mRNA Levels Of Keap1 Research Articles

Carthamus tinctorius L. (Safflower) Flower Extract Attenuates Hepatic Injury and Steatosis in a Rat Model of Type 2 Diabetes Mellitus via Nrf2-Dependent Hypoglycemic, Antioxidant, and Hypolipidemic Effects.

This study aimed to examine the hepatic and anti-steatotic protective effects of methanolic extract from Carthamus tinctorius (safflower) flowers (SFFE), using a rat model of type 2 diabetes mellitus (T2DM), and to examine the molecular mechanisms underlying these effects. Adult male Wistar rats were used for this study. First, T2DM was induced in some rats by feeding them a high-fat diet (HFD) for 4 weeks, followed by a single dose of streptozotocin (STZ) (35 mg/kg, i.p.). Experimental groups included the following five groups (n = 8 in each): control, control + SFFE, T2DM, T2DM + SFFE, and T2DM + SFFE + brusatol (an Nrf2 inhibitor, 2 mg/kg, i.p.). SFFE was administered at a concentration of 300 mg/kg, and all experiments concluded after 8 weeks. Treatments with SFFE significantly reduced fasting blood glucose levels, free fatty acids (FFAs), cholesterol, triglycerides, and low-density lipoprotein cholesterol in both the control and T2DM rats, but they failed to reduce fasting insulin levels in these groups. SFFE treatments also improved the liver structure and reduced hepatocyte vacuolization and hepatic levels of triglycerides and cholesterol in T2DM rats, in addition to increasing the hepatic mRNA levels of keap1 and the cytoplasmic levels and nuclear activities of Nrf2 in both the control and T2DM rats. SFFE also stimulated the expression levels of PPARα and CPT-1 but reduced the malondialdehyde (MDA), mRNA levels of SREBP1, fatty acid synthase, and acetyl CoA carboxylase in both the control and T2DM rats; meanwhile, it reduced hepatic mRNA and the nuclear activities of NF-κB and increased levels of glutathione, superoxide dismutase, and heme oxygenase-1 in the livers of both groups of treated rats. Furthermore, SFFE suppressed the levels of caspase-3, Bax, tumor necrosis factor-α, and interleukin-6 in the T2DM rats. Treatment with brusatol prevented all of these effects of SFFE. In conclusion, SFFE suppresses liver damage and hepatic steatosis in T2DM through Nrf2-dependent hypoglycemic, antioxidant, anti-inflammatory, and hypolipidemic effects.

Ameliorative Effect of Lycopene on Follicular Reserve Depletion, Oxidative Damage, Apoptosis Rate, and Hormonal Profile during Repeated Superovulations in Mice.

Superovulation is a crucial step in assisted reproductive technology that involves the administration of gonadotrophins. Repeated superovulations result in severe ovarian damage. The present study investigated the effect of in vivo administration of lycopene on ovarian damage induced by four successive cycles of superovulation. Superovulated mice were simultaneously administered intraperitoneally with saline (R4) or 5 mg/kg lycopene (R4-Lyc). The evaluated parameters were the count of different types of follicles, expression of ovarian antioxidant- and apoptosis-related genes, and serum concentrations of estradiol, progesterone, and inhibin-B. Increased numbers of healthy follicles and a decreased count of atretic follicles were observed in mice of the R4-Lyc group compared to those of the R4 group. Moreover, significantly higher mRNA levels of Sod3, Cat, and Nrf2 and lower mRNA levels of Keap1, Tnf, Nfkb, and Casp3, together with decreased H2O2 concentrations and increased total antioxidant capacity, were detected in the ovaries of lycopene-treated mice. Regarding serum reproductive hormones, elevated concentrations of estradiol, progesterone, and inhibin-B were evident in lycopene-administered mice. The present study reports a significant role of lycopene in alleviating the ovarian damage induced by multiple hormonal superstimulations, which could help to improve the outcomes of in vitro embryo production.

Excessive Replacement of Fish Meal by Soy Protein Concentrate Resulted in Inhibition of Growth, Nutrient Metabolism, Antioxidant Capacity, Immune Capacity, and Intestinal Development in Juvenile Largemouth Bass (Micropterus salmoides).

