Kaempferitrin attenuates unilateral ureteral obstruction-induced renal inflammation and fibrosis in mice by inhibiting NOX4-mediated tubular ferroptosis.

Tubular ferroptosis significantly contributes to renal inflammation and fibrosis, critical factors in chronic kidney disease (CKD). This study aims to investigate Kaempferitrin, a potent flavonoid glycoside from Bauhinia forficata leaves, renowned for its anti-inflammatory and antitumor effects, and to elucidate its potential mechanisms in mitigating inflammation and fibrosis induced by tubular ferroptosis. The study investigated Kaempferitrin's impact on tubular ferroptosis using a unilateral ureteral obstruction (UUO) model-induced renal inflammation and fibrosis. In vitro, erastin-induced ferroptosis in primary tubular epithelial cells (TECs) was utilized to further explore Kaempferitrin's effects. Additionally, NADPH oxidase 4 (NOX4) transfection in TECs and cellular thermal shift assay (CETSA) were conducted to identify Kaempferitrin's target protein. Kaempferitrin effectively improved renal function, indicated by reduced serum creatinine and blood urea nitrogen levels. In the UUO model, it significantly reduced tubular necrosis, inflammation, and fibrosis. Its renoprotective effects were linked to ferroptosis inhibition, evidenced by decreased iron, 4-hydroxynonenal (4-HNE), and malondialdehyde (MDA) levels, and increased glutathione (GSH). Kaempferitrin also normalized glutathione peroxidase 4 (GPX4) and Solute Carrier Family 7 Member 11(SLC7A11) expression, critical ferroptosis mediators. In vitro, it protected TECs from ferroptosis and consistently suppressed NOX4 expression. NOX4 transfection negated Kaempferitrin's antiferroptosis effects, while CETSA confirmed Kaempferitrin-NOX4 interaction. Kaempferitrin shows promise as a nephroprotective agent by inhibiting NOX4-mediated ferroptosis in tubular cells, offering potential therapeutic value for CKD.

Neutrophil membrane-engineered Panax ginseng root-derived exosomes loaded miRNA 182-5p targets NOX4/Drp-1/NLRP3 signal pathway to alleviate acute lung injury in sepsis: experimental studies.

BACKGROUND: The purpose of this study was to prepare neutrophil membrane-engineered Panax ginseng root-derived exosomes (N-exo) and investigate the effects of N-exo microRNA (miRNA) 182-5p (N-exo-miRNA 182-5p) on acute lung injury (ALI) in sepsis. METHODS: Panax ginseng root-derived exosomes were separated by differential centrifugation. Neutrophil membrane engineering was performed on exo to obtain N-exo. miRNA182-5p was transmitted into N-exo by electroporation technology to obtain N-exo-miRNA 182-5p. LPS was used to establish an in-vivo and in-vitro model of ALI of sepsis to evaluate the anti-inflammatory effect of N-exo-miRNA 182-5p. RESULTS: The results of transmission electron microscope showed that exo was a double-layer membrane structure like a saucer. Nanoparticle size analysis showed that the average particle size of exo was 129.7 nm. Further, compared with exo, the level of miRNA182-5p was significantly increased in N-exo. The experimental results showed that N-exo-miRNA 182-5p significantly improved ALI via target regulation of NOX4/Drp-1/NLRP3 signal pathway in vivo and in vitro . CONCLUSION: In conclusion, this study prepared a novel engineered exosome (N-exo and N-exo-miRNA 182-5p significantly improved ALI in sepsis via target regulation of NOX4/Drp-1/NLRP3 signal pathway, providing new ideas and methods for treatment of ALI in sepsis.

Natural Bioactive Compounds Targeting NADPH Oxidase Pathway in Cardiovascular Diseases.

Cardiovascular disease (CVD) is the leading cause of death worldwide, in both developed and developing countries. According to the WHO report, the morbidity and mortality caused by CVD will continue to rise with the estimation of death going up to 22.2 million in 2030. NADPH oxidase (NOX)-derived reactive oxygen species (ROS) production induces endothelial nitric oxide synthase (eNOS) uncoupling and mitochondrial dysfunction, resulting in sustained oxidative stress and the development of cardiovascular diseases. Seven distinct members of the family have been identified of which four (namely, NOX1, 2, 4 and 5) may have cardiovascular functions. Currently, the treatment and management plan for patients with CVDs mainly depends on the drugs. However, prolonged use of prescribed drugs may cause adverse drug reactions. Therefore, it is crucial to find alternative treatment options with lesser adverse effects. Natural products have been gaining interest as complementary therapy for CVDs over the past decade due to their wide range of medicinal properties, including antioxidants. These might be due to their potent active ingredients, such as flavonoid and phenolic compounds. Numerous natural compounds have been demonstrated to have advantageous effects on cardiovascular disease via NADPH cascade. This review highlights the potential of natural products targeting NOX-derived ROS generation in treating CVDs. Emphasis is put on the activation of the oxidases, including upstream or downstream signalling events.

