Glutathione Supplementation Prevents Neonatal Parenteral Nutrition-Induced Short- and Long-Term Epigenetic and Transcriptional Disruptions of Hepatic H2O2 Metabolism in Guinea Pigs.

The parenteral nutrition (PN) received by premature newborns is contaminated with peroxides that induce global DNA hypermethylation via oxidative stress. Exposure to peroxides could be an important factor in the induction of chronic diseases such as those observed in adults who were born preterm. As endogenous H2O2 is a major regulator of glucose-lipid metabolism, our hypothesis was that early exposure to PN induces permanent epigenetic changes in H2O2 metabolism. Three-day-old guinea pigs were fed orally (ON), PN or glutathione-enriched PN (PN+GSSG). GSSG promotes endogenous peroxide detoxification. After 4 days, half the animals were sacrificed, and the other half were fed ON until 16 weeks of age. The liver was harvested. DNA methylation and mRNA levels were determined for the SOD2, GPx1, GCLC, GSase, Nrf2 and Keap1 genes. PN induced GPx1 hypermethylation and decreased GPx1, GCLC and GSase mRNA. These findings were not observed in PN+GSSG. PN+GSSG induced Nrf2 hypomethylation and increased Nrf2 and SOD2 mRNA. These observations were independent of age. In conclusion, in neonatal guinea pigs, PN induces epigenetic changes, affecting the expression of H2O2 metabolism genes. These changes persist for at least 15 weeks after PN. This disruption may signify a permanent reduction in the capacity to detoxify peroxides.

Porous Se@SiO2 nanospheres alleviate diabetic retinopathy by inhibiting excess lipid peroxidation and inflammation.

BACKGROUND: Lipid peroxidation is a characteristic metabolic manifestation of diabetic retinopathy (DR) that causes inflammation, eventually leading to severe retinal vascular abnormalities. Selenium (Se) can directly or indirectly scavenge intracellular free radicals. Due to the narrow distinction between Se's effective and toxic doses, porous Se@SiO2 nanospheres have been developed to control the release of Se. They exert strong antioxidant and anti-inflammatory effects. METHODS: The effect of anti-lipid peroxidation and anti-inflammatory effects of porous Se@SiO2 nanospheres on diabetic mice were assessed by detecting the level of Malondialdehyde (MDA), glutathione peroxidase 4 (GPX4), decreased reduced/oxidized glutathione (GSH/GSSG) ratio, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL) -1ß of the retina. To further examine the protective effect of porous Se@SiO2 nanospheres on the retinal vasculopathy of diabetic mice, retinal acellular capillary, the expression of tight junction proteins, and blood-retinal barrier destruction was observed. Finally, we validated the GPX4 as the target of porous Se@SiO2 nanospheres via decreased expression of GPX4 and detected the level of MDA, GSH/GSSG, TNF-α, IFN-γ, IL -1ß, wound healing assay, and tube formation in high glucose (HG) cultured Human retinal microvascular endothelial cells (HRMECs). RESULTS: The porous Se@SiO2 nanospheres reduced the level of MDA, TNF-α, IFN-γ, and IL -1ß, while increasing the level of GPX4 and GSH/GSSG in diabetic mice. Therefore, porous Se@SiO2 nanospheres reduced the number of retinal acellular capillaries, depletion of tight junction proteins, and vascular leakage in diabetic mice. Further, we identified GPX4 as the target of porous Se@SiO2 nanospheres as GPX4 inhibition reduced the repression effect of anti-lipid peroxidation, anti-inflammatory, and protective effects of endothelial cell dysfunction of porous Se@SiO2 nanospheres in HG-cultured HRMECs. CONCLUSION: Porous Se@SiO2 nanospheres effectively attenuated retinal vasculopathy in diabetic mice via inhibiting excess lipid peroxidation and inflammation by target GPX4, suggesting their potential as therapeutic agents for DR.

Lizhong decoction ameliorates ulcerative colitis by inhibiting ferroptosis of enterocytes via the Nrf2/SLC7A11/GPX4 pathway.

ETHNOPHARMACOLOGY RELEVANCE: Traditional herbal medicines have been considered as a novel and effective way to treat many diseases. Lizhong decoction (LZD), a classical prescription composed of Zingiber officinale Rosc., Panax ginseng C. A. Mey., Atractylodes macrocephala Koidz., and Glycyrrhiza uralensis Fisch., has been used to treat gastrointestinal disorders in clinical practices for thousands of years. However, the mechanism of LZD in alleviating ulcerative colitis (UC) is still unclear. AIM OF THE STUDY: The purpose of this study was to clarify the potential molecular mechanism of LZD in improving UC. MATERIALS AND METHODS: The amelioration of LZD on dextran sodium sulfate (DSS)-induced UC mice was evaluated by body weight, colon length, pathology of colon tissues, pro-inflammatory cytokines, and intestinal tight junction (TJ) proteins. Moreover, the gene expression profiles of UC patients were extracted to investigate potential pathological mechanisms of UC. The influence of LZD on ferroptosis was analyzed by iron load, malondialdehyde (MDA), and the expression of ferroptosis-associated proteins. Meanwhile, the inhibition of LZD on oxidative stress (OS) was assessed by the superoxide dismutase (SOD) activity, as well as the expression levels of glutathione (GSH) and glutathione disulfide (GSSG). Furthermore, the influence of LZD on ferroptosis was assessed by inhibiting nuclear factor (erythroid-derived-2)-like 2 (Nrf2). RESULTS: LZD showed significant therapeutic effects in UC mice, including reduction of intestinal injury and inflammation. Moreover, LZD treatment notably upregulated the expression of TJ proteins. Further investigation indicated that LZD significantly inhibited the ferroptosis of enterocytes by decreasing iron load and MDA, and increasing the expression levels of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in colon tissues. Furthermore, the decreased activity of SOD, reduced level of GSH, and increased content of GSSG in UC mice were notably reversed by LZD. Consistent with in vivo results, LZD could markedly inhibit ferroptosis and OS in RSL3-induced Caco-2 cells. Mechanistically, LZD alleviated ferroptosis by suppressing OS through the activation of Nrf2 signaling. CONCLUSIONS: Collectively, LZD remarkably improved intestinal pathological injury in UC mice, and its potential mechanism was the suppression of ferroptosis in enterocytes by the Nrf2/SLC7A11/GPX4 pathway.

