Curdione inhibits ferroptosis in isoprenaline-induced myocardial infarction via regulating Keap1/Trx1/GPX4 signaling pathway.

Myocardial infarction (MI) is a common disease with high morbidity and mortality. Curdione is a sesquiterpenoid from Radix Curcumae. The current study is aimed to investigate the protective effect and mechanism of curdione on ferroptosis in MI. Isoproterenol (ISO) was used to induce MI injury in mice and H9c2 cells. Curdione was orally given to mice once daily for 7 days. Echocardiography, biochemical kits, and western blotting were performed on the markers of cardiac ferroptosis. Curdione at 50 and 100 mg/kg significantly alleviated ISO-induced myocardial injury. Curdione and ferrostatin-1 significantly attenuated ISO-induced H9c2 cell injury. Curdione effectively suppressed cardiac ferroptosis, evidenced by decreasing malondialdehyde and iron contents, and increasing glutathione (GSH) level, GSH peroxidase 4 (GPX4), and ferritin heavy chain 1 expression. Importantly, drug affinity responsive target stability, molecular docking, and surface plasmon resonance technologies elucidated the direct target Keap1 of curdione. Curdione disrupted the interaction between Keap1 and thioredoxin1 (Trx1) but enhanced the Trx1/GPX4 complex. In addition, curdione-derived protection against ISO-induced myocardial ferroptosis was blocked after overexpression of Keap1, while enhanced after Keap1 silence in H9c2 cells. These findings demonstrate that curdione inhibited ferroptosis in ISO-induced MI via regulating Keap1/Trx1/GPX4 signaling pathway.

The Impact of Severe COVID-19 on Plasma Antioxidants.

Several studies suggested the association of COVID-19 with systemic oxidative stress, in particular with lipid peroxidation and vascular stress. Therefore, this study aimed to evaluate the antioxidant signaling in the plasma of eighty-eight patients upon admission to the Clinical Hospital Dubrava in Zagreb, of which twenty-two died within a week, while the other recovered. The differences between the deceased and the survivors were found, especially in the reduction of superoxide dismutases (SOD-1 and SOD-2) activity, which was accompanied by the alteration in glutathione-dependent system and the intensification of the thioredoxin-dependent system. Reduced levels of non-enzymatic antioxidants, especially tocopherol, were also observed, which correlated with enhanced lipid peroxidation (determined by 4-hydroxynonenal (4-HNE) and neuroprostane levels) and oxidative modifications of proteins assessed as 4-HNE-protein adducts and carbonyl groups. These findings confirm the onset of systemic oxidative stress in patients with severe SARS-CoV-2, especially those who died from COVID-19, as manifested by strongly reduced tocopherol level and SOD activity associated with lipid peroxidation. Therefore, we propose that preventive and/or supplementary use of antioxidants, especially of lipophilic nature, could be beneficial for the treatment of COVID-19 patients.

The Effects of TRX Suspension Training Combined with Taurine Supplementation on Body Composition, Glycemic and Lipid Markers in Women with Type 2 Diabetes.

BACKGROUND: We aimed to investigate the effects of an 8-week total-body resistance exercise (TRX) suspension training intervention combined with taurine supplementation on body composition, blood glucose, and lipid markers in T2D females. METHODS: Forty T2D middle-aged females (age: 53 ± 5 years, body mass = 84.3 ± 5.1 kg) were randomly assigned to four groups, TRX suspension training + placebo (TP; n = 10), TRX suspension training + taurine supplementation (TT; n = 10), taurine supplementation (T; n = 10), or control (C; n = 10). Body composition (body mass, body mass index (BMI), body fat percentage (BFP)), blood glucose (fasting blood sugar (FBS)), hemoglobin A1c (HbA1c), Insulin, and Insulin resistance (HOMA-IR), and lipid markers (low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglyceride (TG), and total cholesterol (TC)) were evaluated prior to and after interventions. RESULTS: All three interventions significantly decreased body mass, BMI, and BFP with no changes between them for body mass and BMI; however, BFP changes in the TT group were significantly greater than all other groups. FBS was significantly reduced in TP and TT. Insulin concentrations' decrement were significantly greater in all experimental groups compared to C; however, no between group differences were observed between TT, TP, and T. In regards to HOMA-IR, decreases in TT were significantly greater than all other groups TG, HbA1c, and LDL were reduced following all interventions. HDL values significantly increased only in the TT group, while TC significantly decreased in TP and TT groups. Changes in HbA1c, TG, HDL, and TC were significantly greater in the TT compared to all other groups. CONCLUSIONS: TRX training improved glycemic and lipid profiles, while taurine supplementation alone failed to show hypoglycemic and hypolipidemic properties. Notably, the synergic effects of TRX training and taurine supplementation were shown in HbA1c, HOMA-IR, TG, TC, HDL, and BFP changes. Our outcomes suggest that TRX training + taurine supplementation may be an effective adjuvant therapy in individuals with T2D.