[Effect of electroacupuncture combined with Zhuang-medicine-thread moxibustion on oxidative stress in gastric antrum of diabetic gastroparesis rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) combined with Zhuang-medicine-thread moxibustion on oxidative stress-related indicators in diabetic gastroparesis (DGP) rats, so as to explore its mechanism underlying improvement of DGP. METHODS: Male SD rats were randomly divided into normal, model, medication, EA, Zhuang-medicine-thread moxibustion (moxibustion) and EA+Zhuang-medicine-thread moxibustion (combination) groups (15 rats in each group). The DGP model was established by intraperitoneal injection of streptozotocin (STZ). Rats of the medication group were treated by gavage of 0.15 mg/mL mosapride citrate suspension（10 mL/kg）. EA (10 Hz/50 Hz, 2 mA, 20 min) or Zhuang-medicine-thread moxibustion (3 cones) was applied to "Zhongwan" (CV12), bilateral "Neiguan" (PC6) and bilateral "Sanyinjiao" (SP6) of the related groups, once a day for 3 weeks. The body weight, blood glucose, gastric emptying rate and intestinal propulsion rate of rats were measured. The serum malondialdehyde (MDA) content was measured by thiobarbituric acid method, the serum supero-xide dismutase (SOD) activity was measured by xanthine oxidase method, and the serum reactive oxygen species (ROS) activity was detected by ELISA. HE staining was used to observe the pathological changes of gastric antrum. The expression of heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), nicotin-amide adenine dinucleotide phosphate oxidases (NOX4), peroxisome proliferators activated receptor-gamma coactivator 1α(PGC-1α) proteins and mRNAs in gastric antrum was detected by Western blot and real-time quantitative PCR, respectively. RESULTS: Compared with the normal group, the body weight, gastric emptying rate, intestinal propulsion rate, serum SOD activity, the expressions of HO-1, PGC-1α, total Nrf2 proteins and mRNAs, and Nrf2 nuclear translocation in gastric antrum were decreased (P<0.01), while the blood glucose, serum MDA content and ROS activity, NOX4 protein and mRNA expressions in gastric antrum were increased (P<0.01) in the model group. Compared with the model group, the blood glucose was decreased in the EA, moxibustion and combination groups (P<0.01); the body weight, gastric emptying rate, intestinal propulsion rate, and the expressions of HO-1 and PGC-1α mRNAs in gastric antrum were all increased in the four treatment groups (P<0.01, P<0.05), while the serum MDA content and ROS activity, NOX4 protein and mRNA expressions in gastric antrum were all decreased (P<0.01); the serum SOD activity and total Nrf2 protein expression in gastric antrum were increased in medication, moxibustion and combination groups (P<0.01, P<0.05); the expressions of HO-1 and PGC-1α proteins, and Nrf2 nuclear translocation in gastric antrum were increased in medication and combination groups (P<0.05, P<0.01); the expression of Nrf2 mRNA was increased in the medication, EA and combination groups (P<0.01, P<0.05). Compared with the combination group, the body weight, gastric emptying rate and intestine propulsion rate were decreased in the medication, EA and moxibustion groups(P<0.01, P<0.05), and the blood glucose increased (P<0.01); the serum MDA content and ROS activity, NOX4 protein and mRNA expressions in gastric antrum were increased (P<0.01, P<0.05), serum SOD activity, and the expressions of total Nrf2 protein, PGC-1α protein and mRNA, HO-1 mRNA in gastric antrum were decreased (P<0.01, P<0.05) in the EA and moxibustion groups; the expression of Nrf2 mRNA was decreased in the moxibustion group (P<0.05). HE staining showed a large number of inflammatory cell infiltration in the lamina propria and submucosa, and the gastric glands in the lamina propria were significantly expanded, the submucosa was severely edematous in the model group, which were relative milder in the four treatment groups. CONCLUSION: EA combined with Zhuang-medicine-thread moxibustion can effectively improve the activity of antioxidant enzymes, reduce the production of lipid peroxide, and regulate the expression of antioxidant related proteins and genes, which may be one of the mechanisms in treating DGP.

Complanatoside A targeting NOX4 blocks renal fibrosis in diabetic mice by suppressing NLRP3 inflammasome activation and autophagy.

