Nrf2 mRNA Research Articles

New insights into the cytotoxic effects of Thymus vulgaris essential oil on the human triple-negative breast cancer cell line MDA-MB-231

Essential oils (EOs) are natural products that have gained wide interest due to their biological activities and anticancer properties through various mechanisms. The present study aimed to test the cytotoxicity of Thymus vulgaris L. (thyme) EO of Italian origin, rich in thymol (49.6%) and p-cymene (18.8%), towards the triple-negative breast cancer cell line MDA-MB-231 and to investigate the biochemical mechanisms underlying its antitumor activity. Thyme EO reduced cancer cell viability in a dose-dependent manner after 24 h treatment, with an IC50 value equal to 75.1 ± 15.2 μg/ml; simultaneously, the inhibition of cancer cell migration and colony formation capacity was evidenced. Thyme EO antiproliferative effects were related to the induction of apoptosis as demonstrated by the increased expression of the pro-apoptotic proteins Bax, cleaved caspase-3, phospho-p53, and SMAC/Diablo and by the reduction of the anti-apoptotic proteins Bcl-2, cIAP-1, cIAP-2, HIF-1α, survivin, and XIAP. Thyme EO administration led to the early formation of intracellular ROS, followed by the increment of MDA as an index of lipid peroxidation and by the decreased expression of the antioxidant enzymes catalase and PON2. The upregulation of Nrf2 mRNA expression and the strong induction of HO-1 sustained the activation of the Nrf2 pathway by thyme EO. These data showed that the EO from Thymus vulgaris L. might inhibit the malignant phenotype of MDA-MB-231, thus suggesting potential benefits against human triple-negative breast cancer.

Taify Pomegranate Juice Extract Abrogates Testicular Dysfunction Induced by Acrylamide: Role of Inflammatory, Antioxidants, and Oxidative Stress Biomarkers

High altitude (HA) poses various dangers to living organisms, the most serious of which are oxidative stress and its accompanying metabolic problems, as well as hypoxia and its associated metabolic abnormalities. Acrylamide is a poisonous chemical produced under the oxidative stress as a result of intracellular reactive oxygen species formation and toxicity. Heating carbohydrates-rich meals give acrylamide that is widely used in the industry. The precise mechanism of acrylamide toxicity is unknown. The current study aimed to examine the impacts of Taify pomegranate juice (TPJ) from Taif area (Punica granatum L.) on acrylamide-induced testicular stress and dysfunction. Twenty four male of adult Wistar rats, were divided into four groups: Group 1 was a negative-control received only saline; Group 2 was a positive-control that received orally acrylamide (20 mg/kg/bw) for consecutive 4 weeks; and Group 3 received TPJ; 2 mL/kg/bw, orally for 4 weeks. Rats in Group 4 received pomegranate juice and acrylamide as described in Groups 2 and 3, with the TPJ delivered 1 week before the acrylamide. Acrylamide elevated serum levels of inflammatory cytokines (interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α)). Moreover, acrylamide decreased concentrations of follicle stimulating hormone (FSH), leutinizing hormone (LH), and testosterone and altered semen characteristics (decreased sperm viability and concentration, and increased sperm abnormalities). Acrylamide increased malondialdehyde levels (MDA), while it decreased antioxidant activities (superoxide dismutase; SOD and reduced glutathione; GSH). The inflammation associated cytokines were restored by TPJ administration to acrylamide rats. GSH and SOD were significantly recovered to near control levels in TPJ plus acrylamide group compared to acrylamide-administered rats. TPJ preadministration to rats restored semen profiles, alteration in testis pathology, and normalized the changes on the male reproductive hormones affected by acrylamide. Furthermore, the TPJ reversed the upregulation in caspases-3 and the decrease in B-cell lymphoma-2 (Bcl-2) gene expressions affected by acrylamide, with significant upregulation of hemoxygenase-1 (HO-1) and nuclear factor erythroid-2 (Nrf2) mRNA expression. These findings collectively revealed that TPJ possesses anti-inflammatory, potent antioxidant, and antiapoptotic effect against acrylamide-induced testicular damage.

Climbazole causes cell apoptosis and lipidosis in the liver of grass carp

Climbazole, an azole, is widely used in personal care products, pharmaceuticals, and pesticides and is frequently detected in surface water. Climbazole has showed endocrine-disrupting effects. However, the effects of climbazole in fish are still largely unclear. In this study, grass carp (Ctenopharyngodon idella) and liver cell lines (L8824 cells) were treated with climbazole at concentrations ranging from 0.2 to 20 μg/L for 42 days in vivo and 24 h in vitro to evaluate the effects on the liver, respectively. Pathological, biochemical, and gene transcription and expression analyses were conducted to examine the hepatotoxicity. Our results showed that climbazole significantly decreased the hepatosomatic index, caused cell apoptosis in vivo and in vitro, and finally accumulated lipids in the liver. Beside, climbazole increased ROS levels, reduced Nrf2 and Keap1 mRNA and protein levels, and further decreased transcription of Nrf2-dependent downstream antioxidant enzyme genes, causing oxidative stress. Moreover, climbazole increased transcription and protein levels of apoptosis-related genes. Finally, climbazole damaged mitochondrial function and structure, disrupted liver lipid metabolism. Overall, climbazole caused hepatotoxicity, leading to a high ecological risk for aquatic organisms.

