OBJECTIVE: To explore the mechanism by which ω-3 polyunsaturated fatty acids (hereinafter referred to as "ω-3") exert antioxidant stress protection and promote osteogenic differentiation in MC3T3-E1 cells, and to reveal the relationship between ω-3 and the key antioxidant stress pathway involving nuclear factor E2-related factor 2 (Nrf2) and NAD (P) H quinone oxidoreductase 1 (NQO1) in MC3T3-E1 cells. METHODS: The optimal concentration of H ₂O ₂ (used to establish the oxidative stress model of MC3T3-E1 cells in vitro) and the optimal intervention concentrations of ω-3 were screened by cell counting kit 8. MC3T3-E1 cells were divided into blank control group, oxidative stress group (H ₂O ₂), low-dose ω-3 group (H ₂O ₂+low-dose ω-3), and high-dose ω-3 group (H ₂O ₂+high-dose ω-3). After osteoblastic differentiation for 7 or 14 days, the intracellular reactive oxygen species (ROS) level was measured by fluorescence staining and flow cytometry, and the mitochondrial morphological changes were observed by biological transmission electron microscope; the expression levels of Nrf2, NQO1, heme oxygenase 1 (HO-1), Mitofusin 1 (Mfn1), and Mfn2 were detected by Western blot to evaluate the cells' antioxidant stress capacity; the expression levels of Runt-related transcription factor 2 (RUNX2) and osteocalcin (OCN) were detected by immunofluorescence staining and Western blot; osteogenic potential of MC3T3-E1 cells was evaluated by alkaline phosphatase (ALP) staining and alizarin red staining. RESULTS: Compared with the oxidative stress group, the content of ROS in the low and high dose ω-3 groups significantly decreased, and the protein expressions of Nrf2, NQO1, and HO-1 significantly increased ( P<0.05). At the same time, the mitochondrial morphology of MC3T3-E1 cells improved, and the expressions of mitochondrial morphology-related proteins Mfn1 and Mfn2 significantly increased ( P<0.05). ALP staining and alizarin red staining showed that the low-dose and high-dose ω-3 groups showed stronger osteogenic ability, and the expressions of osteogenesis-related proteins RUNX2 and OCN significantly increased ( P<0.05). And the above results showed a dose-dependence in the two ω-3 treatment groups ( P<0.05). CONCLUSION ω-3 can enhance the antioxidant capacity of MC3T3-E1 cells under oxidative stress conditions and upregulate their osteogenic activity, possibly through the Nrf2/NQO1 signaling pathway.
Oxidative Stress/drug effects* ; NF-E2-Related Factor 2/metabolism* ; NAD(P)H Dehydrogenase (Quinone)/metabolism* ; Animals ; Mice ; Osteogenesis/drug effects* ; Cell Differentiation/drug effects* ; Fatty Acids, Omega-3/pharmacology* ; Signal Transduction/drug effects* ; Osteoblasts/drug effects* ; Reactive Oxygen Species/metabolism* ; Cell Line ; Hydrogen Peroxide/pharmacology* ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Antioxidants/pharmacology* ; Heme Oxygenase-1/metabolism*

β-Nicotinamide mononucleotide (NMN), as the precursor of nicotinamide adenine dinucleotide (NAD), plays an important role in enhancing NAD levels. Intake of NMN can alter the composition and vitality of gut microbiota, restore mitochondrial function, inhibit inflammatory pathways, improve metabolism, counteract oxidative stress, and alleviate inflammation. NMN significantly improves recovery from aging-related diseases, such as diminished heart function, reduced fertility, memory decline, and diabetes. NMN demonstrates both efficacy and safety in anti-aging. The use of NMN in China has gradually gained acceptance, highlighting the importance of exploring the mechanism of NMN in anti-aging effects and improving the biosynthesis of NMN. In addition, NMN in combination with stem cells hold promise in the treatment of aging-related degenerative diseases and promote overall human and animal health.
Humans ; Nicotinamide Mononucleotide/pharmacology* ; Aging/physiology* ; Animals ; Oxidative Stress/drug effects* ; Gastrointestinal Microbiome ; NAD/metabolism*

Bacterial Proteins ; Humans ; Metronidazole/pharmacology* ; Mycobacterium tuberculosis ; NAD ; Nitroreductases ; Tuberculosis, Lymph Node

