Objective: To explore the mechanism of reactive oxygen species/thioredoxin-interacting protein/nucleotide-binding oligomerization domain-like receptor 3 (ROS/TXNIP/NLRP3) pathway in the skin injury of trichloroethylene (TCE) sensitized mice. Methods: In August 2020, 40 female BALB/c mice were randomly divided into control group (n=5) , solvent control group (n=5) , TCE treatment group (n=15) and TCE+(2-(2, 2, 6, 6-Tetrameyhylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (Mito TEMPO) treatment group (n=15) . The TCE sensitization model was established. Mice in the TCE treatment group and TCE+Mito TEMPO treatment group were divided into the sensitized positive group and the sensitized negative group according to the skin erythema and edema reactions on the back of the mice 24 h after the last stimulation. The mice were sacrificed 72 h after the last stimulation, the back skin of the mice was taken, and the skin lesions were observed. Immunohistochemistry (IHC) was used to detect the expression level of NLRP3, and the Western Blot was performed to detect the expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) , cysteinyl aspartate specific proteinase 1 (Caspase 1) , Interleukin-1β (IL-1β) and TXNIP proteins in the skin of the mice, the reactive oxygen species (ROS) kit was used to detect the level of intracellular ROS in the back skin tissue. Results: The sensitization rates of TCE treatment group and TCE+Mito TEMPO treatment group were 40.0% (6/15) and 33.3% (5/15) , respectively, and there was no significant difference between the two groups (P>0.05) . The back skin of the mice in the TCE sensitized positive group was thickened and infiltrated by a large number of inflammatory cells. The number of mitochondria in the epidermis cells was significantly reduced, the mitochondrial crest disappeared and vacuolar degeneration occurred. TCE+Mito TEMPO sensitized positive group had less damage, more mitochondria and relatively normal cell structure. Compared with the solvent control group and corresponding sensitized negative groups, the expression levels of NLRP3, ASC, Caspase 1, IL-1β, TXNIP proteins and the content of ROS in the TCE sensitized positive group and TCE+Mito TEMPO sensitized positive group were significantly increased (P<0.05) . Compared with TCE sensitized positive group, the expression levels of NLRP3, ASC, Caspase 1, IL-1β, TXNIP proteins and the content of ROS in the TCE+Mito TEMPO sensitized positive group were significantly decreased (P<0.05) . Conclusion: ROS/TXNIP/NLRP3 pathway was activated and then encouraged the release of IL-1β, finally aggravated the TCE-induced skin injury.
Animals ; Carrier Proteins ; Caspase 1/metabolism* ; Female ; Inflammasomes/metabolism* ; Mice ; Mice, Inbred BALB C ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Reactive Oxygen Species/metabolism* ; Solvents ; Thioredoxins/metabolism* ; Trichloroethylene/toxicity*

Animals ; Lung/metabolism* ; Rats ; Silicon Dioxide/toxicity* ; Thioredoxins/metabolism*

To investigate the role of thioredoxin interacting protein(TXNIP)/ nucleotides-binding oligomerization domain-like receptor protein(NLRP)3 inflammasome in the sciatic nerve of streptozotocin(STZ)-induced diabetic rats. The diabetic rat model was established by single intraperitoneal injection of STZ.The rats with matched sex and age were taken as normal control group.The blood glucose and body weight were monitored.The mechanical withdrawal threshold was measured by von Frey filaments at 12 weeks after the model was established.At 12 weeks,the rats were sacrificed and the sciatic nerves were separated for Luxol fast blue staining,the expressions of TXNIP,NLRP3,caspase-1,and interleukin(IL)-1β were detected by immunohistochemistry and Western blot method,and the levels of IL-1β and IL-18 in serum were measured by enzyme-linked immunosorbent assay(ELISA). The expression of TXNIP protein in the sciatic nerve of diabetic rats was 3.78±0.08,which significantly increased than that in the normal control group(0.99±0.06)(=26.980,<0.0001).Compared with the normal control group(0.97±0.05),the expression of NLRP3 protein in the diabetic group(2.44±0.16)was significantly higher(=8.885,<0.0001).The expression of cleaved caspase-1 was 4.45±0.19 in the diabetic group and 1.08±0.06 in the normal control group,and the difference was significant(=16.900,<0.0001).The expression of IL-1β protein in the diabetic group(4.50±0.16)was significantly higher than that(1.19±0.08)in the normal control group(=18.630,<0.0001).Compared with the normal control group,the levels of IL-1β [(110.50±8.80)pg/ml (17.97±3.18)pg/ml,=9.892,<0.0001] and IL-18 [(591.70±8.78)pg/ml (160.70±8.33)pg/ml,=35.620,<0.0001] in the serum of diabetic rats significantly increased. The pathogenesis of diabetic peripheral neuropathy may be related to increased expression of TXNIP,activation of NLRP3 inflammasome,and downstream inflammation,which may provide a new target for diabetic peripheral neuropathy therapy.
Animals ; Diabetes Mellitus, Experimental ; Inflammasomes ; Nucleotides ; Rats ; Sciatic Nerve ; Streptozocin ; Thioredoxins

