Therapeutic potential of NADH: in neurodegenerative diseases characterizde by mitochondrial dysfunction / 临床耳鼻咽喉头颈外科杂志

Nicotinamide adenine dinucleotide（NADH） in its reduced form of is a key coenzyme in redox reactions, essential for maintaining energy homeostasis.NADH and its oxidized counterpart, NAD+, form a redox couple that regulates various biological processes, including calcium homeostasis, synaptic plasticity, anti-apoptosis, and gene expression. The reduction of NAD+/NADH levels is closely linked to mitochondrial dysfunction, which plays a pivotal role in the cascade of various neurodegenerative disorders, including Parkinson's disease and Alzheimer's disease.Auditory neuropathy（AN） is recognized as a clinical biomarker in neurodegenerative disorders. Furthermore, mitochondrial dysfunction has been identified in patients with mutations in genes like OPA1and AIFM1. However, effective treatments for these conditions are still lacking. Increasing evidence suggests that administratering NAD+ or its precursors endogenously may potentially prevent and slow disease progression by enhancing DNA repair and improving mitochondrial function. Therefore, this review concentrates on the metabolic pathways of NAD+/NADH production and their biological functions, and delves into the therapeutic potential and mechanisms of NADH in treating AN.

Electroacupuncture Improves Blood-Brain Barrier and Hippocampal Neuroinflammation in SAMP8 Mice by Inhibiting HMGB1/TLR4 and RAGE/NADPH Signaling Pathways / 中国结合医学杂志

OBJECTIVE@#To investigate the molecular mechanisms underlying the beneficial effect of electroacupuncture (EA) in experimental models of Alzheimer's disease (AD) in vivo.@*METHODS@#Senescence-accelerated mouse prone 8 (SAMP8) mice were used as AD models and received EA at Yingxiang (LI 20, bilateral) and Yintang (GV 29) points for 20 days. For certain experiments, SAMP8 mice were injected intravenously with human fibrin (2 mg). The Morris water maze test was used to assess cognitive and memory abilities. The changes of tight junctions of blood-brain barrier (BBB) in mice were observed by transmission electron microscope. The expressions of fibrin, amyloid- β (Aβ), and ionized calcium-binding adapter molecule 1 (IBa-1) in mouse hippocampus (CA1/CA3) were detected by reverse transcription-quantitative polymerase chain reaction (qRT-PCR), Western blot or immunohistochemical staining. The expression of fibrin in mouse plasma was detected by enzyme-linked immunosorbent assay. The expressions of tight junction proteins zonula occludens-1 and claudin-5 in hippocampus were detected by qRT-PCR and immunofluorescence staining. Apoptosis of hippocampal neurons was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining.@*RESULTS@#Fibrin was time-dependently deposited in the hippocampus of SAMP8 mice and this was inhibited by EA treatment (P<0.05 or P<0.01). Furthermore, EA treatment suppressed the accumulation of Aβ in the hippocampus of SAMP8 mice (P<0.01), which was reversed by fibrin injection (P<0.05 or P<0.01). EA improved SAMP8 mice cognitive impairment and BBB permeability (P<0.05 or P<0.01). Moreover, EA decreased reactive oxygen species levels and neuroinflammation in the hippocampus of SAMP8 mice, which was reversed by fibrin injection (P<0.05 or P<0.01). Mechanistically, EA inhibited the promoting effect of fibrin on the high mobility group box protein 1 (HMGB1)/toll-like receptor 4 (TLR4) and receptor for advanced glycation end products (RAGE)/nicotinamide adenine dinucleotide phosphate (NADPH) signaling pathways (P<0.01).@*CONCLUSION@#EA may potentially improve cognitive impairment in AD via inhibition of fibrin/A β deposition and deactivation of the HMGB1/TLR4 and RAGE/NADPH signaling pathways.

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.