Alleviative effect of poly-β-hydroxybutyrate on lipopolysaccharide-induced oxidative stress, inflammation and cell apoptosis in Cyprinus carpio

In this study, the alleviative effects of poly-β-hydroxybutyrate (PHB) in bioflocs on oxidative stress, inflammation and apoptosis of common carp (Cyprinus carpio) induced by lipopolysaccharide (LPS) were evaluated. Common carp were irregularity divided into 5 groups and fed five diets with 0 % (CK), 2 %, 4 %, 6 % and 8 % PHB. After 8-week feeding trial, LPS challenge was executed. Results showed that appropriate level of PHB enhanced serum immune function by reversing LPS-induced the decrease of C3, C4, IgM, AKP, ACP and LZM in serum, alleviated LPS-induced intestinal barrier dysfunction by decreasing the levels of 5-HT, D-LA, ET-1 and DAO in serum, increasing ZO-1, Occludin, Claudin-3 and Claudin-7 mRNA, improving intestinal morphology. Moreover, dietary PHB reversed LPS-induced the decrease of AST and ALT in hepatopancreas, while in serum exhibited the opposite trend. Suitable level of PHB reversed LPS-induced the reduction of GSH-PX, CAT, T-SOD and T-AOC in intestines and hepatopancreas, whereas MDA showed the opposite result. PHB alleviated LPS-induced the decrease of Nrf2, HO-1, CAT, SOD and GSH-PX mRNA, the increase of Keap1 mRNA. Appropriate level of PHB alleviated LPS-induced inflammation and apoptosis by up-regulating TGF-β, IL-10 and Bcl-2 mRNA, down-regulating NF-κB, TNF-α, IL-6, Bax, Caspase-3, Caspase-8 and Caspase-9 mRNA. Furthermore, PHB inhibited activation of NLRP3 inflammasomes by reducing the levels of NLRP3, Caspase-1, ASC, IL-1β and IL-18 mRNA and protein. In addition, the increases of dietary PHB linearly and quadratically affected LPS-induced adverse effects on common carp. Summary, this study suggested that appropriate level of dietary PHB alleviated LPS-induced oxidative stress, inflammation, apoptosis and the activation of NLRP3 inflammasome in common carp. And the appropriate level of PHB in common carp diets was 4 %.

The Effects of 1-Deoxynojirimycin from Mulberry on Oxidative Stress and Inflammation in Laying Hens and the Direct Effects on Intestine Epithelium Cells In Vitro.

The intestine is highly vulnerable to various factors and has been proposed as a promising determinant for poultry health. Phytogenic or plant-derived feed additives can be used to help improve intestinal health. In this study, we aimed to investigate the effects of DNJ on the antioxidative parameters, including malondialdehyde (MDA), total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and inflammatory cytokines (IL-6, IL-1β, and TNF-α), in plasma and intestinal tissues using layers supplemented with or without the DNJ extract of mulberry leaves (DNJ-E) via the ELISA method. A total of 192 healthy Hy-Line Brown layers, aged 47 weeks old, were used to conduct a 56-day study. All hens were randomly separated into four groups as follows: a basal diet containing 0 mg/kg DNJ-E(CON), 50 mg/kg, 100 mg/kg, and 150 mg/kg DNJ-E. Furthermore, the potential mechanism by which DNJ influences intestinal function was also investigated in in vitro cultured intestinal epithelium cells (IEC) with quantification methods including the use of a cell counting kit-8 (CCK8), ELISA, qRT-PCR, and ROS detection. The results showed that CAT in plasma significantly increased following 50 mg/kg DNJ-E supplementation. Moreover, 50 mg/kg DNJ-E supplementation was associated with increases in T-SOD in the jejunum and ileum. However, there was no significant difference in inflammatory cytokines between groups in in vivo experiments. Subsequent in vitro IEC studies revealed that cell viability increased significantly following 5 µM and 10 µM DNJ treatments while decreasing significantly following 20 µM DNJ treatment. Antioxidative parameters improved at 5 µM and 10 µM DNJ concentrations. However, there were no ameliorative effects on antioxidant parameters observed under 20 µM DNJ treatment. The expression levels of Nrf2 mRNA increased significantly under DNJ treatment. DNJ treatment was associated with significant changes in the expression of genes of inflammatory cytokines. In conclusion, our study revealed that DNJ could improve oxidative stress and inflammation responses in the chicken intestine. These findings provide a theoretical reference for the development of functional feed additives that regulate intestinal health and lay the foundation for systematically revealing the mechanism of DNJ.

Alantolactone triggers oxeiptosis in human ovarian cancer cells via Nrf2 signaling pathway

IntroductionA growing body of evidence indicated that Alantolactone (ALT) promotes Reactive Oxygen Species (ROS) generation exclusively in cancer cells. Therefore, the aim of this study was to investigate the effect of ALT on the molecular mechanism of oxeiptosis, as a novel cell death pathway due to the high levels of intracellular ROS in ovarian cancer. MethodsMTT assay was used to evaluate the effect of ALT on SKOV3 cell viability. mRNA and protein expression levels of Nrf2 (nuclear factor erythroid 2–related factor 2), KEAP1 (Kelch-like ECH-associated protein 1), PGAM5 (phosphoglycerate mutase family member 5), AIFM1 (Mitochondrial Apoptosis-Inducing Factor), Glutathione synthetase (GSS) and glutathione peroxidase (GPX) were analyzed by real time PCR and western blotting methods respectively. ResultsOur findings showed that ALT inhibits the proliferation of skov3 cells in a time and dose dependent manner and IC50 was 32 μM at 24h.A significant down-regulation of Nrf2, GSH and GPX mRNA levels was seen in skov3 cells incubated with 32 and 64 μM of ALT in comparison with control group, while, mRNA expression levels of PGAM5 and KEAP1 were increased.Western blot analysis showed that ALT significantly decreases protein levels of Nrf2 and increases PGAM5 and KEAP1.ALT dephosphorylated PS116-AIFM1 and total AIFM1 protein level was elevated. ConclusionOur results provided evidence that ALT could be a potential option for ovarian cancer treatment by ROS-mediated oxeiptosis.

