Glutathione Peroxidase mRNA Levels Research Articles

Baicalin induces ferroptosis in HepG2 cells by inhibiting ROS-mediated PI3K/Akt/FoxO3a signaling pathway

This study aims to investigate whether baicalin induces ferroptosis in HepG2 cells and decipher the underlying mechanisms based on network pharmacology and cell experiments. HepG2 cells were cultured in vitro and the cell viability was detected by the cell counting kit-8(CCK-8). The transcriptome data of hepatocellular carcinoma were obtained from the Cancer Genome Atlas(TCGA), and the ferroptosis gene data from FerrDb V2. The DEG2 package was used to screen the differentially expressed genes(DEGs), and the common genes between DEGs and ferroptosis genes were selected as the target genes that mediate ferroptosis to regulate hepatocellular carcinoma progression. The functions and structures of the target genes were analyzed by Gene Ontology(GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment with the thresholds of P<0.05 and |log_2(fold change)|>0.5. DCFH-DA probe was used to detect the changes in the levels of cellular reactive oxygen species(ROS) in each group. The reduced glutathione(GSH) assay kit was used to measure the cellular GSH level, and Fe~(2+) assay kit to determine the Fe~(2+) level. Real-time quantitative PCR(RT-PCR) was employed to measure the mRNA levels of glutathione peroxidase 4(GPX4) and solute carrier family 7 member 11(SLC7A11) in each group. Western blot was employed to determine the protein levels of GPX4, SLC7A11, phosphatidylinositol 3-kinase(PI3K), p-PI3K, protein kinase B(Akt), p-Akt, forkhead box protein O3a(FoxO3a), and p-FoxO3a in each group. The results showed that treatment with 200 μmol·L~(-1) baicalin for 48 h significantly inhibited the viability of HepG2 cells. Ferroptosis in hepatocellular carcinoma could be regulated via the PI3K/Akt signaling pathway. The cell experiments showed that baicalin down-regulated the expression of SLC7A11 and GPX4, lowered the GSH level, and increased ROS accumulation and Fe~(2+) production in HepG2 cells. However, ferrostatin-1, an ferroptosis inhibitor, reduced baicalin-induced ROS accumulation, up-regulated the expression of SLC7A11 and GPX4, elevated the GSH level, and decreased PI3K, Akt, and FoxO3a phosphorylation. In summary, baicalin can induce ferroptosis in HepG2 cells by inhibiting the ROS-mediated PI3K/Akt/FoxO3a pathway.

Effect and mechanism of Jiedu Huoxue Decoction in regulating YAP/ACSL4 pathway to inhibit ferroptosis in treatment of acute kidney injury

Jiedu Huoxue Decoction(JDHX), first recorded in the Correction on Errors in Medical Works by WANG Qing-ren, is an effective formula screened out from ancient formulas by the traditional Chinese medicine(TCM) master ZHANG Qi to treat acute kidney injury(AKI) caused by heat, toxicity, stasis, and stagnation. This paper elucidated the therapeutic effect of JDHX on AKI and probed into the potential mechanism from ferroptosis. Thirty-two male C57BL/6 mice were randomized into four groups(n=8): normal, model, and low-and high-dose JDHX. Since the clinical treatment of AKI depends on supportive or alternative therapies and there is no specific drug, this study did not include a positive drug group. The low dose of JDHX corresponded to half of clinically equivalent dose, while the high dose corresponded to the clinically equivalent dose. Mice were administrated with JDHX by gavage daily for 7 consecutive days, while those in the normal group and the model group were administered with the corresponding volume of distilled water. On day 5 of drug administration, mice in other groups except the normal group were injected intraperitoneally with cisplatin solution at a dose of 20 mg·kg~(-1) to induce AKI, and the normal group was injected with saline. All of the mice were sacrificed 72 h after modeling, blood and kidney samples were collected for subsequent analysis. The levels of serum creatine(Scr) and blood urea nitrogen(BUN) were measured by the commercial kits. The expression level of kidney injury molecule 1(KIM-1) in the serum was measured by enzyme-linked immunosorbent assay. Hematoxylin-eosin(HE) staining, periodic acid-Schiff(PAS) staining, and Prussian blue staining were employed to observe the pathological changes, glycogen deposition, and iron deposition, respectively, in the renal tissue. In addition, the levels of glutathione(GSH), superoxide dismutase(SOD), and catalase(CAT) in the renal tissue were examined by biochemical colorimetry. Western blot was performed to determine the protein levels of acyl-CoA synthetase long chain family member 4(ACSL4), lysophosphatidylcholine acyltransferase 3(LPCAT3), and Yes-associated protein(YAP, a key molecule in the Hippo pathway) in the renal tissue. Immunohistochemistry was then employed to detect the location and expression of YAP in the renal tissue. Real-time fluorescence quantitative polymerase chain reaction(qRT-PCR) was performed to measure the mRNA levels of ACSL4 and glutathione peroxidase 4(GPX4). Compared with the normal group, the model group showed elevated serum levels of Scr, BUN, and KIM-1. In the AKI model group, the tubular epithelial cells underwent atrophy and necrotic detachment, disappearance of brush border, and some tubules became protein tubules or experienced vacuole-like degeneration. In addition, this group presented widening of the interstitium or even edema, increased renal tubule injury score, and obvious glycogen and iron deposition in parts of the renal tissue. Moreover, the model group had lower GSH, SOD, and CAT levels, higher ASCL4 and LPCAT3 levels, and lower GPX4 expression and higher YAP expression than the normal group. Compared with the model group, high dose of JDHX effectively protected renal function, lowered the levels of Scr, BUN and KIM-1, alleviated renal pathological injury, reduced glycogen and iron deposition, and elevated the GSH, SOD, and CAT levels in the renal tissue. Furthermore, JDHX down-regulated the protein levels of ACSL4, LPCAT3, and YAP and up-regulated the level of GPX4, compared with the model group. In conclusion, JDHX can protect mice from cisplatin-induced AKI by inhibiting ferroptosis via regulating the YAP/ACSL4 signaling pathway.

