Mechanism of Modified Zhenwutang in Delaying Renal Interstitial Fibrosis in Chronic Renal Failure Based on Ang Ⅱ/AT1R/NOX4 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the underlying mechanism of modified Zhenwutang in delaying renal interstitial fibrosis in chronic renal failure （CRF） by observing the effects of modified Zhenwutang on the expression of angiotensin Ⅱ （Ang Ⅱ）， angiotensin Ⅱ type 1 receptor （AT1R）， nicotinamide adenine dinucleotide phosphate （NADPH） oxidase 4 （NOX4）， transforming growth factor-β1 （TGF-β1）， type I collagen （COL1A1）， and type Ⅲ collagen （COL3A1） in the serum and renal tissues of adenine-induced CRF rats. MethodFifty male SPF-grade SD rats were randomly divided into a normal group （n=10） and an experimental group （n=40） using a random number table. After one week of adaptive feeding， the experimental CRF model was established in rats by administering adenine at 150 mg·kg-1·d-1 orally. Three rats from each group were randomly selected to evaluate the model induction. After successful modeling， rats in the experimental group were randomly divided into a model group， low-， medium， and high-dose modified Zhenwutang groups， and a benazepril hydrochloride group， with six rats in each group. The rats were orally administered the corresponding drugs once daily for four weeks. At the end of the first week， 13th week， and 17th week of the experiment， 24 hour urinary protein quantification （24 h-UTP） was measured. At the end of the 17th week， the rats were euthanized， and blood samples were collected from the abdominal aorta for the measurement of total protein （TP）， albumin （ALB）， creatinine （Cr）， and blood urea nitrogen （BUN） in the serum. Enzyme-linked immunosorbent assay （ELISA） was used to measure the expression levels of serum Ang Ⅱ. Hematoxylin-eosin （HE） staining and Masson's trichrome staining were performed to observe the pathological changes in renal tissues. Immunohistochemistry （IHC） was performed to observe the expression of AT1R， NOX4， TGF-β1， COL1A1， and COL3A1. Real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） was used to observe the mRNA expression levels of AT1R， NOX4， and TGF-β1. Western blot was conducted to measure the protein expression levels of AT1R， NOX4， and TGF-β1. Result① Compared with the normal group， the model group showed a significant increase in 24 h-UTP （P<0.01）. The levels of Cr and BUN in the model group were significantly higher （P<0.01）， while the levels of TP and ALB were significantly lower （P<0.01）. The serum Ang Ⅱ level in the model group was significantly elevated （P<0.01）. The model group exhibited widening of the renal glomerular mesangial space， necrotic glomeruli， increased interstitial width with extensive inflammatory cell infiltration， brownish precipitates blocking the renal tubular lumens， irregular renal tubules， and significant deposition of collagen fibers in the renal interstitium. Additionally， the collagen fibers around the renal vessels， outside the parietal layer of the renal sacs， glomerular basement membrane， and tubular basement membrane increased significantly. The expression of AT1R and NOX4 in the glomeruli and renal tubules of the model group was significantly enhanced， and TGF-β1 expression also significantly increased in the renal tubules. The expression of COL1A1 and COL3A1 in the renal interstitium significantly increased. The mRNA expression of AT1R and TGF-β1 in the model group significantly increased （P<0.01）， while NOX4 mRNA expression significantly decreased （P<0.01）. The protein expression of AT1R， NOX4， and TGF-β1 was significantly enhanced （P<0.01）. ② Compared with the model group， modified Zhenwutang significantly reduced 24h-UTP （P<0.01）， decreased levels of Cr and BUN （P<0.01）， increased levels of TP and ALB （P<0.01）， reduced serum Ang Ⅱ level （P<0.01）， alleviated renal pathological damage， reduced expression of AT1R， NOX4， TGF-β1， COL1A1， and COL3A1 in the glomeruli， renal tubules， and renal interstitium， reduced mRNA expression of AT1R and TGF-β1 （P<0.01）， increased NOX4 mRNA expression （P<0.01）， and weakened protein expression of AT1R， NOX4， and TGF-β1 （P<0.01）. The modified Zhenwutang groups showed a significant dose-effect trend. ConclusionModified Zhenwutang may delay renal interstitial fibrosis in CRF rats by reducing the expression of Ang Ⅱ， AT1R， NOX4， and TGF-β1 in the serum and renal tissues， thereby alleviating renal pathological damage， reducing proteinuria， protecting renal function， and delaying the progression of CRF. The modified Zhenwutang group exhibited a dose-effect trend.