BACKGROUND: Diabetic nephropathy (DN) is an important cause of end-stage renal disease. Complanatoside A (CA), an active component from Semen Astragali Complanati, has been reported to be a potential candidate for the treatment of kidney diseases. However, the underlying mechanisms and protective effects of CA in DN remain unclear. PURPOSE: In this paper, the effects and the mechanism of CA against ameliorating DN were investigated in vivo and in vitro. STUDY DESIGN: Here, a high-fat diet/streptozotocin-induced diabetic model and TGF-ß1-induced HK-2 cells were used to explore the protective effects and mechanisms of CA on DN in vivo and in vitro. METHODS: Major biochemical indexes, Histopathological morphology, and Immunohistochemistry have explored the therapeutic effect of CA on DN. Subsequently, TGF-ß1-induced HK-2 cells were utilized to investigate the anti-renal fibrosis effect of CA. Finally, the mechanism of CA against renal fibrosis was studied via western blotting, immunofluorescence, transfection, and molecular docking. RESULTS: The results showed that CA attenuated glomerular hypertrophy, collagen matrix deposition, and tubular interstitial fibrosis in diabetic mice. Moreover, the activation of TGF-ß1-inducible epithelial-mesenchymal transition (EMT) was hindered by CA treatment in HK-2 cells. Mechanistically, the data suggested that upregulated NOX4 during diabetes and TGF-ß1 in HK-2 cells was prominently diminished after CA treatment. Furthermore, CA exposure inhibited NLRP3 inflammasome activation and downstream inflammation gene expression such as IL-18 and IL-1ß in vivo and vitro. These findings indicated that CA obstructed the EMT to protect renal tubular epithelial cells against fibrosis via blocking NLRP3 activation, which was associated with inhibiting NOX4. Besides, the markedly raised autophagy levels in the diabetic model characterized by increasing LC3II/LC3I and Beclin1 were reversed after CA treatment, which is also a pivotal mechanism against renal fibrosis. More importantly, specific NOX4 overexpressed in HK-2 cells abolished that CA exposure blocked TGF-ß1-induced-EMT, ROS generation, NLRP3, and autophagy activation. Meanwhile, the inhibition of cell migration, ROS generation, autophagy, and renal inflammation after CA treatment was more pronounced in NOX4-deficient HK-2 cells. CONCLUSION: Our findings provided evidence that CA might be a potential therapeutic agent for DN by ameliorating NLRP3 inflammasome and autophagy activation via targeting NOX4 inhibition.

NADPH and xanthine oxidases control induction of inflammatory mediator expression by organic dust in the lung.

Exposure to organic dust in animal and agricultural farms and the ensuing lung inflammation are linked to the development of respiratory diseases. We found previously that elevated production of reactive oxygen species (ROS) by aqueous poultry organic dust extract (hereafter referred to as dust extract) mediates induction of proinflammatory mediators in airway epithelial cells. In the present study, we investigated whether ROS generated by NADPH oxidases (NOX) and xanthine oxidase (XO) controls induction of inflammatory mediators by dust extract and the underlying mechanisms in bronchial epithelial cells. Using chemical inhibitors and siRNA targeted knockdown, we found that NOX1, NOX2, NOX4, and XO-derived ROS regulates induction of proinflammatory mediator levels. Like airway epithelial cells in vitro, NOX inhibitor VAS2870 reduced keratinocyte chemoattractant (KC), IL-6, and TNF-α production and 4-hydroxynonenal (4-HNE) staining induced by dust extract in mouse lungs. VAS2870 inhibition of proinflammatory mediators was associated with reduced NFκB and Stat3 activation indicating that NOX generated ROS activates NFκB and Stat3 to induce proinflammatory gene expression. Dust extract increased the membrane association of p47phox in airway epithelial cells indicating NOX2 activation but had no effect on NOX2 protein levels. In summary, our studies have shown that NOX and XO generated ROS control organic dust induction of proinflammatory mediators in airway epithelial cells via NFκB and Stat3 activation.

GABA and Fermented Curcuma longa L. Extract Enriched with GABA Ameliorate Obesity through Nox4-IRE1α Sulfonation-RIDD-SIRT1 Decay Axis in High-Fat Diet-Induced Obese Mice.