Denervation aggravates renal ischemia reperfusion injury via BMAL1-mediated Nrf2/ARE pathway

AimTo explore the change of clock gene rhythm under renal denervation (RDN) and its effect on renal function and oxidative stress during renal ischemia-reperfusion (IR) injury. MethodC57/BL6 mice were randomly divided into 4 groups at daytime 7 A M (zeitgeber time [ZT] 0) or at nighttime 7 P M (ZT12) in respectively: Sham (S) group, RDN group, IR group and RDN + IR (DIR) group. Renal pathological and functional changes were assessed by H&E staining, and serum creatinine, urea nitrogen and neutrophil gelatinase-associated lipocalin levels. Renal oxidative stress was detected by SOD and MDA levels, and renal inflammation was measured by IL-6, IL-17 A F and TNF-ɑ levels. BMAL1, CLOCK, Nrf2 and HO-1 mRNA and protein expressions were tested by qPCR and Western Blot. ResultCompared with S groups, the rhythm of BMAL1, CLOCK and Nrf2 genes in the kidney were disordered in RDN groups, while renal pathological and functional indexes did not change significantly. Compared with IR groups, renal pathological and functional indexes were significantly higher in the DIR groups, as well as oxidative stress and inflammation in renal tissues. The nocturnal IR injury in the RDN kidney was the worst while the BMAL1, Nrf2 and HO-1 expressions were the highest. In DIR groups, renal injury was aggravated after the Brusatol treatment, but there was no significant improvement after the t-BHQ treatment at night, which might be consistent with the changes of Nrf2 and HO-1 protein expressions. ConclusionRDN lead to the disruption of BMAL1-mediated Nrf2 rhythm accumulation in the kidney, which reduced the renal ability to resist oxidative stress and inflammation, due to the impaired effect of activating Nrf2/ARE pathway in renal IR injury at nighttime.

Herbal cake-separated moxibustion relieves aspirin induced gastric mucosal injury by activating Nrf2/ARE/HO-1 signaling in rats

To investigate whether herbal cake-separated moxibustion can activate nuclear factor erythroid 2-related factor 2 （Nrf2） / antioxidant responsive element （ARE） /hemeoxygenase-1 （HO-1） signaling pathway to repair aspirin induced gastric mucosal injury （GMI） in rats. SD rats （half male and half female） were randomly divided into blank control, model, moxibustion, inhibitor of HO-1 （inhibitor）, model+inhibitor, moxibustion + inhibitor groups, with 20 rats in each group. The GMI model was established by gavage of aspirin 150 mg/kg（1 mL/100 g）. Herbal cake-separated moxibustion was alternatively applied to bilateral "Zusanli" （ST36） and "Zhongwan" （CV12） and bilateral "Pishu" （BL20） and "Weishu" （BL21） for 30 min, once daily for 8 days. The rats in the three inhibitor groups received intraperitoneal injection of HO-1 inhibitor zinc protoporphyrin （5 mg/kg）. The rats' behavior score, emotional response score, skin hair score, diet score and stool state score were given. The GMI index was calculated according to Guth's methods. Histopathological changes of gastric mucosa were observed by H.E. staining. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the gastric mucosal tissue and serum were detected by ELISA. The levels of Nrf2/ARE/HO-1 signaling pathway-related factors Keap1, Nrf2, HO-1, CAT, GST, and NQO1 in the gastric mucosal tissue were detected by quantitative real-time PCR and Western blot, separately. Compared with the blank control group, the behavior score, emotional response score, skin hair score, diet score and stool state score, GMI index, MDA contents of gastric mucosal tissue and serum, expression le-vels of Keap1 mRNA and protein were significantly increased （P<0.01）, while the activity of gastric mucosal and serum SOD, the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins were considerably decreased （P<0.01） in the model group. Compared with the model group, moxibustion obviously reversed the increase of emotional response score, skin hair score, stool state score, GMI index, MDA levels of gastric mucosal tissue and serum, and expression levels of Keap1 mRNA and protein （P<0.01, P<0.05）, and the decrease of activity of SOD of gastric mucosal and serum, and the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. After administration of antagonist of HO-1, the effects of moxibustion were eliminated or weakened pronouncedly in reducing skin hair score, GMI index, contents of gastric mucosal and serum MDA, and expression of Keap1 mRNA and protein, and in up-regulating gastric mucosal and serum SOD, and expression of HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. Herbal-cake separated moxibustion can improve the GMI in rats, which may be associated with its effects in reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathway.