Heixiaoyao Powder interferes with microglia polarization in AD model mice by regulating NOX2/ROS/NF-κB signaling pathway / 中国中药杂志

The effect and mechanism of Heixiaoyao Powder on the polarization of microglia(MG) in APP/PS1 double transgenic mice were explored based on NADPH oxidase 2(NOX2)/reactive oxygen species(ROS)/nuclear factor kappaB(NF-κB) signaling pathway. Fifty 4-month-old male APP/PS1 mice were randomly divided into a model group, an MCC950 group(10 mg·kg~(-1)), and low-, medium-, and high-dose Heixiaoyao Powder groups(6.45, 12.89, and 25.78 g·kg~(-1)). Thirty male C57BL/6J mice of the same age and strain were randomly divided into a blank group, a blank + intragastric intervention group, and a blank + intraperitoneal injection group. Drug intervention lasted 90 days. Morris water maze test was used to detect learning and cognitive ability. Nissl staining and transmission electron microscopy were used to observe the pathological morphology and ultrastructure of hippocampal neurons. Immunofluorescence was used to detect the positive expression of M1-type marker CD16/32~+/Iba-1~+, M2-type marker CD206~+/Iba-1~+ of MG and the expression of hippocampal ROS. The colorimetric method was used to detect the content of malondialdehyde(MDA) and superoxide dismutase(SOD) in the hippocampus. Enzyme linked immunosorbent assay(ELISA) was used to detect the levels of inflammatory factors, including interleukin-6(IL-6), interleukin-8(IL-8), and tumor necrosis factor-α(TNF-α), in the hippocampus. Western blot was used to detect the protein expression of β-amyloid protein(Aβ), Iba-1, CD16/32, CD206, NOX2, NF-κB, p-NF-κB, NF-κB inhibitor alpha(IκBα), and p-IKBα in the hippocampus. The results showed that as compared with the blank group, the model group showed prolonged target quadrant movement distance and escape latency(P<0.01), shortened target quadrant retention time and percentage(P<0.01), disorganized neuronal cells with swelling, nuclear disappearance or bias, reduced number of cells, dissolved or absent Nissl bodies, and a clear area in the cytoplasm, damaged and shrunk cell membrane with abnormal cell morphology, few organelles in the cytoplasm, reduced and swollen mitochondria, increased MG M1-type marker CD16/32~+/Iba-1~+(P<0.01), decreased M2-type marker CD206~+/Iba-1~+(P<0.01), increased ROS activity and MDA content(P<0.01), decreased SOD level(P<0.01), elevated inflammatory factors IL-6, IL-8, and TNF-α(P<0.01), up-regulated protein expression and phosphorylation of Aβ, CD16/32, Iba-1, NOX2, NF-κB, and IKBα(P<0.01), and down-regulated CD206(P<0.01). There was no statistically significant difference between the blank group, the blank + intragastric intervention group, and the blank + intraperitoneal injection group. After the intervention of Heixiaoyao Powder, the Heixiaoyao Powder groups showed shortened target quadrant movement distance and escape latency(P<0.01), prolonged target quadrant retention time and percentage(P<0.01), increased and neatly arranged cells with relieved swelling, increased Nissl bodies, regular cell morphology, and intact cell membrane, relieved swelling of mitochondria, slightly expanded endoplasmic reticulum, decreased CD16/32~+/Iba-1~+(P<0.05 or P<0.01), increased CD206~+/Iba-1~+(P<0.01), decreased ROS activity and MDA content(P<0.01), increased SOD level(P<0.01), decreased content of inflammatory factors IL-6, IL-8, and TNF-α(P<0.01), down-regulated protein expression and phosphorylation of Aβ, CD16/32, Iba-1, NOX2, NF-κB, and IKBα(P<0.01), and up-regulated CD206(P<0.01). In conclusion, Heixiaoyao Powder can alleviate neuronal damage and improve the learning and memory abilities of APP/PS1 mice. The mechanism of action may be related to the inhibition of NOX2/ROS/NF-κB signaling pathway, regulating the polarization of MG, increasing the expression of M2 type, inhibiting the expression of M1 type, and reducing the release of inflammatory factor.

Dapagliflozin regulates myocardial SK2 protein expression in diabetic rats / 中国药理学通报

Aim To investigate the effect of dapagliflozin on the small conductance calcium-activated potassium channel 2 (SK2 channel) protein in the myocardium of diabetic rats and its possible mechanism of action. Methods In vivo: type 2 diabetes model was established by high-glucose and high-fat diet combined with intraperitoneal injection of low-dose streptozotocin (35 mg

Swertiamarin attenuates paraquat-induced pulmonary epithelial-like cell apoptosis via NOX4-mediated regulation of redox and mitochondrial function

Abstract The aim of the present study was to investigate the effect of swertiamarin (STM) in attenuating paraquat (PQ)-induced human lung alveolar epithelial-like cell (A549) apoptosis and the underlying mechanisms. A549 cells were pretreated with different concentrations of STM for 2 hr and then cultured with or without PQ (700 µM) for 24 hr. Cell survival was determined using the CCK8 assay. Morphological changes, MDA content, inflammatory factors, fibrogenesis parameters, apoptosis rates, redox status and mitochondrial membrane potential (MMP) were evaluated. The expression of several genes involved in the modulation of redox status was measured by Western blotting. Cell viability and MMP were decreased, but the apoptosis rate and DCFH oxidation were elevated by PQ exposure. STM pretreatment notably increased cell viability and MMP and reduced the apoptosis rate and DCFH oxidation. Furthermore, TLR4- NOX4 signaling was significantly inhibited by STM. The downregulation of NOX4 by siRNA exerted the same protective effects as STM. This study provides the first evidence that STM attenuates PQ-induced pulmonary epithelial-like cell apoptosis via NOX4-mediated regulation of redox and mitochondrial function

Effect of TRPV4-Nox2 complex on aortic vasodilatory function of obese mice / 中国药理学通报