This study investigated the effects of replacing 0% (SPC0), 25% (SPC25), 50% (SPC50), 75% (SPC75), and 100% (SPC100) of fish meal (FM) with soy protein concentrate (SPC) on the growth, nutritional metabolism, antioxidant capacity, and inflammatory factors in juvenile largemouth bass (Micropterus salmoides) (17.03 ± 0.01 g). After 56 days of culturing, various growth parameters including FW, WGR, and SGR were not significantly different among SPC0, SPC25, and SPC50 groups; however, they were significantly higher than those in SPC75 and SPC100 groups. Conversely, significantly lower FCR were determined for the SPC0, SPC25, and SPC50 groups compared with that for the SPC100 group; specifically, no significant difference among SPC0, SPC25, and SPC50 groups was found. Moreover, compared with SPC75 and SPC100 groups, a significantly higher FI was observed in the SPC0 group, whereas a significantly lower SR was observed in SPC100 compared with that in SPC0 and SPC25 groups. Compared with the SPC0 group, significantly lower mRNA levels of tor, rps6, 4ebp1, pparγ, and fas were found in SPC75 and SPC100. Additionally, the mRNA levels of cpt were significantly higher in SPC0, SPC25, and SPC50 groups than in SPC75 and SPC100 groups. Moreover, the mRNA levels of scd and acc remained unchanged for all the groups. Replacement of FM with SPC did not significantly affect the mRNA levels of gk, pk, and pepck. Compared with the SPC0 group, significantly decreased activities of CAT were observed in the SPC50, SPC75, and SPC100 groups, and significantly decreased activities of GSH-Px were observed in the SPC75 and SPC100 groups. In addition, significantly lower activity of SOD was observed in SPC100 compared with the other groups. Moreover, compared with the other groups, the SPC75 and SPC100 groups had significantly decreased and increased contents of GSH and MDA, respectively, while significantly lower mRNA levels of nrf2, cat, sod, and gsh-px were found in SPC50, SPC75, and SPC100; however, significantly higher mRNA levels of keap1 were observed in SPC75 and SPC100 groups. Additionally, significantly higher mRNA levels of il-8 and nf-κb were found in the SPC50, SPC75, and SPC100 groups compared with the SPC0 group. Conversely, significantly lower mRNA levels of il-10 and significantly higher mRNA levels of tnf-α were found in the SPC75 and SPC100 groups compared with the other groups. Compared with the SPC0 group, mucosal thickness and villus height were significantly decreased in the SPC75 and SPC100 groups. Collectively, SPC replacing 50% FM did not affect its growth of juvenile largemouth bass. However, SPC replacing 50% or more FM might inhibit antioxidant capacity and immune capacity to even threaten the SR, resulting in impaired intestinal development in replacing FM level of 75% or more.

Protective effects of nodosin against lipopolysaccharide-induced acute kidney injury through regulation of oxidative stress, inflammation, and ferroptosis in rats.

This research aimed to explore the impact of nodosin on lipopolysaccharide (LPS)-induced acute kidney injury (AKI) in rats. The study involved administering nodosin orally at doses of 2 and 4mg/kg body weight orally to rats for 7 days before induction of AKI. Toward the end of the study, urine, blood, and kidneys were gathered from the rats to undergo biochemical and molecular examination after sacrificing them. Serum Scr, BUN, urine NGAL, and KIM-1 levels were significantly decreased in nodosin-treated AKI rats. Besides, nodosin administration resulted in a significant reduction in kidney MDA and 4-HNE levels. In contrast, antioxidant enzymes such as SOD, CAT, GPx, and GST levels increased, as well as Nrf2, NQO1, and HO-1 levels increased, while Keap-1 mRNA levels decreased in AKI rats. In addition, AKI rats treated with nodosin reversed excessive ferroptosis in the kidneys of LPS-induced AKI rats, as evidenced by increased mRNA and protein levels of GPX4, SLC7A11, and FTH-1. The administration of nodosin significantly reduced levels of inflammatory markers including TLR4, MYD88, NF-κB p65, IkKβ, and IL-1β, while IL-10 levels increased in the AKI-induced rats. Besides, histopathological changes were reduced in AKI-induced rats treated with nodosin. Nodosin proves highly beneficial in safeguarding the kidney from AKI by regulating oxidative stress, inflammation, and ferroptosis. The treatment of AKI could greatly benefit from this option.

Partridge tea polyphenols alleviated STZ-induced diabetic nephropathy by regulating Keap1/Nrf2/ARE signaling pathway in C57BL/6 mice

The present work investigated the effect of Partridge tea polyphenols (PTP) on streptozotocin (STZ)‑induced diabetic mice as well as the underlying mechanism with rosiglitazone as positive control. Administration with PTP (250 or 500 mg/kg) for 5 weeks, compared to the diabetic nephropathy control group (DC group), PTP significantly decreased the renal index, creatinine in serum and urine, urea nitrogen and proteinuria and alleviated histopathological injury of renal tissue. The levels of TNF-α, IL-1β and IL-6 were decreased and SOD, CAT activities were increased by PTP. PTP significantly reduced the FN and COL-IV levels. Moreover, PTP significantly inhibited the Keap1 mRNA level of Keap1 and the corresponding protein expression, while increased the mRNA levels of Nrf2, HO-1, NQO1 and the corresponding protein expression. Therefore, PTP could reverse renal injury, enhance renal functions, inhibit renal oxidative stress, and activate Keap1/Nrf2/ARE signaling pathway to alleviative diabetic nephropathy.

Novel hub genes and regulatory network related to ferroptosis in tetralogy of Fallot.