Lipid changes and molecular mechanism inducing cuproptosis in Cryptocaryon irritans after copper–zinc alloy exposure

Cryptocaryons irritans is a ciliate parasite responsible for cryptocaryoniasis, leading to considerable economic losses in aquaculture. It is typically managed using a copper–zinc alloy (CZA), effectively diminishing C. irritans infection rates while ensuring the safety of aquatic organisms. Nevertheless, the precise mechanism underlying cuproptosis induced C. irritans mortality following exposure to CZA remains enigmatic. Therefore, this study delves into assessing the efficacy of CZA, investigate cuproptosis as a potential mechanism of CZA action against C. irritans, and determine the alterations in antioxidant enzymes, peroxidation, and lipid metabolism. The mRNA expression of dihydrolipoamide S-acetyltransferase was upregulated after 40 and 70 min, while aconitase 1 was implicated in cuproptosis following 70 min of CZA exposure. Furthermore, the relative mRNA levels of glutathione reductase experienced a significant increase after 40 and 70 min of CZA exposure. In contrast, the relative mRNA levels of glutathione S-transferase and phospholipid-hydroperoxide glutathione peroxidase were significantly decreased after 70 min, suggesting a disruption in antioxidant defense and an imbalance in copper ions. Lipidomics results also unveiled an elevation in glycerophospholipids metabolism and the involvement of the lipoic acid pathway, predominantly contributing to cuproptosis. In summary, exposure to CZA induces cuproptosis in C. irritans, impacts glutathione-related enzymes, and alters glycerophospholipids, consequently triggering lipid oxidation.

Effects of dietary tributyrin supplementation in low fish meal diet containing high-Clostridium autoethanogenum protein on the growth performance, antioxidative capacity, lipid metabolism and intestinal microbiota of largemouth bass (Micropterus salmoides)

This study aimed to investigate the impact of dietary tributyrin (TB) in the low fish meal (high-Clostridium autoethanogenum protein, CAP) diet on the growth, antioxidation, lipid metabolism and intestinal microbiota of largemouth bass (Micropterus salmoides). For eight weeks, largemouth bass (15.05 ± 0.58 g) were fed on eight isonitrogenous and isolipid diets with graded levels of dietary TB (0 g/kg, 0.2 g/kg, 0.4 g/kg, 0.8 g/kg, 1.6 g/kg, 3.2 g/kg, 4.0 g/kg and 8.0 g/kg). The findings showed that the weight gain rate (WGR) and specific growth rate (SGR) were increased by 1.6 g/kg of dietary TB. The protein efficiency and deposition ratios were linearly affected by dietary TB. The broken-line model based on the WGR revealed that 1.617 g/kg of dietary TB was sufficient for optimal growth. The digestive enzyme activities and villus length of the intestine were increased by dietary TB (1.6 g/kg). Total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and catalase (CAT) activities were significantly improved, and the malondialdehyde (MDA) and protein carbonyl (PC) contents were significantly decreased after dietary TB. The expressions of nuclear factor erythroid 2-related factor 2 (nrf2) and kelch like ech associated protein 1 (keap1) in the liver and intestine were significantly increased and decreased by 1.6 g/kg of dietary TB, respectively. The mRNA levels of copper-zinc superoxide dismutase (cuznsod), cat, heme oxygenase-1 (ho-1) and glutathione peroxidase (gpx) were significantly upregulated. Dietary TB could significantly affect the total protein (TP) and albumin (ALB) content in the serum. The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were the lowest at 1.6 g/kg of TB group. Besides, the hepatocyte vacuolation and nuclear translocation were alleviated after supplementation of 0.2‰− 3.0‰ of dietary TB. The total cholesterol (T-CHO), high density lipoprotein cholesterol (HDLC) and low density lipoprotein cholesterol (LDLC) were significantly increased by dietary TB. The triglycerides (TG) content was decreased by dietary TB. The mRNA levels of sterol regulatory element-binding protein 1-c (srebp1-c), acetyl-coa carboxylase alpha (accα), fatty acid synthase (fasn) and accβ in the liver were significantly downregulated, and peroxisome proliferator-activated receptor alpha (pparα) and carnitine palmitoyltransferase 1 (cpt-1) were upregulated by 1.6 g/kg of dietary TB. Dietary TB had effects on the intestine microbes of largemouth bass. The Cetobacterium was present in the highest percentage and increased from 41% (0 g/kg TB) to 59% (1.6 g/kg TB). The Mycoplasmataceae was the second most predominant genus and decreased from 90% in the 0 g/kg of TB group to 10% in the 1.6 g/kg of TB group. In summary, the dietary TB could be supplemented in the CAP-based diet for better growth and antioxidation, lipid metabolism and intestinal microbiota of largemouth bass.