Dietary glycine supplementation enhances glutathione availability in tissues of pigs with intrauterine growth restriction.

This study tested the hypothesis that dietary supplementation with glycine enhances the synthesis and concentrations of glutathione (GSH, a major antioxidant) in tissues of pigs with intrauterine growth restriction (IUGR). At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of two groups, representing supplementation with 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Blood and other tissues were obtained from the pigs within 1 wk after the feeding trial ended at 188 d of age to determine GSH, oxidized GSH (GSSG), and activities of GSH-metabolic enzymes. Results indicated that concentrations of GSH + GSSG or GSH in plasma, liver, and jejunum (P < 0.001) and concentrations of GSH in longissimus lumborum and gastrocnemius muscles (P < 0.05) were lower in IUGR pigs than in NBW pigs. In contrast, IUGR increased GSSG/GSH ratios (an indicator of oxidative stress) in plasma (P < 0.001), jejunum (P < 0.001), both muscles (P < 0.05), and pancreas (P = 0.001), while decreasing activities of γ-glutamylcysteine synthetase and GSH synthetase in liver (P < 0.001) and jejunum (P < 0.01); and GSH reductase in jejunum (P < 0.01), longissimus lumborum muscle (P < 0.01), gastrocnemius muscle (P < 0.05), and pancreas (P < 0.01). In addition, IUGR pigs had greater (P < 0.001) concentrations of thiobarbituric acid reactive substances (TBARS; an indicator of lipid peroxidation) in plasma, jejunum, muscles, and pancreas than NBW pigs. Compared with isonitrogenous controls, dietary glycine supplementation increased concentrations of GSH plus GSSG and GSH in plasma (P < 0.01), liver (P < 0.001), jejunum (P < 0.001), longissimus lumborum muscle (P = 0.001), and gastrocnemius muscle (P < 0.05); activities of GSH-synthetic enzymes in liver (P < 0.01) and jejunum (P < 0.05), while reducing GSSG/GSH ratios in plasma (P < 0.001), jejunum (P < 0.001), longissimus lumborum muscle (P < 0.001), gastrocnemius muscle (P = 0.01), pancreas (P < 0.05), and kidneys (P < 0.01). Concentrations of GSH plus GSSG, GSH, and GSSG/GSH ratios in kidneys were not affected (P > 0.05) by IUGR. Furthermore, glycine supplementation reduced (P < 0.001) TBARS concentrations in plasma, jejunum, muscles, and pancreas. Collectively, IUGR reduced GSH availability and induced oxidative stress in pig tissues, and these abnormalities were prevented by dietary glycine supplementation in a tissue-specific manner.

Pigs have the highest rate of intrauterine growth restriction (IUGR) among livestock species. These pigs, which have low birth weights (<1.1 kg) and account for ~15% to 20% of newborn pigs, are often culled after birth because they have lower growth performance and feed efficiency due to multiple factors (including oxidative stress in tissues), when compared with litter mates with normal birth weights (NBW). Much evidence shows that glutathione, which is a tripeptide synthesized from glutamate, glycine, and cysteine via enzymes (biological catalysts, γ-glutamylcysteine synthetase, and glutathione synthetase), is a major low-molecular-weight antioxidant in animal cells. Based on the findings of our recent study that dietary glycine supplementation enhanced the growth performance of IUGR pigs from weaning to market weight, the current study tested the hypothesis that this nutritional strategy increased the synthesis and availability of glutathione in their tissues. Our results indicated that the key organs of the digestive system (the small intestine, liver, and pancreas) as well as both longissimus lumborum and gastrocnemius muscles of IUGR pigs had lower concentrations of glutathione as compared with NBW pigs, due to reductions in both the activities of glutathione-synthetic enzymes and the availability of glycine. Dietary supplementation with 1% glycine prevented these metabolic deficiencies in tissues of IUGR pigs. Our findings support the notion that IUGR pigs fed conventional corn- and soybean meal-based diets do not synthesize adequate glutathione and that dietary glycine supplementation plays an important role in enhancing the availability of glutathione and mitigating oxidative stress to improve health and growth in these compromised animals.

Beta vulgaris-derived exosome-like nanovesicles alleviate chronic doxorubicin-induced cardiotoxicity by inhibiting ferroptosis.

Dose-dependent heart failure is a major complication of the clinical use of doxorubicin (Dox), one of the most potent chemotherapeutic agents. Effective adjuvant therapy is required to prevent Dox-induced cardiotoxicity. Currently, plant-derived exosome-like nanovesicle (PELNV) has revealed their salubrious antioxidant and immunological regulating actions in various disease models. In this study, we isolated, purified and characterized Beta vulgaris-derived exosome-like nanovesicle (BELNV). Dox or normal saline was given to HL-1 cells (3 µM) and 8-week C57BL/6N mice (5 mg/kg bodyweight per week for 4 weeks) to establish the in vitro and in vivo model of Dox-induced cardiotoxicity. Administration of BELNV significantly alleviated chronic Dox-induced cardiotoxicity in terms of echocardiographic and histological results. A reduced malondialdehyde (MDA), increased ratio of glutathione (GSH) to oxidized glutathione (GSSG) and levels of system xc- and glutathione peroxidase 4 were observed, indicating that DOX-stimulated ferroptosis was reversed by BELNV. Besides, the safety of BELNV was also validated since no liver, spleen, and kidney toxicity induced by BELNV was observed. These findings provide evidence that BELNV may act as a novel therapeutic biomaterial for patients undergoing adverse effects of Dox, at least partly mediated by inhibiting Dox-induced ferroptosis.