Aim To investigate the effects of TRPV4-Nox2 complex on ROS production and aortic vasodilatory function in mice fed with high-fat diet.Methods Male C57 BL/6J mice and TRPV4 KO mice were randomly divided into seven groups, with 10 mice in each group: normal diet group(ND), high-fat diet group(HFD), TRPV4 KO mice fed with high-fat diet group(TRPV4 KO-HFD), HFD+AAV-Flt1-Vector/Nox2 ▵3 group, TRPV4 KO-HFD+AAV-Flt1 -Vector/Nox2 ▵3 group.Body weight and blood pressure were recorded.14 weeks later primary aortic endothelial cells were isolated for CM-H2DCFDA staining and immuno-FRET assay, and aortic rings were isolated for vascular tone assay.Results ① Obesity significantly increased ROS production, triggered vasodilatory dysfunction and increased the strength of physical coupling between TRPV4-Nox2 complex(P<0.05); ② Decreasing the physical association of TRPV4-Nox2 complex could help reduce obesity-induced increased ROS production and vasodilatory dysfunction(P<0.05); ③ Entrectinib had no effect on the expression and function of TRPV4 and Nox2, but only decreased the physical association of the TRPV4-Nox2, which in turn improved obesity-induced oxidative stress and restored vasodilatory function.Conclusions Reducing the physical association of TRPV4 and Nox2 through Entrectinib can help reduce obesity-induced increase in ROS production and improve vasodilatory function of obese mice.

Effect of methyl eugenol on hypoxia/reoxygenation injury of human renal tubular epithelial cells and its mechanism / 中国中药杂志

This study aimed to investigate the effect of methyl eugenol(ME) on hypoxia/reoxygenation(H/R)-induced injury of human renal tubular epithelial HK-2 cells and its mechanism. The viability of HK-2 cells cultured with different concentrations of ME and exposed to H/R was detected by cell counting kit-8(CCK-8) assay. The effect of ME on the morphology of HK-2 cells was observed under an inverted microscope. The content of intracellular reactive oxygen species in different groups was detected after 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) fluorescence staining. Cell apoptosis was determined by flow cytometry. Changes in mitochondrial membrane potential were monitored by JC-1 dye. The concentrations of nuclear factor erythroid 2 related factor 2(Nrf2), heme oxygenase-1(HO-1), and nicotinamide adenine dinucleotide phosphatase oxidase 4(Nox4) were measured by Western blot, followed by the assay of Nrf2 concentration changes in cytoplasm and nucleus by confocal fluorescence staining. The results showed that when the concentration of ME was 0-40 μmol·L~(-1), the activity of HK-2 cells was not affected. Compared with the model group, ME enhanced the activity of HK-2 cells and the cell morphology was normal. As revealed by further experiments, ME inhibited the release of reactive oxygen species and the decline in mitochondrial membrane potential of HK-2 cells after H/R injury, promoted Nrf2/HO-1 expression and Nrf2 translocation to the nucleus, and down-regulated the expression of Nox4, thereby significantly reducing apoptosis. This protective effect of ME could be reversed by the specific Nrf2 inhibitor ML385. These findings have preliminarily proved that ME effectively protected HK-2 cells against H/R injury, which might be related to its promotion of Nrf2/HO-1 signaling pathway and inhibition of Nox4. Such exploration on the possible mechanism of ME in the treatment of renal ischemia-reperfusion injury(IRI) and protection of organ function from the perspective of antioxidant stress has provided reference for related research on the treatment of acute kidney injury with traditional Chinese medicine.

Effect of Modified Shengjiangsan on Oxidative Stress and Apoptosis in MN Rats via Regulating HIF-1α/NOX4 Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the intervention effect of modified Shengjiangsan on hypoxia-inducible factor-1<italic>α </italic>（HIF-1<italic>α</italic>）/nicotinamide adenine dinucleotide phosphate oxidase 4 （NOX4） signaling pathway in membranous nephropathy （MN） rats and to explore its mechanism to reduce oxidative stress and apoptosis in renal tissues. Method:Cationized bovine serum albumin （C-BSA） was injected into the tail vein of rats to replicate the MN model. Rats were randomly divided into a model group， a modified Shengjiangsan group， and a benazepril group after modeling， and administered by gavage once a day accordingly. At the end of the 4^th week， the 24-h urine total protein （UTP）， urea nitrogen （BUN）， and serum creatinine （SCr） levels of each group were detected. Enzyme-linked immunosorbent assay（ELISA）was used to detect the levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， and reactive oxygen species （ROS） in renal tissues of rats. In situ end labeling（TUNEL） staining was used to detect the cell apoptosis rate. The mRNA and protein expression levels of HIF-1<italic>α</italic> and NOX4 were detected by real-time fluorescence-based quantitative polymerase chain reaction（Real-time PCR）and Western blot， respectively. The immunohistochemistry method was used to detect the protein expression levels of B-cell lymphomas -2 （Bcl-2）， B-cell lymphomas xl （Bcl-xl）， Bcl-2 associated X protein （Bax）， Bcl-2 cell death regulator antibody （Bim）. Result:Compared with the normal group， the model group showed increased UTP （<italic>P</italic><0.05）， decreased SOD， elevated MDA and ROS （<italic>P</italic><0.05）， up-regulated mRNA and protein expression of HIF-1<italic>α</italic> and NOX4 （<italic>P</italic><0.05）， enhanced protein expression of Bax and Bim， declining protein expression of Bcl-xl and Bcl-2 （<italic>P</italic><0.05）， and increased cell apoptosis in renal tissues. Compared with the model group， the modified Shengjiangsan group and the benazepril group displayed declining UTP （<italic>P</italic><0.05）， up-regulated SOD， decreased MDA and ROS （<italic>P</italic><0.05）， down-regulated mRNA and protein expression of HIF-1<italic>α</italic> and NOX4 （<italic>P</italic><0.05）， diminished protein expression of Bax and Bim， elevated protein expression of Bcl-xl and Bcl-2 （<italic>P</italic><0.05）， and reduced cell apoptosis in renal tissues （<italic>P</italic><0.05）. Conclusion:The protective effect of modified Shengjiangsan on the kidney is presumedly achieved by reducing the oxidative stress and apoptosis in renal tissues of MN rats via inhibiting the HIF-1<italic>α</italic>/NOX4 signaling pathway.

