mRNA Levels Of Nrf2 Research Articles

6-shogaol suppresses oxidative damage in L6 muscle cells

Ginger (Zingiber Officinale Roscoe) has been known reduce muscle pain after exercise, and 6-shogaol {(E)-1-(4-Hydroxy-3-methoxyphenyl)dec-4-en-3-one)} is the major essential oil contained in ginger. In this study, the protective effect of 6-shogaol on L6 muscle cells against oxidative damage was measured. 6-shagol inhibited the damage of L6 cell induced by H2O2, and allowed the increase in mRNA and protein expression levels of intracellular HO-1 and NRF2. 6-shogaol also reduced the production of intracellular ROS. These results suggested that 6-shagol effectively inhibits oxidative damage of skeletal muscle cell.

Astragaloside IV-induced Nrf2 nuclear translocation ameliorates lead-related cognitive impairments in mice

Recently, oxidative stress is a common denominator in the pathogenesis of metal-induced neurotoxicity. Thus, antioxidant therapy is considered as a promising strategy for treating lead-related cognitive impairment. Here, we tested the hypothesis that astragaloside IV (AS-IV) ameliorates lead-associated cognitive deficits through Nrf2-dependent antioxidant mechanisms. Male Nrf2-KO and WT mice received drinking water with 2000 ppm lead and/or AS-IV by gavage for 8 weeks starting at 4 weeks of age. Morris water maze test and biochemical assays were employed to study cognition-enhancing and antioxidant effects of AS-IV. The signaling pathways involved were analyzed using RT-PCR and western blot technology. Significantly, AS-IV attenuated Morris water maze-based cognitive impairment in lead-intoxicated mice. Importantly, cognition-enhancing effect of AS-IV was lost in Nrf2-KO mice. In parallel, AS-IV suppressed lead acetate (PbAc)-induced oxidative stress, as measured by MDA. Mechanistically, AS-IV can up-regulate the expressions of the GCLc and HO-1 at the level of transcription and translation, but not SOD, TrxR activity, GCLm, Trx1, and NQO1 expression. Interestingly, AS-IV induced accumulation of Nrf2 in the nucleus, whereas Nrf2 mRNA levels were unchanged. Furthermore, AS-IV treatment resulted in elevated levels of phosphorylated Akt (active form) and phosphorylated GSK-3β (inactive forms) but decreased level of phosphorylated Fyn. Collectively, our findings indicate that AS-IV may target Nrf2 to attenuate lead-triggered oxidative stress and subsequent cognitive impairments, suggesting that AS-IV is a potential candidate for the treatment of lead-associated cognitive diseases.

Condensed tannins enhanced antioxidant capacity and hypoxic stress survivability but not growth performance and fatty acid profile of juvenile Japanese seabass (Lateolabrax japonicus)

Condensed tannins (CT) have been shown a promising alternative to antibiotics for livestock, but little information is available about the effect of purified CT on the growth and health of fish. A 56-day feeding trial with 640 juvenile Japanese seabass (Lateolabrax japonicus) was conducted to assess the effects of CT on the growth performance, fatty acid composition, antioxidant capacity and hypoxic stress of L. japonicus. Four diets were formulated to contain 0 (CT0), 100 (CT100), 200 (CT200) and 400 (CT400) mg/kg CT isolated from grape seed, quadruplicate groups of 40 fish with initial body weight of 10.1 ± 0.11 g were fed to apparent satiation two times daily. Fish were dissected at the end of experiment to evaluate the somatic indexes. Blood samples were collected to analyze serum metabolites, antioxidant enzymes and non-specific immune indices. Muscle and hepatopancreas were sampled to determine fatty acid composition. At the end of the feeding trail, 15 fish from each tank were suffered with a 10-hr hypoxic stress. Blood and hepatopancreas of the suffered fish were collected to analyze antioxidant enzymes, and hepatic mRNA levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), catalase (CAT) and superoxide dismutase (SOD) were analyzed. Results indicated that growth performance, whole body compositions, serum biochemical parameters, and fatty acid profile in muscle and hepatopancreas of fish were not affected by dietary treatments. Intraperitoneal fat ratio of the fish was linearly decreased (P < 0.01) as the dietary CT increasing from 0 to 400 mg/kg. Serum total antioxidant capacity (TAOC) and CAT were linearly increased (P < 0.01), but tumor necrosis factor was linearly decreased (P < 0.05) and IL-6 was linearly and quadratically decreased (P < 0.001) with increasing CT. There was a linear increase (P < 0.05) in hepatic TAOC and anti-superoxide anion with increased dietary CT. Hepatic CAT was quadratically increased (P < 0.01), and GSH-Px and SOD were linearly and quadratically decreased (P < 0.05) with increasing CT. Cumulative mortality rate (CMR) after 10 h of hypoxic stress and serum MDA were linearly and quadratically decreased (P < 0.001), whereas serum CAT and GSH-Px were linearly increased (P < 0.01) and TAOC was linearly and quadratically increased (P < 0.05) with increasing dietary CT. Hepatic CAT and SOD after 10 h of hypoxic stress were linearly increased (P < 0.05) with CT increasing. The Nrf2 and HO-1 mRNA levels were up-regulated (P < 0.05) with increasing dietary CT, and CAT and SOD mRNA levels were up-regulated (P < 0.05) in CT400 than CT0-CT200. In conclusion, dietary CT up to 400 mg/kg can not only improve the antioxidant status of L. japonicus without affecting growth performance, but also reduce the CMR under hypoxic stress by activating Nrf2 signaling pathway.