Recombinant production of Trx-Ib-AMP4 and Trx-E50-52 antimicrobial peptides and antimicrobial synergistic assessment on the treatment of methicillin-resistant Staphylococcus aureus under in vitro and in vivo situations.

PURPOSE: The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA). METHOD: At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested. RESULTS AND CONCLUSION: The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log10 CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects.

Anti-oxidant, anti-inflammatory and anti-fibrosis effects of ganoderic acid A on carbon tetrachloride induced nephrotoxicity by regulating the Trx/TrxR and JAK/ROCK pathway.

Ganoderic acid A (GAA), one of the major triterpenoid components extracted from Ganoderma mushroom has been shown to possess numerous important pharmacological activities. The present study was aimed to investigate the mechanisms of GAA on carbon tetrachloride (CCl4)-induced kidney inflammation, fibrosis and oxidative stress in mice. The male mice were treated with 25 and 50 mg/mg GAA after stimulated with CCl4. Our results showed that GAA improved renal damage by decreasing the serum levels of creatinine, urea, uric acid and alleviating kidney fibrosis. GAA ameliorated CCl4-induced indices of inflammation. GAA suppressed oxidative stress by regulating the glutathione antioxidant system and the thioredoxin antioxidant system. GAA increased the activations of thioredoxin reductase (TrxR), Trx, GSH, SOD, GPx. Furthermore, GAA supplementation inhibited the JAK and STAT3 pathway. GAA inhibited the activations of RhoA, ROCK, NF-κB, TGF-ß and Smad3. Thus, this study demonstrated that GAA possesses immune-protective properties through regulating the Trx/TrxR, JAK2/STAT3 and RhoA/ROCK pathways.

Sodium tanshinone IIA sulfonate attenuates silica-induced pulmonary fibrosis in rats via activation of the Nrf2 and thioredoxin system.

Silicosis is characterized by pulmonary fibrosis due to long-term inhalation of silica particles. Although the cause of this serious disease is known, its pathogenesis remains unclear and there are currently no specific treatments. Recent studies have shown that the anti-oxidant transcription factor Nrf2 is expressed at reduced levels in fibrotic foci, which may be related to disease progression. However, the molecular mechanisms by which this might occur have yet to be elucidated. Sodium tanshinone IIA sulfonate (STS), an extract of Salvia miltiorrhiza, is used in traditional Chinese medicine in the treatment of coronary heart disease. STS has been shown to play a strong anti-oxidative role in various organs. Here, we employed a rat model to explore the effects of STS on oxidative stress and the progression of fibrosis in silicosis. STS significantly reduced collagen deposition in the lungs, thereby antagonising silicosis. Immunohistochemical and immunofluorescence staining showed that Nrf2 was differentially expressed in lung cells during silica induced fibrosis, and chromatin immunoprecipitation-sequencing experiments demonstrated that Nrf2 promoted the expression of the antioxidant proteins thioredoxin and thioredoxin reductase. Our results suggest that the anti-fibrotic effects of STS may be related to upregulation of Nrf2 nuclear expression, especially in fibrotic lesions, and the promotion of thioredoxin and thioredoxin reductase expression. Our findings may open up new avenues for the development of STS as a treatment for silicosis.

Enhanced Keap1-Nrf2/Trx-1 axis by daphnetin protects against oxidative stress-driven hepatotoxicity via inhibiting ASK1/JNK and Txnip/NLRP3 inflammasome activation.