Glutamate leads to neuronal cell damage by generating neurotoxicity during brain development. The objective of this study is to identify proteins that differently expressed by glutamate treatment in neonatal cerebral cortex. Sprague-Dawley rat pups (post-natal day 7) were intraperitoneally injected with vehicle or glutamate (10 mg/kg). Brain tissues were isolated 4 h after drug treatment and fixed for morphological study. Moreover, cerebral cortices were collected for protein study. Two-dimensional gel electrophoresis and mass spectrometry were carried out to identify specific proteins. We observed severe histopathological changes in glutamate-exposed cerebral cortex. We identified various proteins that differentially expressed by glutamate exposure. Identified proteins were thioredoxin, peroxiredoxin 5, ubiquitin carboxy-terminal hydrolase L1, proteasome subunit alpha proteins, isocitrate dehydrogenase, and heat shock protein 60. Heat shock protein 60 was increased in glutamate exposed condition. However, other proteins were decreased in glutamate-treated animals. These proteins are related to anti-oxidant, protein degradation, metabolism, signal transduction, and anti-apoptotic function. Thus, our findings can suggest that glutamate leads to neonatal cerebral cortex damage by regulation of specific proteins that mediated with various functions.
Animals ; Brain ; Cerebral Cortex ; Chaperonin 60 ; Electrophoresis, Gel, Two-Dimensional ; Glutamic Acid ; Humans ; Infant, Newborn ; Isocitrate Dehydrogenase ; Mass Spectrometry ; Metabolism ; Neurons ; Peroxiredoxins ; Proteasome Endopeptidase Complex ; Proteolysis ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Thioredoxins ; Ubiquitin Thiolesterase

Oxidized low-density lipoprotein (ox-LDL)-induced macrophage foam cell formation and apoptosis play critical roles in the pathogenesis of atherosclerosis. Thioredoxin-1 (Trx) is an antioxidant that potently protects various cells from oxidative stress-induced cell death. However, the protective effect of Trx on ox-LDL-induced macrophage foam cell formation and apoptosis has not been studied. This study aims to investigate the effect of recombinant human Trx (rhTrx) on ox-LDL-stimulated RAW264.7 macrophages and elucidate the possible mechanisms. RhTrx significantly inhibited ox-LDL-induced cholesterol accumulation and apoptosis in RAW264.7 macrophages. RhTrx also suppressed the ox-LDL-induced overproduction of lectin-like oxidized LDL receptor (LOX-1), Bax and activated caspase-3, but it increased the expression of Bcl-2. In addition, rhTrx markedly inhibited the ox-LDL-induced production of intracellular reactive oxygen species (ROS) and phosphorylation of p38 mitogen-activated protein kinases (MAPK). Furthermore, anisomycin (a p38 MAPK activator) abolished the protective effect of rhTrx on ox-LDL-stimulated RAW264.7 cells, and SB203580 (a p38 MAPK inhibitor) exerted a similar effect as rhTrx. Collectively, these findings indicate that rhTrx suppresses ox-LDL-stimulated foam cell formation and macrophage apoptosis by inhibiting ROS generation, p38 MAPK activation and LOX-1 expression. Therefore, we propose that rhTrx has therapeutic potential in the prevention and treatment of atherosclerosis.
Anisomycin ; Apoptosis* ; Atherosclerosis ; Caspase 3 ; Cell Death ; Cholesterol ; Foam Cells* ; Humans* ; Lipoproteins ; Macrophages* ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Reactive Oxygen Species ; Receptors, Oxidized LDL ; Thioredoxins*

Helicobacter pylori infection is related to the development of gastric diseases. Our previous studies showed that high thioredoxin-1 (Trx1) expression in H. pylori can promote gastric carcinogenesis. To explore the underlying molecular mechanisms, we performed an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis of stomach tissues from Mongolian gerbil infected with H. pylori expressing high and low Trx1. Differences in the profiles of the expressed proteins were analyzed by bioinformatics and verified using Western blot analysis. We found three candidate proteins, 14-3-3α/β, glutathione-S-transferase (GST), and heat shock protein 70 (HSP70), in high Trx1 tissues compared with low Trx1 tissues and concluded that cellular stress and redox activity-related proteins were involved in the pathogenesis of gastric cancer associated with H. pylori Trx1.
14-3-3 Proteins/physiology* ; Animals ; Computational Biology ; Gerbillinae ; Glutathione Transferase/physiology* ; HSP70 Heat-Shock Proteins/physiology* ; Helicobacter Infections/complications* ; Helicobacter pylori ; Oxidation-Reduction ; Stomach Neoplasms/etiology* ; Stress, Physiological ; Thioredoxins/physiology*