AIFM1 variants associated with auditory neuropathy spectrum disorder cause apoptosis due to impaired apoptosis-inducing factor dimerization / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Auditory neuropathy spectrum disorder (ANSD) represents a variety of sensorineural deafness conditions characterized by abnormal inner hair cells and/or auditory nerve function, but with the preservation of outer hair cell function. ANSD represents up to 15% of individuals with hearing impairments. Through mutation screening, bioinformatic analysis and expression studies, we have previously identified several apoptosis-inducing factor (AIF) mitochondria-associated 1 (AIFM1) variants in ANSD families and in some other sporadic cases. Here, to elucidate the pathogenic mechanisms underlying each AIFM1 variant, we generated AIF-null cells using the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and constructed AIF-wild type (WT) and AIF-mutant (mut) (p.‍T260A, p.‍R422W, and p.‍R451Q) stable transfection cell lines. We then analyzed AIF structure, coenzyme-binding affinity, apoptosis, and other aspects. Results revealed that these variants resulted in impaired dimerization, compromising AIF function. The reduction reaction of AIF variants had proceeded slower than that of AIF-WT. The average levels of AIF dimerization in AIF variant cells were only 34.5%‍‒‍49.7% of that of AIF-WT cells, resulting in caspase-independent apoptosis. The average percentage of apoptotic cells in the variants was 12.3%‍‒‍17.9%, which was significantly higher than that (6.9%‍‒‍7.4%) in controls. However, nicotinamide adenine dinucleotide (NADH) treatment promoted the reduction of apoptosis by rescuing AIF dimerization in AIF variant cells. Our findings show that the impairment of AIF dimerization by AIFM1 variants causes apoptosis contributing to ANSD, and introduce NADH as a potential drug for ANSD treatment. Our results help elucidate the mechanisms of ANSD and may lead to the provision of novel therapies.

Roles of NAD / 中国生物化学与分子生物学报

Nicotinamide adenine dinucleotide (NAD

Protective Effects of Hu-Lu-Ba-Wan () against Oxidative Stress in Testis of Diabetic Rats through PKCα/NAPDH Oxidase Signaling Pathway / 中国结合医学杂志

OBJECTIVE@#To explore the protective effect and the underlying mechanism of Hu-Lu-Ba-Wan (, HLBW) on the testis of diabetic rats.@*METHODS@#Twenty-four male Wistar rats (160-180 g) were randomly divided into 3 groups according to a random number table, including a control group (n=8), diabetic group (n=8), and HLBW group (n=8). Diabetic rat model was established by high-fat-diet administration and single intravenous injection of streptozotocin (26 mg/kg). Then HLBW granule was administrated for 12 weeks. Fasting blood glucose and insulin levels as well as serum total testosterone level and testicular testosterone content were examined. Oxidative stress markers in both serum and testis were tested. Meanwhile, testicular morphology was observed under hematoxylin and eosin (HE) and the ultrastructure of Leydig cell was observed by electron microscope. The superoxide anion level was detected by DHE, and TUNEL-positive cells of testis was evaluated by TUNEL assay. The gene and protein expression of protein kinase C (PKCα), phosphorylated PKCα (P-PKCα) and P47phox in testicular tissues were determined by quantitative RT-PCR analysis and Western bolt analysis.@*RESULTS@#Compared with the diabetic group, HLBW treatment significantly reduced the fasting glucose levels and increased the levels of fasting insulin and testosterone in serum (P<0.01). HLBW administration also reduced the levels of reactive oxygen species (ROS) in plasma and alleviated the damage of oxidative stress in the testis of diabetic rats. Additionally, HLBW down-regulated the protein and mRNA levels of PKCα, P-PKCα and P47phox in testicular tissues.@*CONCLUSION@#HLBW may attenuate the oxidative stress in the testis of diabetic rats via PKCα /NAPDH oxidase signaling pathway.