Curcumin improves necrotising microscopic colitis and cell pyroptosis by activating SIRT1/NRF2 and inhibiting the TLR4 signalling pathway in newborn rats

This study aimed to explore comprehensively the biological function of curcumin, and its underlying mechanism, in protecting from necrotising microscopic colitis in newborn rats. A total of 20 normal healthy rats were selected, and a necrotising enterocolitis (NEC) model was established. After hypoxia and hypothermia stimulation, these rats were treated with different doses of curcumin (control group, NEC model group, NEC+20 mg/kg curcumin and NEC+50 mg/kg curcumin). Inflammation was identified using hematoxylin and eosin staining, and inflammatory factors were detected via ELISA. The mRNA and protein levels of SIRT1, NRF2, TLR4, NLRP3 and caspase-1 were determined by quantitative RT-PCR and Western blotting, respectively. Curcumin improved the inflammatory condition of NEC and inhibited the expression of inflammatory factors in NEC newborn rat intestinal tissue. Furthermore, the SIRT1/NRF2 pathway was inhibited in the intestinal tissue of NEC newborn rats, whereas curcumin treatment induced the activation of the SIRT1/NRF2 pathway and inhibited TLR4 expression in these animals. In addition, curcumin could also inhibit the expression of inflammatory factors and alleviate the LPS/ATP-induced focal death pathway in intestinal epithelial cells through the SIRT1 pathway. Curcumin can improve necrotising microscopic colitis and cell pyroptosis by attenuating NEC-induced inhibition of SIRT1/NRF2 and inhibiting the TLR4 signalling pathway in newborn rats.

Aerobic exercise combined with huwentoxin-I upregulates phase-Ⅱ detoxification enzymes to alleviate obstructive jaundice-induced central nervous system injury in mice

To explore the effects of aerobic exercise combined with huwentoxin-I (HWTX-I)-mediated Keap1-Nrf2-ARE pathway on phase II detoxification enzymes HO-1 and NQO1 and their protective effects against obstructive jaundice (OJ)-induced central nervous system injury in mice. 50 male KM mice were randomly divided into blank group (GO), model group (M), aerobic exercise group (T), HWTX-I group (H), and aerobic exercise combined with HWTX-I group (TH). Mouse models of OJ were established with surgical suture for 72 h in the mice in all the groups except for the blank control group. The mice received interventions by aerobic exercise and tail vein injection of HWTX-I (0.05 μg/g) and were assessed by behavioral observation, Clark's neurological function scores, enzyme-linked immunosorbent assay (ELISA), brain tissue Nissl staining, hippocampal tissue Western blotting, and liver tissue mRNA expression profiling and sequencing. The mice in group M had obvious jaundice symptoms after the operation with significantly increased Clark's neurological score (P &lt; 0.01). Compared with those in group M, the mice in group T, group H, and group TH showed significantly decreased serum levels of ALT, AST, TBIL, and TBA (P &lt; 0.01) with increased contents of 5-HT and BDNF and decreased contents of S100B and NSE in the hippocampus (P &lt; 0.01). Synergistic effects between aerobic exercise and HWTX-I were noted on the above parameters except for the liver function indicators. Interventions with aerobic exercise and HWTX-I, alone or in combination, obviously lessened pathologies in the brain tissue induced by OJ, and the combined treatment produced the strongest effect. The treatment also increased the expression levels of Nrf2, HO-1, and NQO1 mRNA and protein in brain tissues (P &lt; 0.01 or 0.05) with a synergistic effect between aerobic exercise and HWTX-I. Illumina high-throughput sequencing showed that the differentially expressed factors participated mainly in such neural regulatory pathways as neuroactive ligand-receptor interaction, GABAergic synapses, dopaminergic synapses, synaptic vesicle circulation, and axon guidance, involving tissue cell neuronal signal transduction, apoptosis inhibition, immune response, and toxicity. Aerobic exercise and HWTX-I synergistically increased the accumulation of the signal pathways related with neuron damage repair and proliferation. Aerobic exercise combined with HWTX-I can up-regulate the expression of phase Ⅱ detoxification enzymes HO-1 and NQO1 through the Keap1-Nrf2-ARE pathway to protect the central nervous system against OJ-induced damage in mice.