Herbal cake-separated moxibustion relieves aspirin induced gastric mucosal injury by activating Nrf2/ARE/HO-1 signaling in rats

To investigate whether herbal cake-separated moxibustion can activate nuclear factor erythroid 2-related factor 2 （Nrf2） / antioxidant responsive element （ARE） /hemeoxygenase-1 （HO-1） signaling pathway to repair aspirin induced gastric mucosal injury （GMI） in rats. SD rats （half male and half female） were randomly divided into blank control, model, moxibustion, inhibitor of HO-1 （inhibitor）, model+inhibitor, moxibustion + inhibitor groups, with 20 rats in each group. The GMI model was established by gavage of aspirin 150 mg/kg（1 mL/100 g）. Herbal cake-separated moxibustion was alternatively applied to bilateral "Zusanli" （ST36） and "Zhongwan" （CV12） and bilateral "Pishu" （BL20） and "Weishu" （BL21） for 30 min, once daily for 8 days. The rats in the three inhibitor groups received intraperitoneal injection of HO-1 inhibitor zinc protoporphyrin （5 mg/kg）. The rats' behavior score, emotional response score, skin hair score, diet score and stool state score were given. The GMI index was calculated according to Guth's methods. Histopathological changes of gastric mucosa were observed by H.E. staining. The activity of superoxide dismutase （SOD） and the content of malondialdehyde （MDA） in the gastric mucosal tissue and serum were detected by ELISA. The levels of Nrf2/ARE/HO-1 signaling pathway-related factors Keap1, Nrf2, HO-1, CAT, GST, and NQO1 in the gastric mucosal tissue were detected by quantitative real-time PCR and Western blot, separately. Compared with the blank control group, the behavior score, emotional response score, skin hair score, diet score and stool state score, GMI index, MDA contents of gastric mucosal tissue and serum, expression le-vels of Keap1 mRNA and protein were significantly increased （P<0.01）, while the activity of gastric mucosal and serum SOD, the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins were considerably decreased （P<0.01） in the model group. Compared with the model group, moxibustion obviously reversed the increase of emotional response score, skin hair score, stool state score, GMI index, MDA levels of gastric mucosal tissue and serum, and expression levels of Keap1 mRNA and protein （P<0.01, P<0.05）, and the decrease of activity of SOD of gastric mucosal and serum, and the expression levels of Nrf2, HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. After administration of antagonist of HO-1, the effects of moxibustion were eliminated or weakened pronouncedly in reducing skin hair score, GMI index, contents of gastric mucosal and serum MDA, and expression of Keap1 mRNA and protein, and in up-regulating gastric mucosal and serum SOD, and expression of HO-1, CAT, GST and NQO1 mRNAs and proteins （P<0.01, P<0.05）. Herbal-cake separated moxibustion can improve the GMI in rats, which may be associated with its effects in reducing oxidative stress and activating Nrf2/ARE/HO-1 signaling pathway.

Remote ischemic conditioning attenuates oxidative stress and inflammation via the Nrf2/HO-1 pathway in MCAO mice

The protective effects of remote ischemic conditioning (RIC) on acute ischemic stroke have been reported. However, the protective mechanisms of RIC have not been fully elucidated. This study aimed to investigate whether RIC could reduce oxidative stress and inflammatory responses in middle cerebral artery occlusion (MCAO)-reperfusion mice via the nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. C57BL/6 mice were subjected to MCAO and underwent RIC twice daily at 1, 3, and 7 days after MCAO. ML385 was used to specifically inhibit Nrf2 in MCAO mice. Neurological deficit scores, infarct volume, and hematoxylin-eosin (HE) staining were assessed. Oxidative stress levels were assessed based on total antioxidant capacity (TAC), malonaldehyde (MDA), superoxide dismutase (SOD), and glutathione/glutathione disulfide (GSH/GSSG). mRNA levels were detected using real-time polymerase chain reaction (PCR), and protein levels were detected using western blotting and enzyme-linked immunosorbent assay (ELISA). Protein localization was investigated using immunofluorescence staining. RIC significantly reduced infarct volume and improved neurological function and histological changes after MCAO. RIC significantly increased TAC, SOD, and GSH/GSSG levels and decreased MDA levels. RIC significantly increased Nrf2 and HO-1 mRNA levels and decreased Keap1, NLRP3, and Cleaved Caspase-1 mRNA levels. RIC significantly increased Nrf2, HO-1, and NQO1 protein expression and decreased Keap1, NLRP3, Cleaved Caspase-1, Cleaved IL-1β, IL-6, and TNF-α protein expression. RIC promoted the activation and translocation of Nrf2 into the nucleus. The protective effects of RIC were abolished by ML385 treatment. In conclusion, our findings suggest that RIC alleviates oxidative stress and inflammatory responses via the Nrf2/HO-1 pathway, which in turn improves neurobehavioral function. RIC may provide novel therapeutic options for acute ischemic stroke.

Involvement of Nrf2 Activation and NF-kB Pathway Inhibition in the Antioxidant and Anti-Inflammatory Effects of Hesperetin in Activated BV-2 Microglial Cells.