Effects of NF-κB/NOX1 signaling pathway in TNF-α-induced apoptosis of A549 cells / 中国职业医学

OBJECTIVE: To investigate the effects of nuclear transcription factor-κB(NF-κB)/amide adenine dinucleotide phosphate oxidase 1(NOX1) signaling pathway in tumor necrosis factor-α(TNF-α) induced apoptosis of A549 cells. METHODS: i) A549 cells were stimulated with TNF-α at the concentrations of 0.00, 0.25, 0.50, and 1.00 nmol/L. CCK-8 assay was used to detect the cell viability to screen the optimal stimulating concentration of TNF-α. ii) A549 cells at logarithmic growth stage were randomly divided into four groups, the control group, the TNF-α group, the BAY11-7082(NF-κB inhibitor) group and the TNF-α+BAY11-7082 group. The cells in the control group were not treated. The TNF-α and BAY11-7082 groups were stimulated with 0.50 nmol/L TNF-α and 5 μmol/L BAY11-7082, respectively. The TNF-α+BAY11-7082 group was stimulated by both TNF-α and BAY11-7082. After 24 hours of culture, the cell survival rate was detected by CCK-8 assay. Flow cytometry was used to detect cell apoptotic rate, and Western blotting was used to detect the relative expression of NF-κB(p65) and NOX1 proteins. RESULTS: i) When A549 cells were stimulated with TNF-α at the concentration of 0.50 nmol/L, the cell proliferative activity was reduced and the cell apoptosis was promoted. This concentration was selected as the stimulation dose of TNF-α in subsequent experiments. ii) The survival rate of A549 cells in the TNF-α group decreased(P<0.05), the apoptotic rate and the protein expressions of NF-κB(p65) and NOX1 increased in TNF-α group(all P<0.05) compared with the control group. In BAY11-7082 group, the survival rate and the relative expression of NF-κB(p65) and NOX1 of A549 cells were decreased(all P<0.05), and the apoptotic rate of A549 cells was increased(P<0.05) compared with the control group. A549 cells in TNF-α+BAY11-7082 group changed from a long spindle shape to an irregular one. The cell survival rate increased(P<0.05), the apoptotic rate and the relative expression of NF-κB(p65) and NOX1 decreased(all P<0.05) compared with the TNF-α group. CONCLUSION: NF-κB/NOX1 signaling pathway is involved in A549 cells apoptosis induced by TNF-α.

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.

Inhibitory effects of NADPH oxidase 4 inhibitor on VEGF expression in hypoxia-induced human RPE cells / 中华实验眼科杂志

Objective@#To observe the inhibitory effects of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) on vascular endothelial growth factor (VEGF) expression in hypoxia-induced human retinal pigment epithelial cells.@*Methods@#The cultured APRE-19 cells were divided into Avastin group and VAS2870 group, and then the Avastin group was subdivided into the normoxic control group, hypoxia control group, 0.25 mg/ml Avastin intervention group, 0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group, and the VAS2870 group was subdivided into 1 μmol/ml VAS2870 intervention group, 3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group.CoCl2 of final concentration of 300 mol/L was added to the medium to establish the cytochemical hypoxia model.The expressions of NOX4 and VEGF in human retinal pigment epithelial cells were located and evaluated by immunofluorescence staining, and relative expressing levels of NOX4 and VEGF proteins were compared by Western blot assay.@*Results@#The relative expression of NOX4 was 0.657±0.153, 1.000±0.200, 1.206±0.300, 1.260±0.200 and 1.413±0.273, and the relative expression of VEGF-A was 0.821±0.110, 1.210±0.100, 0.672±0.100, 0.340±0.120 and 0.300±0.130 in the normoxic control group, hypoxia control group, 0.25 mg/ml Avastin intervention group, 0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group, respectively, with statistically significant differences among the groups (F=17.631, P<0.001; F=4.777, P<0.05). The relative expression of NOX4 protein in 0.75 mg/ml Avastin intervention group was significantly lower than that in normoxia control group (P<0.001). The relative expression of VEGF-A protein in the cells of the 0.25, 0.50 and 0.75 mg/ml Avastin intervention group was significantly lower than that in hypoxia control group (P<0.05). The expression of NOX4 protein in the cells was 0.970±0.120, 1.060±0.130, 0.880±0.130, 0.567±0.135 and 0.450±0.120, and the relative expression of VEGF-A protein was 0.387±0.135, 0.627±0.125, 0.370±0.140, 0.363±0.140 and 0.160±0.100 in the normoxia control group, hypoxia control group, 1 μmol/ml VAS2870 intervention group, 3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group, respectively, with statistically significant differences among them (F=12.933, P<0.001; F=4.948, P<0.05). The relative expression of VEGF-A protein in the 1, 3 and 5 μmol/ml VAS2870 intervention group was significantly lower than that in hypoxia control group (P<0.05).@*Conclusions@#NOX4 inhibitor can inhibit the expression of VEGF-A protein in hypoxia-induced human RPE cells by down-regulating the NOX4 level.

