treated with XN4 to analyse the mechanism of XN4. Results XN4 increased cell death (IC50 values of XN4 on SGC-7901 and BGC-823 cells: 1.592 ± 0.14 μmol/L and 2.022 ± 0.19 μmol/L) and Fe2+ levels in SGC-7901 and BGC-823 cells. These effects of 2.0 μmol/L XN4 were abolished by 100 μmol/L DFO treatment. XN4 enhanced transferrin and transferrin receptor expression to induce Fe2+ accumulation. XN4 decreased mitochondrial membrane potentials in GC cells, similar to erastin. Additionally, XN4 increased MDA, hydrogen peroxide, and ROS levels, but diminished total glutathione levels. Liproxstatin-1 (200 nmol/L) nullified the effects of XN4 (2.0 μmol/L) on MDA levels and cell death. Moreover, GPX4 levels decreased, but NOX4 and ferroptosis-related protein PTGS2 levels increased in GC cells following XN4 treatment, which was nullified by NOX4 knockdown. Discussion and conclusions The pro-ferroptotic role of XN4 in GC might enable it to become a promising drug for GC treatment in the future despite the need for extensive research.

NOX4 promotes Kupffer cell inflammatory response via ROS-NLRP3 to aggravate liver inflammatory injury in acute liver injury.

Aim: This work aimed to investigate the mechanism of NOX4 in promoting Kupffer cells (KCs) activation and tissue inflammatory response in acute liver injury.Methods: Initially, the mouse KCs were cultured in vitro. Thereafter, the NOX4 overexpression plasmid was transfected into KCs to construct the overexpression cell line. Then, KCs inflammatory response was induced by LPS + Nigericin treatment. CCK-8 assay was performed to detect cell viability, flow cytometry (FCM) was conducted to measure cell apoptosis, enzyme-linked immunosorbent assay (ELISA) was performed to detect inflammatory factor levels in the culture medium, NLRP3 and ASC expression in cells was detected by immunofluorescence (IF) staining, and ROS expression was detected by the DCFH-DA probe. Furthermore, the expression levels of NLRP3, ASC and Caspase-1 proteins were detected by Western-Blot (WB) assay. Furthermore, cells were pre-treated with NOX inhibitor or NAC to suppress NOX4 expression or ROS production, aiming to further investigate the effect on KCs inflammatory response. In mouse experiments, the NOX4 knockdown mice and wild-type (WT) mice were adopted for carrying out experiments. The mouse model of ALI was constructed with LPS and D-GalN treatment. Thereafter, the changes in tissue samples were detected by H&E staining, NLRP3 expression was measured by histochemical staining, inflammatory factors in tissues were analyzed by ELISA, and the levels of NLRP3, ASC and Caspase-1 proteins in tissues were detected by WB assay.Results: LPS induced KCs inflammatory response. NOX4 overexpression decreased the mouse viability and increased the apoptosis rate. The levels of inflammatory factors were up-regulated in the culture medium. In addition, ROS were activated, and the positive cell number increased. Moreover, NOX4 promoted NLRP3 activation and significantly increased the expression of NLRP3 and ASC. Pretreatment with NOX4 inhibitor or NAC antagonized the effects of NOX4 and suppressed the KCs inflammatory response. In the mouse model, NOX4 knockdown significantly suppressed the activation and inflammatory response of microglial cells in tissues, reducing the NLRP3 expression in tissues.Conclusion: NOX4 activates the NLRP3 inflammasome via ROS to promote inflammatory response in KCs and the release of inflammatory factors, suppressing NOX4 can improve ALI in mice, and NOX4 is promising as a new target for ALI treatment.

Induction of reactive oxygen species by mechanical stretch drives endothelin production in neonatal pig renal epithelial cells