Ferroptosis is a newly discovered type of cell death mainly triggered by uncontrolled lipid peroxidation, and it could potentially have a significant impact on the development and progression of tetralogy of Fallot (TOF). Our project aims to identify and validate potential genes related to ferroptosis in TOF. We obtained sequencing data of TOF from the GEO database and ferroptosis-related genes from the ferroptosis database. We employed bioinformatics methods to analyze the differentially expressed mRNAs (DEmRNAs) and microRNAs between the normal control group and TOF group and identify DEmRNAs related to ferroptosis. Protein-protein interaction analysis was conducted to screen hub genes. Furthermore, a miRNA-mRNA-TF co-regulatory network was constructed to utilize prediction software. The expression of hub genes was further validated through quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). After conducting the differential gene analysis, we observed that in TOF, 41 upregulated mRNAs and three downregulated mRNAs associated with ferroptosis genes were found. Further Gene Ontology/Kyoto Encyclopedia of Genes and Genomes analysis revealed that these genes were primarily involved in molecular functions and biological processes related to chemical stress, oxidative stress, cellular response to starvation, response to nutrient levels, cellular response to external stimulus, and cellular response to extracellular stimulus. Furthermore, we constructed a miRNA-mRNA-TF co-regulatory network. qRT-PCR analysis of the right ventricular tissues from human cases showed an upregulation in the mRNA levels of KEAP1 and SQSTM1. Our bioinformatics analysis successfully identified 44 potential genes that are associated with ferroptosis in TOF. This finding significantly contributes to our understanding of the molecular mechanisms underlying the development of TOF. Moreover, these findings have the potential to open new avenues for the development of innovative therapeutic approaches for the treatment of this condition.

N-Acetylcysteine Administration Improves the Redox and Functional Gene Expression Levels in Spleen, Mesenteric Lymph Node and Gastrocnemius Muscle in Piglets Infected with Porcine Epidemic Diarrhea Virus.

Our previous study reported that N-acetylcysteine (NAC) administration improved the function of intestinal absorption in piglets infected with porcine epidemic diarrhea virus (PEDV). However, the effects of NAC administration on the functions of other tissues and organs in PEDV-infected piglets have not been reported. In this study, the effects of NAC on the liver, spleen, lung, lymph node, and gastrocnemius muscle in PEDV-infected piglets were investigated. Thirty-two 7-day-old piglets with similar body weights were randomly divided into one of four groups: Control group, NAC group, PEDV group, and PEDV+NAC group (eight replicates per group and one pig per replicate). The trial had a 2 × 2 factorial design consisting of oral administration of 0 or 25 mg/kg body weight NAC and oral administration of 0 or 1.0 × 104.5 TCID50 PEDV. The trial lasted 12 days. All piglets were fed a milk replacer. On days 5-9 of the trial, piglets in the NAC and PEDV + NAC groups were orally administered NAC once a day; piglets in the control and PEDV groups were orally administered the same volume of saline. On day 9 of trial, piglets in the PEDV and PEDV+NAC groups were orally administrated 1.0 × 104.5 TCID50 PEDV, and the piglets in the control and NAC groups were orally administrated the same volume of saline. On day 12 of trial, samples, including of the liver, spleen, lung, lymph node, and gastrocnemius muscle, were collected. PEDV infection significantly increased catalase activity but significantly decreased the mRNA levels of Keap1, Nrf2, HMOX2, IFN-α, MX1, IL-10, TNF-α, S100A12, MMP3, MMP13, TGF-β, and GJA1 in the spleens of piglets. NAC administration ameliorated abnormal changes in measured variables in the spleens of PEDV-infected piglets. In addition, NAC administration also enhanced the antioxidant capacity of the mesenteric lymph nodes and gastrocnemius muscles in PEDV-infected piglets. Collectively, these novel results revealed that NAC administration improved the redox and functional gene expression levels in the spleen, mesenteric lymph nodes, and gastrocnemius muscle in PEDV-infected piglets.

Asiatic acid alleviates LPS-induced acute kidney injury in broilers by inhibiting oxidative stress and ferroptosis via activation of the Nrf2 pathway

Asiatic acid (AA), a triterpenoid compound isolated from Centella asiatica, has anti-inflammatory, antioxidant and anticancer biological characteristics. To explore the effect of AA on LPS-induced acute kidney injury (AKI) in broilers, a total of 60 one-day-old broilers were randomly divided into 6 groups, including the normal group, AKI model group, AKI+AA 15mg/kg group, AKI+AA 30mg/kg group, AKI+AA 60mg/kg group and normal+AA 60mg/kg group. Hematoxylin-eosin staining was used to observe the histopathology in kidney tissue, and the mRNA and protein expressions related to oxidative stress and ferroptosis were tested by qPCR and western blotting respectively. AA mitigated vacuolar degeneration and enlarged glomerular space caused by LPS in kidney tissue. Additionally, AA significantly increased the mRNA levels of Nrf2, HO-1, NQO1, GCLC, GCLM, GPX4, SLC7A11 and FTH1, and decreased the mRNA levels of Keap1 and PTGS2 in LPS-induced AKI. Likewise, AA significantly upregulated the protein expressions of Nrf2, HO-1, NQO1, GPX4, SLC7A11 and FTH1, and downregulated the protein expressions of Keap1 and PTGS2 in LPS-induced AKI. These results suggested that AA alleviated LPS-induced AKI by inhibiting oxidative stress and ferroptosis through targeting regulation of the Nrf2 pathway in broilers.