Alzheimer's disease is a progressive neurodegenerative disorder leading to cognitive decline and memory loss. The incidence of this disease continues to increase due to the limited number of novel therapeutics that prevent or slow down its progression. Flavonoids have been investigated for their potential effects on cellular damage triggered by excessive reactive oxygen species (ROS) and neuroinflammatory conditions. This study investigated the effect of the flavonoid hesperetin on LPS-activated murine BV-2 microglial cells. Results show that hesperetin reduced nitric oxide levels and increased catalase, glutathione, and superoxide dismutase levels, suggesting its potential to reduce neuroinflammation and oxidative stress. Moreover, RT-PCR arrays showed that hesperetin modulated multiple genes that regulate oxidative stress. Hesperetin downregulated the mRNA expression of ERCC6, NOS2, and NCF1 and upregulated HMOX1 and GCLC. RT-PCR results showed that hesperetin-induced Nrf2 mRNA and protein expression in LPS-activated BV-2 microglial cells is involved in the transcription of several antioxidant genes, suggesting that hesperetin's antioxidant effects may be exerted via the Keap1/Nrf2 signaling pathway. Furthermore, the data demonstrated that hesperetin reduced the gene expression of PD-L1, which is upregulated as an individual ages and during chronic inflammatory processes, and inhibited the expression of genes associated with NF-kB signaling activation, which is overactivated during chronic inflammation. It was concluded from this investigation that hesperetin may have therapeutic potential to prevent or slow down the progression of neurodegenerative diseases, such as Alzheimer's disease, by reducing chronic oxidative stress and modulating neuroinflammation.

Amelioration of oxidative kidney damage in offspring by maternal trans-fatty acid exposure in mice by secoisolariciresinol diglucoside.

Trans-fatty acids (TFAs), primarily derived from the food industry's production processes, have become a globally recognized public health issue due to the detrimental impact they have on human well-being. Secoisolariciresinol diglucoside (SDG) is a polyphenolic compound derived from flax lignans, possessing antioxidative properties. This study aims to investigate the protective effect of SDG on kidney oxidative damage in offspring of mice caused by maternal exposure to TFA during pregnancy and lactation. A total of 30 c57BL/6 female rats were randomly divided into 5 groups: a control group, a TFA-exposed group, a low-(TFA+LSDG) group, a medium-(TFA+MSDG) group, and a high-(TFA+HSDG) group (n=6 in each group). With the exception of the control group, the maternal mice in the remaining 4 groups received a daily oral gavage of TFA at a dosage of 60 mg/(kg·BW) throughout the experimental period. The mothers in the control group were administered physiological saline via oral gavage once daily. Meanwhile, the 3 SDG intervention groups were provided with ad libitum access to SDG feed containing 10 mg/kg (low), 20 mg/kg (medium), and 30 mg/kg (high) of SDG. The female mice were conceived overnight. If the vaginal plug appeared in the next morning, the female mice were conceived and included in the experimental stage until the end of the 21th day lactation period. The body weight and kidney mass of offspring were recorded, and the kidney coefficient was calculated. The kidney was detected by HE staining to observe the histopathological changes, and the level of reactive oxidative species (ROS) was detected by fluorescence probe-dihydroethidium (DHE) staining; the expression levels of total superoxide dismutase (T-SOD) and malondialdehyde (MDA) in renal homogenate were detected, and the expression of nuclear factor E2-related fator2 (Nrf2) and hemeoxygenase-1 (HO-1) protein was analyzed by immunohistochemistry (IHC) staining. The mRNA expressions of Nrf2 and HO-1 were detected by real-time PCR, and the protein expression of Cu/Zn-superoxide dismutase (Cu/Zn-SOD), Mn-superoxide dismutase (Mn-SOD), glutathione peroxidase-1 (GPx-1), Nrf2 and HO-1 were detected by Western blotting. Compared with the control group, the kidney coefficient in the TFA-exposed group was increased, the morphology and structure of kidney tissue was abnormal; the activity of T-SOD enzyme was decreased, and the content of MDA was increased, the level of ROS was increased; the expressions of Cu/Zn-SOD, Mn-SOD, GPx1 protein were decreased, and the mRNA and protein expressions of Nrf2 and HO-1 were decreased, there were all significant difference (all P<0.05). Compared with the TFA-exposed group, the ROS levels were reduced, and the T-SOD enzyme activity as well as the protein expression of Cu/Zn-SOD, GPx-1, Mn-SOD, Nrf2 and HO-1 were up-regulated in the low, middle and high dose SDG intervention groups; the kidney coefficient and MDA content were decreased in the middle and high dose SDG groups; the Nrf2 mRNA expression in the high dose SDG group was up-regulated, there were all significant difference (all P<0.05). Maternal exposure to TFA during pregnancy and lactation can lead to oxidative damage in the kidney of offspring, and the SDG intervention may alleviate TFA-induced oxidative damage by up-regulating the expression of Nrf2 and HO-1 signal pathway.

Intervention of Dietary Protein Levels on Muscle Quality, Antioxidation, and Autophagy in the Muscles of Triploid Crucian Carp (Carassius carassius Triploid).