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.

Effects of nicotinamide mononucleotide adenylyl transferase 3 on mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells via regulating nicotinamide adenine dinucleotide levels / 中国修复重建外科杂志

Objective: To investigate the effect of nicotinamide mononucleotide adenosyl transferase 3 (NMNAT3) on the mitochondrial function and anti-oxidative stress of rabbit bone marrow mesenchymal stem cells (BMSCs) under oxidative stress in vitro by regulating nicotinamide adenine dinucleotide (NAD +) levels. Methods: The bone marrow of femur and tibia of New Zealand white rabbits were extracted. BMSCs were isolated and cultured in vitro by density gradient centrifugation combined with adherent culture. The third generation cells were identified by flow cytometry and multi-directional induction. Overexpression of NMNAT3 gene was transfected into rabbit BMSCs by enhanced green fluorescent protein (EGFP) labeled lentivirus (BMSCs/Lv-NMNAT3-EGFP), and then the expression of NMNAT3 was detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot and cell proliferation by cell counting kit 8 (CCK-8) method. BMSCs transfected with negative lentivirus (BMSCs/Lv-EGFP) and untransfected BMSCs were used as controls. The oxidative stress injury cell model was established by using H 2O 2 to treat rabbit BMSCs. According to the experimental treatment conditions, they were divided into 4 groups: Group A was normal BMSCs without H 2O 2 treatment; untransfected BMSCs, BMSCs/Lv-EGFP, and BMSCs/Lv-NMNAT3-EGFP in groups B, C, and D were treated with H 2O 2 simulated oxidative stress, respectively. The effects of NMNAT3 on the mitochondrial function of BMSCs under oxidative stress [changes of mitochondrial membrane potential, NAD + and adenosine triphosphate (ATP) levels], the changes of anti-oxidative stress ability of BMSCs [reactive oxygen species (ROS) and malondialdehyde (MDA) levels, manganese superoxide dismutase (Mn-SOD) and catalase (CAT) activities], and the effects of BMSCs on senescence and apoptosis [senescence associated-β-galactosidase (SA-β-gal) staining and TUNEL staining] were detected after 24 hours of treatment. Results: The rabbit BMSCs were successfully isolated and cultured in vitro. The stable strain of rabbit BMSCs with high expression of NMNAT3 gene was successfully obtained by lentiviral transfection, and the expressions of NMNAT3 gene and protein significantly increased ( P<0.05). There was no significant difference in the trend of cell proliferation compared with normal BMSCs. After treatment with H 2O 2, the function of mitochondria was damaged and apoptosis increased in all groups. However, compared with groups B and C, the group D showed that the mitochondrial function of BMSCs improved, the membrane potential increased, the level of NAD + and ATP synthesis of mitochondria increased; the anti-oxidative stress ability of BMSCs enhanced, the levels of ROS and MDA decreased, and the activities of antioxidant enzymes (Mn-SOD, CAT) increased; and the proportion of SA-β-gal positive cells and the rate of apoptosis decreased. The differences in all indicators between group D and groups B and C were significant ( P<0.05). Conclusion: NMNAT3 can effectively improve the mitochondrial function of rabbit BMSCs via increasing the NAD + levels, and enhance its anti-oxidative stress and improve the survival of BMSCs under oxidative stress conditions.