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.

Effect of electroacupuncture pretreatment on ferroptosis in neurons of rats with cerebral ischemia-reperfusion injury

To observe the effect of electroacupuncture（EA）preconditioning on ferroptosis in rats with cerebral ischemia-reperfusion injury （CIRI）, so as to explore the neuroprotective mechanism of EA preconditioning. Male SD rats were randomly divided into sham operation, model, EA, inhibitor and inducer groups with 20 rats in each group. The CIRI model was established by modified Zea Longa occlusion of the middle cerebral artery. Before modeling, EA treatment （2 Hz/15 Hz, 1-2 mA） was applied to "Baihui"（GV20）, "Fengfu"（GV16） and "Dazhui"（GV14） for rats of the EA group, 20 min a day for 7 consecutive days. Rats of the inhibitor group were intraperitoneally injected with ferristatin-1（25 mg/kg）at a slow and uniform rate. Rats of the inducer group were intraperitoneally injected with Erastin（100 mg/kg） after 7 days of EA preconditioning, once every 2 h for a total of 4 times. The CIRI models were prepared 2 d later after the above interventions finished by thread-occlusion. The degree of neurological impairment was evaluated by modified Zea Longa score. The percentage of infarct size was calculated by TTC staining. The ultrastructure of neurons in hippocampus was observed by transmission electron microscope. The contents of ferrous ion （Fe2+）, malondialdehyde （MDA） and glutathione （GSH） in cerebral tissue and reactive oxygen species （ROS） in serum were determined by biochemical method. The changes of mitochondrial membrane potential in rats brain tissues were detected by flow cytometry. The mRNA and protein expression levels of glutathione peroxidase 4 （GPX4）, acyl-CoA synthetase long-chain family member 4 （ACSL4）, transferrin receptor （TFRC）, 15-lipoxygenase （15-LOX） and cyclooxygenase-2 （COX-2） in the ischemic hippocampal region of CIRI rats were detected by real-time quantitative PCR and Western blot, respectively. Compared with the sham operation group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression levels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01）, and mitochondria in brain tissue were significantly damaged （P<0.01） in the model group. Compared with the model group, the above indexes were all reversed （P<0.05, P<0.01） in the EA group and inhibitor group. Compared with the EA group, the neurological impairment score, the percentage of cerebral infarction area, the contents of MDA and Fe2+ in cerebral tissue as well as ROS in serum, the protein and mRNA expression le-vels of ACSL4, TFRC, 15-LOX, COX-2 in hippocampal tissue were increased （P<0.05, P<0.01）, while the content of GSH in cerebral tissue, the protein and mRNA expression levels of GPX4 in hippocampal tissue were decreased （P<0.01, P<0.05）, and mitochondria in brain tissue were significantly damaged （P<0.05） in the inducer group. EA preconditioning has neuroprotective effect on CIRI rats, which may be related to inhibiting ACSL4/TFRC/15-LOX/COX-2 expression and increasing GSH/GPX4 expression.

Contribution of Oxidative Stress, Apoptosis, Endoplasmic Reticulum Stress and Autophagy Pathways to the Ameliorative Effects of Hesperidin in NaF-Induced Testicular Toxicity.

The ameliorative effects of hesperidin (HES) on the toxicities created by sodium fluoride (NaF) in the testes tissue of rats were studied via oxidative stress, apoptosis and endoplasmic reticulum (ER) stress pathways. The animals were divided into five distinct groups (7 rats in each group). Group 1 was control group, group 2 received NaF-only (600 ppm), group 3 received HES-only (200 mg/kg bw); group 4 received NaF (600 ppm)+HES (100 mg/kg bw) and group 5 received NaF (600 ppm)+HES (200 mg/kg bw) for 14 days. NaF-induced testes tissue damage by reducing activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) and levels of glutathione (GSH), and increasing lipid peroxidation levels. NaF treatment significantly downregulated the mRNA levels of SOD1, CAT and GPx. NaF supplementation caused apoptosis in the testes by upregulating p53, NFkB, caspase-3, caspase-6, caspase-9, and Bax and downregulating Bcl-2. Furthermore, NaF caused ER stress via increasing mRNA transcript levels of PERK, IRE1, ATF-6 and GRP78. NaF treatment led to autophagy via upregulation of Beclin1, LC3A, LC3B and AKT2. In testes tissue, however, co-treatment with HES at doses of 100 and 200 mg/kg significantly reduced oxidative stress, apoptosis, autophagy and ER stress. Overall, the findings of this study suggest that HES may help to reduce testes damage caused by NaF toxicity.

High‑throughput screening identification of a small‑molecule compound that induces ferroptosis and attenuates the invasion and migration of hepatocellular carcinoma cells by targeting the STAT3/GPX4 axis.