Effects of chronic glyphosate exposure on antioxdative status, metabolism and immune response in tilapia (GIFT, Oreochromis niloticus)

Glyphosate (Gly) is an active ingredient of herbicide, its underlying toxicity on fish is still unclear. The aim of this study was to evaluate chronic toxicity of Gly on tilapia via determining antioxidative status, metabolism, inflammation and immune response. The fish were exposed to different concentrations of Gly (0, 0.2, 0.8, 4 and 16 mg/L) for 80 days. The blood, liver, gills and spleen were collected to assay biochemical parameters and genes expression after 80 days of exposure. The results showed that treatments with higher Gly (4 and/16 mg/L) significantly increased the levels of TC, TG, AST, ALT, LDL-C and MDA, and apparently decreased the levels of SOD, GSH, CAT, HDL-C, HK, G3PDH, FBPase and G6PD in serum, liver and/or gills. The gene expression data showed that the treatments with Gly adversely affected Nrf2 pathway in liver, gills and spleen, as shown by significant changes of nrf2, keap1, ho-1, nqo1 and gsta mRNA levels. Meanwhile, inflammatory response was activated via enhancing the mRNA levels of nf-κb2, rel, rela tnf-α, and il-1β, and immunotoxicity was caused through downregulating the genes expression of c-lzm, hep, igm, hsp70 and c3 in liver, gills and/or spleen of tilapia after Gly exposure. Moreover, the mRNA levels of cyp1a and cyp3a were upregulated in 16 or 0.2 mg/kg Gly group in liver. Overall results suggested chronic Gly exposure reduced antioxidative ability, disturbed liver metabolism, promoted inflammation and suppressed immunity. Interestingly, the Nrf2 and NF-κB signaling pathways played key roles in Gly chronic toxicity.

Effects of dietary astaxanthin on the growth, innate immunity and antioxidant defence system of Paramisgurnus dabryanus

The effects of astaxanthin supplementationon the growth, innate immunity and antioxidant defence system of loach (Paramisgurnus dabryanus) were investigated in this study. A total of 450 fish (initial average weights of 3.00 ± 0.10 g) were fed five diets with graded levels of astaxanthin (0.00, 50.00, 100.00, 150.00 and 200.00 mg/kg) for 56 days. The results showed that astaxanthin supplementation significantly stimulated the growth (FBW, WG, FER, HSI), innate immunity (AKP activity in the hepatopancreas, intestinal tract, muscle and skin and LZM activity in the hepatopancreas, intestinal tract and skin), antioxidant ability (T-SOD, GSH-PX and CAT activities and T-AOC, GSH and MDA contents in the hepatopancreas, intestinal tract, muscle and skin) of loach. Moreover, dietary astaxanthin supplementation significantly up-regulated the relative mRNA levels of Nrf2 and Maf, and down-regulated the relative mRNA level of Keap1 in the hepatopancreas when the supplementation levels of astaxanthin were 50–200 mg/kg. In conclusion, the diets with 100–151.06 mg/kg astaxanthin supplementation were optimal for loach, which based on the growth, immunity and antioxidant-related indicators, and the astaxanthin supplementation regulated the antioxidant ability partially referring to Keap1-Nrf2 signallings.