Inhibitory effects of NADPH oxidase 4 inhibitor on VEGF expression in hypoxia-induced human RPE cells / 中华实验眼科杂志

Objective To observe the inhibitory effects of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) on vascular endothelial growth factor (VEGF) expression in hypoxia-induced human retinal pigment epithelial cells.Methods The cultured APRE-19 cells were divided into Avastin group and VAS2870 group,and then the Avastin group was subdivided into the normoxic control group,hypoxia control group,0.25 mg/ml Avastin intervention group,0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group,and the VAS2870 group was subdivided into 1 μmol/ml VAS2870 intervention group,3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group.CoCl2 of final concentration of 300 mol/L was added to the medium to establish the cytochemical hypoxia model.The expressions of NOX4 and VEGF in human retinal pigment epithelial cells were located and evaluated by immunofluorescence staining,and relative expressing levels of NOX4 and VEGF proteins were compared by Western blot assay.Results The relative expression of NOX4 was 0.657± 0.153,1.000±0.200,1.206 ± 0.300,1.260± 0.200 and 1.413 ± 0.273,and the relative expression of VEGF-A was 0.821±0.110,1.210±0.100,0.672±0.100,0.340±0.120 and 0.300±0.130 in the normoxic control group,hypoxia control group,0.25 mg/ml Avastin intervention group,0.50 mg/ml Avastin intervention group and 0.75 mg/ml Avastin intervention group,respectively,with statistically significant differences among the groups (F =17.631,P＜ 0.001;F=4.777,P＜0.05).The relative expression of NOX4 protein in 0.75 mg/ml Avastin intervention group was significantly lower than that in normoxia control group (P＜0.001).The relative expression of VEGF-A protein in the cells of the 0.25,0.50 and 0.75 mg/ml Avastin intervention group was significantly lower than that in hypoxia control group (P＜0.05).The expression of NOX4 protein in the cells was 0.970±0.120,1.060±0.130,0.880±0.130,0.567±0.135 and 0.450±0.120,and the relative expression of VEGF-A protein was 0.387±0.135,0.627±0.125,0.370±0.140,0.363±0.140 and 0.160±0.100 in the normoxia control group,hypoxia control group,1 μmol/ml VAS2870 intervention group,3 μmol/ml VAS2870 intervention group and 5 μmol/ml VAS2870 intervention group,respectively,with statistically significant differences among them (F =12.933,P＜ 0.001;F =4.948,P＜ 0.05).The relative expression of VEGF-A protein in the 1,3 and 5 μmol/ml VAS2870 intervention group was significantly lower than that in hypoxia control group (P＜0.05).Conclusions NOX4 inhibitor can inhibit the expression of VEGF-A protein in hypoxia-induced human RPE cells by down-regulating the NOX4 level.

‘Obesageing’: Linking obesity & ageing

Obesity is one of the leading causes of preventable mortalities in many parts of the globe. The rise in geriatric population due to better treatment opportunities has also emerged as a major public health challenge. Both of these health challenges have impacted developed as well as developing countries. Obesity is attributed as a powerful risk factor of a variety of health problems such as cardiovascular diseases, hypertension, type 2 diabetes, dementia, neuropsychiatric diseases and many more. On the other hand, ageing is a natural process involving a gradual decline in physiological functions and is associated with similar co-morbidities as obesity. This review discusses about the commonalities (termed as ‘Obesageing') between the pathological phenomenon of obesity and normal physiological process of ageing. A unique rodent model of obesageing has been developed (WNIN/Ob) that has characteristics of morbid obesity as well as premature ageing. Such a novel animal model would facilitate the understanding of the complex interplay of different mechanisms that are common to obesity and ageing and help to devise strategies in future to tackle the growing burden of obesity and ageing.