Hepatocellular carcinoma (HCC) is a lethal malignancy. Although considerable efforts have been made in recent years regarding treatments, effective therapeutic drugs for HCC remain insufficient. In the present study, polyphyllin VI was identified as a potential therapeutic drug for HCC by screening natural herbal compounds. The therapeutic effects of polyphyllin VI were assessed using Cell Counting Kit‑8, lactate dehydrogenase release and colony formation assays. The occurrence of ferroptosis was determined by assessing lipid peroxidation by reactive oxygen species, malondialdehyde levels, intracellular ferrous iron levels, and the mRNA and protein levels of glutathione peroxidase 4 (GPX4). The migratory and invasive abilities of HCC cells were examined using wound healing and Transwell assays. The results revealed that polyphyllin VI inhibited the proliferation, invasion and metastasis of HCC cells (HCCLM3 and Huh7 cells) by inducing ferroptosis. In addition, through a network pharmacology‑based approach and molecular docking analyses, it was found that polyphyllin VI may target the signal transducer and activator of transcription 3 (STAT3). HCC cells were treated with polyphyllin VI or a STAT3 inhibitor (Stattic), both of which exerted similar inhibitory effects on protein expression. Furthermore, immunofluorescence staining revealed that polyphyllin VI significantly inhibited the nuclear translocation of p‑STAT3 in HCC cells. Mechanistically, by the overexpression of STAT3, it was confirmed that STAT3 binds to GPX4 and promotes its protein expression and transcription, whereas polyphyllin VI induces ferroptosis by inhibiting the STAT3/GPX4 axis. Subsequently, invivo experiments revealed that polyphyllin VI inhibited the growth of subcutaneously transplanted tumors. On the whole, findings of the present study suggest that polyphyllin VI inhibits STAT3 phosphorylation, which inhibits GPX4 expression and induces the ferroptosis of HCC cells, eventually inhibiting their invasion and metastasis. These data suggest that polyphyllin VI may be a candidate for the prevention and treatment of HCC.

H2O2 Mediates VEGF- and Flow-Induced Dilations of Coronary Arterioles in Early Type 1 Diabetes: Role of Vascular Arginase and PI3K-Linked eNOS Uncoupling

In diabetes, the enzyme arginase is upregulated, which may compete with endothelial nitric oxide (NO) synthase (eNOS) for their common substrate L-arginine and compromise NO-mediated vasodilation. However, this eNOS uncoupling can lead to superoxide production and possibly vasodilator hydrogen peroxide (H2O2) formation to compensate for NO deficiency. This hypothesis was tested in coronary arterioles isolated from pigs with 2-week diabetes after streptozocin injection. The NO-mediated vasodilation induced by flow and VEGF was abolished by NOS inhibitor L-NAME and phosphoinositide 3-kinase (PI3K) inhibitor wortmannin but was not affected by arginase inhibitor Nω-hydroxy-nor-L-arginine (nor-NOHA) or H2O2 scavenger catalase in control pigs. With diabetes, this vasodilation was partially blunted, and the remaining vasodilation was abolished by catalase and wortmannin. Administration of L-arginine or nor-NOHA restored flow-induced vasodilation in an L-NAME sensitive manner. Diabetes did not alter vascular superoxide dismutase 1, catalase, and glutathione peroxidase mRNA levels. This study demonstrates that endothelium-dependent NO-mediated coronary arteriolar dilation is partially compromised in early type 1 diabetes by reducing eNOS substrate L-arginine via arginase activation. It appears that upregulated arginase contributes to endothelial NO deficiency in early diabetes, but production of H2O2 during PI3K-linked eNOS uncoupling likely compensates for and masks this disturbance.

Glutathione metabolism in Cryptocaryon irritans involved in defense against oxidative stress induced by zinc ions

BackgroundCryptocaryon irritans is a fatal parasite for marine teleosts and causes severe economic loss for aquaculture. Galvanized materials have shown efficacy in controlling this parasite infestation through the release of zinc ions to induce oxidative stress.MethodsIn this study, the resistance mechanism in C. irritans against oxidative stress induced by zinc ions was investigated. Untargeted metabolomics analysis was used to determine metabolic regulation in C. irritans in response to zinc ion treatment by the immersion of protomonts in ZnSO4 solution at a sublethal dose (20 μmol). Eight differential metabolites were selected to assess the efficacy of defense against zinc ion stimulation in protomonts of C. irritans. Furthermore, the mRNA relative levels of glutathione metabolism-associated enzymes were measured in protomonts following treatment with ZnSO4 solution at sublethal dose.ResultsThe results showed that zinc ion exposure disrupted amino acid metabolism, carbohydrate metabolism, lipid metabolism, and nucleotide metabolism in C. irritans. Four antioxidants, namely ascorbate, S-hexyl-glutathione, syringic acid, and ubiquinone-1, were significantly increased in the Zn group (P < 0.01), while the glutathione metabolism pathway was enhanced. The encystment rate of C. irritans was significantly higher in the ascorbate and methionine treatment (P < 0.05) groups. Additionally, at 24 h post-zinc ion exposure, the relative mRNA level of glutathione reductase (GR) was increased significantly (P < 0.01). On the contrary, the relative mRNA levels of glutathione S-transferase (GT) and phospholipid-hydroperoxide glutathione peroxidase (GPx) were significantly decreased (P < 0.05), thus indicating that the generation of reduced glutathione was enhanced.ConclusionsThese results revealed that glutathione metabolism in C. irritans contributes to oxidative stress resistance from zinc ions, and could be a potential drug target for controlling C. irritans infection.Graphical

Dietary effects of aucubin on growth and flesh quality of grass carp (Ctenopharyngodon idellus) based on metabolomics.