Acute deltamethrin exposure induces oxidative stress, triggers endoplasmic reticulum stress, and impairs hypoxic resistance of crucian carp

Deltamethrin (Del) has been widely used for effectively controlling ectoparasites of crucian carp and was also strictly prohibited in a hypoxic environment. A previous study indicated that Del exposure causes gill injury in Carassius auratus, which is associated with oxidative stress and endoplasmic reticulum stress (ER stress), but the precise mechanism is not well understood. Here, crucian carp were exposed to Del (0.61, 1.22, 2.44, 4.88 μg/L) for 24 h and then subjected to acute hypoxia challenge (1.0 mg/L) for 24 h. The results revealed that acute exposure to Del notably increased MDA content but markedly decreased CAT activities. Moreover, the T-AOC and SOD activities first increased and then decreased in the 4.88 μg/L Del group. Likewise, the mRNA levels of Nrf2 signaling and its target genes (ho-1, mt, sod, cat, and gpx1) were significantly downregulated in the high concentration exposure groups, while the mRNA levels of keap1 showed the opposite change trend. Meanwhile, Del exposure evoked the PERK-ATF4-CHOP and IRE1 signaling pathways and triggered ER stress in a dose-dependent manner in crucian carp. Importantly, we found that Del exposure significantly decreased the survival rate of crucian carp after hypoxia challenge by reducing oxygen uptake, modifying energy metabolism, and promoting lactate accumulation. Additionally, Del exposure aggravated gill damage and apoptosis under hypoxic stress, which was confirmed by histological assays. Collectively, we inferred that acute exposure to deltamethrin induces oxidative stress and ER stress and impairs hypoxic resistance of crucian carp.

Rhizoma curcumae Longae ameliorates high dietary carbohydrate-induced hepatic oxidative stress, inflammation in golden pompano Trachinotus ovatus

In general, starch, as a complex carbohydrate, is the most economical energy source in aquaculture for its relatively low cost. However, excessive dietary levels of carbohydrate result in pathological conditions. An 8-week feeding trial with CT (control diet, containing 21% carbohydrate), HC (a high-carbohydrate diet, containing 50% carbohydrate) and HCR (a HC diet supplemented with 0.015% Rhizoma curcumae Longae) was performed to investigate the protective effect of curcumin on high-carbohydrate-induced hepatic oxidative stress and intestine lesion in juvenile Trachinotus ovatus. In the current study, HC group significantly decreased WGR, SGR, plasma CAT activity, intestinal C4 levels, hepatic Nrf2, Keap1, Bach1, HO1, CAT, and GPX mRNA expression as well as ZO-1, Occludin, and Claudin-3, TGF-β mRNA transcription levels, while the opposite was true for plasma AST activity, hepatic MDA contents, intestinal Claudin-15, NF-κB, IL-1β, IL-6, and TNF-α mRNA expression. In contrast with the HC group, the HCR group significantly increased the activities of hepatic CAT, SOD, intestinal C3, C4, IgG and LZM levels, hepatic Nrf2, Bach1, CAT, and GPX mRNA expression as well as intestinal ZO-1, Occludin, Claudin-3, TGF-β and IL-10 mRNA expression levels, but the opposite trend was found in plasma triglyceride content, hepatic lipid deposition, hepatic Keap1 mRNA level as well as intestinal NF-κB, IL-6. In conclusion, high-carbohydrate diet can cause detrimental effect on physiological health status in Trachinotus ovatus, while adding Rhizoma curcumae Longae can improve hepatic and intestinal health status via attenuating the oxidative stress, inflammation, and reducing lipid deposition.

The Protective Effect of α-Lipoic Acid against Gold Nanoparticles (AuNPs)-Mediated Liver Damage Is Associated with Upregulating Nrf2 and Suppressing NF-κB.

This study examined if regulating the keap-1? Nrf2 antioxidant pathway mediated gold nanoparticles (AuNPs) induced liver damage, and examined the protective effect of co-supplement of α-lipoic acid (α-LA). Rats were separated into 4 groups (n = 8/each) as control, α-LA (200 mg/kg), AuNPs (5 µg/2.85 × 1011), and AuNPs (5 µg/2.85 × 1011) + α-LA (200 mg/kg). After 7 days, AuNPs induced severe degeneration in the livers of rats with the appearance of some fatty changes. In addition, it increased serum levels of alanine aminotransferase (ALT) and gamma-glutamyl transferase (ɣ-GTT), and aspartate aminotransferase (AST), as well as liver levels of malondialdehyde (MDA). Concomitantly, AuNPs significantly depleted hepatic levels of total glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) but increased hepatic levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). It also reduced mRNA levels of B-cell lymphoma 2 (Bcl2) and heme oxygenase-1 (HO-1) but significantly increased those of Bax and cleaved caspase-3, as well as the ratio of Bax/Bcl2. In addition, AuNPs enhanced the total and nuclear levels of NF-κB p65 but reduced the mRNA and total and nuclear protein levels of Nrf2. Of note, AuNPs did not affect the mRNA levels of keap-1. All these events were reversed by α-LA in the AuNPs-treated rats. In conclusion, α-LA attenuated AuNPs-mediated liver damage in rats by suppressing oxidative stress and inflammation, effects that are associated with upregulation/activation of Nrf2.