Effect of Albiziae Flos and Polygalae Radix Alone and Their Combination on Depression-like Behavior and CREB and NOX2 Expression in Hippocampus of Chronic Unpredictable Stress-induced Rats / 中国实验方剂学杂志

Objective:To observe the effects of Albiziae Flos （AF） and Polygalae Radix （PR） alone and their combination on the improvement of depression-like behavior in rats with chronic unpredictable stress （CUS） as well as on hippocampal ultrastructure and the expression of cyclic adenosine monophosphate response element binding protein （CREB） and nicotinamide adenine dinucleotide phosphate oxidase 2 （NOX2）， to explore their action mechanisms. Method:Seventy-two Wistar rats were randomly divided into the normal group， model group， AF group， PR group， AF-PR group， and fluoxetine group. Rats in all groups except for the normal group were exposed to CUS and separated feeding to induce depression. Since the first day of modeling， rats in the AF group， PR group， AF-PR group were provided with the corresponding decoction containing 1.05 g·kg^-1 total crude drug by gavage， the ones in the fluoxetine group with 2.1 mg·kg^-1 fluoxetine hydrochloride aqueous solution， and those in the normal group and model group with the distilled water， for 28 successive days. The open field test and forced swimming test were performed 1 d before modeling and 7， 14， 21， 28 d after modeling， respectively. The morphological changes in hippocampus were observed under an electron microscope on the 28^th day. The superoxide dismutase （SOD） and malondialdehyde （MDA） levels in hippocampus were detected by ultraviolet spectrophotometry， and the expression levels of CREB and NOX2 were detected by real-time polymerase chain reaction （Real-time PCR） and Western blot. Result:The behavioral experiment results showed that the number of horizontal activities and sugar water consumption in the model group declined as compared with those in the normal group， while the immobility time in the forced swimming test was prolonged （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the model group， the AF group， PR group， and AF-PR group exhibited elevated number of horizontal activities， increased sugar water consumption but shortened immobility time in the forced swimming test （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the AF group or PR group， the AF-PR group showed significantly different behavioral indexes （<italic>P</italic><0.05）. Morphological results showed that the mitochondria of the model group were obviously swollen and the ultrastructure of the hippocampus was destroyed. By contrast， the hippocampal ultrastructure in each administration group was close to normal. The comparison with the normal group revealed that the activity of SOD in the hippocampus of the model group was significantly reduced， whereas the content of MDA was elevated （<italic>P</italic><0.01）. Compared with the model group， the AF group， PR group， and AF-PR group displayed increased activity of SOD and decreased content of MDA in the hippocampal tissues （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with AF or PR alone， the herbal pair AF-PR resulted in significant differences in the above-mentioned indexes （<italic>P</italic><0.05， <italic>P</italic><0.01）. The results of Real-time PCR and Western blot demonstrated that NOX2 expression in the hippocampus of the model group was up-regulated in comparison with that in the normal group， while the CREB expression was down-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the model group， the AF group， PR group， and AF-PR group all showed diminished NOX2 expression but elevated CREB expression in the hippocampal tissues （<italic>P</italic><0.05， <italic>P</italic><0.01）. The protein expression levels of NOX2 and CREB in the AF group or PR group were significantly different from those in the AF-PR group （<italic>P</italic><0.01）. Conclusion:AF and PR alone and their combination improve the depression-like behavior of rats exposed to CUS， which may be related to the reduction of oxidative stress， the up-regulation of CREB expression， and the down-regulation of NOX2 expression in hippocampus.