This aim of this study was to investigate the effects of dietary aucubin on the growth, flesh quality, and metabolomics of grass carp (Ctenopharyngodon idella). Five diets were designed with the aucubin inclusion of 0 (control diet), 0.2, 0.4, 0.6, and 0.8 g/kg (Auc-0.2, Auc-0.4, Auc-0.6, Auc-0.8) and were fed to grass carp with an initial body weight of 17.0 ± 0.2 g for 60 d. The results indicated that dietary aucubin did not significantly affect the growth performance of grass carp (P > 0.05). Compared to the control, dietary supplementation with 0.2 to 0.8 g/kg aucubin increased flesh hardness, chewiness, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and the contents of total free amino acids (TFAA) and n-3 polyunsaturated fatty acids (n-3 PUFA) (P < 0.05). The contents of malondialdehyde (MDA) and lactic acid (LD) in the flesh were significantly decreased by the addition of 0.4 to 0.6 g/kg aucubin and by the addition of 0.6 to 0.8 g/kg aucubin (P < 0.05), respectively, while the content of delicious amino acids (DAA) was significantly enhanced by the addition of 0.4 to 0.8 g/kg aucubin (P < 0.05). Moreover, the contents of collagen and C22:6n3 (DHA) in the flesh of the Auc-0.8 group were significantly higher than those of the control (P < 0.05). In the metabolomics profiling of flesh, 133 and 135 named differential metabolites were identified in the Auc-0.4 and Auc-0.8 groups, respectively, compared to the control, and these metabolites were found to be involved in the second-grade pathways of "lipid metabolism" and "amino acid metabolism". Regarding gene expression, the mRNA levels of CuZn-SOD, CAT, COL1A1, COL1A2, Smad4, and FAS in flesh were upregulated in the Auc-0.4 and Auc-0.8 groups, and the expression levels of GPx, Nrf2, and TGF-β1 mRNA were also upregulated in the Auc-0.8 group (P < 0.05). In summary, dietary aucubin did not promote growth, but improved the flesh quality of grass carp, which might be associated with the TGF-β/Smad and Nrf2 pathways. The recommended supplementation level of aucubin in the diet of grass carp was 0.6 to 0.8 g/kg.

Dietary lysine levels modulate the lipid metabolism, mitochondrial biogenesis and immune response of grass carp, Ctenopharyngodon idellus

A 10-week experiment was conducted to evaluate the effects of dietary lysine levels on growth performance, lipid metabolism, anti-oxidant capacity, mitochondrial biogenesis and immune response of grass carp, Ctenopharyngodon idella. Fish (mean initial weight 51.01 ± 0.17 g) were fed seven diets containing graded levels of lysine (1.69 %, 1.79 %, 1.91 %, 2.01 %, 2.11 %, 2.24 % and 2.31 %). Results showed that fish fed the control diet (1.69 %) showed lower weight gain (WG) and higher feed conversion rate FCR than those fed 2.01–2.31 % lysine (P < 0.05). Viscerosomatic index decreased when dietary lysine increasing from 1.69 % to 2.11 % (P < 0.05). Suitable dietary lysine levels enhanced the lipolysis and mitochondrial biogenesis of grass carp. Total antioxidant capacity (T-AOC) in the liver was higher in fish fed 2.24 % dietary lysine than those fed diets containing 1.69 % and 1.79 % lysine (P < 0.05). The malonaldehyde (MDA) contents in liver decreased when dietary lysine increased from 1.69 % to 2.01 % (P < 0.05). Relative mRNA levels of glutathione peroxidase and catalase in liver and intestine were affected by dietary lysine levels (P < 0.05). B-cell lymphoma 2 (Bcl-2) mRNA level in liver was up-regulated and inhibitor kappa B kinase β (IKKβ) mRNA level in the intestine was down-regulated by increasing dietary lysine levels (P < 0.05). Second-order regression based on WG indicated the suitable level of lysine in feed of grass carp was 2.24 % (6.19 % protein).