Objective: A gradient stress model of PC12 cells induced by corticosterone was established to provide a basis for the evaluation and regulation of cell stress. Methods: The effect of corticosterone on cell viability was observed by measuring PC12 cell viability at different concentrations of corticosterone (0~1 000 μmol/L) after different intervention times (8~48 h) to screen the cell models for optimal intervention conditions. Key stress indicators (MDA, SOD, NADH, LDH) were measured spectrophotometrically and microscopically to evaluate the models. Results: When the concentration of corticosterone was below 200 μmol/L and the intervention time was 12 h, the cell viability was below half inactivation rate, which could reduce the confounding factors due to the decrease of cell viability in each group. Compared with the blank control group, corticosterone increased the levels of MDA, NADH and LDH,and decreased the levels of SOD in the model group in a concentration-dependent manner (P＜0.01), which was consistent with the construction of the gradient stress model. Conclusion: A gradient stress injury model of PC12 cells was successfully established, with intervention concentrations of 0 μmol/L, 25 μmol/L, 50 μmol/L, 100 μmol/L, 150 μmol/L and 200 μmol/L corticosterone at an intervention time of 12 h. The degree of stress injury of the cell model was increased gradually, which could be used as a basis and object for conducting cell stress injury assessment and regulation experiments.
Animals ; Cell Survival ; Corticosterone/pharmacology* ; NAD/pharmacology* ; PC12 Cells ; Rats ; Superoxide Dismutase

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD(+), essential for a number of enzymes and regulatory proteins involved in a variety of cellular processes, including deacetylation enzyme SIRT1 which modulates several tumor suppressors such as p53 and FOXO. Herein we report that NQO1 substrates Tanshione IIA (TSA) and β-lapachone (β-lap) induced a rapid depletion of NAD(+) pool but adaptively a significant upregulation of NAMPT. NAMPT inhibition by FK866 at a nontoxic dose significantly enhanced NQO1-targeting agent-induced apoptotic cell death. Compared with TSA or β-lap treatment alone, co-treatment with FK866 induced a more dramatic depletion of NAD(+), repression of SIRT1 activity, and thereby the increased accumulation of acetylated FOXO1 and the activation of apoptotic pathway. In conclusion, the results from the present study support that NAMPT inhibition can synergize with NQO1 activation to induce apoptotic cell death, thereby providing a new rationale for the development of combinative therapeutic drugs in combating non-small lung cancer.
Abietanes ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; enzymology ; genetics ; physiopathology ; Cell Line, Tumor ; Cytokines ; antagonists & inhibitors ; genetics ; metabolism ; Enzyme Inhibitors ; pharmacology ; Humans ; NAD ; metabolism ; NAD(P)H Dehydrogenase (Quinone) ; genetics ; metabolism ; Naphthoquinones ; pharmacology ; Nicotinamide Phosphoribosyltransferase ; antagonists & inhibitors ; genetics ; metabolism

PURPOSE: To investigate the effects of anthocyanins extracted from black soybean, which have antioxidant activity, on apoptosis in vitro (in hormone refractory prostate cancer cells) and on tumor growth in vivo (in athymic nude mouse xenograft model). MATERIALS AND METHODS: The growth and viability of DU-145 cells treated with anthocyanins were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis was assessed by DNA laddering. Immunoblotting was conducted to evaluate differences in the expressions of p53, Bax, Bcl, androgen receptor (AR), and prostate specific antigen (PSA). To study the inhibitory effects of anthocyanins on tumor growth in vivo, DU-145 tumor xenografts were established in athymic nude mice. The anthocyanin group was treated with daily oral anthocyanin (8 mg/kg) for 14 weeks. After 2 weeks of treatment, DU-145 cells (2x106) were inoculated subcutaneously into the right flank to establish tumor xenografts. Tumor dimensions were measured twice a week using calipers and volumes were calculated. RESULTS: Anthocyanin treatment of DU-145 cells resulted in 1) significant increase in apoptosis in a dose-dependent manner, 2) significant decrease in p53 and Bcl-2 expressions (with increased Bax expression), and 3) significant decrease in PSA and AR expressions. In the xenograft model, anthocyanin treatment significantly inhibit tumor growth. CONCLUSION: This study suggests that anthocyanins from black soybean inhibit the progression of prostate cancer in vitro and in a xenograft model.
Animals ; Anthocyanins/*pharmacology ; Apoptosis/*drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Male ; Mice, Inbred C57BL ; Mice, Nude ; NAD/metabolism ; Prostate-Specific Antigen/metabolism ; Prostatic Neoplasms/genetics/*pathology ; Receptors, Androgen/metabolism ; Tumor Suppressor Protein p53/metabolism ; *Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein/genetics/metabolism

OBJECTIVETo measure the levels of ghrelin-induced expression or activation of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and

NAD(P)Hquinone oxidoreductase 1 (NQO1) in the PQ-injured lungs of mice and to evaluate the protective effect of ghrelin against paraquat (PQ)-induced acute lung injury in mice.