The role of nicotinamide-adenine dinucleotide phosphate oxidase NOX family in acute lung injury / 中华危重病急救医学

Acute?lung?injury?(ALI)?and?its?severe?form,?acute?respiratory?distress?syndrome?(ARDS),?are?common?critical?syndromes.?The?causes?of?the?syndrome?are?complex?and?diverse.?The?main?pathological?features?are?the?diffuse?inflammatory?and?protein-rich?pulmonary?edema?caused?by?destruction?of?the?blood-air?barrier.?Reactive?oxygen?species?(ROS)?mediate?oxidative?damage?by?oxidizing?bio-macromolecules,?including?lipids,?proteins?and?nucleic?acid.?Among?many?systems?producing?ROS,?nicotinamide-adenine?dinucleotide?phosphate?(NADPH)?oxidase-mediated?ROS?is?the?main?source,?and?its?functional?subunit?is?the?transmembrane?subunit?NOX?family.?The?distribution?of?NOX?family?proteins?in?lung?tissue?is?cell?type?dependent.?NOX-derived?ROS?is?involved?in?the?defense?function?of?lung?tissue?and?related?to?the?occurrence?and?development?of?ALI/ARDS.?This?review?mainly?describes?the?cell?distribution,?activation?factors,?and?its?relationship?with?the?occurrence?and?development?of?ALI?of?the?NOX?family.

Adrenomedullin and bronchopulmonary dysplasia / 国际儿科学杂志

Bronchopulmonary dysplasia (BPD) is a common respiratory disease in premature infants characterized by alveolar and pulmonary vascular dysplasia.There are currently no effective preventive and therapeutic measures.Adrenomedullin (ADM) is a vasodilator peptide produced by endothelial and smooth muscle cells of the systemic and pulmonary circulation,which promotes alveolar growth and pulmonary angiogenesis.ADM protects the respiratory system through three signaling pathways:(1) it promotes pulmonary vascular endothelial cell proliferation through extracellular regulated protein kinases (ERK);(2) attenuates lung cell apoptosis and improves cell proliferation through protein kinase B (PKB) pathway;(3) it down-regulates NADPH oxidase 4 (NOX4) expression in lung tissue to inhibit oxidative stress and improves BPD.So ADM signaling axis may be a potential therapeutic target for human infants with BPD.It provides theoretical basis for clinical prevention and treatment of BPD in premature infants.

Effect of benazepril on nuclear factor E2 related factor 2, nicotinamide adenine dinucleotide phosphate oxidase and reactive oxygen species in rats with hepatic fibrosis / 中华肝脏病杂志