Astragalin inhibits airway inflammation in asthma through NOX2/ROS/NF-κB signaling pathway / 中国药理学通报

Aim To investigate the effect of astragalin (AG) on airway inflammation in asthmatic mice and its mechanism. Methods Fifty SPF male mice were randomly divided into normal group, asthma model group, and astragalin low (AG25), medium (AG50), and high (AG100) dose groups. A mouse model of asthma was prepared by egg albumin inhalation, the number of cells was counted by Diff-Quik staining after collecting bronchoalveolar lavage fluid (BALF), and IL4, 5, and 13 levels in BALF were measured by ELISA. The inflammatory changes in mouse lung tissues were observed using HE staining. Reactive oxygen species (ROS) levels were measured using a DCFH-DA probe, and superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured by colorimetry. IL-4, IL-5, IL-13, NOX2, p47phox, p-NF-κBp65, NF-κBp65, IκBα, p-IκBα and β-actin expressions in lung tissues were detected by Western blot. Results AG significantly reduced the number of inflammatory cells and total cells in BALF, decreased IL-4, IL-5, and IL-13 contents in BALF and lung tissues, and reduced inflammatory cell infiltration in lung tissues. AG inhibited nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) and p47phox expression, decreased ROS levels and MDA levels, increased SOD activity, and inhibited IκBα and NF-κBp65 phosphorylation. Conclusion AG attenuates airway inflammation in asthma by modulating the oxidative stress response through the NOX2/ROS/NF-κB signaling pathway.

Coffee beverage reduces ROS production and does not affect the organism's response against Candida albicans

Coffee is a mixture of substances with potential beneficial and adverse health effects. Several studies demonstrate the antioxidant effect of the phenolics compounds present in coffee. Neutrophils produce reactive oxygen species (ROS) by activating of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2), which plays a key role in organism defenseagainst microbial pathogens. Diabetes mellitus patients are more susceptible to bacterial and fungal infections. The present study evaluated the influence of coffee beverage on NOX2 activity and ROS generation and the impact of this effect on phagocytosis and killing of Candida albicansby neutrophils from diabetic and non-diabetic animals. Diabetes mellitus was induced in male Wistar rats using 2% alloxan. Diabetic and non-diabetic animals were divided into groups treated and untreated with coffee drink (7.2 mL/kg/day) or apocyanine (16 mg/kg/day) for 50 days. After 50 days, the animals' glycemic profile was measured by blood glucose and glycated hemoglobin (HbA1c) tests. The generation of ROS in neutrophilic cells was measured by chemiluminescence and cytochrome C reduction assays. C. albicans phagocytosis and death were evaluated by optical microscopy using the May-Grunwald-Giemsa staining method. The coffee drink has not altered the glycemic profile and NOX2 activity of the animals. However, coffee reduced the ROS pool in non-diabetic and diabetic animals, but this activity did not harm the phagocytosis or killing of neutrophils. Treatment with apocyanin decreased ROS production and killing capacity of neutrophils from non-diabetic animals against C. albicans. We suggest that the coffee drink intake prevents oxidative damage and does not impair response of the organism against opportunistic microorganism.

Huidouba Ameliorates Kidney Oxidative Stress Injury by Down-regulating Nox4 Expression in Rats with Diabetic Nephropathy / 中国实验方剂学杂志

Objective:From a new perspective，to explore therapeutic effect of Huidouba (HDB) on alleviating kidney oxidative damage in rats with diabetic nephropathy (DN) and provide a scientific basis for developing HDB as a potential Tibetan medicine for treatment of DN. Method:Rats were fed with high-fat diet (HFD) and injected with streptozocin (STZ, 65 mg·kg-1) intraperitoneally to induce DN model, while rats in Blank group were injected with an equal volume of vehicle and fed with normal chow. The successfully modeling DN rats were randomly divided into three groups, 8 rats per group, DN model group (10 mL·kg-1·d-1), Metformin group (0.045 g·kg-1·d-1) and HDB group (0.18 g·kg-1·d-1). Monitor body weight (BW) and fasting blood glucose (FBG) weekly, and collect 24 hours urine before and after medication to examine microalbuminuria (mAlb). Calculate kidney index (KI) after sacrificing, analyze mAlb, serum creatinine (SCr) and blood urea nitrogen (BUN) with a fully automatic biochemical analyzer. Histopathology of kidney was observed by Masson staining. Lipid peroxidation malondialdehyde (MDA) assay kit was used to examine MDA content in kidney tissue. Nox4, as a subtype of triphosphopyridine nucleotide (NADPH) oxidase family was determined by Western blot and immunofluorescence assay of kidney tissue. Result:Compared with blank group, levels of FBG, 24 h mAlb, SCr, BUN and MDA in DN model group were increased (P<0.01), tissue damage was obvious and Nox4 expression in glumeruli was increased significantly (P<0.01). Compared with DN model group, levels of FBG, 24 h mAlb, SCr, BUN and MDA in drug administration groups were decreased (P<0.01), kidney injury was alleviated and Nox4 expression was down-regulated（P<0.01）. Conclusion:HDB as a Yiqiyangyin Tibetan medicine, could ease oxidative stress injury of kidney and reduce proteinuria in DN rats, thus prevent the development of DN. Its mechanism is closely related to down-regulating Nox4 expression of kidney tissue in DN rats.

Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Production of reactive oxygen species (ROS) is a conserved immune response primarily mediated by NADPH oxidases (NOXs), also known in plants as respiratory burst oxidase homologs (RBOHs). Most microbe-associated molecular patterns (MAMPs) trigger a very fast and transient ROS burst in plants. However, recently, we found that lipopolysaccharides (LPS), a typical bacterial MAMP, triggered a biphasic ROS burst. In this study, we isolated mutants defective in LPS-triggered biphasic ROS burst (delt) in Arabidopsis, and cloned the DELT1 gene that was shown to encode RBOHD. In the delt1-2 allele, the antepenultimate residue, glutamic acid (E919), at the C-terminus of RBOHD was mutated to lysine (K). E919 is a highly conserved residue in NADPH oxidases, and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease. Consistently, we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure. It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein’s stability and complex assembly. However, we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association, suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs. Taken together, our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.