The aim of this study is to investigate the effect of dietary protein levels on flesh quality, oxidative stress, and autophagy status in the muscles of triploid crucian carp (Carassius carassius triploid), and the related molecular mechanisms. Six experimental diets with different protein levels (26%, 29%, 32%, 35%, 38%, 41%) were formulated. A total of 540 fish with an initial weight of 11.79 ± 0.09 g were randomly assigned to 18 cages and six treatments with three replicates of 30 fish each for 8 weeks feeding. It could be found that the whole-body ash content significantly increased in high protein level groups (p < 0.05). The 29% dietary protein level group exhibited the highest muscle moisture, although there was an inconspicuous decrease in the chewiness of the muscles when compared with the other groups. The dietary protein level influenced the content of free amino acids and nucleotides, especially the content of flavor amino acids, which exhibited an increasing tendency along with the increasing protein level, such as alanine and glutamic acid, while the flavor nucleotides showed different fluctuation trends. Moreover, the genes related to muscle development were shown to be influenced by the dietary protein level, especially the expression of MRF4, which was up-regulated with the increasing dietary protein levels. The 29% dietary protein level promoted the majority of analyzed muscle genes expression to the highest level when compared to other dietary levels, except the Myostain, whose expression reached its highest at 38% dietary protein levels. Furthermore, the effect of dietary protein levels on antioxidant signaling pathway genes were also examined. High protein levels would boost the expression of GSTα; GPX1 and GPX4α mRNA expression showed the highest level at the 32% dietary protein group. The increasing dietary protein level decreased both mRNA and protein expressions of Nrf2 by up-regulating Keap1. Autophagy-related gene expression levels reached the peak at 32% dietary protein level, as evidenced by a similar change in protein expression of FoxO1. In summary, muscle nutritional composition, antioxidative pathways, and autophagy levels were affected by the dietary protein levels. A total of 29-32% dietary protein level would be the appropriate level range to improve muscle quality and promote the antioxidant and autophagy capacity of triploid crucian carp muscles.

Heat-tonifying acupuncture relieves pain and synovial inflammatory injury by regulating Keap1-Nrf2/ARE/ HO-1 signaling pathway in rabbits with cold syndrome type rheumatoid arthritis

To observe the effect of heat-tonifying needling on Keap1-Nrf2/ARE/HO-1 signal transduction pathway in knee synovium in rabbits with cold syndrome type rheumatoid arthritis (RA), so as to explore its mechanisms underl-ying improvement of RA. New Zealand rabbits were randomly divided into normal control, RA model, uniform reinforcing-reducing acupuncture, twisting reinforcing acupuncture and heat-tonifying acupuncture groups, with 6 rabbits in each group. The cold syndrome type RA model was established by subcutaneous injection of mixture fluid of ovalbumin and Freund's complete adjuvant at the shoulder-back as well as injection of mixture of ovalbumin and normal saline into knee-joint cavity combined with ice-compress freezing. Acupuncture stimulation (uniform reinforcing-reducing, or twisting reinforcing or heat-tonifying) was applied to bilateral "Zusanli"(ST36) for 1 min with the needle retained for 30 min, once a day for 7 consecutive days. The general conditions of rabbits in each group were recorded, the thermal pain threshold (TPT) and perimeter of knee joints was measured. Conditions of the synovium in the knee cavity, hydrops, blood flow signal, articular surface, and related muscles were observed by using a color Doppler ultrasonic diagnostic apparatus, and the blood flow signals inside the synovium (image scores) were divided into 0 (no signals), I (1 or 2 dot-like signal), II (less than half) ad III (more than half). After H.E. staining, the pathological changes (0-3 points) were assessed according to the state of inflammatory cell infiltration, and hyperplasia of synovial matrix and coating cells. The expression levels of Keap1, Nrf2, HO-1 and GSH-PX1 mRNAs in the knee synovium were detected by quantitative real-time PCR, and the expression of knee synovial HO-1 protein was measured by Western blot. In comparison with the normal control group, the model group had a significant increase in the perimeter, pathological score, expression of Nrf2, HO-1 mRNAs and HO-1 protein (P<0.05), and an obvious decrease in the TPT, expression levels of Keap1 and GSH-PX1 mRNAs (P<0.05). Relevant to the model group, all the three acupuncture maneuvers reversed modeling-induced increase of perimeter and pathological score (P<0.05), decrease of TPT and expression of GSH-PX1 mRNA(P<0.05), further down-regulated expression of Keap1 mRNA (P<0.05), further up-regulated the expression of Nrf2, HO-1 mRNAs and HO-1 protein (P<0.05). The heat-reinforcing manipulation was significantly superior to uniform reinforcing-reducing and twirling reinforcing manipulations in up-regulating TPT, and expression of Nrf2 mRNA, GSH-PX1 mRNA, HO-1 mRNA and protein (P<0.05), and in down-regulating pathological score and Keap1 mRNA expression (P<0.05). Heat-tonifying, uniform reinforcing-reducing and twirling reinforcing needling manipulations may relieve pain and improve pathological state in RA rabbits, which may be associated with their functions in raising the ability of anti-oxidative stress by regulating Keap1-Nrf2/ARE/ HO-1 signaling pathway, the therapeutic effect of heat-tonifying needling is superior to that of uniform reinforcing-reducing and twirling reinforcing needling.

ETS1 Ameliorates Hyperoxia-Induced Bronchopulmonary Dysplasia in Mice by Activating Nrf2/HO-1 Mediated Ferroptosis.