Yi-qi-hua-yu-jie-du decoction induces ferroptosis in cisplatin-resistant gastric cancer via the AKT/GSK3ß/NRF2/GPX4 axis.

BACKGROUND: Resistance to chemotherapy in gastric cancer (GC) is a ubiquitous challenge for its treatment. Yi-qi-hua-yu-jie-du decoction (YJD), an empirical formula in Traditional Chinese Medicine (TCM), demonstrated survival-prolonging functions in patients with GC. Previous research has shown that YJD could also inhibit drug resistance in GC. However, the precise mechanisms for how YJD accomplishes this remain incompletely explained. PURPOSE: The research aimed to identify differential metabolic characteristics in cisplatin-resistant GC and investigate whether YJD can target these differences to suppress GC drug resistance. METHODS: Metabolomic analysis was conducted to identify metabolic disparities between cisplatin-resistant and parental GC cells, as well as metabolic modifications resulting from YJD intervention in cisplatin-resistant GC cells. The effect of YJD on ferroptosis stimulation was assessed by measuring the levels of reactive oxygen species (ROS), malondialdehyde (MDA), iron ions, the reduced glutathione (GSH) to oxidised glutathione (GSSG) ratio, and alterations in mitochondrial morphology. Western blotting and quantitative real-time polymerase chain reaction (Q-PCR) were employed to verity the mechanisms of YJD-triggered ferroptosis through GPX4 and NRF2 overexpression models, alongside the AKT activator SC79. In vivo validation was conducted using nude mouse xenograft models. RESULTS: Cisplatin-resistant GC exhibited altered GSH/GPX4 metabolism, and ferroptosis was a significantly enriched cell death pattern with YJD treatment in cisplatin-resistant GC cells. Ferroptosis biomarkers, including ROS, MDA, iron ions, the GSH/GSSG ratio, and mitochondrial morphology, were remarkably changed with the YJD intervention. Mechanistic experiments demonstrated that YJD inhibited the phosphorylation cascade activity of the AKT/GSK3ß pathway, thereby reducing NRF2 expression. The level of GPX4, a crucial enzyme involved in glutathione metabolism, was attenuated, facilitating ferroptosis induction in cisplatin-resistant GC. CONCLUSION: The research reveals, for the first time, changes in GSH/GPX4 metabolism in cisplatin-resistant GC cells based on metabolomic analysis. YJD induced ferroptosis in cisplatin-resistant GC by inhibiting GPX4 through the AKT/GSK3ß/NRF2 pathway, thus attenuating the cisplatin drug resistance in GC. Our findings identify metabolic changes in cisplatin-resistant GC and establish a theoretical framework for YJD on tackling drug resistance in GC through ferroptosis.

Effect of Antioxidant Supplementation on NET Formation Induced by LPS In Vitro; the Roles of Vitamins E and C, Glutathione, and N-acetyl Cysteine.

Neutrophil extracellular traps (NETs) require reactive oxygen species (ROS) to eliminate pathogens by inducing oxidative stress. However, this process can also cause tissue damage to the host. Neutrophils contain high concentrations of vitamin C (1.5 mM) compared to the bloodstream (0.1 mM), and this antioxidant can interact with vitamin E and glutathione (GSH) inside the cell to maintain the redox balance. Previous studies have investigated the effect of vitamins E or C and N-acetyl cysteine (NAC) on NET formation, but the interactions of these molecules in neutrophils remain unknown. In this study, we investigated the effect of antioxidants alone and two combinations on NET formation and oxidative stress. Neutrophils were pre-loaded with GSH + NAC or vitamin E + vitamin C + GSH + NAC (termed ALL), and LPS-induced NET formation was assessed using fluorometry and immunofluorescence. Antioxidant effects were evaluated by measuring the total antioxidant capacity (TAC), GSH/GSSG ratio, ROS production, nitrite + nitrate levels, and lipid peroxidation. Our results showed that even low doses of antioxidants are capable of decreasing NETs. Furthermore, the combinations augmented TAC and GSH/GSSG ratio and decreased ROS, nitrites + nitrates, and malondialdehyde (MDA) levels in supplemented neutrophils in vitro.

Dietary lysolecithin supplementation improves growth performance of weaned piglets via improving nutrients absorption, lipid metabolism, and redox status.

Lysolecithin is widely used as emulsifier to improve the digestibility and retention of fat. The current study aimed to investigate the effects of dietary lysolecithin supplementation on growth performance, nutrients absorption, lipid metabolism, and redox status of weaned pigs. A total of 60 weaned piglets were assigned into 2 dietary treatments in a randomized complete block design, receiving basal diet with 0 or 1,000 mg/kg lysolecithin for a period of 28 d. Each dietary treatment had 10 replicates with 3 piglets per replicate. Growth performance and fecal score were monitored during trial. Samples of blood, ileum, and liver tissues were collected and analyzed for serology, intestinal histomorphology, and lipid metabolism-related gene and protein expressions. Dietary lysolecithin supplementation increased average daily gain (+15%, P < 0.05) and tended to increase average daily feed intake (+14%, P = 0.08) in overall experimental period. At final, the average body weight of piglets in lysolecithin group was 10% greater than that of control group (P = 0.09). In addition, dietary lysolecithin supplementation improved the ability of nutrients absorption as indicated by the higher d-xylose level in plasma (P < 0.05). Moreover, piglets from lysolecithin group had higher concentration of high-density lipoprotein (P < 0.05), but lower triglyceride (P < 0.05) in plasma. The inclusion of lysolecithin in diet increased the level of reduced glutathione (GSH) and GSH to oxidized glutathione (GSSG) ratio in plasma and liver (P < 0.05), but attenuated the levels of malondialdehyde and GSSG in ileum (P < 0.05). The upregulation of lipogenesis-related genes (FAS and ACC), downregulation of lipolysis (PNPLA2 and PABP1), and lipid mobilization (PGC-1α and SRIT1) genes were observed in lysolecithin relative to control piglets. Compared with control group, dietary lysolecithin supplementation upregulated protein expressions of GPX4, SREBP1, and LPL in liver and LPL in ileum (P < 0.05). Collectively, our study indicates that dietary lysolecithin supplementation improved growth performance of weaned piglets, which may be associated with the improved nutrients absorption, redox status, and lipid metabolism.