Objective@#To study the effect of benazepril on the expression of nuclear factor E2 related factor 2 (Nrf2), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and reactive oxygen species (ROS) concentration in rats with hepatic fibrosis and to explore the possible antifibrotic mechanism of benazepril.@*Methods@#Twenty-two healthy male Sprague-Dawley rats were randomly divided into 3 groups: control group (6 rats), model group (8 rats) and benazepril treatment group (8 rats). Two rats died during modeling and treatment in the model group and the benazepril treatment group, and a model of hepatic fibrosis induced by carbon tetrachloride (CCL4) was established. The rats in benazepril group were given benazepril for 8 weeks by gastric gavage. The assessment of liver tissue damage in each group was measured using conventional hematoxylin-eosin and Masson staining. The mRNA level of Nrf2, NOX4 in liver tissue was detected by RT-PCR, and serum ROS concentration was determined by colorimetry. All data were expressed in mean ± standard deviations, and were analyzed using SPSS21.0 statistical software. The data were compared using one-way analysis of variance, and the LSD-t method was used for pairwise comparison between the two groups. The correlation analysis was performed by Spearman’s correlation analysis.@*Results@#In the liver of the model group, with the aggravation of liver fibrosis the expression of Nrf2mRNA, NOX4 mRNA and ROS concentration were higher than control group [(4.01 ± 3.40), (31.78 ± 3.96), (1.82 ± 0.46) μg/ ml vs. (0.12 ± 0.11), (2.03 ± 0.31), (1.56±0.84) μg/ml, P < 0.05]. After benazepril treatment, NOX4 mRNA expression and ROS concentration were decreased than the model group [(15.93 ± 5.01), (0.78 ± 0.44) μg/ml vs. (31.78 ± 3.96), (1.82 ± 0.46) μg /ml, P < 0.05], while Nrf2 mRNA expression was higher than the model group [(6.69 ± 4.86) vs. (4.01 ± 3.40), P < 0.05]. There was a positive correlation between Nrf2 and NOX4, Nrf2 and ROS, and NOX4 and ROS (r = 0.616, 0.411, 0.802, P < 0.05).@*Conclusion@#Benazepril may exert an anti-hepatic fibrosis effect by activating Nrf2 expression, or may inhibit the ROS-mediated oxidative stress in response to NOX4.

Recent trends in two-photon auto-fluorescence lifetime imaging (2P-FLIM) and its biomedical applications

Two photon fluorescence microscopy and the numerous technical advances to it have served as valuable tools in biomedical research. The fluorophores (exogenous or endogenous) absorb light and emit lower energy photons than the absorption energy and the emission (fluorescence) signal is measured using a fluorescence decay graph. Additionally, high spatial resolution images can be acquired in two photon fluorescence lifetime imaging (2P-FLIM) with improved penetration depth which helps in detection of fluorescence signal in vivo. 2P-FLIM is a non-invasive imaging technique in order to visualize cellular metabolic, by tracking intrinsic fluorophores present in it, such as nicotinamide adenine dinucleotide, flavin adenine dinucleotide and tryptophan etc. 2P-FLIM of these molecules enable the visualization of metabolic alterations, non-invasively. This comprehensive review discusses the numerous applications of 2P-FLIM towards cancer, neuro-degenerative, infectious diseases, and wound healing.

Enzyme histochemical detection of cell viability in ablation area of thyroid nodules at 6 months after microwave ablation / 第二军医大学学报

Objective To explore the cell viability in the ablation area of thyroid nodules at 6 months after microwave ablation by enzyme histochemical staining. Methods Twenty-four ablation areas of thyroid nodules were selected from 20 patients who underwent histopathological assessment of the ablation area by core needle biopsy at 6 months after microwave ablation between Dec. 2017 and Sep. 2018. Core needle biopsy was performed at the central and marginal regions of the ablation area with a cutting biopsy needle. The specimens were obtained and placed in liquid nitrogen to make frozen sections. Enzyme histochemical staining was used to detect the activities of succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), and the difference of cell morphology and histological structure was compared with H-E staining results. Results The specimens of the central and marginal regions of 24 ablation areas were successfully obtained. The histochemical staining of SDH and NADPH-d in the central region of ablation area had good consistency, and the negative rates were both 95.83% (23/24). The histochemical staining of SDH and NADPH-d in the marginal region of ablation area also had good consistency, and the negative rates were both 91.67% (22/24). H-E staining of 23 central regions and 22 marginal regions showed pink amorphous mass of necrosis. H-E staining of 1 central region and 2 marginal regions showed partly necrotic and fibrous tissue hyperplasia. The location of fibrous tissue hyperplasia was consistent with the location of the positive region of enzyme histochemical staining. Conclusion At 6 months after microwave ablation, the tissue in the ablation area of thyroid nodules is consistent with coagulative necrosis, and is still inactivated. SDH or NADPH-d enzyme histochemical staining combined with H-E staining can objectively evaluate the old ablation area.