BACKGROUND: Oxidative stress-triggered fatal hepatotoxicity is an essential pathogenic factor in acute liver failure (ALF). AIMS: To investigate the protective effect of daphnetin (Daph) on tert-butyl hydroperoxide (t-BHP) and acetaminophen (APAP)-induced hepatotoxicity through altering Nrf2/Trx-1 pathway activation. MATERIALS AND METHODS: In vivo, male C57BL/6 mice with Wild-type (WT) and Nrf2-/- were divided into five groups and acute liver injury model were established by APAP or LPS/GalN after injection with Daph (20, 40, or 80 mg/kg), seperately. Then, liver tissue and serum were collected for biochemical determination, TUNEL and H & E staining, and western blot analysis. In vitro, HepG2 cells were used to investigate the protective effect and mechanism of daphnetin against ROS and apoptosis induced by t-BHP via apoptosis detection, western blot, immunofluorescence analysis, and sgRNA transfection. RESULTS: Our results indicated that Daph efficiently inhibited t-BHP-stimulated hepatotoxicity, and modulated Trx-1 expression and Nrf2 activation which decreased Keap1-overexpression in HepG2 cells. Moreover, Daph inhibited t-BHP-excited hepatotoxicity and enhanced Trx-1 expression, which was reversed in Nrf2-/- HepG2 cells. In vivo, a survival rate analysis first suggested that Daph significantly reduced the lethality induced by APAP or GalN/LPS in a Nrf2-dependent or -independent manner by using Nrf2-/- mice, respectively. Next, further results implicated that Daph not only effectively alleviated APAP-induced an increase of ALT and AST levels, histopathological changes, ROS overproduction, malondialdehyde (MDA) formation and GSH/GSSG reduction, but it also relieved hepatic apoptosis by strengthening the suppression of cleaved-caspase-3 and expression of P53 protein. Additionally, Daph attenuated mitochondrial dysfunction by suppressing ASK1/JNK activation and decreasing apoptosis-inducing factor (AIF) and Cytochrome c release and Bax mitochondrial translocation. Daph inhibited inflammatory responses by inactivating the thioredoxin-interacting protein (Txnip)/NLRP3 inflammasome. Furthermore, Daph efficiently enhanced Nrf2 nuclear translocation and Trx-1 expression. However, these effects in WT mice were eliminated in Nrf2-/- mice. CONCLUSIONS: These investigations demonstrated that Daph treatment has protective potential against oxidative stress-driven hepatotoxicity by inhibition of ASK1/JNK and Txnip/NLRP3 activation, which may be strongly related to the Nrf2/Trx-1 upregulation.

Effect of Bugu granules in a drug-containing serum on chondrocyte apoptosis and the Trx2 signaling pathway.

Traditional Chinese medicine for invigorating the kidney and promoting blood circulation is commonly prescribed for the treatment of osteoarthritis associated with kidney deficiency and blood stasis. However, the specific mechanisms of these medicines are still unclear. The present study aimed to evaluate the protective effects of Bugu granules against sodium nitroprusside-induced chondrocyte apoptosis and elucidate the underlying molecular mechanisms. Drug-containing serum was prepared by administering rats with Bugu granules and harvesting the serum. Chondrocytes were exposed to different dilutions of serum, and apoptosis assessed by flow cytometry after staining with annexin V­FITC/PI. Flow cytometry showed that chondrocyte apoptosis increased significantly after incubation with 2 mol/L sodium nitroprusside for 24 h (t = -48.221, P = 0.000), and the apoptotic rate of chondrocytes decreased with increasing concentrations of drug-containing serum (F = 33.965, P = 0.000). Cellular levels of Trx2, ASK1, caspase­3, and reactive oxygen species (ROS) were detected by enzyme-linked immunosorbent assay. The cellular content of Trx2 increased gradually with increasing concentrations of drug-containing serum (F = 2610.593, P = 0.000), while that of ASK1 (F = 2473.545, P = 0.000), caspase­3 (F = 209.921, P = 0.000), and ROS (F = 1666.435, P = 0.000) all decreased significantly. The mRNA expression levels were analyzed by RT-qPCR, which revealed that expression levels of Trx2 and caspase­3 mRNA increased and decreased significantly, respectively, following exposure to Bugu granules in the drug-containing serum (F = 6.974, P = 0.003 and F = 3.691, P = 0.191; respectively), but the expression of ASK1 mRNA was not significantly different between treatment groups (F = 1.784, P = 0.191). Taken together, these results support the hypothesis that the Trx2 signaling pathway is activated by Bugu granules, which in turn inhibits chondrocyte apoptosis. This may play a role in preventing the development of osteoarthritis.

Regulation of hyperglycemia in diabetic mice by autolysates from ß-mannanase-treated brewer's yeast.