Early weaning has been widely adopted to maximize productivity for swine production. However, the weaned piglets suffer from insufficient energy intake due to the reduced feed intake caused by weaning stress, which compromises the growth rate of piglets. In the present study, dietary lysolecithin supplementation increased average daily gain and average daily feed intake in overall experimental period, thereby showed positive effect on final body weight of weaned piglets. In addition, lysolecithin supplementation improved adipogenesis and anti-oxidant capacity, but suppressed lipolysis and pro-oxidant factors.

Nanomicelle curcumin-induced testicular toxicity: Implications for altered mitochondrial biogenesis and mitophagy following redox imbalance.

The purpose of this study was to examine the effects of nano-micelle curcumin (NMC)-induced redox imbalance on mitochondrial biogenesis and mitophagy. For this purpose, 24 mature male Wistar rats were divided into control and NMC-received groups (7.5, 15, and 30 mg/kg) groups. After 48 days, the Nrf1, Nrf2, and SOD (Cu/Zn) expression levels, as well as GSH/GSSG, NADP+ /NADPH relative balances (elements involved in redox homeostasis) were analyzed. Moreover, to explore the effect of NMC on mitochondrial biogenesis, the expression levels of Mfn1, Mfn2, OPA1, Fis1, and Drp1 were investigated. Finally, the expression levels of Parkin/PARK and PINK (genes involved in mitochondrial quality control), as well as LC3-I/II (mitophagy marker), were analyzed. Observations showed that NMC, dose-dependently, altered GSH/GSSG, NADP+ /NADPH relative balances, suppressed SOD expression and diminished its biochemical level, and repressed Nrf1 and Nrf2 expression levels. Moreover, it could change the Mfn1, Mfn2, OPA1, Fis1, and Drp1 expression pattern and stimulate the Parkin/PARK and PINK as well as LC3-I/II expression levels, dose-dependently. In conclusion, chronic and high-dose NMC is able to suppress the redox capacity by down-regulating the Nrf1 and Nrf2 expression. Finally, at high-dose levels, it is able to trigger mitophagy signaling in the testicles.

Hyperthermia-triggered NO release based on Cu-doped polypyrrole for synergistic catalytic/gas cancer therapy.

Nitric oxide (NO) is a crucial gaseous medium for tumor growth and progression, but it may also cause mitochondrial disorder and DNA damage by drastically increasing its concentration in tumor. Due to its challenging administration and unpredictable release, NO based gas therapy is difficult to eliminate malignant tumor at low safe doses. To address these issues, herein, we develop a multifunctional nanocatalyst called Cu-doped polypyrrole (CuP) as an intelligent nanoplatform (CuP-B@P) to deliver the NO precursor BNN6 and specifically release NO in tumors. Under the aberrant metabolic environment of tumors, CuP-B@P catalyzes the conversion of antioxidant GSH into GSSG and excess H2O2 into ·OH through Cu+/Cu2+ cycle, which results in oxidative damage to tumor cells and the concomitant release of cargo BNN6. More importantly, after laser exposure, nanocatalyst CuP can absorb and convert photons into hyperthermia, which in turn, accelerates the aforesaid catalytic efficiency and pyrolyzes BNN6 into NO. Under the synergistic effect of hyperthermia, oxidative damage, and NO burst, almost complete tumor elimination is achieved in vivo with negligible toxicity to body. Such an ingenious combination of NO prodrug and nanocatalytic medicine provides a new insight into the development of NO based therapeutic strategies. STATEMENT OF SIGNIFICANCE: A hyperthermia-responsive NO delivery nanoplatform (CuP-B@P) based on Cu-doped polypyrrole was designed and fabricated, in which CuP catalyzed the conversion of H2O2 and GSH into ·OH and GSSG to induce intratumoral oxidative damage. After laser irradiation, hyperthermia ablation and responsive release of NO further coupled with oxidative damage to eliminate malignant tumors. This versatile nanoplatform provides new insights into the combined application of catalytic medicine and gas therapy.