Bone morphogenetic protein 4 alleviates nonalcoholic steatohepatitis by inhibiting hepatic ferroptosis

Nonalcoholic steatohepatitis (NASH) is a state of simple steatosis that progresses to inflammation and liver injury accompanied by ferroptosis. Bone morphogenetic protein 4 (BMP4) plays an important role in adipogenesis and differentiation, as well as in hepatic steatosis and iron regulation. However, the direct impact of BMP4 on NASH remains unclear. In this study, our aim was to investigate the effect of BMP4 on NASH and its underlying mechanism. We first explored BMP4 expression in vivo in mice and patients and in vitro in HepG2 and LO2 cell lines, and then, determined whether ferroptosis occurs in NASH. Further overexpression or inhibition of BMP4 was induced to observe the effect of BMP4 on liver ferroptosis in NASH. BMP4 expression was upregulated in patients and mice with nonalcoholic fatty liver disease, and free fatty acid (FFA)-induced HepG2 and LO2 cell lines. We observed ferroptosis in high-fat diet and high-fructose diet-fed mice and FFA-induced HepG2 and LO2 cell lines. BMP4 overexpressing plasmid was constructed and the HepG2 and LO2 cells were transfected with lentivirus (oe-BMP4), or treated with exogenously added recombinant human BMP4 or BMP antagonist noggin. BMP4 suppressed the markers of hepatic steatosis, liver inflammation, and liver injury. Upregulated BMP4 expression in HepG2 and LO2 cells reduced reactive oxygen species and malondialdehyde content and relieved ferroptosis. Mechanistically, BMP4 overexpression in hepatocytes upregulated the mRNA and protein levels of glutathione peroxidase 4 (GPX4), a central regulator of ferroptosis, while exogenous inhibition of BMP4 by noggin decreased their levels. Immunoprecipitation assays demonstrated a physical interaction between BMP4 and GPX4 in HepG2 and LO2 cells, and confocal imaging confirmed colocalization of BMP4 and GPX4. Consistently, BMP4 overexpression plays an important role in NASH by increasing GPX4 expression, therefore decreasing hepatic ferroptosis. This study proposes BMP4 as a therapeutic target for preventing steatohepatitis.

Effects of L-carnitine Supplementation in High-Fat Diet on Growth, Antioxidant Capacity and Lipid Metabolism of Golden Pompano (Trachinotus ovatus)

A 6-week feeding trial was carried out to investigate whether dietary L-carnitine supplementation could alleviate the adverse effects of high-fat diet on the growth, antioxidation and lipid metabolism of Trachinotus ovatus. Four isonitrogenous experimental diets were formulated supplementing with or without L-carnitine (low fat diet (D1): 13% lipid; high fat diet (D2): 20% lipid; D3: 20% lipid with 0.02% L-carnitine; D4: 20% lipid with 0.06% L-carnitine). The final body weight, weight gain rate, specific growth ratio and feed intake in fish fed with D2 diet were significantly lower than that of fish fed with D1, D3 and D4 diets (P &amp;lt; 0.05). The weight gain rate and specific growth ratio of D2 diet were as low as 167.21% and 2.41% initial body weight/d, respectively. However, experimental diets were unable to change the feed conversion ratio and survival rate of fish (P &amp;gt; 0.05). The lowest value of crude protein and the highest value of crude lipid of whole-body were found in fish fed with D2 diet (P &amp;lt; 0.05), which were 31.35% and 17.41% wet weight respectively. Meantime, the level of crude lipid of whole-body in fish fed with D3 and D4 diets was significantly higher than that of fish fed with D2 diet (P &amp;lt; 0.05). Besides, there were significantly higher levels of triglyceride and total cholesterol in the D2 diet when compared to the other diets (P &amp;lt; 0.05). Additionally, the superoxide dismutase activity, malondialdehyde content and the mRNA levels of manganese superoxide dismutase and glutathione peroxidase of the liver in fish fed with D2 diet were significantly higher than those fed with D1, D3 and D4 diets (P &amp;lt; 0.05). The mRNA levels of carnitine palmitoyl transferase 1 and peroxisome proliferator-activated receptors-alpha of the liver in fish fed with D2 diet were significantly higher than those fed with D1, D3 and D4 diets (P &amp;lt; 0.05). In conclusion, L-carnitine supplementation in high-fat diet improved the growth performance and health of T. ovatus by promoting lipid hydrolysis, improving cholesterol transport and antioxidant capacity. Therefore, we recommended the 0.02% addition level of L-carnitine for T. ovatus diet.

Anhydroicaritin Inhibits EMT in Breast Cancer by Enhancing GPX1 Expression: A Research Based on Sequencing Technologies and Bioinformatics Analysis.