Bronchopulmonary dysplasia (BPD) is associated with hyperoxia-induced oxidative stress-associated ferroptosis. This study examined the effect of E26 oncogene homolog 1 (ETS1) on oxidative stress-associated ferroptosis in BPD. Hyperoxia-induced A549 cells and neonatal mice were used to establish BPD models. The effects of ETS1 on hyperoxia-induced ferroptosis-like changes in A549 cells were investigated by overexpression of ETS1 plasmid transfection and erastin treatment. Glucose consumption, lactate production, and NADPH levels were assessed by the glucose, lactate, and NADP+/NADPH assay kits, respectively. The potential regulatory relationship between ETS1 and Nrf2/HO-1 was examined by treating hyperoxia-induced A549 cells with the Nrf2 inhibitor ML385. ETS1 effect on the Nrf2 promoter was explored by dual-luciferase reporter and chromatin immunoprecipitation assay. The effect of ETS1 on the symptoms of BPD mice was examined by injecting an adenovirus overexpressing ETS1. ETS1 overexpression increased hyperoxia-induced cell viability, glucose consumption, lactate production, and NADPH levels and reduced inflammation and apoptosis in A549 cells. In animal experiments, ETS1 overexpression prevented weight loss, airway enlargement, and reductions in radial alveolar counts in BPD mice, while reducing the mean linear intercept, mean alveolar diameter and inflammation. ETS1 overexpression suppressed PTGS2 and CHAC1 expression, reduced ROS, MDA and ferrous iron (Fe2+) production and increased GSH levels in hyperoxia-induced A549 cells and BPD mice. In addition, ETS1 can bind to the Nrf2 promoter region and thus promote Nrf2 transcription. ETS1 overexpression increased the mRNA and protein levels of Nrf2, HO-1, xCT, and GPX4 in hyperoxia-induced A549 cells and BPD mice. In hyperoxia-induced A549 cells, erastin and ML385 treatment abolished the effect of ETS1 overexpression. ETS1 is important in oxidative stress-related ferroptosis in a hyperoxia-induced BPD model, and the effect is partially mediated by the Nrf2/HO-1 axis.

Effects of sinapic acid on lead acetate-induced oxidative stress, apoptosis and inflammation in testicular tissue.

In this study, the effect of lead acetate (PbAc) and sinapic acid (SNP) administration on oxidative stress, apoptosis, inflammation, sperm quality and histopathology in testicular tissue of rats was tried to be determined. PbAc was administered at a dose of 30 mg/kg/bw for 7 days to induce testicular toxicity in rats. Oral doses of 5 and 10 mg/kg/bw SNP were administered to rats for 7 days after PbAc administration. According to our findings, while PbAc administration increased MDA content in rats, it decreased GPx, SOD, CAT activity and GSH content. NF-kB, IL-1β, TNF-α, and COX-2, which are among the inflammation parameters that increased due to PbAc, decreased with the administration of SNP. Nrf2, HO-1, and NQO1 mRNA transcript levels decreased with PbAc, but SNP treatments increased these mRNA levels in a dose-dependent manner. RAGE and NLRP3 gene expression were upregulated in PbAc treated rats. MAPK14, MAPK15, and JNK relative mRNA levels decreased with SNP treatment in PbAc treated rats. While the levels of apoptosis markers Bax, Caspase-3, and Apaf-1 increased in rats treated with PbAc, the level of Bcl-2 decreased, but SNP inhibited this apoptosis markers. PbAc caused histopathological deterioration in testis tissue and negatively affected spermatogenesis. When the sperm quality was examined, the decrease in sperm motility and spermatozoon density caused by PbAc, and the increase in the ratio of dead and abnormal spermatozoa were inhibited by SNP. As a result, while PbAc increased apoptosis and inflammation by inducing oxidative stress in testicles, SNP treatment inhibited these changes and increased sperm quality.

Neurotoxicity of 4-nonylphenol in adult zebrafish: Evaluation of behaviour, oxidative stress parameters and histopathology of brain

Nonylphenol and its derivatives use as plasticizer or additives in manufacturing industries. Effluents originated from industrial areas are being added to soil, ground water, river and marine water intentionally or unintentionally. Complex mixture of these contaminants enter the food chain and produce sub-lethal deleterious effects mainly on nervous and reproductive systems of aquatic animals and human beings. The information pertaining to oxidative stress-mediated alterations in brain of zebrafish would be helpful to understand the toxicity potential of such compounds in aquatic animals. The aim of the present study was to evaluate the behavioural changes, status of oxidative stress markers; sod, cat, and NF-E2-related factor 2 (nrf2) mRNA gene expression profile; and histopathological changes in the brain of adult zebrafish exposed to 4-nonylphenol (4NP) at concentration of 100 and 200 μg/L of water for 21 days. Zebrafish were divided into four groups viz; control (C1), vehicle (C2, ethanol 10 μg/L of water), treatment 1 (T1, 4-NP, 100 μg/L) and treatment 2 (T2, 4-NP, 200 μg/L). Both exposure levels of 4-NP adversely affected the exploratory behaviour of zebrafish and produced anxiety-like symptom. Concentration-dependent reduction in activity of superoxide dismutase and catalase; and glutathione level, with increased level of malondialdehyde recorded in the brain of exposed zebrafish. Gene expression analysis showed down regulation of sod, cat, nrf2 genes in brain of zebrafish from toxicity groups indicating 4-NP induced oxidative stress in brain. However, noticeable histological alterations were not observed in 4-NP exposed brain of zebrafish.

Huangqin Decoction Delays Progress of Colitis-Associated Carcinogenesis by Regulating Nrf2/HO-1 Antioxidant Signal Pathway in Mice.