Gamma-aminobutyric acid (GABA) is a natural amino acid with antioxidant activity and is often considered to have therapeutic potential against obesity. Obesity has long been linked to ROS and ER stress, but the effect of GABA on the ROS-associated ER stress axis has not been thoroughly explored. Thus, in this study, the effect of GABA and fermented Curcuma longa L. extract enriched with GABA (FCLL-GABA) on the ROS-related ER stress axis and inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) sulfonation were examined with the HFD model to determine the underlying anti-obesity mechanism. Here, GABA and FCLL-GABA supplementations significantly inhibited the weight gain in HFD fed mice. The GABA and FCLL-GABA supplementation lowered the expressions of adipogenic transcription factors such as PPAR-γ, C/EBPα, FAS, and SREBP-1c in white adipose tissue (WAT) and liver from HFD-fed mice. The enhanced hyper-nutrient dysmetabolism-based NADPH oxidase (Nox) 4 and the resultant IRE1α sulfonation-RIDD-SIRT1 decay under HFD conditions were controlled with GABA and FCLL-GABA. Notably, GABA and FCLL-GABA administration significantly increased AMPK and sirtuin 1 (SIRT1) levels in WAT of HFD-fed mice. These significant observations indicate that ER-localized Nox4-induced IRE1α sulfonation results in the decay of SIRT1 as a novel mechanism behind the positive implications of GABA on obesity. Moreover, the investigation lays a firm foundation for the development of FCLL-GABA as a functional ingredient.

Shikonin attenuates kidney tubular epithelial cells apoptosis, oxidative stress, and inflammatory response through nicotinamide adenine dinucleotide phosphate oxidase 4/PTEN pathway in acute kidney injury of sepsis model.

Natural compounds were used in the treatment of acute kidney injury (AKI) caused by sepsis. This study investigated the function of shikonin from the roots of Arnebia purpurea in sepsis-induced AKI model. The target genes of shikonin were predicted by traditional Chinese medicine integrative database (TCMID). The markers of kidney injury, oxidative stress, and inflammatory factors were measured by enzyme-linked immunosorbent assay (ELISA). The pathological changes of kidney tubules were assessed by Hematoxylin and Eosin staining. Apoptosis of kidney tubular epithelial cells (KTECs) was detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Protein expression was measured by western blot. Shikonin significantly improved kidney injury induced by cecal ligation and perforation (CLP). Besides, shikonin reduced KTECs apoptosis, malondialdehyde (MDA), reactive oxygen species (ROS), interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) levels, while augmented SOD and IL-10 levels. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase4 (NOX4) was predicted a target gene of shikonin. The expression of NOX4 was significantly inhibited in shikonin-treated group and the levels of phosphatidylinositol 3,4,5-trisphosphate 3-phosphate and dual specificity protein phosphate (PTEN) and p-p65 were decreased, while level of p-Akt was elevated. In vitro experiments, shikonin inhibited cell apoptosis, inflammatory, and ROS in human HK-2 cells and rat TECs. Shikonin downregulated expression of NOX4, PTEN and p-p65, and upregulated p-AKT and Bcl-2 expression in HK2 cells treated with lipopolysaccharide (LPS). Moreover, overexpression of NOX4 enhanced the effect of LPS on the expression level of PTEN, p-p65, p-AKT, and Bcl-2, which was reversed by the addition of shikonin. Taken together, shikonin could improve sepsis-induced AKI in rats, and attenuate the LPS induced KTECs apoptosis, oxidative stress, and inflammatory reaction via modulating NOX4/PTEN/AKT pathway.

Hyperoside exerts osteoprotective effect on dexamethasone-induced osteoblasts by targeting NADPH Oxidase 4 (NOX4) to inhibit the reactive oxygen species (ROS) accumulation and activate c-Jun N-terminal kinase (JNK) pathway.

Hyperoside (Hyp) is a flavonoid active compound deriving from Chinese herbal medicines. Increasing studies have implicated that Hyp may serve as a predominant promoting factor in osteoblast differentiation. This paper investigates whether Hyp could relieve glucocorticoid-induced osteonecrosis of the femoral head (GONFH) via promoting osteoblast survival and differentiation as well as to uncover its potential mechanism. GONFH cell model was induced by treating MC3T3-E1 cells with dexamethasone (DEX). The viability, apoptosis, and osteogenic differentiation of DEX-induced cells with the presence or absence of Hyp were assessed by CCK-8, Tunel, ALP assay, and ARS staining, respectively. The NADPH Oxidase 4 (NOX4) overexpression was performed by transfection with overexpression vector. Besides, western blot was used to determine the levels of apoptosis-, osteogenic differentiation-, and c-Jun N-terminal kinase (JNK) signaling-related proteins. It was noticed that Hyp caused no significant effects on the viability of MC3T3-E1 cells without any treatment but significantly enhanced the viability of DEX-induced cells. Besides, Hyp inhibited the apoptosis in DEX-induced cells but enhanced ALP activity and calcium nodule formation. Additionally, Hyp declined NOX4 expression in DEX-induced cells. However, NOX4 overexpression partially reversed the impacts of Hyp on DEX-exposed MC3T3-E1 cells. Finally, Hyp suppressed the activation of ROS/JNK pathway in DEX-induced cells, which was then counteracted by NOX4 overexpression. In conclusion, Hyp could promote the survival and differentiation of DEX-induced osteoblasts by targeting NOX4 to inhibit the ROS/JNK pathway. These results provide evidence for the application of Hyp in treating GONFH.