Background: Breast cancer (BC) is the leading cause of cancer-related deaths among women worldwide. The application of advanced technology has promoted accurate diagnosis and treatment of cancer. Anhydroicaritin (AHI) is a flavonoid with therapeutic potential in BC treatment. The current study aimed to determine AHI’s mechanism in BC treatment via RNA sequencing, comprehensive bioinformatics analysis, and experimental verification. Methods: Network pharmacology and MTT (3-(4,5)-dimethylthiazolyl-3,5- diphenyltetrazolium bromide) experiments were conducted to first confirm AHI’s anti-BC effect. RNA sequencing was performed to identify the genes affected by AHI. Differential expression analysis, survival analysis, gene set enrichment analysis, and immune infiltration analysis were performed via bioinformatics analysis. Western blot analysis, reverse transcription–polymerase chain reaction (RT-PCR) experiment, molecular docking, and drug affinity responsive target stability (DARTS) experiments were also performed to confirm AHI’s direct effect on glutathione peroxidase 1 (GPX1) expression. Confocal immunofluorescence analysis was conducted to verify AHI’s effect on the occurrence and development of epithelial–mesenchymal transition (EMT). Finally, BC nude mouse xenografts were established, and AHI’s molecular mechanism on BC was explored. Results: Network pharmacology results demonstrated that AHI’s therapeutic targets on BC were related to the proliferation, invasion, and metastasis of BC cells. AHI significantly inhibited the proliferation of 4T1 and MDA-MB-231 BC cells in the MTT experiments. RNA sequencing results showed that AHI upregulated the GPX1 expression in the 4T1 and MDA-MB-231 BC cells. Next, bioinformatics analysis revealed that GPX1 is less expressed in BC than in normal breast tissues. Patients with high GPX1 expression levels tended to have prolonged overall survival and disease-free survival than patients with low GPX1 expression levels in BC. Western blot and RT-PCR experiments revealed that AHI increased the protein and mRNA levels of GPX1. Molecular docking and DARTS experiments confirmed the direct binding combination between AHI and GPX1. After the evaluation of the EMT scores of 1,078 patients with BC, we found a potential anti-BC role of GPX1 possibly via suppression of the malignant EMT. The confocal immunofluorescence analysis showed that AHI increased E-cadherin expression levels and reduced vimentin expression levels in BC cells. Animal experiments showed that AHI significantly inhibited tumor growth. AHI also inhibited EMT by enhancing GPX1 and caspase3 cleavage, hence inhibiting EMT markers (i.e., N-cadherin and vimentin) and Ki-67. Conclusion: GPX1 plays a critical role in BC, which may be a biomarker for the prognosis. In addition, AHI suppressed EMT by increasing GPX1 expression, which may serve as a potential therapy for BC treatment.

Mitochondrial oxidative stress mediated Fe-induced ferroptosis via the NRF2-ARE pathway

Ferroptosis is a regulated form of cell death induced by iron (Fe)-dependent lipid peroxidation. At present, the underlying molecular mechanisms remain elusive. Herein, we hypothesized that mitochondria and the NRF2 (transcription factor nuclear factor E2-related factor 2) are potential mediators of ferroptosis, considering their well-established involvement in the oxidative stress pathway. We found that a high iron diet increased hepatic iron content and promoted glutathione (GSH) depletion, lipid peroxidation and oxidative stress. Dietary iron overload also decreased mRNA and protein expression levels of glutathione peroxidase 4 (GPX4) and cystine-glutamate antiporter (SLC7A11), and increased mRNA and protein expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), which are all markers of ferroptosis. Consistent with ferroptosis, iron overload promoted lipid peroxidation and the generation of mitochondrial reactive oxygen species (ROS), and decreased the mitochondrial membrane potential (MMP). Pre-treatment with deferoxamine mesylate (DFO, an iron chelator) alleviated ROS generation and lipid peroxidation, indicating a causative link between iron overload and lipid peroxidation. Suppression of mitochondrial oxidative stress attenuated ferroptosis. Experiments with HEK293T cells revealed that Fe-induced ferroptosis involved direct inhibition of NRF2 binding to antioxidant response elements (AREs) within the promoters of the gpx4 and slc7a11 genes, which in turn induced transcriptional silencing. In conclusion, our study provided a direct link between mitochondrial oxidative stress and ferroptosis via the NRF2-ARE pathway.

Differential expression of antioxidant genes during clinical mastitis of cow caused by Staphylococcus aureus and Escherichia coli

There is considerable interest in the hypothesis that oxidative stress is enhanced in the pathophysiology of clinical mastitis. The main goal of this research was to establish profiles of antioxidant gene expression in the milk of cows with clinical mastitis. Standard bacteriology was conducted on pretreatment milk specimens from 77 cows with clinical mastitis between 15 and 70 days in milk (DIM). Examinations were performed on mRNA expression of antioxidant genes, such as catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD). Additionally, levels of lipid peroxidation were measured in milk samples from healthy cows and those with clinical mastitis. The isolated bacteria consisted of Staphylococcus aureus (S. aureus, 10.48%), Streptococcus agalactiae (7.69%), Streptococcus dysagalactiae (6.29%), and Escherichia coli (E. coli, 29.37%). E. coli was the most prevalent pathogen found in the milk of cows with clinical mastitis in early lactation. The mean level of malondialdehyde (MDA) in clinical mastitis samples was significantly higher (P&lt;0.05) than that of healthy cows. The results revealed that the expression profiles of SOD in mastitis milk induced by S. aureus were significantly (P&lt;0.0001) up-regulated compared with E.coli. In addition, the mRNA levels of GPx in mastitis milk due to E.coli were significantly (P&lt;0.0001) over expressed compared to S. aureus. CAT gene expression had a tendency to be enhanced in mastitis milk induced by S. aureus compared with mastitis in cows due to E.coli. These results showed that the mRNA levels of antioxidant genes may differ depending on the type of bacteria, and diminished expression of antioxidant genes might increase susceptibility to mastitis.