Vasoactive endothelin (ET) is generated by ET converting enzyme (ECE)-induced proteolytic processing of pro-molecule big ET to biologically active peptides. H2O2 has been shown to increase the expression of ECE1 via transactivation of its promoter. The present study demonstrates that H2O2 triggered ECE1-dependent ET1-3 production in neonatal pig proximal tubule (PT) epithelial cells. A uniaxial stretch of PT cells decreased catalase, increased NADPH oxidase (NOX)2 and NOX4, and increased H2O2 levels. Stretch also increased cellular ECE1, an effect reversed by EUK-134 (a synthetic superoxide dismutase/catalase mimetic), NOX inhibitor apocynin, and siRNA-mediated knockdown of NOX2 and NOX4. Short-term unilateral ureteral obstruction (UUO), an inducer of renal tubular cell stretch and oxidative stress, increased renal ET1-3 generation and vascular resistance (RVR) in neonatal pigs. Despite removing the obstruction, UUO-induced increase in RVR persisted, resulting in early acute kidney injury (AKI). ET receptor (ETR)-operated Ca2+ entry in renal microvascular smooth muscle (SM) via transient receptor potential channel 3 (TRPC3) channels reduced renal blood flow and increased RVR. Although acute reversible UUO (rUUO) did not change protein expression levels of ETR and TRPC3 in renal microvessels, inhibition of ECE1, ETR, and TRPC3 protected against renal hypoperfusion, RVR increase, and early AKI. These data suggest that mechanical stretch-driven oxyradical generation stimulates ET production in neonatal pig renal epithelial cells. ET activates renal microvascular SM TRPC3, leading to persistent vasoconstriction and reduction in renal blood flow. These mechanisms may underlie rUUO-induced renal insufficiency in infants.

LncRNA MALAT1 Aggravates Renal Tubular Injury via Activating LIN28A and the Nox4/AMPK/mTOR Signaling Axis in Diabetic Nephropathy.

BackgroundDiabetic nephropathy (DN) is a serious complication among patients with diabetes. Elucidating its pathogenesis is crucial for identifying novel biomarkers and therapeutic targets for DN.MethodsDN tissues were harvested for examining MALAT1, LIN28A and Nox4. Human kidney-2 (HK-2) cells were treated with high glucose (HG) for establishing a cell model of DN. Cell viability was examined by MTT assay. HG-induced cell apoptosis and secretion of TNF-α and IL-6 were analyzed by TUNEL and ELISA assays, respectively. RIP and RNA pull-down assays were applied to analyze the interaction between MALAT1, LIN28A and Nox4 in HK-2 and human embryonic kidney 293T (HEK-293T) cells. A rat model of DN was established to determine the role of MALAT1 in DN in vivo.ResultsMALAT1, LIN28A and Nox4 were upregulated in DN tissues and HG-treated HK-2 cells. Overexpression of MALAT1, LIN28A or Nox4 reduced cell viability and enhanced cell apoptosis, ROS generation and secretion of inflammatory cytokines in HG-treated HK-2 cells, whereas knockdown of MALAT1, LIN28A or Nox4 exerted opposite effects. Furthermore, MALAT1 directly interacted with LIN28A. Moreover, MALAT1 facilitated the interaction between LIN28A and Nox4 to increase Nox4 stability. Knockdown of Nox4 relieved HG-induced injury by suppressing the AMPK/mTOR signaling in HK-2 cells. Knockdown of MALAT1 alleviated renal tubular epithelial injury by suppressing LIN28A and the Nox4/AMPK/TOR signaling in DN.ConclusionMALAT1 activates the AMPK/mTOR signaling via interacting with LIN28A to stabilize Nox4 mRNA, thereby aggravating high glucose-induced renal tubular epithelial injury. Our findings provide potential therapeutic targets for DN.

Empagliflozin inhibits intermittent hypoxia‐induced TRAF3IP2/NF‐κB/HIF‐1α/IL‐6‐dependent human aortic smooth muscle cell proliferation

Chronic intermittent hypoxia (IH), a characteristic feature of obstructive sleep apnea and chronic obstructive pulmonary disease, contributes to cardiovascular diseases, including atherosclerosis, through persistent oxidative stress and inflammation. During atherogenesis, vascular smooth muscle cells acquire a proinflammatory and a proliferative phenotype. TRAF3IP2 (TRAF3 Interacting Protein 2) is an oxidative stress‐responsive proinflammatory adapter molecule and plays a causal role in a preclinical model of atherosclerosis. Therefore, we tested the hypothesis that IH stimulates human aortic smooth muscle cell (SMC) proliferation via TRAF3IP2, and this effect is inhibited by the SGLT2 (Sodium/Glucose Cotransporter 2) inhibitor empagliflozin. Results show that IH upregulates TRAF3IP2 expression, TRAF3IP2‐dependent superoxide, hydrogen peroxide and nitric oxide production, HIF‐1α and NF‐κB activation, IL‐6 expression, and SMC proliferation. These effects were inhibited by TRAF3IP2, NOX2, NOX4, p65 or IL6R knockdown, or pre‐exposure to potent and specific iNOS inhibitor AMT or 1400W, the JAK inhibitor Tofacitinib, the STAT3‐specific inhibitor HO‐3867, or the gp130 inhibitor SC144. Importantly, SMC express SGLT2, and pretreatment with empagliflozin abrogated IH‐induced TRAF3IP2 induction, nitroxidative stress, HIF‐1α and NF‐κB activation, IL‐6 expression, and SMC proliferation. In summary, these results suggest the therapeutic potential of empagliflozin in IH‐associated inflammatory vascular proliferative diseases.