Oxidative Injury in Ischemic Stroke: A Focus on NADPH Oxidase 4.

Ischemic stroke is a leading cause of disability and mortality worldwide. Thus, it is urgent to explore its pathophysiological mechanisms and find new therapeutic strategies for its successful treatment. The relationship between oxidative stress and ischemic stroke is increasingly appreciated and attracting considerable attention. ROS serves as a source of oxidative stress. It is a byproduct of mitochondrial metabolism but primarily a functional product of NADPH oxidases (NOX) family members. Nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is most closely related to the formation of ROS during ischemic stroke. Its expression is significantly upregulated after cerebral ischemia, making it a promising target for treating ischemic stroke. Several drugs targeting NOX4, such as SCM-198, Iso, G-Rb1, betulinic acid, and electroacupuncture, have shown efficacy as treatments of ischemic stroke. MTfp-NOX4 POC provides a novel insight for the treatment of stroke. Combinations of these therapies also provide new approaches for the therapy of ischemic stroke. In this review, we summarize the subcellular location, expression, and pathophysiological mechanisms of NOX4 in the occurrence and development of ischemic stroke. We also discuss the therapeutic strategies and related regulatory mechanisms for treating ischemic stroke. We further comment on the shortcomings of current NOX4-targeted therapy studies and the direction for improvement.

Forsythiaside A Regulates Activation of Hepatic Stellate Cells by Inhibiting NOX4-Dependent ROS.

Hepatic stellate cells (HSCs) activation is an important step in the process of hepatic fibrosis. NOX4 and reactive oxygen species expressed in HSCs play an important role in liver fibrosis. Forsythiaside A (FA), a phenylethanoid glycoside extracted and isolated from Forsythiae Fructus, has significant antioxidant activities. However, it is not clear whether FA can play a role in inhibiting the HSCs activation through regulating NOX4/ROS pathway. Therefore, our purpose is to explore the effect and mechanism of FA on HSCs activation to alleviate liver fibrosis. LX2 cells were activated by TGF-ß1 in vitro. MTT assay and Wound Healing assay were used to investigate the effect of FA on TGF-ß1-induced LX2 cell proliferation and migration. Elisa kit was used to measure the expression of MMP-1 and TIMP-1. Western blot and RT-qPCR were used to investigate the expression of fibrosis-related COLI, α-SMA, MMP-1 and TIMP-1, and inflammation-related TNF-α, IL-6 and IL-1ß. The hydroxyproline content was characterized using a biochemical kit. The mechanism of FA to inhibit HSCs activation and apoptosis was detected by DCF-DA probe, RT-qPCR, western blot and flow cytometry. NOX4 siRNA was used to futher verify the effect of FA on NOX4/ROS pathway. The results showed that FA inhibited the proliferation and migration of LX2 cells and adjusted the expression of MMP-1, TIMP-1, COLI, α-SMA, TNF-α, IL-6 and IL-1ß as well as promoted collagen metabolism to show potential in anti-hepatic fibrosis. Mechanically, FA down-regulated NOX4/ROS signaling pathway to improve oxidation imbalances, and subsequently inhibited PI3K/Akt pathway to suppress proliferation. FA also promoted the apoptosis of LX2 cells by Bax/Bcl2 pathway. Furthermore, the effects of FA on TGF-ß1-induced increased ROS levels and α-SMA and COLI expression were weaken by silencing NOX4. In conclusion, FA had potential in anti-hepatic fibrosis at least in part by remolding of extracellular matrix and improving oxidation imbalances to inhibit the activation of HSCs and promote HSCs apoptosis.

Main active components of Si-Miao-Yong-An decoction (SMYAD) attenuate autophagy and apoptosis via the PDE5A-AKT and TLR4-NOX4 pathways in isoproterenol (ISO)-induced heart failure models.