The role of Nox-4 in gastric mucosal inflammation induced by chronic restraint stress in mice / 中华行为医学与脑科学杂志

Objective To investigate the expression of NADPH oxidase Nox-4 induced by stress in gastric mucosa and its role in inflammation.Methods Twenty male SPF Kunming mice were randomly divided into chronic restraint stress group(stress group) and control group.Stress mice were restrained in selfmade restraint device for 2 hours each day.The rest of the time,the mice in the two groups had free access to food and water normally,experiment lasted 14 days.The histopathological changes of gastric mucosa were assessed by HE staining under light microscope.The expression of Nox-4 in gastric mucosa of mice was carried out by immunohistochemical method.The relative expression levels of Nox-4,antioxidant protein (Mn-SOD,GSH,Catalase) and inflammatory factors(IL-8,IL-1β,TNF-α) in gastric mucosa were detected by real-time quantitative RT-PCR and ELISA.Results Basal cell proliferation,neutrophil,eosinophil and plasma cell infiltration and inflammatory changes were observed in the lamina propria and glandular epithelium of stress mice,while no obvious abnormalities were found in control mice.The expression of Nox-4 in stress group was deeper and more abundant than that in control group,mainly expressed in lamina propria and glandular epithelium.The mRNA expression levels of Nox-4 in gastric mucosa of stress group was(2.42±0.51) times higher than that of control group,and blood concentration of stress group was(2.23±0.67) times higher than that of control group(t=-46.32,P＜0.001).The RT-PCR of antioxidant proteins in gastric mucosa showed that the transcription levels of Mn SOD,GSH and Catalase in stress group were significantly lower than that of control group (Mn-SOD:0.59± 0.10,GSH:0.58± 0.11,Catalase:0.57± 0.09),and there were significant differences between the two groups(t=13.57,11.67,15.01,P＜0.01).RT-PCR results showed that the transcription levels of IL-8,IL-1β,TNF-α in stress group were significantly higher than those in control group (IL-8:1.47±0.34,IL-1β:1.48 ± 0.42,TNF-α:1.51 ± 0.37),and there were significant differences in two groups(t=-18.45,-19.14,-20.85,P＜0.01).ELISA results showed that the serum levels of inflammatory factors in stress group were significantly higher than those in control group(2.25±0.37,3.59±0.45,3.41±0.34),and the differences were statistically significant(t=-47.11,-79.36,-96.32,P＜0.01).Pearson correlation analysis showed that there was a positive correlation between serum concentration of Nox-4 and inflammatory factors(IL-8,IL-1β,TNF-αt) in stress group(r=0.97,0.99,0.98,P＜0.01).Spearman rank correlation analysis showed that the grade of gastric mucosal inflammation was positively correted with serum levels of Nox-4 and inflammatory factors (IL-8,IL-1β,TNF-α) (r =0.96,0.92,0.91,0.94,all P＜ 0.01)Conclusion Stress may lead to gastric mucosal lesion by overexpression of proinflammatory factors through destroying the balance of oxidation/antioxidant system in gastric mucosa.

Perivascular Cells and NADPH Oxidase Inhibition Partially Restore Hyperglycemia-Induced Alterations in Hematopoietic Stem Cell and Myeloid-Derived Suppressor Cell Populations in the Bone Marrow

BACKGROUND AND OBJECTIVES: Patients suffer from long-term diabetes can result in severe complications in multiple organs through induction of vascular dysfunctions. However, the effects of chronic hyperglycemic conditions on hematopoiesis and the microenvironment in the bone marrow (BM) are not yet well understood. METHODS: BM cells were harvested from femurs of mice and analyzed using flow cytometry. Human PVCs were cultured in serum-free α-MEM. After 24hrs, PVC-CM was collected and filtered through a 0.22 μm filter. RESULTS: In this study, we showed that hyperglycemia alters hematopoietic composition in the BM, which can partially be restored via paracrine mechanisms, including perivascular cells (PVCs) and NADPH oxidase (NOX) inhibition in mice with streptozotocin-induced diabetes. Prolonged hyperglycemic conditions resulted in an increase in the frequency and number of long-term hematopoietic stem cells as well as the number of total BM cells. The altered hematopoiesis in the BM was partially recovered by treatment with PVC-derived conditioned medium (CM). Long-term diabetes also increased the number of myeloid-derived suppressor cells in the BM, which was partially restored by the administration of PVC-CM and diphenyleneiodonium (DPI), a NOX inhibitor. We further showed the downregulation of ERK and p38 phosphorylation in BM cells of diabetic mice treated with PVC-CM and DPI. This may be associated with dysfunction of hematopoietic cells and promotion of subsequent diabetic complications. CONCLUSIONS: Our data suggested that alterations in BM hematopoietic composition due to prolonged hyperglycemic conditions might be restored by improvement of the hematopoietic microenvironment and modulation of NOX activity.