Resveratrol Attenuates Heat Stress-Induced Impairment of Meat Quality in Broilers by Regulating the Nrf2 Signaling Pathway.

Simple SummaryBroilers easily experience heat stress (HS), especially in summer, due to a lack of sweat glands. HS has unfavorable effects on the production efficiency, meat quality, antioxidant capacity, and welfare of broilers, which can be mitigated with nutritional strategies. Resveratrol (RES) has been found to mitigate the HS-induced decrease of antioxidant capacity in muscle and, therefore, improve the meat quality of broilers under HS. Nevertheless, there are few reports on the mechanism of action of RES on meat quality and antioxidant status in the muscle of broilers subjected to HS. Our study demonstrated that RES could improve the antioxidant ability of muscle in broilers under HS by activating the Nrf2 signaling pathway and ultimately relieving the HS-induced deterioration of meat quality.Studies have indicated that dietary resveratrol (RES) improves the meat quality of broilers subjected to heat stress (HS), but the mechanism of action remains unclear. Therefore, the main purpose of this study was to investigate the effect of RES on meat quality, muscle antioxidant status, and its mechanism of action in broilers under HS. A total of 162 male AA broilers at 21 days old with similar weight were randomly assigned to 3 treatment groups with 6 replicates each. The control group (ambient temperature: 22 ± 1 °C) and HS group (ambient temperature: 33 ± 1 °C for 10 h a day from 8:00 to 18:00 and 22 ± 1 °C for the remaining time) were fed a basal diet and the HS + RES group was fed a basal diet with 400 mg/kg RES. The feeding was conducted for 21 continuous days. The results indicated that HS decreased final body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), breast and leg muscle yield, a*24h, pH24h, the activities of catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GSH-Px), and mRNA levels of nuclear factor erythroid 2–related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1), and GSH-Px (p < 0.05). HS also increased b*45min, L*24h, drip loss, malondialdehyde (MDA) content, and kelch-like epichlorohydrin-associated protein 1 (Keap1) mRNA level (p < 0.05). Compared with the HS group, the HS + RES group exhibited a higher ADG, breast and leg muscle yield, a*24h, pH24h, activities of GST and GSH-Px, and mRNA levels of Nrf2, HO-1, and NQO1 but had lower drip loss and Keap1 mRNA level (p < 0.05). RES can improve meat quality and the muscle antioxidant ability of heat-stressed broilers by activating the Nrf2 signaling pathway.

Effects of Dietary Supplementation with Epigallocatechin Gallate on Meat Quality and Muscle Antioxidant Capacity of Broilers Subjected to Acute Heat Stress.

Simple SummaryBroilers are readily affected by acute heat stress (AHS), and the development of intensive and high-density management and the occurrence of high temperatures during the summer exacerbate this problem. AHS has undesirable effects on animal immunity, meat quality, antioxidant capacity, and welfare of broilers, which can be alleviated by nutrition regulation. Epigallocatechin gallate (EGCG) has been found to reduce the damage of AHS on growth performance, antioxidant capacity, and the expression of nuclear factor erythroid 2-related 2 (Nrf2) in liver and jejunum. However, there are few reports on the effect and mechanism of action of EGCG on meat quality and antioxidant function in broilers under AHS. We demonstrated that EGCG protects against AHS-impaired meat quality by improving muscle antioxidant capacity, which seems to be associated with the activation of the Nrf2 signaling pathway. Moreover, these findings suggested that EGCG could be an effective additive to improve meat quality and muscle redox balance by regulating the Nrf2 signaling pathway.This study evaluated epigallocatechin gallate’s (EGCG’s, 400 mg/kg) effect on meat quality and muscle antioxidant status of broilers under acute heat stress (AHS). A total of 144 21-day-old male Huainan partridge chickens were randomly allocated to the EGCG-free group (12 replicates) and the EGCG group (6 replicates). On day 94, the EGCG-free group was divided into the control group (CON) and the AHS group, and then AHS group and EGCG group (identified as AHS + EGCG group) were treated with AHS (33 ± 1 °C for 12 h). AHS increased (p < 0.05) L*24h, drip loss, muscle lactic acid, malondialdehyde (MDA) contents, and kelch-like ECH-associated protein 1 (Keap1) mRNA level, and decreased (p < 0.05) eviscerated percentage, pH24h, a*, muscle total superoxide dismutase (T-SOD) activity, the ratio of T-SOD/MDA and glutathione peroxidase /MDA, glycogen content, and nuclear factor erythroid 2-related 2 (Nrf2), catalase (CAT), NAD(P)H/quinone dehydrogenase 1 (NQO1) mRNA levels. The AHS + EGCG group exhibited lower (p < 0.05) L*24h, drip loss, muscle lactic acid, MDA contents and Keap1 mRNA level, and greater (p < 0.05) eviscerated percentage, pH24h, a*, muscle T-SOD activity, the ratio of T-SOD/MDA, Nrf2, and NQO1 mRNA levels compared with the AHS group. In conclusion, EGCG protects against AHS-impaired meat quality by improving muscle antioxidant capacity, which seems to be associated with the activation of the Nrf2 signaling pathway.