Heart failure (HF), the main cause of death in patients with many cardiovascular diseases, has been reported to be closely related to the complicated pathogenesis of autophagy, apoptosis, and inflammation. Notably, Si-Miao-Yong-An decoction (SMYAD) is a traditional Chinese medicine (TCM) used to treat cardiovascular disease; however, the main active components and their relevant mechanisms remain to be discovered. Based on our previous ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) results, we identified angoriside C (AC) and 3,5-dicaffeoylquinic acid (3,5-DiCQA) as the main active components of SMYAD. In vivo results showed that AC and 3,5-DiCQA effectively improved cardiac function, reduced the fibrotic area, and alleviated isoproterenol (ISO)-induced myocarditis in rats. Moreover, AC and 3,5-DiCQA inhibited ISO-induced autophagic cell death by inhibiting the PDE5A/AKT/mTOR/ULK1 pathway and inhibited ISO-induced apoptosis by inhibiting the TLR4/NOX4/BAX pathway. In addition, the autophagy inhibitor 3-MA was shown to reduce ISO-induced apoptosis, indicating that ISO-induced autophagic cell death leads to excess apoptosis. Taken together, the main active components AC and 3,5-DiCQA of SMYAD inhibit the excessive autophagic cell death and apoptosis induced by ISO by inhibiting the PDE5A-AKT and TLR4-NOX4 pathways, thereby reducing myocardial inflammation and improving heart function to alleviate and treat a rat ISO-induced heart failure model and cell heart failure models. More importantly, the main active components of SMYAD will provide new insights into a promising strategy that will promote the discovery of more main active components of SMYAD for therapeutic purposes in the future.

The effect of Kappaphycus alvarezii active fraction on oxidative stress and inflammation in streptozotocin and nicotinamide-induced diabetic rats.

BACKGROUND: High glucose concentration increases the glycation process which leads to oxidative stress and inflammation, that can cause complications in diabetes. Several medicinal plants have been used in the treatment of diabetes and its complications. One of them is Kappaphycus alvarezii, an algae that has known antidiabetic abilities. This study aimed to examine the effect of K. alvarezii active fraction on plasma hydrogen peroxide (H2O2) and Tumor Necrosis Factor α (TNFα) levels, renal NADPH oxidase 4 (NOX4) and Nuclear Factor κ B (NFκB) gene expressions. METHODS: Active fraction was obtained from bioassay-guided fractionation with antiglycation ability. In vivo study was performed on twenty Wistar male rats. The level of H2O2 was measured using H2O2 Assay Kit, the Optical Density value measured using spectrophotometer at a wavelength of 405 nm. Plasma TNFα level was measured using ELISA. Renal NOX4 and NFκB gene expression was analyzed using qPCR. RESULTS: Active fraction significantly reduced plasma H2O2 but not TNFα levels. Furthermore, renal NOX4 gene expression was lower in the diabetic rat group treated with active fraction compared to the untreated group but not NFκB gene expression. CONCLUSIONS: K. alvarezii active fraction has an activity to reduce plasma H2O2 as well as renal NOX4 gene expression. Therefore, this fraction could be developed as a potential candidate for diabetes treatment through oxidative stress mechanisms.

JI017, a Complex Herbal Medication, Induces Apoptosis via the Nox4-PERK-CHOP Axis in Ovarian Cancer Cells.

Many anti-cancer drugs, including paclitaxel and etoposide, have originated and been developed from natural products, and traditional herbal medicines have fewer adverse effects and lesser toxicity than anti-tumor reagents. Therefore, we developed a novel complex herbal medicine, JI017, which mediates endoplasmic reticulum (ER) stress and apoptosis through the Nox4-PERK-CHOP signaling pathway in ovarian cancer cells. JI017 treatment increases the expression of GRP78, ATF4, and CHOP and the phosphorylation of PERK and eIF2α via the upregulation of Nox4. Furthermore, it increases the release of intracellular reactive oxygen species (ROS), the production of intracellular Ca2+, and the activation of exosomal GRP78 and cell lysate GRP78. Combination treatment using the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (TG) and JI017 reportedly induces increased ER stress and cell death in comparison to the control; however, knockdown experiments of PERK and CHOP indicated suppressed apoptosis and ER stress in JI017-treated ovarian cancer cells. Furthermore, targeting Nox4 using specific siRNA and pharmacological ROS inhibitors, including N-acetylcystein and diphenylene iodonium, blocked apoptosis and ER stress in JI017-treated ovarian cancer cells. In the radioresistant ovarian cancer model, when compared to JI017 alone, JI017 co-treatment with radiation induced greater cell death and resulted in overcoming radioresistance by inhibiting epithelial-mesenchymal-transition-related phenomena such as the reduction of E-cadherin and the increase of N-cadherin, vimentin, Slug, and Snail. These findings suggest that JI017 is a powerful anti-cancer drug for ovarian cancer treatment and that its combination treatment with radiation may be a novel therapeutic strategy for radioresistant ovarian cancer.

Bioactive Compounds From Coptidis Rhizoma Alleviate Pulmonary Arterial Hypertension by Inhibiting Pulmonary Artery Smooth Muscle Cells' Proliferation and Migration.