[Electroacupuncture promotes expression of glutathione related regulatory enzymes in ovary tissue of rats with diminished ovarian reserve].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on ovarian function and expression of glutathione (GSH) related regulatory enzymes γ-glutamylcysteine synthetase (γ-GCS), glutathione reductase (GR) protein and gene in rats with diminished ovarian reserve (DOR), so as to explore its mechanisms underlying up-regulation of antioxidant stress ability. METHODS: A total of 30 female SD rats with normal estrous cycle were randomly divided into blank control, model and EA groups, with 10 rats in each group. The DOR model was established by gavage of tripterygium wilfordii polyglycoside suspension (50 mg·kg-1·d-1) for 14 consecutive days, while the rats in the blank group were given equal volume of 0.9% sodium chloride solution. One hour after daily gavage, EA (1.0 mA, 100 Hz) was applied alternately to bilateral "Shenshu"(BL23), and "Zhongwan"(CV12)+"Guanyuan"(CV4) for 10 min, for 14 consecutive days. Estrous cycles of rats in each group were observed and recorded daily during intervention.After the intervention, H.E.staining was used to observe histopathological changes of the ovarian tissue. The contents of serum sex hormones ï¼»follicle stimulating hormone (FSH), anti-mullerian hormone (AMH), estradiol (E2)ï¼½ and oxidative damage markers ï¼»8-hydroxydeoxyguanosine (8-OHDG) and nitrotyrosine (NTY)ï¼½ were determined by ELISA. The contents of GSH and oxidized glutathione (GSSG) in the liver tissue were determined by colorimetry, and their ratios were calculated. Immunohistochemistry and real-time fluorescence quantitative PCR were used to detect the immunoactivity and gene expression levels of γ-GCS and GR in the ovarian tissues, respectively. RESULTS: Compared with the blank group, the model group had a marked increase in the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents (P<0.01) and a considerable decrease in the levels of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number as well as the expression of γ-GCS and GR mRNAs (P<0.05, P<0.01). After EA intervention, the increase of the disorder rate of estrous cycle, serum FSH, 8-OHDG and NTY contents and the decrease of serum AMH and E2, liver GSH and GSSG contents and GSH/GSSG ratio, ovarian optical density and cell number of γ-GCS and GR as well as the expression of γ-GCS genes were all reversed (P<0.01, P<0.05). H.E. staining showed degenerative changes of the ovarian tissue, fewer follicles at every level and increase of atretic follicles, disarrangement and layer number decrease of granulosa cells, and atrophy of corpus luteum in the model group, which were relatively milder in the EA group. CONCLUSION: EA can improve ovarian function, and reduce oxidative stress damage in DOR rats, which may be associated with its functions in up-regulating the expression of γ-GCS and GR protein and gene in the ovarian tissue.

Cystine/Glutamate Xc- Antiporter Induction Compensates for Transsulfuration Pathway Repression by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) to Ensure Cysteine for Hepatic Glutathione Biosynthesis.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been associated with the induction of oxidative stress and the progression of steatosis to steatohepatitis with fibrosis. It also disrupts metabolic pathways including one-carbon metabolism (OCM) and the transsulfuration pathway with possible consequences on glutathione (GSH) levels. In this study, complementary RNAseq and metabolomics data were integrated to examine the hepatic transsulfuration pathway and glutathione biosynthesis in mice following treatment with TCDD every 4 days for 28 days. TCDD dose-dependently repressed hepatic cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH) mRNA and protein levels. Reduced CBS and CTH levels are also correlated with dose-dependent decreases in hepatic extract hydrogen sulfide (H2S). In contrast, cysteine levels increased consistent with the induction of Slc7a11, which encodes for the cystine/glutamate Xc- antiporter. Cotreatment of primary hepatocytes with sulfasalazine, a cystine/glutamate Xc- antiporter inhibitor, decreased labeled cysteine incorporation into GSH with a corresponding increase in TCDD cytotoxicity. Although reduced and oxidized GSH levels were unchanged following treatment due to the induction of GSH/GSSG efflux transporter by TCDD, the GSH:GSSG ratio decreased and global protein S-glutathionylation levels in liver extracts increased in response to oxidative stress along with the induction of glutamate-cysteine ligase catalytic subunit (Gclc), glutathione synthetase (Gss), glutathione disulfide reductase (Gsr), and glutathione transferase π (Gstp). Furthermore, levels of ophthalmic acid, a biomarker of oxidative stress indicating GSH consumption, were also increased. Collectively, the data suggest that increased cystine transport due to cystine/glutamate Xc- antiporter induction compensated for decreased cysteine production following repression of the transsulfuration pathway to support GSH synthesis in response to TCDD-induced oxidative stress.

Baicalin induces ferroptosis in osteosarcomas through a novel Nrf2/xCT/GPX4 regulatory axis.

BACKGROUND: Osteosarcomas (OS) is a kind of malignant bone tumor which occurs primarily in children and adolescents, and the clinical therapeutics remain disappointing. As a new programmed cell death, ferroptosis is characterized by iron dependent and intracellular oxidative accumulation, which provides a potential alternative intervene for the OS treatment. Baicalin, a major bioactive flavone derived from traditional Chinese medicine Scutellaria baicalensis, has been proved to have anti-tumor properties in OS. Whether ferroptosis participated in the baicalin mediated anti-OS activity is an interesting project. PURPOSE: To explore the pro-ferroptosis effect and mechanisms of baicalin in OS. METHODS/STUDY DESIGN: Pro-ferroptosis effect of baicalin on cell death, cell proliferation, iron accumulation, lipid peroxidation production was determined in MG63 and 143B cells. The levels of glutathione (GSH), oxidized (GSSG) glutathione and malondialdehyde (MDA) were determined by enzyme linked immunosorbent assay (ELISA). The expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2), Glutathione peroxidase 4 (GPX4) and xCT were detected by western blot in baicalin-mediated ferroptosis regulation. In vivo, a xenograft mice model was adopted to explore the anticancer effect of baicalin. RESULTS: In the present study, it was found that baicalin significantly suppress tumor cell growth in vitro and in vivo. By promoting the Fe accumulation, ROS formation, MDA production and suppressing the ratio of GSH/GSSG, baicalin was found to trigger ferroptosis in OS and ferroptosis inhibitor ferrostatin-1 (Fer-1) successfully reversed these suppressive effects, indicating that ferroptosis participated in the baicalin mediated anti-OS activity. Mechanistically, baicalin physically interacted with Nrf2, a critical regulator of ferroptosis, and influenced its stability via inducing ubiquitin degradation, which suppressed the Nrf2 downstream targets GPX4 and xCT expression, and led to stimulating ferroptosis. CONCLUSIONS: Our findings for the first time indicated that baicalin exerted anti-OS activity through a novel Nrf2/xCT/GPX4-dependent ferroptosis regulatory axis, which hopefully provides a promising candidate for OS treatment.

Dachaihu decoction ameliorates septic intestinal injury via modulating the gut microbiota and glutathione metabolism as revealed by multi-omics.