P–227 Fatty acid regulation of Nrf2/Keap1 pathway during mouse preimplantation embryo development

Abstract Study question Our objective is determining whether supplementing embryo culture media with palmitic acid and/or oleic acid impacts Nrf2/Keap1 antioxidant response pathways during preimplantation mouse embryo development. Summary answer Supplementation of embryo culture media with palmitic acid increases cellular Nrf2 levels per embryo after 48-hour culture, while oleic acid reverses this effect. What is known already Obese women experience higher incidence of infertility than women with healthy BMIs. The obese reproductive tract environment supporting preimplantation embryo development is likely to include enhanced free fatty acid (FFA) levels and increased accumulation of reactive oxygen species. Exposure to palmitic acid (PA) in vitro significantly impairs mouse embryo development while increasing ER stress mRNAs. Oleic acid (OA) reverses these effects. To further define effects of FFA exposure, we are characterizing the influence of FFAs on the Nrf2–Keap1 pathway and its downstream antioxidant defense systems. We hypothesize that PA treatment induces Nrf2-Keap1 activity, while OA treatment alleviates pathway activity. Study design, size, duration Female CD–1 mice (4–6 weeks) were super-ovulated via intraperitoneal injections of PMSG, followed 48 hours later by hCG. Female mice were mated with male CD–1 mice (6–8 months) overnight. Females were euthanized using CO2 and two-cell embryos were collected by flushing oviducts. Two-cell embryos were placed into KSOMaa-based treatment groups: 1) BSA (control); 2) 100µM PA; 3) 100µM OA; 4) 100µM PA+OA, and cultured for 48 hours (37 °C; 5% O2, 5% CO2, 90% N2). Participants/materials, setting, methods After 48-hour embryo culture, developmental stages of all mouse embryos were recorded. Immunofluorescence analysis of Nrf2 and Keap1 localization was performed for embryo treatments (BSA, 100µM PA, 100µM OA &amp; 100µM PA+OA) using rabbit polyclonal anti-Nrf2 antibody, with Rhodamine-Phalloidin and DAPI staining. Embryos were imaged using confocal microscopy and Nrf2-positive cells were counted using ImageJ. Nrf2 and Keap1 mRNA abundances were assessed after culture in each treatment condition using RT-qPCR and the delta-delta Ct method. Main results and the role of chance Inclusion of 100µM PA in embryo culture significantly decreased blastocyst development frequency from 70.06±16.38% in the BSA (control) group to 11.61±8.19% in the PA-treated group (p &amp;lt; 0.0001). Embryo culture with 100µM OA and 100µM PA+OA co-treatment did not significantly impair blastocyst development (OA: 61.59±8.07%, p = 0.4053; PA+OA: 63.53±7.63%, p = 0.6204). Embryo culture with PA treatment significantly increased the mean percentage of Nrf2-positive cells to 56.83±30.49% compared with 21.22±15.63% in the control group (p &amp;lt; 0.0001). Conversely, 100µM OA and 100µM PA+OA treatments did not significantly affect Nrf2-positive cell frequencies compared with the control group (OA: 33.28±21.83%, p = 0.1825; PA+OA: 34.84±12.66%, p = 0.0691). Immunofluorescence results show that treating embryos with 100µM PA for 48 hours results in increased levels of cellular Nrf2, while combining 100µM PA with 100µM OA reversed these effects. Preliminary qPCR analysis showed no significant differences in Nrf2 or Keap1 relative transcript abundance between any embryo treatment groups. Nrf2 and Keap1 mRNA levels were both higher after embryo culture with 100µM OA than all other culture groups (p = 0.6268; p = 0.3201). Notably, Keap1 relative transcript levels dropped to undetectable levels after culture with 100µM PA, which suggests an increase in Nrf2 activation.Limitations, reasons for caution: While immunofluorescence localization of Nrf2/Keap1 provides insight into how the proteins behave during preimplantation embryo development, confocal images cannot determine protein-protein interactions or activity levels. Similarly, transcript information from RT-qPCR analysis only provides information about Nrf2 and Keap1 at the transcript level. Nrf2 activity will be assessed via downstream targets. Wider implications of the findings: The Nrf2–Keap1 pathway coordinates numerous cellular defence mechanisms, and is implicated in various diseases, including cancer. Establishing an impact of free fatty acid exposure on Nrf2–Keap1 during preimplantation embryo development will provide valuable information regarding the effects of maternal obesity on outcomes for embryos produced from these patients. Trial registration number Not applicable

Effects of Resveratrol on Growth Performance, Intestinal Development, and Antioxidant Status of Broilers under Heat Stress.