ABSTRACT: Pulmonary arterial hypertension (PAH) is a devastating disorder characterized by excessive proliferation and vasoconstriction of small pulmonary artery vascular smooth muscle cells (PASMCs). Coptidis rhizoma (CR) because of the complexity of the components, the underlying pharmacological role and mechanism of it on PAH remains unknown. In this article, the network pharmacological analysis was used to screen the main active constituents of CR and the molecular targets that these constituents act on. Then, we evaluated the importance of berberine and quercetin (biologically active components of CR) on the proliferation and migration of PASMCs and vascular remodeling in experimental models of PAH. Our results showed that berberine and quercetin effectively inhibited the proliferation and migration of hypoxia-induced PASMCs in a manner likely to be mediated by the suppression of MAPK1, NADPH oxidase 4 (NOX4), and cytochrome P450 1B1 (CYP1B1) expression. Furthermore, berberine and quercetin treatment attenuates pulmonary hypertension, reduces right ventricular hypertrophy, and improves pulmonary artery remodeling in monocrotaline-induced pulmonary hypertension in rat models. In conclusion, this research demonstrates CR might be a promising treatment option for PAH, and the network pharmacology approach can be an effective tool to reveal the potential mechanisms of Chinese herbal medicine.

Clitoria ternatea L. extract prevents kidney damage by suppressing the Ang II/Nox4/oxidative stress cascade in l-NAME-induced hypertension model of rats.

Clitoria ternatia L. (CT) has been reported to have anti-inflammatory and antioxidant effects. This study investigated the effect of CT aqueous flower extract on blood pressure and renal alterations in Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced hypertensive rats. Male Sprague Dawley rats received l-NAME in drinking water and were treated with CT flower extract or lisinopril. CT aqueous flower extract and lisinopril alleviated l-NAME-induced hypertension (p < 0.05). Glomerular extracellular matrix accumulation, renal fibrosis, and increased serum creatinine levels were observed in l-NAME-induced hypertensive rats and attenuated by CT flower extract or lisinopril co-treatment (p < 0.05). High levels of plasma angiotensin II (Ang II) and upregulated nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) protein expression in the kidneys induced by l-NAME were alleviated by CT flower extract or lisinopril co-treatment (p < 0.05). Furthermore, CT flower extract and lisinopril treatment reduced lipid peroxidation and elevated plasma and kidney malondialdehyde levels in l-NAME-induced hypertensive rats (p < 0.05). In conclusion, CT flower extract prevented l-NAME-induced renal injury and dysfunction in rats. The possible mechanism may be related to the suppression of Ang II-mediated Nox4 expression and the oxidative stress cascade in rats.

A small molecule inhibitor of Nox2 and Nox4 improves contractile function after ischemia-reperfusion in the mouse heart.

The NADPH oxidase enzymes Nox2 and 4, are important generators of Reactive oxygen species (ROS). These enzymes are abundantly expressed in cardiomyocytes and have been implicated in ischemia-reperfusion injury. Previous attempts with full inhibition of their activity using genetically modified animals have shown variable results, suggesting that a selective and graded inhibition could be a more relevant approach. We have, using chemical library screening, identified a new compound (GLX481304) which inhibits Nox 2 and 4 (with IC50 values of 1.25 µM) without general antioxidant effects or inhibitory effects on Nox 1. The compound inhibits ROS production in isolated mouse cardiomyocytes and improves cardiomyocyte contractility and contraction of whole retrogradely (Langendorff) perfused hearts after a global ischemia period. We conclude that a pharmacological and partial inhibition of ROS production by inhibition of Nox 2 and 4 is beneficial for recovery after ischemia reperfusion and might be a promising venue for treatment of ischemic injury to the heart.

Therapeutic potential of diosmin, a citrus flavonoid against arsenic-induced neurotoxicity via suppression of NOX 4 and its subunits.

OBJECTIVES: Water contaminated with arsenic affected millions of people worldwide and arsenic exposure is related to various neurological disorders. Hence, the current study was planned to investigate the neuroprotective activity of diosmin (DSN) against arsenic induced neurotoxicity as an attempt to identify therapeutic intervention to combat arsenicism. MATERIALS AND METHODS: Sodium arsenite an inducer of neurotoxicity was administered orally (13 mg/kg) and DSN treatment at two selected doses (50 and 100 mg/kg) was done for 21 days. Behavioral and biochemical variations were examined by various parameters. Furthermore, histopathological and immunohistochemistry studies were done with the brain sections. RESULTS: The behavioral studies evidenced that arsenic has suppressed the exploratory behavior and motor coordination in rats and DSN treatment has recovered the behavioral changes to normal. Arsenic administration has also found to induce oxidative stress and DSN co-treatment has ameliorated the oxidative stress markers. Interestingly, depleted levels of neurotransmitters were observed with the arsenic and it was restored back by the DSN treatment. Histopathological alterations like pyknosis of the neuronal cells were identified with arsenic exposure and subsided upon DSN co administration. Immunohistochemical studies have revealed the expression of NOX4 and its gp91phox and P47phox subunits and its suppression by DSN treatment may be the key therapeutic factor of it. CONCLUSIONS: Treatment with DSN showed a beneficial effect in protecting against arsenic-induced neurotoxicity by suppressing the toxicity changes and the antioxidant effect of DSN might be attributed to its ability of suppressing NOX4 and its subunits.