MiRNAs-Modulation of Nrf2 Signaling Networks in Regulation Oxidative Stress of Chinese Perch Skeletal Muscle After Fasting Treatment.

Nrf2 is an important transcription factor involved in the antioxidant response and is widely expressed in animal tissues. The function of Nrf2 is regulated by its negative regulator Keap1 by inducing its cytoplasmic degradation. Recent studies have suggested that Nrf2 is also regulated post-transcriptionally via miRNAs. However, to date, how miRNAs regulate Nrf2 in fish skeletal muscles is unknown. In this study, the full-length cDNAs with 2398bp of the Nrf2 was firstly cloned by SMART RACE amplification tools from Chinese perch. The Nrf2 gene structure and its 3'-UTR region for possible miRNA binding sites, as well as its spatial expression profile were assayed. Then, we employed TargetScan Fish tool MiRNAnome to predict putative sites for five miRNAs including miR-181a-5p, MiR-194a, MiR-216a, miR-459-5p, and miR-724. Using qRT-PCR assay, we found that Nrf2 mRNA levels have negative correlation with all five miRNAs expression in muscle of nutritionally deprived fish, and that ectopic expression of miR-181a-5p alone reduces Nrf2 mRNA levels. Luciferase reporter assay in a heterologous cell system revealed that each of the five miRNAs reduced Nrf2 expression, suggesting a direct regulatory mechanism. Moreover, the miR-181a-5p suppression using specific antagomir led to a significant increase in Nrf2 expression in vivo. At the same time, the expression levels of the antioxidant enzymes CAT, ZnSOD, GPx, GSTA, and GSTA genes increased significantly after injecting miR-181a-5p antagomir. Taken together, these findings provide evidence that miRNAs are involved in the Nrf2 signaling networks in regulation of oxidative stress in fish, at least in Chinese perch muscle.

Polysaccharides from Hemp Seed Protect against Cyclophosphamide-Induced Intestinal Oxidative Damage via Nrf2-Keap1 Signaling Pathway in Mice.

Hemp seed has been used as a traditional oriental medicine and health food in China for centuries. Polysaccharides from hemp seed (HSP) exhibit important properties of intestinal protection, but there are limited data on the specific underlying mechanism. The primary objective of this study was to investigate the protective effect of HSP on intestinal oxidative damage induced by cyclophosphamide (Cy) in mice. The results showed that pretreatment with HSP significantly increased the average daily gain, thymus index, spleen index, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activity in serum and ileal homogenate and significantly reduced malondialdehyde (MDA) content in ileal homogenate. In addition, the expression levels of SOD, GSH-Px, Nrf2, heme oxidase-1 (HO-1), and quinoneoxidoreductase-1 (NQO1) mRNA in ileal homogenate were significantly increased. Western blot results showed that HSP significantly upregulated the expression of Nrf2 protein and downregulated the expression of Keap1 protein in the ileum. Collectively, our findings indicated that HSP had protective effects on intestinal oxidative damage induced by Cy in mice, and its mechanism might be related to the activation of Nrf2-Keap1 signaling pathway.

Rosemary extract reverses oxidative stress through activation of Nrf2 signaling pathway in hamsters fed on high fat diet and HepG2 cells

• Diet rosemary extract (RE) alleviated high-fat induced oxidative damage in vivo and in vitro. • Diet RE enhanced the antioxidant enzyme activity, gene and protein expression level. • The protective effect of diet RE was achieved by regulating Nrf2 pathway. Rosemary extract (RE), a useful component isolated from Rosmarinus officinalis L. , was recently reported to protect cells against the harmful effects of ROS. However, the underlying mechanisms of this action remained unknown. We investigated the defensive effect of RE on the oxidative stress injury resulted from high fat diet in hamsters and oleic acid (OA) in HepG2 cells respectively, and its underlying mechanisms. Treatment with RE alleviated liver steatosis and reduced plasma and liver content of TG and TC, as well as plasma content of AST and ALT. RE apparently increased the activity of SOD, GSH-Px, CAT and decreased MDA content. RE notably increased CuZn-SOD, Mn-SOD, CAT, GSH-Px, GCLC, GCLM and Nrf2 mRNA and protein expression level. Nrf2 shRNA knockdown in HepG2 cells increased OA-induced ROS generation, and effectively altered OA-induced cytotoxicity, suggesting that Nrf2 is a crucial mediator against high-fat induced oxidative damage.