Knockdown of NADPH oxidase 4 reduces mitochondrial oxidative stress and neuronal pyroptosis following intracerebral hemorrhage.

Intracerebral hemorrhage is often accompanied by oxidative stress induced by reactive oxygen species, which causes abnormal mitochondrial function and secondary reactive oxygen species generation. This creates a vicious cycle leading to reactive oxygen species accumulation, resulting in progression of the pathological process. Therefore, breaking the cycle to inhibit reactive oxygen species accumulation is critical for reducing neuronal death after intracerebral hemorrhage. Our previous study found that increased expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NADPH oxidase 4, NOX4) led to neuronal apoptosis and damage to the blood-brain barrier after intracerebral hemorrhage. The purpose of this study was to investigate the role of NOX4 in the circle involving the neuronal tolerance to oxidative stress, mitochondrial reactive oxygen species and modes of neuronal death other than apoptosis after intracerebral hemorrhage. We found that NOX4 knockdown by adeno-associated virus (AAV-NOX4) in rats enhanced neuronal tolerance to oxidative stress, enabling them to better resist the oxidative stress caused by intracerebral hemorrhage. Knockdown of NOX4 also reduced the production of reactive oxygen species in the mitochondria, relieved mitochondrial damage, prevented secondary reactive oxygen species accumulation, reduced neuronal pyroptosis and contributed to relieving secondary brain injury after intracerebral hemorrhage in rats. Finally, we used a mitochondria-targeted superoxide dismutase mimetic to explore the relationship between reactive oxygen species and NOX4. The mitochondria-targeted superoxide dismutase mimetic inhibited the expression of NOX4 and neuronal pyroptosis, which is similar to the effect of AAV-NOX4. This indicates that NOX4 is likely to be an important target for inhibiting mitochondrial reactive oxygen species production, and NOX4 inhibitors can be used to alleviate oxidative stress response induced by intracerebral hemorrhage.

Glyphosate damages blood-testis barrier via NOX1-triggered oxidative stress in rats: Long-term exposure as a potential risk for male reproductive health.

Blood-testis barrier (BTB) creates a privileged niche indispensable for spermatogenesis. Glyphosate (GLY), the most commonly used herbicide worldwide, has been reported to decrease sperm quality. However, whether and how GLY destroys the BTB to affect sperm quality remains to be elucidated. Herein, this study was designed to investigate the influence of GLY on the BTB in vivo and in vitro experiments. The results showed that male rats exposed to GLY for 4months exhibited a decrease in sperm quality and quantity, accompanied by BTB integrity disruption and testicular oxidative stress. Additionally, GLY-induced reactive oxygen species (ROS) contributed to the downregulation of BTB-related proteins in primary Sertoli cells (SCs). Intriguingly, we identified a marked upregulation of oxidative stress-related gene NOX1 in GLY-exposed testis based on transcriptome analysis. NOX1 knockdown blocked the GLY-induced oxidative stress, as well as prevented BTB-related protein decrease in SCs. Furthermore, the estrogen receptor (ER)-α was significantly upregulated in vivo and in vitro models. An ER-α inhibitor decreased the expression levels of both ER-α and NOX1. Mechanistically, GLY directly interacted with ER-α at the site of Pro39 and Lys401 to promote ER-α activation, which boosted NOX1 expression to trigger ROS accumulation. Collectively, these results demonstrate that long-term GLY exposure adversely affects BTB integrity, which disrupts spermatogenesis via activation of ER-α/NOX1 axis. This study presents a better understanding of the risk of long-term GLY exposure to male fertility.

Neuronal NOX4 knockdown alleviates pathological tau-related alterations in a humanized mouse model of tauopathy

Neuronal NOX4 knockdown alleviates pathological tau-related alterations in a humanized mouse model of tauopathy

Nox4 Promotes RANKL-Induced Autophagy and Osteoclastogenesis via Activating ROS/PERK/eIF-2α/ATF4 Pathway.