Simple SummaryBroilers have unique physiological characteristics, no sweat glands and full of feathers, which makes it difficult to dissipate heat in high-temperature environments and is prone to heat stress (HS). HS has strong adverse effects on the meat production, growth performance, intestinal morphology, mortality and welfare of broilers, which can be alleviated by nutrition regulation. Resveratrol has been found to reduce the damage of HS on meat quality, immune and inflammatory response of broilers. However, there are few reports on the effects of resveratrol on the intestinal development and antioxidant capacity of broilers under HS. We demonstrated that resveratrol could improve the intestinal development and growth performance of broilers under HS. Besides, these findings suggest that resveratrol may offer an effective nutritional strategy to improve intestinal antioxidant function by regulating the expression of critical factors in the Nrf2 signaling pathway.The study investigated resveratrol’s effect on growth performance, intestinal development, and antioxidant capacity of broilers subjected to heat stress (HS). A total of 162 21-day-old male AA broilers were randomly divided into 3 treatment groups with 6 replicates of 9 birds each. The 3 treatment groups were as follows: the control (CON), in which broilers were housed at 22 ± 1 °C for 24 h day−1, and the HS and HS + resveratrol (400 mg/kg) groups, in which broilers were housed at 33 ± 1 °C for 10 h a day from 8:00 to 18:00 and 22 ± 1 °C for rest of the time. Results indicated that birds in the HS group exhibited lower (p < 0.05) final body weight (BW) and average daily gain (ADG) compared with birds in the CON group. HS birds also had lower (p < 0.05) relative jejunum weight, relative ileum and jejunum length, jejunal villus height, and villus height to crypt depth ratios than the CON group. The activities of glutathione peroxidase (GPX), glutathione S-transferase (GST), superoxide dismutase (SOD), and the mRNA levels of NF-E2-related factor 2 (Nrf2), SOD1, and GPX were also lower (p < 0.05) in the HS than CON group. The HS group had higher (p < 0.05) protein carbonyl (PC) contents and Kelch-like ECH-associated protein 1 (Keap1) mRNA levels. Compared with HS group, the HS + resveratrol group exhibited higher (p < 0.05) BW and ADG, relative jejunum weight, relative length of ileum, jejunal villus height, activities of GPX and GST, and mRNA levels of Nrf2 and SOD1, but they had lower (p < 0.05) PC content and Keap1 mRNA levels. In conclusion, resveratrol can improve the intestinal development and antioxidant function of broilers under HS, and therefore improve growth performance. The mechanism by which resveratrol enhances the intestinal antioxidant capacity is mediated by Nrf2 signaling pathway.

Effects of dietary curcumin inhibit deltamethrin-induced oxidative stress, inflammation and cell apoptosis in Channa argus via Nrf2 and NF-κB signaling pathways

The current study evaluated the ameliorative effects of dietary curcumin (CUR) on oxidative stress, inflammation and cell apoptosis in Channa argus exposed to deltamethrin (DEL) for 28 d. This study was divided into five groups: the first group served as a control group, and the second, third, fourth and fifth groups were exposed to DEL (0.242 ppb) in water and fed CUR-supplemented diets at 0, 100, 200 and 400 mg/kg, respectively. The results showed that the biochemical parameter levels (ADA, COR, LDH, AST and ALT) and the innate immune parameter levels (LYS, CRP, MPO, C3, C4 and IgM) in the serum of fish exposed to DEL were notably increased (P < 0.05), and dietary supplementation with 200 and 400 mg/kg CUR in the DEL exposure period significantly reduced the levels of these parameters (P < 0.05). The levels of ROS, CAT, GSH, SOD, GSH-Px, GST and GSH-Rt in the gill, kidney, spleen, and liver of fish exposed to DEL were significantly decreased (P < 0.05), and there were notable increases (P < 0.05) in the 200 and 400 mg/kg CUR treatments. MDA content exhibited an opposite trend. The results of RT-qPCR analysis showed that dietary supplementation with CUR attenuated the inflammatory response of fish after DEL exposure by downregulating the expression of pro-inflammatory genes (NF-κB p65, IL-1β, IL-6, IL-8, IL-12 and TNF-α) in the liver and spleen and upregulating the expression of anti-inflammatory genes (TGF-β, IκBα and IL-10). Dietary supplementation with CUR was observed to improve the level of oxidative stress of fish after DEL exposure by downregulating the mRNA levels of Keap1 in the liver and spleen and upregulating the mRNA levels of Nrf2, Cu/Zn SOD, GSH-Px, GST and CAT. Dietary supplementation with CUR was determined to improve the cell apoptosis of fish after DEL exposure by downregulating the expression of pro-apoptotic genes (Bcl-2) in the liver and spleen and upregulating the expression of anti-apoptotic genes (Bax, Cas-3, Cas-8, Cas-9, and p53). In brief, the results of our study indicated that supplying optimal dietary CUR could attenuate deltamethrin-induced oxidative stress, inflammation and cell apoptosis in C. argus via the Nrf2 and NF-κB signaling pathways.