Astragalus polysaccharide alleviates cognitive impairment and β-amyloid accumulation in APP/PS1 mice via Nrf2 pathway

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, and its etiology and pathogenesis are not fully understood. Astragalus polysaccharide (APS) has many pharmacological activities, but there are few reports about its role in AD. Using the common AD model APP/PS1 mice, it was found that the expression of Keap1 (a negative regulatory factor of Nrf2), the protein level of cytoplasmic Nrf2 and the content of MDA were increased significantly, while the mRNA level of Nrf2, the expression of Nrf2 in nucleus and the contents of SOD and GSH-Px were decreased significantly. APS treatment significantly increased the expression of Nrf2 in the nucleus but decreased its expression in the cytoplasm, and restored the expression levels of Keap1, SOD, GSH-Px and MDA. When APP/PS1 mice were treated with APS and injected with Nrf2 siRNA, the down-regulation of Nrf2 expression significantly blocked the regulation of APS on oxidative stress. Continuing to test the physiological function of AD mice showed that the spatial learning and memory abilities of APP/PS1 mice were impaired, the apoptosis of brain cells and the content of β-amyloid (Aβ) were significantly increased. APS treatment significantly improved the cognitive ability of APP/PS1 mice, reduced apoptosis and the accumulation of Aβ, but the above effects of APS were blocked by Nrf2 siRNA injection. Therefore, APS can activate Nrf2 pathway to improve the physiological function of AD mice, which may have important clinical application value.

Curcumin and Baicalin ameliorate ethanol-induced liver oxidative damage via the Nrf2/HO-1 pathway.

One of the key mechanisms of alcoholic liver disease is oxidative stress. Both Curcumin and Baicalin exert antioxidant effects, but the mechanism of their combined effects of ethanol-induced liver injury is still unclear. This study was conducted to evaluate the dual antioxidant activity of Curcumin combined with Baicalin against ethanol-induced liver injury in rats. Rats were divided into five groups, a control, ethanol, ethanol+Curcumin (50mg/kg), ethanol+Baicalin (50mg/kg), and ethanol+Curcumin +Baicalin group with ten rats per group. The effects of ethanol on liver enzymes, oxidative stress indicators and the levels of Nrf2/HO-1 pathway related proteins and mRNA were observed along with liver histopathology in rats. Our results found that the serum ALT and AKP levels were increased in ethanol-treated rats, which also showed a rising trend of 8-OHdG and LPO levels while hydroxyl radical scavenging ability, T-AOC, and the activities of SOD and GSH-Px were decreased in liver. The mRNA levels of Nrf2 and HO-1, the ratio of p-Nrf2/Nrf2, the protein level of HO-1 were decreased while NQO1 mRNA level, Nrf2, p-Nrf2, and NQO1 protein levels were increased in ethanol-treated rats. Combination treatment of Curcumin and Baicalin significantly reversed the ethanol-induced liver oxidative damage and further activate the Nrf2/HO-1 pathway, which was more effective than each drug alone. In conclusion, evidence has shown for the first time in this study that Curcumin combined with Baicalin ameliorated ethanol-induced liver oxidative damage in rats and revealed liver-protection. PRACTICAL APPLICATIONS: Many drugs for treating alcoholic liver disease are available commercially, but some adverse effects they have may cause secondary damage to the liver. At present, the combined treatment of different natural phytochemicals has attracted special attention in modern medicine. Curcumin, a kind of phytochemicals, is extracted from turmeric rhizome. Baicalin is one of the major active components of Scutellaria Baicalensis. The current research is to explore the antioxidant effect of Curcumin and Baicalin in ethanol-induced liver injury in rats. Our research proves that Curcumin combined with Baicalin on ethanol-induced liver oxidative damage is superior to single drug treatment. Therefore, the combination of Curcumin and Baicalin may provide a more prospective natural remedy to combat ethanol-induced liver injury.