BACKGROUND: Diabetes mellitus is a serious chronic disease, characterized by hyperglycemia. This study administered either ß-mannanase-treated yeast cell autolysis supernatant (YCS) or yeast cell-wall residues after autolysis (YCR) to investigate their influence on the alleviation of diabetes in a diabetic mouse model. RESULTS: Application of either YCS or YCR led to body weight gain, blood glucose reduction, and an improvement in lipid composition in the diabetic mice. Administration of YCS was more effective in inhibiting oxidative stress than YCR. The expression of PPARα and CPT1α was enhanced, improving lipid biosynthesis, and Trx1 and HIF-1-α genes were downregulated due to the activation of thioredoxin following the interventions, indicating that the processes of lipid metabolism and oxidative stress were heavily involved in the reduction of diabetic characteristics following the interventions. The current study revealed that consumption of YCR also led to a reduction in hyperglycemia, this being associated with its richness in mineral elements, such as chromium and selenium. CONCLUSION: This study may highlight the potential of both YCS and YCR as functional ingredients in dietary formula for improving diabetic syndromes. © 2019 Society of Chemical Industry.

Effects of Marine Oils, Digested with Human Fluids, on Cellular Viability and Stress Protein Expression in Human Intestinal Caco-2 Cells.

In vitro digestion of marine oils has been reported to promote lipid oxidation, including the formation of reactive aldehydes (e.g., malondialdehyde (MDA) and 4-hydroxy-2-hexenal (HHE)). We aimed to investigate if human in vitro digestion of supplemental levels of oils from algae, cod liver, and krill, in addition to pure MDA and HHE, affect intestinal Caco-2 cell survival and oxidative stress. Cell viability was not significantly affected by the digests of marine oils or by pure MDA and HHE (0-90 µM). Cellular levels of HSP-70, a chaperone involved in the prevention of stress-induced protein unfolding was significantly decreased (14%, 28%, and 14% of control for algae, cod and krill oil, respectively; p ≤ 0.05). The oxidoreductase thioredoxin-1 (Trx-1) involved in reducing oxidative stress was also lower after incubation with the digested oils (26%, 53%, and 22% of control for algae, cod, and krill oil, respectively; p ≤ 0.001). The aldehydes MDA and HHE did not affect HSP-70 or Trx-1 at low levels (8.3 and 1.4 µM, respectively), whilst a mixture of MDA and HHE lowered Trx-1 at high levels (45 µM), indicating less exposure to oxidative stress. We conclude that human digests of the investigated marine oils and their content of MDA and HHE did not cause a stress response in human intestinal Caco-2 cells.

Taurine protects against As2O3-induced autophagy in pancreas of rat offsprings through Nrf2/Trx pathway.

Arsenic was increasingly to blame as a risk factor for type 2 diabetes mellitus. In our previous study, we had found iAs stimulated autophagic flux and caused autophagic cell death through ROS pathway in INS-1 cells. Since NF-E2-related factor 2 (Nrf2) and the thioredoxin (Trx) system was a crucial line of defense against ROS, we investigated whether Nrf2/Trx pathway contributed to As2O3-stimulated autophagy and the role of taurine in this study. After treatment with 2 mg/kg BW-8 mg/kg BW As2O3 for 57 d, the expression of Nrf2 protein was decreased significantly in offsprings' pancreas. The expression of Trx gene was decreased significantly in pancreas subsequently. Finally, the generation of reactive oxygen species stimulated autophagy in arsenic-treated pancreas. Taurine could reverse arsenic-inhibited Nrf2 and Trx and inhibit autophagy. In short, inhibition of Nrf2/Trx pathway might play an important role in the pathogenesis of arsenic-related diabetes. Taurine could serve as nutrition supplementation against arsenic-related diabetes in high arsenic exposure area.

Protective Effect of Astragaloside IV Against Paraquat-Induced Lung Injury in Mice by Suppressing Rho Signaling.