Benfotiamine ameliorates high‐carbohydrate diet‐induced hepatic oxidative stress, inflammation and apoptosis in Megalobrama amblycephala

A 12-week nutritional research was conducted to evaluate the influences of benfotiamine on the growth performance, oxidative stress, inflammation and apoptosis in Megalobrama amblycephala (45.25 ± 0.34 g) fed high-carbohydrate (HC) diets. Six diets were prepared, containing the control diet (30% carbohydrate, C), the HC diet (43% carbohydrate) and the HC diet supplemented with four benfotiamine levels (0.7125 (HCB1), 1.425 (HCB2), 2.85 (HCB3) and 5.7 (HCB4) mg/kg). HC diet remarkably decreased DGC, GRMBW, liver T-AOC, SOD and CAT activities, SIRT1 protein expression as well as the mRNA levels of SIRT1, Nrf2, CAT, Mn-SOD and IL10 in liver compared with the C group, but the opposite trend was found in plasma activities of AST and ALT, and contents of IL1β and IL6, liver contents of MDA and mRNA levels of Keap1, NF-κB, TNF α, IL1β, IL6, Bax, caspase 3, caspase 9 and P53. As for benfotiamine supplementation, HCB2 diet remarkably increased DGC, GRMBW, liver T-AOC, SOD and CAT activities, SIRT1 protein expression as well as liver mRNA levels of SIRT1, Nrf2, CAT, Mn-SOD, IL10 and Bcl2, while the opposite was true for plasma AST and ALT activities, and IL1β and IL6 contents, liver MDA contents as well as mRNA levels of Keap1, NF-κB, TNF α, IL1β, IL6, Bax, caspase 3, caspase 9 and P53. In summary, benfotiamine (1.425 mg/kg) promoted the growth, and alleviated the oxidative stress, inflammation and apoptosis of M. amblycephala fed HC diets through the SIRT1-mediated signaling pathway.

Kahweol activates the Nrf2/HO-1 pathway by decreasing Keap1 expression independently of p62 and autophagy pathways.

Kahweol is a diterpene found in coffee beans and unfiltered coffee drinks. Several studies have demonstrated that kahweol induces the nuclear factor erythroid-2 related factor 2/ hemeoxygenase-1 (Nrf2/HO-1) pathway; however, the mechanisms involved are currently unknown. Kelch-like ECH-associated protein 1 (Keap1) is a major regulator of Nrf2 expression and is degraded mostly by autophagy. The p62 protein enhances binding to Keap1 and contributes to the activation of Nrf2. Here, we examined the role of Keap1 regulation in the effect of kahweol on the Nrf2/HO-1 pathway in hepatocytes. In AML12 cells and primary mouse hepatocytes, kahweol increased the levels of Nrf2 and HO-1 protein without increasing expression of the Nrf2 mRNA. In addition, kahweol reduced Keap1 protein levels significantly without decreasing Keap1 mRNA levels. Although regulation of the Keap1-Nrf2-pathway by p62-dependent autophagy is well known, we confirmed here that the reduction of Keap1 protein levels by kahweol does not involve p62-dependent autophagy degradation or ubiquitination. In conclusion, kahweol increases the expression of Nrf2 in hepatocytes by inhibiting translation of the Keap1 mRNA.

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.

Evaluation of antioxidant capacity and immunomodulatory effects of yeast hydrolysates for hepatocytes of blunt snout bream (Megalobrama amblycephala)

An in-vitro study was carried out to examine the effects of yeast hydrolysate (YH) on antioxidant capacity and innate immunity of blunt snout bream (Megalobrama amblycephala) hepatocytes. Fish primary hepatocytes were seeded at a density of 3 × 105 cells mL−1 in 6-well tissue culture plates and treated with two different media including: 1) DMEM/F12 medium (control), and 2) YH medium [DMEM/F12 + 0.1 g L−1 YH]. After incubation for 24 h, the culture medium and primary hepatocytes were collected for subsequent analyses. The results showed no significant (P > 0.05) effect of YH on aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) activities and urea nitrogen (UN) concentration in the conditioned medium. However, significantly (P < 0.05) higher ALT and AST activities were found in YH treated hepatocytes compared to control. Moreover, YH supplementation led to significant enhancement of superoxide dismutase (SOD), catalase (CAT), alternative complement pathway (ACH50) and glutathione peroxidase (GPX) activities and reduction of malondialdehyde (MDA) concentration in the conditioned medium. Furthermore, YH application upregulated the expression of SOD, CAT and NOX2 genes and downregulated mRNA levels of Keap1, Nrf2 and Bach1 in hepatocytes. Also, markedly higher lysozyme activity and albumin concentration were found in the conditioned medium of YH group compared to the control. Additionally, expression of immune-related genes such as antimicrobial peptides 1 (Leap 1) and Leap 2 were significantly upregulated by YH application. Down-regulated expression of NADPH oxidase-2 (NOX2), Kelch-like-ECH-associated protein 1 (Keap1), NF-E2-related factor 2 (Nrf2) and BTB and CNC homolog 1 (Bach1) were observed in YH treated hepatocytes. To conclude, YH supplementation improved antioxidant capacity and innate immunity of blunt snout bream hepatocytes.