Protective role of wogonin against cadmium induced testicular toxicity: Involvement of antioxidant, anti-inflammatory and anti-apoptotic pathways

The present study was conducted to identify possible health - promoting effects of wogonin (Wog) on testicular dysfunction in rats caused by cadmium. Pre-treatment of cadmium chloride (Cd: 5 mg/kg b.wt.) administered rats with wogonin (10 mg/kg b.wt) resulted in significant improvement in Cd-induced decrease in body and organ (testes and epididymides) weights. Wogonin treatment significantly improved Cd-induced reduction in sperm quality and quantity, steroidogenic gene (SFI, StAR, CYP11A1, 3β-HSD, CYP17A1 and 17β-HSD) and protein (SF1, StAR and CYP17A1) expressions and serum testosterone levels. Wogonin treatment provided significant protection to Cd-induced aggression in testicular oxidative (elevated levels of MDA) and anti-oxidative (diminished activities of SOD, CAT and GPx) status. Wog significantly up-regulated mRNA levels of Nrf2, NQO1 and HO-1 and down-regulation of Keap1 in cadmium treated testes. Wogonin administration significantly suppressed Cd-stimulated increase in inflammatory reactions (increase in NF-κB p65 DNA, p-IKKβ, TNF-α levels and decrease in IL-10 levels). Wogonin prevented apoptotic damage by enhanced protein distribution of caspase-9, caspase-3, and Bax due to Cd exposure. Furthermore, Wogonin presented significant protection to histo-morphometric changes resulted after Cd administration. Taken together, the findings of this study provided clear evidence of the therapeutic potential of Cd-induced testicular toxicity at least partly due to its antioxidant, anti-inflammatory and anti-apoptotic properties.

Citrus Flavanones Upregulate Thyrotroph Sirt1 and Differently Affect Thyroid Nrf2 Expressions in Old-Aged Wistar Rats.

A growing population of elderly people consume citrus flavanones, naringenin, and hesperetin in the form of fruits or juices. Flavanones are bioactives with potent antioxidant properties and have potential in slowing down the aging process. Because flavanones exert controversial effects on pituitary-thyroid functioning, our study on the old-aged rat model aimed to elucidate the mechanism by which naringenin and hesperetin affect this axis. Naringenin and hesperetin increased the Sirt1 mRNA level by 91 and 71% (p < 0.05), which was followed by increased Sirt1 expression by 20 and 15% (p < 0.05), respectively. Only naringenin decreased thyroid-stimulating hormone expression by 20% (p < 0.05). Thyroid peroxidase protein expression was upregulated after naringenin or hesperetin by 62 and 43% (p < 0.05), respectively. Naringenin lowered mRNA levels of Tpo, Sod1, Sod2, Cat, and Nrf2 by 50, 32, 45, 35, and 42% (p < 0.05), respectively, and increased Gpx by 54% (p < 0.05), while hesperetin decreased Sod1 and Sod2 mRNA levels by 46 and 55% (p < 0.05), respectively. Naringenin increased the protein expressions of Nrf2 and SOD2 by 58 and 50% (p < 0.05), respectively, and decreased SOD1 expression by 48% (p < 0.05), while hesperetin protein decreased expressions of SOD1 and Nrf2 by 63 and 32% (p < 0.05), respectively. Altogether, our findings suggest that citrus flavanones contribute to restoring the impaired thyroid functioning in the old-aged rats.

SIRT6 as a key event linking P53 and NRF2 counteracts APAP-induced hepatotoxicity through inhibiting oxidative stress and promoting hepatocyte proliferation

Acetaminophen (APAP) overdose is the leading cause of drug-induced liver injury, and its prognosis depends on the balance between hepatocyte death and regeneration. Sirtuin 6 (SIRT6) has been reported to protect against oxidative stress-associated DNA damage. But whether SIRT6 regulates APAP-induced hepatotoxicity remains unclear. In this study, the protein expression of nuclear and total SIRT6 was up-regulated in mice liver at 6 and 48 h following APAP treatment, respectively. Sirt6 knockdown in AML12 cells aggravated APAP-induced hepatocyte death and oxidative stress, inhibited cell viability and proliferation, and downregulated CCNA1, CCND1 and CKD4 protein levels. Sirt6 knockdown significantly prevented APAP-induced NRF2 activation, reduced the transcriptional activities of GSTμ and NQO1 and the mRNA levels of Nrf2, Ho-1, Gstα and Gstμ. Furthermore, SIRT6 showed potential protein interaction with NRF2 as evidenced by co-immunoprecipitation (Co-IP) assay. Additionally, the protective effect of P53 against APAP-induced hepatocytes injury was Sirt6-dependent. The Sirt6 mRNA was significantly down-regulated in P53−/− mice. P53 activated the transcriptional activity of SIRT6 and exerted interaction with SIRT6. Our results demonstrate that SIRT6 protects against APAP hepatotoxicity through alleviating oxidative stress and promoting hepatocyte proliferation, and provide new insights in the function of SIRT6 as a crucial docking molecule linking P53 and NRF2.