ETHNOPHARMACOLOGICAL RELEVANCE: Dachaihu decoction (DCH), a classic formula for Yangming and Shaoyang Syndrome Complex recorded in "Treatise on Cold Damage", has been widely used in treating intestinal disorders and inflammatory diseases with few side effects in China. However, the mechanism of DCH on septic intestinal injury (SII) remains to be explored. AIM OF THE STUDY: This study aimed to clarify the mechanism of DCH on SII. MATERIALS AND METHODS: SII model of rat, established by cecal ligation and puncture (CLP), was used to study the effect of DCH on SII. 24 h mortality was recorded. Histological changes were observed by H&E staining. The expression of tight junction protein ZO-1 (ZO-1) and mucin2 (MUC2) was determined by immunohistochemical analysis. Secretory IgA (sIgA), diamine oxidase (DAO) and intestinal fatty acid binding protein (iFABP) were determined by enzyme-linked immunosorbent assay (ELISA). IL-1ß, IL-6 and TNF-α were measured by ELISA and quantitative Real-time PCR (RT-qPCR). The gut microbiota was analyzed by 16S rRNA sequencing. The potential targets and pathways of DCH in treating SII were analyzed by integrative analysis of transcriptomic and metabolomic methods. Total glutathione (T-GSH), GSH, GSSG (reduced form of GSH), GSH peroxidase (GPX), superoxide dismutase (SOD), malonaldehyde (MDA) and indicators of hepatic and renal function were measured by biochemical kits. RESULTS: Medium dose of DCH improved 24 h mortality of SII rats, reduced the pathological changes of ileum, and increased the expression levels of ZO-1, MUC2 and sIgA. DCH decreased DAO, iFABP of serum and IL-1ß, IL-6, TNF-α of ileum. DCH improved α- and ß-diversity and modulated the structure of gut microbiota, with Escherichia_Shigella decreased and Bacteroides and Ruminococcus increased. GSH metabolism was identified as the key pathway of DCH on SII by integrative analysis of transcriptome and metabolome. GSH/GSSG and the most common indicators of oxidative stress, were validated. Antioxidative T-GSH, GSH, GPX and SOD were increased, while MDA, the mark of lipid peroxidation was downregulated by DCH. Eventually, DCH was proved to be safe and hepato- and nephro-protective. CONCLUSION: DCH ameliorated septic intestinal injury possibly by modulating the gut microbiota and enhancing glutathione metabolism of SII rats, without hepatotoxicity and nephrotoxicity.

Lobetyolin Alleviates Ferroptosis of Skeletal Muscle in 5/6 Nephrectomized Mice via Activation of Hedgehog-GLI1 Signaling.

BACKGROUND: Muscle wasting increases morbidity and mortality and is related to chronic kidney disease (CKD) and dialysis. It is still unclear whether ferroptosis occurs during this progression and whether it is a potential intervention target for the treatment of CKD-related muscle injury. PURPOSE: The objective is to identify potential compounds for treating ferroptosis and muscle wasting and explore the potential mechanisms in vivo/in vitro. METHODS: Initially, we explored whether ferroptosis is present in the skeletal muscle of 5/6 nephrectomized (NPM) mice via RNA-Seq analysis, TUNEL staining, Oil red O staining, MDA/GSH/GSSG level detection and real-time quantitative PCR (qPCR). Subsequently, utilizing our established molecular phenotyping strategy, we screened potential traditional Chinese herb-derived compounds for alleviation of muscle wasting and ferroptosis. HE staining, Oil red O staining, TUNEL staining, immunofluorescence staining, MDA/GSH/GSSG level detection, Fe level detection, western blotting and qPCR were applied to assess the effects of the identified compound on muscle wasting and ferroptosis and explore the potential mechanism. Furthermore, RNA-Seq analysis, ChIP-Seq analysis and further experiments in vitro were performed to determine the role of Hedgehog signaling in the effect of Lobetyolin (LBT) on ferroptosis. RESULTS: In NPM mice, skeletal muscle dysfunction, lipogenesis, reduced GSH/GSSG ratio, decreased GSH content, increased MDA production and and higher levels of ferroptosis markers were observed. LBT treatment (30 mg/kg or 50 mg/kg) significantly alleviates skeletal muscle injury by inhibiting ferroptosis. Additionally, in an in vitro investigation, C2C12 cells exposed to Indolyl sulfate (IS) induced ferroptosis and LBT treatment (20 µM and 50 µM) protected C2C12 from such injury, consistent with the results from the in vivo analysis. Furthermore, it was found LBT increased the levels of protein involving Hedgehog signaling pathway (SMO and GLI1), and rescue analysis revealed that this pathway played a crucial role in the regulation of ferroptosis. Further experiments demonstrated that LBT upregulated a series of suppressors of ferroptosis by activating Gli1 transcription. CONCLUSION: LBT alleviates CKD-induced muscle injury by inhibiting ferroptosis through activation of the Hedgehog signaling pathway.

Potentilla anserina polysaccharide alleviates cadmium-induced oxidative stress and apoptosis of H9c2 cells by regulating the MG53-mediated RISK pathway.