Correlation between mRNA and protein expression of quinine nicotinamide adenine dinucleotide dehydrogenase 1 and heme oxygenase 1 and liver injury due to coal arsenic poisoning / 中华地方病学杂志

Objective To detect the mRNA and protein expression of downstream quinine nicotinamide adenine dinucleotide (NADH) dehydrogenase 1 (NQO1) and heme oxygenase 1 (HO-1) induced by blood nuclearrelated factor E2 (Nrf2) in the peripheral blood of those exposed to arsenic in the endemic area of coal arsenic poisoning in Guizhou Province,and to discuss its role in the process of occurrence and development of liver injury due to coal arsenic poisoning.Methods Jiaole and Changqing villages in coal-burning-borne arsenism areas in Xingren County of Guizhou were selected as the survey sites,and 161 cases of arsenic-exposed residents were selected as the arsenic exposed group based on physical examination.They were divided into non-patient group (21 cases) and patient group (140 cases) according to the Diagnostic Criteria of Endemic Arsenism (WS/T 211-2001),and the patient group was further divided into mild hepatosis group (52 cases),moderately severe hepatosis group (36 cases) and non-apparent hepatosis group (52 cases) according to the Diagnostic Criteria of Occupational Chronic (GBZ 59-2010).Moreover,45 residents from one village neighboring to non-epidemic area were selected as controls.Peripheral blood samples were collected from all subjects.And mRNA expression of NQO1 and HO-1 were detected by RT-qPCR.Content of NQQ1 and HO-1 were detected by enzyme linked immunosorbent assay (ELISA).Results (①)Results of mRNA expression of NQ01 and HO-1:the relative expression level of mRNA of NQO1 and HO-1 in peripheral blood went up gradually as the degree of liver injury of population exposed to arsenic increased (F =5.548,10.961,all P ＜ 0.05);thereinto,the relative expression level of mRNA of NQO1 of mild hepatosis group (median:0.918) was higher than that of the control group (0.576,P ＜ 0.05),the relative expression level of mRNA of NQO1 of moderately severe hepatosis group (1.243) was higher than those of control group,non-patient group (0.653),non-apparent hepatosis group (0.636) and mild hepatosis group (all P ＜ 0.05);the relative expression level of mRNA of HO-1 of non-patient group (1.059),non-apparent hepatosis group (1.225),mild hepatosis group (1.553) and moderately severe hepatosis group (1.604) were higher than that of control group (0.767,all P ＜ 0.05);the relative expression level of mRNA of HO-1 of mild hepatosis group was higher than that of non-patient group (P ＜ 0.05);the relative expression level of mRNA of HO-1 of moderately severe hepatosis group was higher than those of non-patient group and non-apparent hepatosis group (all P ＜ 0.05).②)Results of protein expression of NQO-1 and HO-1:the level of protein expression of NQO1 and HO-1 in serum went up gradually as the degree of liver injury of population exposed to arsenic increased (F =19.685,17.725,all P ＜ 0.05).Thereinto,the protein expression of NQO1 and HO-1 of non-apparent hepatosis group,mild hepatosis group and moderately severe hepatosis group [NQO1:(6.272 ± 0.744),(6.336 ± 0.628),(6.714 ± 0.540) U/L;HO-1:(45.150 ± 4.813),(45.283 ± 5.049),(48.610 ± 5.365) U/L] were higher than those of control group and non-patient group [NQO1:(5.550 ± 0.730),(5.750 ± 0.427) U/L;HO-1:(39.856 ± 5.249),(42.375 ± 3.014) U/L,all P ＜ 0.05],the protein expression of NQO1 and HO-1 of moderately severe hepatosis group were higher than those of non-apparent hepatosis group and mild hepatosis group (all P ＜ 0.05).Conclusion The expression of mRNA and protein of NQO1 and HO-1 is closely related to the occurrence and development of liver injury due to arsenic exposure in coal arsenic poisoning areas.