METHODSAccording to the random number table method, 50 ICR mice of clean grade were assigned to 5 groups: normal control group (n = 10), PQ group (n = 10), and ghrelin intervention groups (n = 30). For PQ group, mice were injected with a single dose of PQ (20 mg/kg, i.p.); for ghrelin intervention groups, mice were injected with a single dose of PQ (20 mg/kg, i.p.), and then ghrelin was injected at three concentrations (16.58, 33.15, and 49.73 µg/kg). Lung tissues were collected and proceeded to the following studies. HE staining was used for histopathological examination under a light microscope, and the changes in nuclear expression of Nrf2 were evaluated by Western blot. The activities of HO-1 and NQO1 were measured by ELISA. Malondialdehyde (MDA) content and MPO activity were measured by colorimetry. Another 40 mice were divided into PQ group (n = 10) and 16.58, 33.15, and 49.73 µg/kg ghrelin intervention groups (n = 10 for each); mortality and clinical manifestations were recorded within 72 h.

RESULTSCompared with the normal control group, the PQ group showed significant increases in nuclear protein level of Nrf2, content of MDA, and activities of HO-1, NQO1, and MPO (P < 0.05 for all). Compared with the PQ group, ghrelin treatment significantly increased the expression of Nrf2 and activities of HO-1 and NQO1 and significantly reduced the content of MDA and activity of MPO (P < 0.01 for all). Histopathological studies indicated that ghrelin showed an antioxidant property that reduced the histological changes induced by PQ in the lungs. The ghrelin intervention groups had a significantly lower mortality than the PQ group, and there was a significant difference between the high-dose ghrelin intervention group and PQ group (P < 0.05).

CONCLUSIONGhrelin can up-regulate nuclear expression of Nrf2, increase the activities of HO-1 and NQO1, and reduce the activity of MPO and content of MDA, thus protecting PQ-exposed mice from acute lung injury.

Acute Lung Injury ; chemically induced ; metabolism ; Animals ; Ghrelin ; pharmacology ; Heme Oxygenase-1 ; metabolism ; Lung ; drug effects ; metabolism ; Male ; Malondialdehyde ; metabolism ; Membrane Proteins ; metabolism ; Mice ; Mice, Inbred ICR ; NAD(P)H Dehydrogenase (Quinone) ; metabolism ; NF-E2-Related Factor 2 ; metabolism ; Oxidative Stress ; Paraquat ; poisoning ; Peroxidase ; metabolism

OBJECTIVETo investigate the effect of Fuzhenghuayu compound (FZHc) on expression of nuclear factor E2-related factor 2 (Nrf2) in hepatocytes under conditions of hepatic fibrosis using a mouse model.

METHODSMice were randomly assigned to a control group and a hepatic fibrosis model group. The control group was further divided into three subgroups for use as normal controls (A1), mineral oil-treated controls (A2), and FZHc-treated controls (A3); the hepatic fibrosis model group was administered carbon tetrachloride (CC14 dissolved in mineral oil and injected intraperitoneally) and further divided into four subgroups for use as 6-weeks models (B1), 10-weeks models (B2), low-dose (L)-FZHc models (C1), and high-dose (H)-FZHc models (C2). The FZHc (capsule powder diluted with double-distilled water to 0.1 g/mL) was administered via gastric perfusion to groups A3, C1, and C2 starting at week 7 of the experiment. At the end of week 6 and 10, hepatic specimens were collected and evaluated for degree of hepatic fibrosis and inflammation using routine haematoxylin-eosin staining and Masson staining. Immunohistochemical analysis was performed to measure the hepatocyte expression of Nrf2, NAD(P)H quinine oxidoreductase 1 (Nqol), a-smooth muscle actin (a-SMA) and fibronectin (FN). Real-time fluorescence quantitative PCR was used to measure Nrf2 mRNA expression. Western blotting was used to detect Nrf2 and Nqol total protein expression and Nrf2 nuclear translocation. F test, LSD test and ridit test were used for statistical analyses.

RESULTSCompared with the B2 group (ridit value: 0.09), the model groups treated with FZHc showed significantly lower degrees of hepatic inflammation and fibrosis for both the low (C1 group, ridit value: 0.32) and high doses (C2 group, ridit value: 0.40) (F =82.927, P less than 0.05). In addition, compared with the B2 group, the model groups treated with FZHc showed significantly decreased expression of a-SMA and FN proteins, with a dose-dependent trend (by immunohistochemistry: C 1 group at the end of 10 weeks, F =77.421, 118.262, P less than 0.05; C2 group, P =0.002, 0.013) and significantly increased expression of Nrf2 and Nqol proteins (by immunohistochemistry:C1 and C2 groups at the end of 10 weeks, F =182.537, 75.615, P less than 0.05 and by westen blotting: F =45.664, 127.673, P less than 0.05), which also showed a dose-dependent trend (C2 group, P =0.000, 0.014; 0.005, 0.014). Western blotting also indicated that the amount of nuclear transported Nrf2 was higher in the C1 and C2 groups at the end of 10 weeks (vs. B2 group, F =94.787, P less than 0.05), and the amount of nuclear transported Nrf2 was significantly higher in the C2 group (vs. C1 group, P =0.044). Nrf2 mRNA expression was significantly higher in the C1 group than in the B2 group (F =3230.105, P less than 0.05), and the C2 group had more substantially increased expression (P =0.001); there was no statistical difference found between groups B1 and B2 (P =0.094).