Antepenultimate residue at the C-terminus of NADPH oxidase RBOHD is critical for its function in the production of reactive oxygen species in Arabidopsis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Production of reactive oxygen species (ROS) is a conserved immune response primarily mediated by NADPH oxidases (NOXs), also known in plants as respiratory burst oxidase homologs (RBOHs). Most microbe-associated molecular patterns (MAMPs) trigger a very fast and transient ROS burst in plants. However, recently, we found that lipopolysaccharides (LPS), a typical bacterial MAMP, triggered a biphasic ROS burst. In this study, we isolated mutants defective in LPS-triggered biphasic ROS burst (delt) in Arabidopsis, and cloned the DELT1 gene that was shown to encode RBOHD. In the delt1-2 allele, the antepenultimate residue, glutamic acid (E919), at the C-terminus of RBOHD was mutated to lysine (K). E919 is a highly conserved residue in NADPH oxidases, and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease. Consistently, we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure. It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein's stability and complex assembly. However, we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association, suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs. Taken together, our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.

Significance of NADPH oxidases Nox2 and Nox4 expression in DSS induced mouse colitis / 安徽医科大学学报

Objective To investigate the expression and significance of NADPH oxidases Nox2 and Nox4 in mouse colitis. Methods Mouse colitis model was established by using six-to-eight-week-old 129S /SV mice. Mice were randomly divided into 3 groups: control group,1. 5％ dextran sulfate sodium (DSS) group and 3. 0％ DSS group (n = 10 for each group). All of them were fed for 7 days to adapt to the environment. After then,the control group was given drinking water only,colitis was induced by giving drinking water consisted of 1. 5％ DSS or 3. 0％ DSS for 6 days. Weight loss,disease activity index (DAI) and histology were used to quantify the severity of colon inflammation. Oxidative stress indicator,malondialdehyde (MDA) in serum was measured by biochemical methods. The mRNA levels of pro-inflammation cytokines (IL-1β,IL-6 and TNF-α) were quantified by real-time PCR. The protein and mRNA expression of Nox2 and Nox4 in colon tissue of mice was evaluated by immunohistochemistry and real-time PCR,respectively. Results There was no colitis in the control group,while mild and severe enteritis was found in mice in the 1. 5％ DSS group and 3. 0％ DSS group,respectively. The number of goblet cells was decreased significantly in the 1. 5％ DSS group than that of control group (P < 0. 05),and further reduced in the 3. 0％ DSS group (P < 0. 05). MDA was enhanced along with the increased concentration of DSS (P < 0. 05 for both). The expression of Nox2 and Nox4 protein and mRNA was different with the severity of inflammation. The expression of protein and mRNA of both Nox2 and Nox4 were increased in 1. 5％ DSS group compared with the control group (P < 0. 05),and further reduced in the 3. 0％ DSS group (P < 0. 05). Nox2 mostly expressed in the phagocytes and neutrophils; Nox4 mostly expressed in the neutrophils and lymphocytes. Conclusion Nox2 and Nox4 play an important role in the occurrence of mouse colitis.

Salvianolic acid B regulates bone metabolism through Nox4 pathway in HFD mice / 中国药理学通报

Aim To evaluate the effects of salvianolic acid B ( Sal B ) on bone metabolism and its potential mechanism in high fat diet ( HFD) mice.Methods Thirty C57BL/6J male mice were divided into three groups with 10 mice each, namely normal , HFD and HFD+Sal B.HFD and HFD+Sal B mice were treated with HFD, and HFD+Sal B group mice were also with Sal B (125 mg· kg -1· d-1).After 12 weeks' treat-ment, femurs were harvested .The effects of Sal B on biomechanical strength were evaluated by biomechani-cal tests, and the effects of Sal B on bone microstruc-ture were evaluated by Safranin O/fast green staining and hematoxylin and eosin staining .The expression of nuclear factor-kappa B ( NF-κB)-p65 and NADPH ox-idase 4 ( Nox4 ) and cathepsin K in femurs was deter-mined by immunohistochemical staining . Results Maximum load and elastic load significantly decreased ,and the trabeculae became thinner and irregular in the femurs of HFD mice , while Sal B treatment could re-verse the descending biomechanical strength and the disorganized femurs bone micro-structures in HFD mice.In addition, the expressions of Nox4, NF-κB-p65 and cathepsin Kmarkedly increased in HFD mice , and Sal B possessed the ability to down-regulate the ex-pression of Nox4, NF-κB-p65, and cathepsin K in the femurs triggered by HFD .Conclusions Sal B treat-ment improves bone metabolism via regulating Nox 4/NF-κB/cathepsin K signaling pathway in HFD mice . The findings contribute to the understanding and exten-sion of the applications of Salvia miltiorrhiza and its constituents on osteoporosis .