The purpose of the present study was to evaluate the protective effects of astragaloside IV (AS IV) against paraquat (PQ)-induced pulmonary injury in vivo. Fifty BALB/C mice were randomized into five groups: (1) control, (2) PQ, (3) PQ + dexamethasone (Dex, 5 mg/kg), (4) PQ + AS IV (50 mg/kg), and (5) PQ + AS IV (100 mg/kg). A single dose of PQ (50 mg/kg, i.p.) was intraperitoneally given to induced acute lung injury. Then, mice were treated with AS IV (50 and 100 mg/kg/day, orally) for 5 days. At the end of the experiment, animals were euthanized; then, the bronchoalveolar lavage fluid (BALF) and lung tissues were collected for histological observation, biochemical assay, and Western blot analysis. Malondialdehyde (MDA), myeloperoxidase (MPO), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) in lung tissues, and interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α) levels in BALF were determined. Histological examination indicated that AS IV attenuated lung damage caused by PQ. Biochemical results showed that AS IV treatment significantly reduced the levels of MDA, MPO, and inflammatory cytokines while increased the levels of SOD, CAT, and GSH-Px compared with those in PQ group. Western blot results revealed that AS IV attenuated the Txnip/Trx expressions and inhibited Rho/ROCK/nuclear factor kappaB (NF-κB) signaling pathway in PQ-challenged mice. These findings suggested the protective effect of AS IV as a natural product on PQ-induced pulmonary injury.

Intake of dietary antioxidants is inversely associated with biomarkers of oxidative stress among men with prostate cancer.

Prostate cancer is the most common non-cutaneous cancer and the second leading cause of cancer-related mortality among men in the USA. Growing evidence suggests that oxidative stress is involved in the development and progression of prostate cancer. In this study, the association between antioxidants from diet and supplements and biomarkers of oxidative stress in blood (n 278), urine (n 298) and prostate tissue (n 55) were determined among men from the North Carolina-Louisiana Prostate Cancer Project. The association between antioxidant intake and oxidative stress biomarkers in blood and urine was determined using linear regression, adjusting for age, race, prostate cancer aggressiveness and smoking status. Greater antioxidant intake was found to be associated with lower urinary 8-isoprostane concentrations, with a 10% increase in antioxidant intake corresponding to an unadjusted 1·1% decrease in urinary 8-isoprostane levels (95% CI -1·7, -0·3%; P value<0·01) and an adjusted 0·6% decrease (95% CI -1·4, 0·2%; P value=0·16). In benign prostate tissue, thioredoxin 1 was inversely associated with antioxidant intake (P=0·02). No significant associations were found for other blood or urinary biomarkers or for malignant prostate tissue. These results indicate that antioxidant intake may be associated with less oxidative stress among men diagnosed with prostate cancer.

A specific and rapid colorimetric method to monitor the activity of methionine sulfoxide reductase A.

Considerable evidence indicates that methionine sulfoxide (MetO) reductase A (MsrA) plays an important role in cytoprotection against oxidative stress and serves as a potential drug target. To screen for MsrA regulators, a rapid and specific assay to monitor MsrA activity is required. Most of current assays for MsrA activity are based on the reduction of radioactive substrates such as [3H]-N-acetyl-MetO or fluorescent derivatives such as dimethylaminoazo-benzenesulfonyl-MetO. However, these assays require extraction procedures and special instruments. Here, we developed a specific colorimetric microplate assay for testing MsrA activity quickly, which was based on the fact that MsrA can catalyze the reduction of methyl sulfoxides and simultaneously oxidize dithiothreitol (DTT), whose color can be produced by reacting with Ellman's reagent (dithio-bis-nitrobenzoic acid, DTNB). The corresponding absorbance change at 412nm was recorded with a microplate reader as the reaction proceeded. This method to monitor MsrA activity is easy to handle. Our findings may serve as a rapid method for the characterization of recombinant enzyme and for the screening of enzyme inhibitors, pharmacological activators, gene expression regulators and novel substrates.

Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 µg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.

Systematic exploration of thioredoxin target proteins in plant mitochondria.

The thioredoxin (Trx) system is known to play a pivotal role in cellular redox regulation, but its target proteins in plant mitochondria remain largely uncharacterized. In this study, we systemically screened Trx target candidates in plant mitochondria. Mitochondrial protein extracts were prepared from Arabidopsis shoots, spinach leaves and potato tubers, and then subfractionated into soluble matrix and insoluble membrane fractions. Protein extracts were loaded onto an affinity column immobilizing Arabidopsis mitochondria-localized o-type Trx mutant protein, in which one of two internal cysteines at the active site was substituted by serine. Proteins forming mixed-disulfide intermediates with the mutated Trx were identified by proteomic approaches. This procedure allowed the determination of 101 Trx target candidate proteins involved in a broad spectrum of mitochondrial processes. Furthermore, biochemical assay revealed that one of the potential Trx target proteins, alternative oxidase, is actually redox regulated by Trx. This study provides insights into the regulatory mechanism of diverse functions in mitochondrial biology that are mediated through the Trx system.