The role of dietary chromium supplementation in relieving heat stress of juvenile blunt snout bream Megalobrama amblycephala

The present study assessed the role of dietary chromium (Cr) supplementation in relieving heat stress (HS) of juvenile blunt snout bream Megalobrama amblycephala. The supplemented Cr contents by chromium picolinate (Cr-Pic) was 0 mg/kg (control group), 0.4 mg/kg, 1.6 mg/kg and 12.0 mg/kg, respectively. The fish continued to be fed four diets at suitable temperatures (26 °C) for 2 weeks, and then the temperature was then heated up to 33 °C through thermo-regulated system. The results showed that Cr supplementation had no significant effect on the immune indices and antioxidant indices before HS (P > 0.05). However, Cr supplementation played an important role in relieving HS. After HS, compared with the control group, 1.6 mg/kg and 12.0 mg/kg Cr supplementation groups significantly lowered the plasma glucose level and aspartate transaminase (AST) activity (P < 0.05), and 0.4 mg/kg and 1.6 mg/kg Cr supplementation groups significantly lowered alanine aminotransferase (ALT) activity (P < 0.05). 0.4 mg/kg and 1.6 mg/kg supplementation groups significantly improved hepatic total superoxide dismutase (T-SOD) activity (P < 0.05). Furthermore, 0.4mg/kg-12.0 mg/kg Cr supplementation groups significantly improved the activities of hepatic glutathione peroxidase (GPx) and catalase (CAT) and lowered hepatic malondialdehyde (MDA) level in liver (P < 0.05). The mRNA levels of hepatic copper zinc superoxide dismutase (Cu/Zn-SOD), CAT and GPx were significantly improved in 0.4mg/kg-12.0 mg/kg supplementation Cr groups (P < 0.05), however, there was no significant variation of hepatic manganese superoxide dismutase (Mn-SOD) mRNA levels under different levels of supplementation (P > 0.05). Significantly lower mRNA levels of hepatic pro-inflammatory cytokines observed in 0.4mg/kg-12.0 mg/kg Cr supplementation groups including tumour necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and interleukin 8 (IL-8) (P < 0.05), and 0.4mg/kg-12.0 mg/kg Cr supplementation significantly improved the relative expressions of hepatic heat shock protein 70 (HSP70) and heat shock protein 90 (HSP90) (P < 0.05). The present study indicated that dietary Cr supplementation might have no significant effect on immune capacity and antioxidant capacity under normal physiological conditions, whereas it played an important role in relieving HS.

γ-Linolenic Acid Prevents Lipid Metabolism Disorder in Palmitic Acid-Treated Alpha Mouse Liver-12 Cells by Balancing Autophagy and Apoptosis via the LKB1-AMPK-mTOR Pathway.

Excessive fat deposition is the main character in nonalcoholic fatty liver disease (NAFLD), while γ-linolenic acid (GLA) is a polyunsaturated fatty acid that can reduce lipid deposition. This study investigated the effect and regulatory mechanism of GLA (100 μM) on lipid metabolism in alpha mouse liver 12 (AML-12) cells treated by 400 μM palmitic acid (PA). GLA reduced lipid content and increased fatty acid β oxidation, as indicated by decreasing triglyceride and cholesterol contents and increasing mRNA and protein expressions of CPT1α and PPARα. GLA relieved oxidative stress caused by PA, upregulated mRNA levels of superoxide dismutase and glutathione peroxidase, and decreased reactive oxygen species content. GLA reduced apoptosis, as indicated by decreases in the BAX/BCL2 expression level and apoptosis percentage. GLA activated autophagy, autophagosome-lysosome fusion, and LKB1-AMPK-mTOR signaling and upregulated mRNA and protein expressions of Beclin-1, autophagy-related 5, and liver kinase B1 (LKB1). These effects of GLA on lipid metabolism disorders of PA-treated hepatocytes were reversed by autophagy inhibitor 3MA and AMPK inhibitor compound C, confirming our conclusions. Overall, GLA can protect AML-12 cells from lipid metabolism disorder caused by PA via balancing autophagy and apoptosis mediated by the LKB1-AMPK-mTOR pathway. Consequently, GLA, as a dietary supplement, can help to prevent and treat NAFLD by regulating lipid metabolism and autophagy.

Effects of phosphatidic acid on growth and antioxidant capacity in juvenile turbot, Scophthalmus maxius L., fed with high plant protein‐based diets

AbstractReplacement of fish meal with high plant protein meal leads to reduced growth performance and health issues in farmed fish. Supplementation of functional additives has been proposed as a potential strategy to alleviate the negative effect of plant protein replacement. In this study, the effects of phosphatidic acid (PA) on growth and antioxidant capacity in juvenile turbot fed with high plant protein‐based diets were evaluated. An 11‐week feeding trial was performed using diets containing 0 (CON group), 0.05, 0.1, or 0.5% PA. The results showed that adding 0.5% PA could significantly improve the growth performance of turbot without affecting feed intake. Moreover, supplementation of 0.5% PA significantly increased the mRNA level of igf‐1 in liver. The condition factor was also enhanced in PA addition groups. The activity of lipase was increased by PA supplementation, while no difference was observed in amylase and trypsin activity. Supplying PA significantly reduced the content of malondialdehyde. Furthermore, the mRNA levels of superoxide dismutase (sod), glutathione peroxidase (gpx), and peroxiredoxin 6 (prx6) were enhanced in 0.1 and 0.5% PA groups. Our results showed that PA might be a functional additive for aquafeed with growth‐promoting and antioxidative effects.