Prolonged-release pirfenidone prevents obesity-induced cardiac steatosis and fibrosis in a mouse NASH model

Obesity is associated with systemic insulin resistance and cardiac hypertrophy with fibrosis. Peroxisome proliferator-activated receptors (PPARs) regulate carbohydrate and lipid metabolism, improving insulin sensitivity, triglyceride levels, inflammation, and oxidative stress. We previously demonstrated that prolonged-release pirfenidone (PR-PFD) is an agonistic ligand for Pparα with anti-inflammatory and anti-fibrotic effects, and might be a promising drug for cardiac diseases-treatment. Here, we investigated the effects of PR-PFD in ventricular tissue of mice with nonalcoholic steatohepatitis (NASH) and obesity induced by high-fat/high-carbohydrate (HFHC) diet. Five male C57BL/6 J mice were fed with normal diet (ND) and ten with HFHC diet for 16 weeks; at 8 weeks of feeding, five mice with HFHC diet were administered PR-PFD (350 mg/kg/day) mixed with HFHC diet. Systemic insulin resistance, heart weight/body weight ratio, myocardial steatosis with inflammatory foci, hypertrophy, and fibrosis were prevented by PR-PFD. In addition, HFHC mice showed significantly increased desmin, Tgfβ1, Timp1, collagen I (Col I), collagen III (Col III), TNF-α, and Nrf2 mRNA levels, including α-SMA, NF-kB, Nrf2, troponin I, Acox1, Cpt1A, and Lxrα protein levels compared with the ND ventricular tissues. Mechanistically, HFHC mice with PR-PFD treatment significantly decreased these genes overexpressed by HFHC diet. Furthermore, PR-PFD overexpressed the Pgc1a mRNA levels and Pparα, Pparγ, Acox1, and Cpt1A protein levels. The results suggest that PR-PFD could be a promising drug for the prevention and treatment of cardiac steatosis and fibrosis induced by obesity.

Identification of the Nrf2 in the fathead minnow muscle cell line: role for a regulation in response to H2O2 induced the oxidative stress in fish cell.

The Nrf2 (nuclear factor erythroid 2-related factor 2) plays a central role in cell protection against a wide variety of environmental stressors through the Nrf2-Keap1 (Kelch-like ECH-associated protein 1) pathway, but its involvement in modulation of antioxidant system of fish cell is still largely unexplored. The present study focused on the molecular cloning and silencing of the Nrf2 in the fathead minnow muscle cell line (FHM) in response to the oxidative stress induced by H2O2. A full-length cDNA of coding Nrf2 was cloned from FHM cells by RT-PCR and RACE approaches. The obtained cDNA covered 2578bp with an open reading frame (1770bp) of encoding 589 amino acids. Sequence alignment and phylogenetic analysis revealed a high degree of conservation (51-86%) among 16 fishes. Based on the cloned Nrf2 sequence, the siRNA-242 of targeting Nrf2 with the best knocking down efficiency was designed and detected. Then, the mRNA levels of Keap1, Nrf2, Maf (musculoaponeurotic fibrosarcoma oncogene), and HO-1 (haemoxygenase-1); the activities of T-SOD (total superoxide dismutase), CAT (catalase), and GSH-PX (glutathione peroxidase); the levels of GSH (glutathione) and MDA (malonaldehyde); and the cell cycle and apoptosis were analyzed to investigate the molecular responses after H2O2 exposure. These results showed a coordinated transcriptional regulation of Keap1, Maf, and HO-1 and antioxidants (T-SOD, GSH, CAT, and GSH-PX) and MDA levels after H2O2 exposure, leading to oxidative damage and apoptosis. These findings provided an insight to understand the mechanisms of Nrf2 against oxidative stress in fish.

Antioxidative Effects of Thymus quinquecostatus CELAK through Mitochondrial Biogenesis Improvement in RAW 264.7 Macrophages.