Cudrania tricuspidata Root Extract Prevents Methylglyoxal-Induced Inflammation and Oxidative Stress via Regulation of the PKC-NOX4 Pathway in Human Kidney Cells.

Diabetic nephropathy is a microvascular complication induced by diabetes, and methylglyoxal (MGO) is a reactive carbonyl species causing oxidative stress that contributes to the induction of inflammatory response in kidney cells. Cudrania tricuspidata (CT), cultivated in Northeast Asia, has been used as traditional medicine for treating various diseases, including neuritis, liver damage, and cancer. In this study, we determined whether a CT root extract (CTRE) can prevent MGO-induced reactive oxygen species (ROS) production and inflammation and assessed underlying mechanisms using a kidney epithelial cell line, HK-2. We observed that CTRE inhibited MGO-induced ROS production. Additionally, CTRE ameliorated the activation of MGO-induced inflammatory signaling pathways such as p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-JUN N-terminal kinase (JNK). Consistent with these results, expressions of p-nuclear factor-kappa B (NFκB) and inflammatory cytokines, tumor necrosis factor-α, interleukin- (IL-) 1ß, and IL-6, were decreased when compared with MGO-only exposed HK-2 cells. CTRE alleviated the MGO-induced decrease in nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and antioxidant enzyme mRNA expressions. MGO induced the expression of NADPH oxidase 4 (NOX4); CTRE pretreatment inhibited this induction. Further studies revealed that the NOX4 expression was inhibited owing to the suppression of MGO-induced protein kinase C (PKC) activation following CTRE treatment. Collectively, our data suggest that CTRE attenuates MGO-induced inflammation and oxidative stress via inhibition of PKC activation and NOX4 expression, as well as upregulating the Nrf2-antioxidant enzyme pathway in HK-2 cells.

Curculigoside attenuates oxidative stress and osteoclastogenesis via modulating Nrf2/NF-κB signaling pathway in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Curculigo orchioides Gaertn is used for the treatment of impotence, atrophic debility of bones (osteoporosis), limb limpness, and arthritis of the lumbar and knee joints in traditional Chinese medicine and Ayurvedic medical system. Curculigoside (Cur) from Curculigo orchioides Gaertn has been shown to have regulatory effects on bone metabolism via anti-oxidative activities in rats and osteoblasts. However, little is known about the molecular pharmacological activity of Cur in osteoclastic bone resorption. AIM: The aim of this work is to investigate the inhibitory effect of Cur against osteoclasts (OCs) under the oxidative stress status, and explore the possible underlying mechanism. MATERIALS AND METHODS: OCs were induced from RAW264.7 cells using RANKL and H2O2. The number of OCs was measured by tartrate-resistant acid phosphatase (TRAP) staining. F-Actin and nuclear translocation of P65 and Nrf2 were stained with immunofluorescence assay and observed under a laser confocal microscope. The biochemical parameters of OCs were detected with an ELISA kit. The expression of Nrf2 and NF-κB pathway-related proteins was analyzed by Western Blot. RESULTS: Cur inhibited the TRAP activity, release of degrading products from bone slices and the expression of NFATc1, c-Fos, Cathepsin K (Ctsk) and matrix metallopeptidase 9 (MMP9) of OCs induced with RANKL and H2O2. In addition, Cur suppressed the ROS level and NADPH oxidase 1(NOX1) and NADPH oxidase 4 (NOX4) activities of OCS. More importantly, Cur enhanced the expression and nucleus translocation of Nrf2 and activities of its regulatory cytoprotective enzymes, and reduced the NF-κB expression and phosphorylation and nucleus translocation of p65 in OCs. Furthermore, the Nrf2 inhibitor ML385 and NF-κB inhibitor Bay11-7082 counteracted the effect of Cur in OCs. CONCLUSION: Cur mitigated oxidative stress and osteoclastogenesis by activating Nrf2 and inhibiting the NF-κB pathway, suggesting that Cur may prove to be a promising candidate for the treatment of osteoporosis. Our findings may also help partially explain the rationale behind the traditional use of Curculigo orchioides Gaertn.