Oxidative stress plays a crucial role in cadmium (Cd)-induced myocardial injury. Mitsugumin 53 (MG53) and its mediated reperfusion injury salvage kinase (RISK) pathway have been demonstrated to be closely related to myocardial oxidative damage. Potentilla anserina L. polysaccharide (PAP) is a polysaccharide with antioxidant capacity, which exerts protective effect on Cd-induced damage. However, it remains unknown whether PAP can prevent and treat Cd-induced cardiomyocyte damages. The present study was desgined to explore the effect of PAP on Cd-induced damage in H9c2 cells based on MG53 and the mediated RISK pathway. For in vitro evaluation, cell viability and apoptosis rate were analyzed by CCK-8 assay and flow cytometry, respectively. Furthermore, oxidative stress was assessed by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) staining and using superoxide dismutase (SOD), catalase (CAT), and glutathione/oxidized glutathione (GSH/GSSG) kits. The mitochondrial function was measured by JC-10 staining and ATP detection assay. Western blot was performed to detect the expression of proteins related to MG53, the RISK pathway, and apoptosis. The results indicated that Cd increased the levels of reactive oxygen species (ROS) in H9c2 cells. Cd decreased the activities of SOD and CAT and the ratio of GSH/GSSG, resulting in decreases in cell viability and increases in apoptosis. Interestingly, PAP reversed Cd-induced oxidative stress and cell apoptosis. Meanwhile, Cd reduced the expression of MG53 in H9c2 cells and inhibited the RISK pathway, which was mediated by decreasing the ratio of p-AktSer473/Akt, p-GSK3ßSer9/GSK3ß and p-ERK1/2/ERK1/2. In addition, Cd impaired mitochondrial function, which involved a reduction in ATP content and mitochondrial membrane potential (MMP), and an increase in the ratio of Bax/Bcl-2, cytoplasmic cytochrome c/mitochondrial cytochrome c, and Cleaved-Caspase 3/Pro-Caspase 3. Importantly, PAP alleviated Cd-induced MG53 reduction, activated the RISK pathway, and reduced mitochondrial damage. Interestingly, knockdown of MG53 or inhibition of the RISK pathway attenuated the protective effect of PAP in Cd-induced H9c2 cells. In sum, PAP reduces Cd-induced damage in H9c2 cells, which is mediated by increasing MG53 expression and activating the RISK pathway.

Regulation of ferroptosis and ACSL4-15LO1 pathway contributed to the anti-asthma effect of acupuncture.

Acupuncture has been frequently used in China for the treatment asthma for thousands of years. Ferroptosis was recently revealed to be involved in several pathological conditions including asthma. However, the detailed links between ferroptosis and airway inflammation in asthma, as well as the detailed regulation of acupuncture on these disorders remains unclear. Our results demonstrated that the non-haem Fe2+ level increased markedly in the lung tissue of mouse asthma model, and positively correlated with RL and IL-4 level in BALF. Furthermore, lipid peroxidation markers MDA and GSSG increased remarkably in OVA-induced experimental asthma mice. Up-regulation of lipid peroxidation associated proteins ACSL4 and15-LO1 was also observed in OVA-induced experimental asthma mice. To demonstrate the role of ferroptosis in asthma and the effect of acupuncture on these disorders, ferroptosis-induction agent erastin and ferroptosis-inhibition agent fer-1 were used, and our data demonstrated that erastin could augment lung inflammation and lipid peroxidation in OVA induced asthma model. Fer-1 was able to relieve AHR, lung inflammation, non-haem Fe2+ level, lipid peroxidation and ferroptosis related pathway ACSL4-15LO1 in OVA-induced experimental asthma mice. Acupuncture treatment alleviated RL, lung inflammation as well as type 2 cytokines IL-4 and IL-13 levels induced by OVA inhalation. What's more, acupuncture significantly reduced the MDA and GSSG levels, the non-haem Fe2+ level and ACSL4-15-LO1 proteins expression. Acupuncture also relieved erastin-induced exacerbation in lung inflammation and lipid peroxidation in ferroptosis. Acupuncture treatment could relieve ferroptosis related exacerbation in airway inflammation. Our study provided insights into the underlying mechanisms for the protective effects of acupuncture and highlighted a therapeutic potential of acupuncture treatment in the attenuation of lipid peroxidation and ferroptosis in asthma.

Delayed administration of Trichilia catigua A. Juss. Ethyl-acetate fraction after cerebral ischemia prevents spatial memory deficits, decreases oxidative stress, and impacts neural plasticity in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Trichilia catigua A. Juss (Meliaceae) is used in Brazilian folk medicine to alleviate fatigue and emotional stress and improve memory. Previous studies from our laboratory reported that an ethyl-acetate fraction (EAF) of T. catigua that was given before cerebral ischemia in vivo prevented memory loss and reduced oxidative stress and neuroinflammation. Despite the value of these findings of a neuroprotective effect of T. catigua, treatment that was given immediately before or immediately after ischemia limits its clinical relevance. Thus, unknown is whether T. catigua possesses a specific time window of efficacy (TWE) when administered postischemia. AIM OF THE STUDY: Given continuity to previous studies, we investigated whether an EAF of T. catigua maintains its neuroprotective properties if treatment begins at different time windows of efficacy after ischemia. We also evaluated, for the first time, whether T. catigua possesses neuroplasticity/neurotrophic properties. MATERIAL AND METHODS: Rats were subjected to transient global brain ischemia (TGCI) and then given a single dose of the EAF (400 mg/kg) or vehicle (1 ml/kg) orally 1, 4, or 6 h postischemia. The levels of protein PCG, GSH, and GSSG, and activity of SOD and CAT were assayed as markers of oxidative stress on the day after ischemia. In another experiment, naive rats underwent spatial learning training in a radial maze task and then subjected to TGCI. Delayed treatment with the EAF began 4 or 6 h later and continued for 7 days. Retrograde memory performance was assessed 10, 17, and 24 days postischemia. Afterward, brains were examined for neurodegeneration and neuronal dendritic morphology in the hippocampus and cerebral cortex. Another group received the EAF at 4 h of reperfusion, and 4 days later their brains were examined for GFAP and Iba-1 immunoreactivity. Lastly, ischemic rats received the EAF 4 h after ischemia and neural plasticity-related proteins, BDNF, SYN, PSD 95, and NeuN were measured in the hippocampus 7 and 14 days after ischemia. RESULTS: A single EAF administration 1, 4, or 6 h postischemia alleviated oxidative stress that was caused by ischemia, expressed as a reduction of the amount of the PCG and GSSG, normalization of the GSH/GSSG ratio, and the restoration of SOD activity. Ischemia caused the persistent loss of memory (i.e., amnesia), an outcome that was consistently ameliorated by treatment with the EAF that was initiated 4 or 6 h postischemia. The 4 h delay in EAF treatment positively impacted dendritic morphology in neurons that survived ischemia. TGCI reduced BDNF, SYN, PSD-95, and NeuN protein levels in the hippocampus and cerebral cortex. The EAF normalized SYN and PSD-95 protein levels. Ischemia-induced neurodegeneration and glial cell activation were not prevented by EAF treatment. CONCLUSION: The present study corroborates prior data that demonstrated the neuroprotective potential of T. catigua and extends these data by showing that the delayed administration of EAF postischemia effectively prevented memory impairment and decreased oxidative stress, dendritic deterioration, and synaptic protein loss within a TWE that ranged from 1 to 6 h. This specific TWE in preclinical research may have clinical relevance by suggesting the possible utility of this plant for the development of neuroprotective strategies in the setting of ischemic brain diseases. Another innovative finding of the present study was the possible neurotrophic/neuroplastic properties of T. catigua.