Oxidative stress plays a key role in the pathogenesis of several diseases, including neurodegenerative diseases. Recent studies have reported that mitochondrial dysfunction is a leading cause of the overproduction of reactive oxygen species and oxidative stress. Mitochondrial changes play an important role in preventing oxidative stress. However, there is a lack of experimental evidence supporting this hypothesis. Thymus quinquecostatus CELAK (TQC) extract is a plant from China belonging to the thymus species, which can mediate the inflammatory response and prevent cell damage through its antioxidant activities. This study examines whether TQC can scavenge excess ROS originating from the mitochondria in RAW 264.7 macrophages. We used lipopolysaccharide (LPS) to induce inflammation and oxidative stress in RAW 264.7 macrophages and performed an immunocytochemistry dot blot of 8-hydroxy-2′-deoxyguanosine (8-OHdG) and real-time PCR to analyze the expression levels of genes involved in mitochondrial biogenesis and oxidative metabolism. TQC was found to significantly reduce the intensity of immunostained MitoSOX and 8-OHdG levels in the total genomic DNA within the mitochondria in RAW 264.7 macrophages. The HO-1 and Nrf2 mRNA levels were also significantly increased in the TQC groups. Therefore, we verified that TQC improves mitochondrial function and attenuates oxidative stress induced by LPS. Our results can provide reference for the effect of TQC to develop new therapeutic strategies for various diseases.

Antiproliferative Activity of Ursolic Acid in MDA-MB-231 Human Breast Cancer Cells through Nrf2 Pathway Regulation.

The potential mechanisms of action of ursolic acid (UA) in regulating cell proliferation in MDA-MB-231 human breast cancer cells through Nrf2 pathway were investigated. UA significantly inhibited the proliferation of MDA-MB-231 cells at a dose ≥10 μM in a dose-dependent manner, and no cytotoxicity was observed at concentrations below 29.87 ± 2.60 μM. The expressions of Nrf2 and p-Nrf2, in whole cell and nucleus, and NQO1 were inhibited by UA treatment, whereas the Keap1 expression was upregulated. No significant difference was observed in the Nrf2 mRNA levels, indicating that UA reduced Nrf2 expression not through mRNA but through a post-translational mechanism. Additionally, EGF-induced p-Nrf2 and its downstream NQO1 and SOD1 enzymes were abolished by UA. However, EGF or p-EGFR had no effect on the expressions of Keap1. These results suggested that the proliferative inhibitory effect of UA might be partially through downregulating Nrf2 via the Keap1/Nrf2 pathway and EGFR/Nrf2 pathway in MDA-MB-231 cells.

Cyanidin-3-glucoside from black rice prevents renal dysfunction and renal fibrosis in streptozotocin-diabetic rats

Abstract Hyperglycemia, increased inflammation, and mitochondrial dysfunction in diabetes may eventually cause kidney damage. Black rice cyanidin-3-glucoside has anti-hyperglycemic and anti-osteoporosis effects. Whether it has preventive effects on renal dysfunction in diabetes has not been reported. In this study, a rat model of diabetic nephropathy was established, and cyanidin-3-glucoside was administered for 56 days, lisinopril was administered as the positive control. The research found that black rice cyanidin-3-glucoside supplementation decreased glucose, prevented insulin resistance, repaired kidney function of diabetic rats, prevented renal interstitial fibrosis, glomerulosclerosis, and oxidative stress of diabetic rats. The inflammatory cytokines were significantly reduced in cyanidin-3-glucoside treated group. The renal Nrf2 mRNA level was evaluated, renal TNF-α mRNA, and NF-κΒ mRNA expression levels was decreased in cyanidin-3-glucoside treated group. Immunohistochemical results showed that cyanidin-3-glucoside inhibited transforming growth factor-β1 (TGF-β1), phosphorylated Smad2 (p-Smad2), phosphorylated Smad3 (p-Smad3), type IV collagen, fibronetin, and laminin expression in the kidney of diabetic rats. Our results indicate cyanidin-3-glucoside can protect against diabetic nephropathy in rats; it is mainly attributed to its anti-insulin resistance, anti-oxidative stress, anti-inflammation, as well as inhibit TGF-β1/p-Smads expression and extracellular matrix (ECM) aggregation.