To investigate the effect of Huangqin Decoction (HQD) on nuclear factor erythroid 2 related-factor 2 (Nrf2)/heme oxygenase (HO-1) signaling pathway by inducing the colitis-associated carcinogenesis (CAC) model mice with azoxymethane (AOM)/dextran sodium sulfate (DSS). The chemical components of HQD were analyzed by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS/MS) to determine the molecular constituents of HQD. Totally 48 C57BL/6J mice were randomly divided into 6 groups by a random number table, including control, model (AOM/DSS), mesalazine (MS), low-, medium-, and high-dose HQD (HQD-L, HQD-M, and HQD-H) groups, 8 mice in each group. Except for the control group, the mice in the other groups were intraperitoneally injected with AOM (10 mg/kg) and administrated with 2.5% DSS orally for 1 week every two weeks (totally 3 rounds of DSS) to construct a colitis-associated carcinogenesis mouse model. The mice in the HQD-L, HQD-M and HQD-H groups were given HQD by gavage at doses of 2.925, 5.85, and 11.7 g/kg, respectively; the mice in the MS group was given a suspension of MS at a dose of 0.043 g/kg (totally 11 weeks). The serum levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were measured by enzyme-linked immunosorbent assay. The mRNA and protein expression levels of Nrf2, HO-1, and inhibitory KELCH like ECH-related protein 1 (Keap1) in colon tissue were detected by quantitative real-time PCR, immunohistochemistry, and Western blot, respectively. LC-Q-TOF-MS/MS analysis revealed that the chemical constituents of HQD include baicalin, paeoniflorin, and glycyrrhizic acid. Compared to the control group, significantly higher MDA levels and lower SOD levels were observed in the model group (P<0.05), whereas the expressions of Nrf2 and HO-1 were significantly decreased, and the expression of Keap1 increased (P<0.01). Compared with the model group, serum MDA level was decreased and SOD level was increased in the HQD-M, HQD-H and MS groups (P<0.05). Higher expressions of Nrf2 and HO-1 were observed in the HQD groups. HQD may regulate the expression of Nrf2 and HO-1 in colon tissue, reduce the expression of MDA and increase the expression of SOD in serum, thus delaying the progress of CAC in AOM/DSS mice.

Xiangshao Decoction alleviates gastric mucosal injury through NRF2 signaling pathway and reduces neuroinflammation in gastric ulcer rats

BackgroundA type of gastric mucosal injury disease known as gastric ulcer (GU) is clearly connected to the aberrant release of gastric acid. Traditional botanicals have the potential for anti-inflammation, anti-oxidation, and other multitarget therapies, as well as being safe. PurposeThe purpose of this study was to investigate the potential effects of Xiangshao Decoction (XST) on gastric mucosal injury in GU rats and to explore the possible molecular mechanisms. MethodsAfter identifying XST and its components, we established GU rats and cell models by acetic acid and H2O2 induction, respectively. SOD and MDA indexes in gastric tissues and GES-1 cells, and the serum levels of BDNF, ALT, and AST were detected with relevant kits, changes of the gastric mucosa were observed and recorded, and gastric tissue pathology was observed by H&E staining. The production of ROS in GES-1 cells was detected by fluorescent probes. Cell transfection techniques were used to silence or overexpress NRF2. The mRNA or protein expressions of NRF2, KEAP1, NQO1, HO-1, SOD2, IL-1β, IL-6, TNF-α, IBA1, GFAP, or γ-H2AX in the gastric tissue, hippocampus, or GES-1 cells were measured via qPCR, Western blot, immunofluorescence staining, or immunohistochemical staining. ResultsThe pH of gastric acid, ulcer score, and pathological damage score in GU rats could be reversed by XST administration. Expressions of IL-1β, IL-6, and TNF-α in the gastric mucosal tissues and the hippocampus of GU rats after administration of XST were down. Expressions of NRF2, NQO1, HO-1, SOD2, etc. in the gastric mucosal tissues and BDNF in the hippocampus were up-regulated. The production of ROS and MDA and the expressions of IL-1β, IL-6, TNF-α, and KEAP1 in H2O2-induced GES-1 cells were significantly reduced after XST intervention, while the activities of SOD and the expression of NRF2, NQO1, HO-1, and SOD2 were significantly increased, and these could be blocked by silencing NRF2 expression. ConclusionsXST can improve oxidative stress injury and inflammatory response in GU rats and cell models, and its mechanism is mediated by the NRF2 signaling pathway.

Effects of resveratrol on HIF-1α/VEGF pathway and apoptosis in vitrified duck ovary transplantation

Preservation of ovarian tissues is an effective way to ensure genetic diversity of susceptible natural bird populations that are in danger of extinction. We examined whether the addition of the plant phenol resveratrol to vitrification solutions ameliorates the damaging effects of tissue hypoxia and reperfusion injury when the tissues are transplanted. Duck ovary tissues were frozen in the presence of varying concentrations of resveratrol in cryopreservation solutions and then transplanted under the renal capsules of 2-day-old Shelducks. Samples of the transplanted tissues were examined on days 3- and 9- post transplantation for activation of hypoxia-, antioxidant- and apoptosis-related gene expression and apoptosis. Resveratrol significantly increased expression of VEGF, HIF-1α, Nrf2, CAT and Bcl-2 mRNA and decreased BAX and Caspase-3 mRNA and reduced numbers of TUNEL-positive cells after vitrification and heterotopic ovarian transplantation. Resveratrol improved the antioxidant capacity, reduced apoptosis and activated the HIF-1α/VEGF pathway to promote angiogenesis 3- and 9-days following transplantation. These results indicated that the addition of resveratrol to vitrification solutions intended for long-term cryopreservation of ovary tissues improves survival in storage and the grafts following transplantation. This study provides a theoretical basis for the successful transplantation of avian ovarian tissue after vitrification.