Lignans with NADPH Oxidase 2 (NOX2)-inhibitory Activity from the Fruits of Schisandra chinensis

An isoform of NADPH oxidase (NOX), NOX2 is a superoxide-generating enzyme involved in diverse pathophysiological events. Although its potential as a therapeutic target has been validated, there is no clinically available inhibitor. Herein, NOX2-inhibitory activity was screened with the constituents isolated from Schisandra chinensis, which has been reported to have antioxidant and reactive oxygen species (ROS)-scavenging effects. Among the partitions prepared from crude methanolic extract, a chloroform-soluble partition showed the highest NOX2-inhibitory activity in PLB-985 cell-based NOX2 assay. A total of twenty nine compounds (1 – 29) were identified from the chloroform fraction, including two first isolated compounds; dimethyl-malate (25) and 2-(2-hydroxyacetyl) furan (27) from this plants. Of these constituents, two compounds (gomisin T, and pregomisin) exhibited an NOX2-inhibitory effect with the IC₅₀ of 9.4 ± 3.6, and 62.9 ± 11.3 µM, respectively. They are confirmed not to be nonspecific superoxide scavengers in a counter assay using a xanthine-xanthine oxidase system. These findings suggest the potential application of gomisin T (6) and other constituents of S. chinensis to inhibit NOX2.

Minimum inhibitory concentration of Brazilian Brachyspira hyodysenteriae strains / Concentração inibitória mínima de cepas Brachyspira hyodysenteriae brasileiras

The objectives of this study were to characterize Brachyspira hyodysenteriae isolates and to evaluate the antimicrobial susceptibility patterns of strains obtained from pigs in Brazil based on the minimal inhibitory concentration test (MIC). The MIC was performed for 22 B. hyodysenteriae isolates obtained from 2011 to 2013 using the following antimicrobial drugs: tylosin, tiamulin, valnemulin, doxycycline, lincomycin and tylvalosin. Outbreaks of swine dysentery were diagnosed based on clinical presentation, bacterial isolation, gross and microscopic lesions, duplex PCR for B. hyodysenteriae and B. pilosicoli and nox gene sequencing. All obtained MIC values were consistently higher or equal to the microbiological cut-off described in the literature. The MIC 90 values for the tested drugs were 8µg/ml for doxycycline, >4µg/ml for valnemulin, 8µg/ml for tiamulin, 32µg/ml for tylvalosin, >64µg/ml for lincomycin and >128µg/ml for tylosin. These results largely corroborate those reported in the literature. Tiamulin, doxycycline and tylvalosin showed the lowest MIC results. All of the samples subjected to phylogenetic analysis based on the nox gene sequence exhibited similar results, showing 100% identity to B. hyodysenteriae. This is the first study describing the MIC pattern of B. hyodysenteriae isolated in Brazil.(AU)

Os objetivos deste trabalho foram a caracterização de isolados de Brachyspira hyodysenteriae e avaliar os padrões de sensibilidade antimicrobiana de isolados obtidos a partir de suínos no Brasil com base no teste de concentração inibitória mínima (MIC). A MIC foi realizada em 22 isolados de B. hyodysenteriae obtidos entre 2011 a 2013 usando os seguintes antimicrobianos: tilosina, tiamulina, valnemulina, doxiciclina, lincomicina e tilvalosina. Surtos de disenteria suína foram diagnosticados com base na apresentação clínica, isolamento bacteriano, lesões macroscópicas e microscópicas, PCR duplex para B. hyodysenteriae e B. pilosicoli e sequenciamento do gene nox. Todos os valores de MIC obtidos foram consistentemente mais elevados ou igual ao ponto de corte microbiológica descrito na literatura. Os valores de MIC 90 para os fármacos testados foram de 8 µg / mL para a doxiciclina, > 4 µg/ml de valnemulina, 8 µg / mL para a tiamulina, 32 µg / ml para tilvalosina, > 64 µg / ml para a lincomicina e > 128 µg / ml de tilosina. Estes resultados corroboram em grande parte com os relatados na literatura. Tiamulina, doxiciclina e tilvalosina apresentaram os menores resultados de MIC. Todas as amostras submetidas à análise filogenética com base na sequência do gene nox exibiram resultados semelhantes, indicando 100% de identidade com B. hyodysenteriae. Este é o primeiro estudo que descreve o padrão MIC de B. hyodysenteriae isoladas no Brasil.(AU)

Expression of NADPH Oxidase2 in gastric cancer and its correlation with the vascular endothelial growth factor level / 中国现代普通外科进展

Objective:To investigate the expression of NADPH Oxidase2 (NOX2) in gastric cancer tissues and its correlation with vascular endothelial growth factor(VEGF) level.Methods:The gastric cancer tissue and the adjacent tumor tissue were obtained from the patients who received radical operation for gastric cancer during July 2014 to July 2015 in Provincial Hospital Affiliated to Shandong University.The expression of NOX2 in the tumor tissue and the adjacent tissue were detected by the immunohistochemistry(IHC) and Western blot.The VEGF level were detected by IHC in gastric cancer tissues.The spearman rank correlation were used to detected the correlation between the NOX2 and VEGF.Results:The positive expression rate of NOX2 in gastric cancer tissue was 47.2％ (58/123),and the positive expression rate in the adjacent tissue was 8.13％ (10/123),mostly expression in the adjacent was weak positive.The outcome of Western blot show that the NOX2 was up-regulated in the tumor tissue compare to the adjacent tissue [39.0％(48/123)].The expression of NOX2 and the relationship of clinical-pathology showed the expression of NOX2 had no correlation with gender,age,differentiation of tumor (X2 value were 0.852,0.150,5.062,P＞0.05).The result of spearman rank correlation showed that the NOX2 was positively correlated with that of VEGF.Conclusion:NOX2 plays an important role in the genesis and development in the gastric cancer,the expression of NOX2 was closely correlated with the VEGF.