Receptor activator of nuclear factor-κB ligand (RANKL) has been found to induce osteoclastogenesis and bone resorption. However, the underlying molecular mechanisms remain unclear. Via conducting a series of biochemical experiments with in vitro cell lines, this study investigated the role and mechanism of NADPH oxidase 4 (Nox4) in RANKL-induced autophagy and osteoclastogenesis. In the current study, we found that RANKL dramatically induced autophagy and osteoclastogenesis, inhibition of autophagy with chloroquine (CQ) markedly attenuates RANKL-induced osteoclastogenesis. Interestingly, we found that the protein level of Nox4 was remarkably upregulated by RANKL treatment. Inhibition of Nox4 by 5-O-methyl quercetin or knockdown of Nox4 with specific shRNA markedly attenuated RANKL-induced autophagy and osteoclastogenesis. Furthermore, we found that Nox4 stimulated the production of nonmitochondrial reactive oxygen species (ROS), activating the critical unfolded protein response (UPR)-related signaling pathway PERK/eIF-2α/ATF4, leading to RANKL-induced autophagy and osteoclastogenesis. Blocking the activation of PERK/eIF-2α/ATF4 signaling pathway either by Nox4 shRNA, ROS scavenger (NAC) or PERK inhibitor (GSK2606414) significantly inhibited autophagy during RANKL-induced osteoclastogenesis. Collectively, this study reveals that Nox4 promotes RANKL-induced autophagy and osteoclastogenesis via activating ROS/PERK/eIF-2α/ATF4 pathway, suggesting that the pathway may be a novel potential therapeutic target for osteoclastogenesis-related disease.

Nox2 impairs VEGF-A-induced angiogenesis in placenta via mitochondrial ROS-STAT3 pathway

Aberrant placental angiogenesis is associated with fetal intrauterine growth restriction (IUGR), but the mechanism underlying abnormal placental angiogenesis remains largely unknown. Here, lower vessel density and higher expression of NADPH oxidases 2 (Nox2) were observed in the placentae for low birth weight (LBW) fetuses versus normal birth weight (NBW) fetuses, with a negative correlation between Nox2 and placental vessel density. Moreover, it was revealed for the first time that Nox2 deficiency facilitates angiogenesis in vitro and in vivo, and vascular endothelial growth factor-A (VEGF-A) has an essential role in Nox2-controlled inhibition of angiogenesis in porcine vascular endothelial cells (PVECs). Mechanistically, Nox2 inhibited phospho-signal transducer and activator of transcription 3 (p-STAT3) in the nucleus by inducing the production of mitochondrial reactive oxygen species (ROS). Dual-luciferase assay confirmed that knockdown of Nox2 reduces the expression of VEGF-A in an STAT3 dependent manner. Our results indicate that Nox2 is a potential target for therapy by increasing VEGF-A expression to promote angiogenesis and serves as a prognostic indicator for fetus with IUGR.

High glucose induces Nox4 expression and podocyte apoptosis through the Smad3/ezrin/PKA pathway.

ABSTRACTPodocytes are the major target in proteinuric kidney diseases such as diabetic nephropathy. The underlying molecular mechanisms by which high glucose (HG) results in podocyte damage remain unclear. This study investigated the regulatory role of Smad3, ezrin, and protein kinase A (PKA) in NADPH oxidase (Nox4) expression, reactive oxidative species (ROS) production, and apoptosis in HG-treated podocytes. A human podocyte cell line was cultured and differentiated, then treated with 30 mM HG. Apoptosis and intracellular ROS levels were assessed using TUNEL and DCF assays, respectively. Expressions of Nox4, phospho-Smad3Ser423/425, phospho-PKAThr197, and phospho-ezrinThr567 were evaluated using western blotting. ELISA was used to quantify intracellular cAMP concentration and PKA activity. Knockdown assay was used to inhibit the expressions of Smad3, Nox4, and ezrin by lentiviral shRNA. In HG-treated podocytes, the level of phospho-Smad3Ser423/425 and phospho-ezrinThr567 was increased significantly, which was accompanied by the reduction of cAMP and phospho-PKAThr197. HG-induced apoptosis was significantly prevented by the Smad3-inhibitor SIS3 or shRNA-Smad3. In podocytes expressing shRNA-ezrin or shRNA-Nox4, apoptosis was remarkably mitigated following HG treatment. HG-induced upregulation of phospho-ezrinThr567 and downregulation of phospho-PKAThr197 was significantly prevented by SIS3, shRNA-ezrin or shRNA-Smad3. Forskolin, a PKA activator, significantly inhibited HG-mediated upregulation of Nox4 expression, ROS generation, and apoptosis. Additionally, an increase in the ROS level was prohibited in HG-treated podocytes with the knockdown of Nox4, Smad3, or ezrin. Taken together, our findings provided evidence that Smad3-mediated ezrin activation upregulates Nox4 expression and ROS production, by suppressing PKA activity, which may at least in part contribute to HG-induced podocyte apoptosis.