Metabolomics of the anti-inflammatory effect of Pueraria lobata and Pueraria lobata var. Thomsonii in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Pueraria lobata (Willd.) Ohwi and Pueraria lobata var. Thomsonii (Benth.) Maesen are essential medicinal and edible homologous plants widely cultivated in Asian countries. Therefore, P. lobata and P. thomsonii are widely used in the food, health products and pharmaceutical industries and have significant domestic and international market potential and research value. P. lobata and P. thomsonii have pharmacological effects in the clinic, such as antipyretic, analgesic, anti-inflammatory and antioxidant effects. These plants are commonly used in the treatment of inflammatory diseases and other related diseases. However, the potential mechanisms of the anti-inflammatory effects of P. lobata and P. thomsonii have not been elucidated. AIM OF THE STUDY: This study aimed to confirm the anti-inflammatory effects of P. lobata and P. thomsonii on inflammatory model diseases and to investigate the mechanism of their anti-inflammatory effects from the perspective of plasma metabolomics. MATERIALS AND METHODS: First, P. lobata and P. thomsonii were identified by high‒performance liquid chromatography (HPLC). Second, we established the following three inflammation models: an acute inflammation model of auricular swelling in mice induced by xylene, an acute inflammation model of foot swelling in rats induced by carrageenan gum, and a chronic inflammation model of cotton ball granuloma in rats. Then we examined the weight and swelling rate of auricular swelling in mice; the residence time, contact area, and mean contact pressure in rats on the gait meter; and the weight of granulomas in rats and the content of IL-1ß and TNF-α in plasma to investigate the anti-inflammatory pharmacodynamics of P. lobata and P. thomsonii. Third, we used LC‒MS‒based plasma metabolomics techniques to obtain potential biomarkers of P. lobata and P. thomsonii related to inflammation. Then, the potential biomarkers were enriched by MetaboAnalyst and KEGG metabolomics analysis tools to obtain metabolic pathways related to inflammation. Finally, we tested the indicators of COX-2, 5-LOX, GSH, GSSG and γ⁃GCL in rat plasma from the granuloma model by enzyme-linked immunosorbent assays (ELISAs) to verify the inflammation-related metabolic pathway. RESULTS: The experimental results showed that P. lobata and P. thomsonii could reduce the swollen weight and swelling rate of the auricle in mice, and could increase the residence time, contact area and mean contact pressure in rats on the gait meter. Moreover, P. lobata and P. thomsonii could inhibit the growth of granulomas and reduce the content of IL-1ß and TNF-α in plasma in rats. The above results preliminarily verified that P. lobata and P. thomsonii have different anti-inflammatory effects. We identified eighteen plasma biomarkers associated with P. lobata and sixteen plasma biomarkers related to P. thomsonii in regulating inflammation by a plasma metabolomics analysis. The following two major metabolic pathways were further screened and enriched: arachidonic acid metabolism and glutathione metabolism. Then we noted that P. lobata and P. thomsonii could reduce the COX-2, 5-LOX and GSSG levels and increase the GSH, GSH/GSSG and γ⁃GCL levels based on the ELISA results, which demonstrated that P. lobata and P. thomsonii affect the anti-inflammatory mechanism through arachidonic acid metabolism and glutathione metabolism. CONCLUSIONS: The results of this study further elucidate the anti-inflammatory mechanism of action of P. lobata and P. thomsonii, providing a scientific basis for developing new drugs for the treatment of inflammation-related diseases and laying a foundation for the development of herbal resources, such as P. lobata and P. thomsonii.

Characterization of polysulfides in Saccharomyces cerevisiae cells and finished wine from a cysteine-supplemented model grape medium.

Polysulfide degradation in wine can result in hydrogen sulfide (H2S) release, imparting a rotten-egg smell that is detrimental to wine quality. Although the presence of wine polysulfides has been demonstrated, their biogenesis remains unclear. This study investigated the role of Saccharomyces cerevisiae in polysulfide formation during fermentation, with and without 5 mM cysteine supplementation as an H2S source. Using an established liquid chromatography-tandem mass spectrometry method, monobromobimane derivatives of hydropolysulfides, including CysSSSH, CysSSSSH and GSSSSH, and two oxidized polysulfides, GSSG and GSSSSG, were detected in yeast cells at the end of fermentation in a grape juice-like medium. Polysulfide production by four S. cerevisiae single deletion mutants (BY4743 Δcys3, Δcys4, Δmet17 and Δtum1) showed no significant differences compared to BY4743, suggesting that uncharacterized pathways maintain cellular polysulfide homeostasis. Five mM cysteine addition increased the formation of shorter sulfur chain species, including GSS-bimane and GSSG, but did not elevate levels of longer sulfur chain species. Additionally, polysulfides with even numbers of sulfur atoms tended to predominate in cellular lysates. Oxidized polysulfides and longer chain hydropolysulfides were not detected in finished wines. This evidence suggests that these polysulfides are unstable in wine-like environments or not transported extracellularly. Collectively, our data illustrate the complexity of yeast polysulfide metabolism under fermentation conditions.