CONCLUSIONFuzhenghuayu compound increased the expression of Nrf2 mRNA and protein under conditions of hepatic fibrosis in mice and stimulated Nrf2 nuclear transport, as well as increased expression of the Nrf2 target gene Nqol that is known to suppress activation of hepatic stellate cells and decrease the deposition of FN. Therefore, Fuzhenghuayu compound may ameliorate hepatocyte injury in hepatic fibrosis in mice by exerting an antihepatic fibrosis effect.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hepatocytes ; drug effects ; metabolism ; Liver Cirrhosis, Experimental ; metabolism ; Mice ; Mice, Inbred Strains ; NAD(P)H Dehydrogenase (Quinone) ; metabolism ; NF-E2-Related Factor 2 ; metabolism

OBJECTIVETo investigate the effect of curcumin on liver injury in rats induced by paraquat-mediated oxidative stress and the mechanism underlying its effect.

METHODSSixty rats were randomly divided into 4 groups: control group, curcumin control group (curcumin 50 mg/kg), paraquat group (2% paraquat solution 100 mg/kg), and curcumin intervention group (curcumin 50 mg/kg at 15 min, 24 h, or 48 h after paraquat exposure). On days 1, 3, or 7 after paraquat administration, and liver tissue was collected thereafter. The content of malonaldehyde (MDA) and the activities of superoxide dismutase activity (SOD) and catalase (CAT) in the liver tissue were determined by chemical colorimetry. The activities of heme oxygenase 1 (HO-1) and quinone oxidoreductase 1 (NQO-1) in the liver tissue were determined by ELISA. The mRNA and protein levels of NF-E2-related factor 2 (Nrf2) were determined by RT-PCR and Western blot, respectively. The pathological changes of liver tissue were examined by optical microscopy.

RESULTSNo significant change was observed between the control group and the curcumin control group in any examination of this study (P > 0.05). Both paraquat group and curcumin intervention group showed increase in MDA content, decreases in SOD and CAT activities, increases in HO-1 and NQO-1 activities, and increases in the protein and mRNA levels of Nrf2, in comparison with the control group (P < 0.05 for all except HO-1 activity in paraquat group on day 7). In comparison with the parquet group on the same day, the curcumin intervention group showed decrease in MDA content, increases in the activities of SOD, CAT, HO-1, and NQO-1, and increases in the mRNA and protein levels of Nrf2 on days 1, 3, and 7 (P < 0.05). The pathological examination revealed that the damage of liver tissue in the paraquat group was the most serious on the 3rd day after paraquat exposure, and the damage was consistently alleviated by curcumin intervention on days 1, 3, and 7, as compared with the paraquat group.

CONCLUSIONOxidative stress plays an important role in paraquat-induced acute liver damage in rats, and curcumin can exert a hepatoprotective effect against oxidative stress by increasing the expression of Nrf2 and the activities of HO-1, NQO-1, SOD, and CAT and reducing the content of MDA.

Animals ; Catalase ; metabolism ; Curcumin ; pharmacology ; Disease Models, Animal ; Heme Oxygenase (Decyclizing) ; metabolism ; Liver ; drug effects ; metabolism ; pathology ; Male ; Malondialdehyde ; metabolism ; NAD(P)H Dehydrogenase (Quinone) ; metabolism ; Oxidative Stress ; drug effects ; Paraquat ; poisoning ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a key enzyme of de novo GMP biosynthesis. The expression and activity of IMPDH can be affected by diseases and physiological process. It is the drug target for anticancer, antiviral, antimicrobial and immunosuppressive therapeutics. Not only catalytic action but the other biological functions of IMPDH also play an important role in diseases. The basic functions, mechanism of catalysis, classification of inhibitors, biological functions and the latest advances to IMPDH will be illustrated in this review. It is expected to be helpful to the discovery of new inhibitors and biological functions of IMPDH.
Animals ; Binding Sites ; Catalysis ; Drug Design ; Drug Discovery ; Enzyme Inhibitors ; classification ; pharmacology ; Humans ; IMP Dehydrogenase ; antagonists & inhibitors ; genetics ; metabolism ; Inosine Monophosphate ; metabolism ; Molecular Structure ; NAD ; metabolism ; Polymorphism, Genetic