Peroxiredoxin-3 (Prdx3) is a mitochondrial protein of the thioredoxin family of antioxidant peroxidases and is the principal peroxidase responsible for metabolizing mitochondrial hydrogen peroxide. Recent reports have shown that mitochondrial reactive oxygen species (mROS) contribute to macrophage-mediated bactericidal activity in response to Toll-like receptors. Herein, we investigated the functional effect of Prdx3 in bactericidal activity. The mitochondrial localization of Prdx3 in HEK293T cells was confirmed by cell fractionation and confocal microscopy analyses. To investigate the functional role of Prdx3 in bactericidal activity, Prdx3-knockdown (Prdx3(KD)) THP-1 cells were generated. The mROS levels in Prdx3(KD) THP-1 cells were significantly higher than those in control THP-1 cells. Moreover, the mROS levels were markedly increased in response to lipopolysaccharide. Notably, the Salmonella enterica serovar Typhimurium infection assay revealed that the Prdx3(KD) THP-1 cells were significantly resistant to S. Typhimurium infection, as compared with control THP-1 cells. Taken together, these results indicate that Prdx3 is functionally important in bactericidal activity through the regulation of mROS.
Cell Fractionation ; Humans ; Hydrogen Peroxide ; Lipopolysaccharides ; Microscopy, Confocal ; Mitochondrial Proteins ; Peroxidase ; Peroxidases ; Reactive Oxygen Species* ; Salmonella enterica ; Serogroup ; Thioredoxins ; Toll-Like Receptors

Generation of reactive oxygen species (ROS) by diverse anti-cancer drugs or phytochemicals has been closely related with the induction of apoptosis in cancers. Also, the downregulation of ROS by these chemicals has been found to block initiation of carcinogenesis. Therefore, modulation of ROS by phytochemicals emerges as a crucial mechanism to regulate apoptosis in cancer prevention or therapy. This review summarizes the current understanding of the selected chemical compounds and related cellular components that modulate ROS during apoptotic process. Metformin, quercetin, curcumin, vitamin C, and other compounds have been shown to downregulate ROS in the cellular apoptotic process, and some of them even induce apoptosis in cancer cells. The cellular components mediating the downregulation of ROS include nuclear factor erythroid 2-related factor 2 antioxidant signaling pathway, thioredoxin, catalase, glutathione, heme oxygenase-1, and uncoupling proteins. The present review provides information on the relationship between these compounds and the cellular components in modulating ROS in apoptotic cancer cells.
Apoptosis* ; Ascorbic Acid ; Carcinogenesis ; Catalase ; Curcumin ; Down-Regulation* ; Glutathione ; Heme Oxygenase-1 ; Metformin ; Negotiating ; Phytochemicals ; Quercetin ; Reactive Oxygen Species* ; Thioredoxins

The objective of this study was to construct an improved thioredoxin fusion protein expression system, and express the cathelicidin-derived peptide, Lf-CATH2. The improved fusion vector Lf-CATH2-pET32α(-TS) was successfully constructed by firstly deleting the thrombin site and S tag from the pET-32α vector, then inserting the Lf-CATH2 plus a thrombin site instead. Afterwards, Lf-CATH2 was expressed in Escherichia coli as fusion protein. After the cleavage by thrombin, Lf-CATH2 was released and subsequently separated using affinity chromatography. The antimicrobial activity of purified Lf-CATH2 was also examined. The improved expression vector significantly increased enzyme cleavage efficiency by 37%, and Lf-CATH2 could be expressed in high yield and maintain the biological activity. This novel thioredoxin fusion protein expression system enables a quick production of high-yield bioactive cationic peptides like cathelicidins.
Cathelicidins ; biosynthesis ; Chromatography, Affinity ; Escherichia coli ; Genetic Vectors ; Recombinant Fusion Proteins ; biosynthesis ; Thioredoxins ; genetics

OBJECTIVETo explore the mechanism of Astragalus polysaccharides (APS) for improving the cardiac function of Sjogren's syndrome (SS) model rats based on Keapl-Nrf2/ARE signaling pathway.

METHODSTotally 48 male Wistar rats were randomly divided into four groups by random digit table, i.e., the blank control group,the model control group,the APS group, and the hydroxychloroquine group, 12 in each group. Except those in the blank control group, 0. 1 mL mixed antigen protein of sufficiently emulsified Freund's complete adjuvant and submandibular gland protein was injected from two feet plantar to induce SS model. The intervention was started from 19th day after inflammation induction. Equal volume of normal saline was given to rats in the blank control group (1 mL/100 g), APS was administered to those in the APS group (1 mg/100 g), and hydroxychloroquine (0.03 125 g/kg) was administered to those in the hydroxychloroquine group. All rats were intervened once per day for 30 consecutive days. Changes of rats' body mass and drinking water quantity, submandibular gland index, spleen index, histological changes of glands were observed. Changes of the heart function were monitored using invasive hemodynamics. Serum reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), total antioxidant capacity (TAC), tumor necrosis factor alpha (TNF-alpha), and interleukin-35 (IL-35)were detected using ELISA method. The pathological changes were observed using HE staining. The protein expression of ROS, reactive nitrogen species (RNS), glutathione (GSH), and thioredoxin (TRX) were observed by immunohistochemical staining. The mRNA expression of Keap1, Nrf2, and ARE was detected using real time fluorescent quantitative PCR. The protein expression levels of gamma-glutamic acid and a half long glycine synthetase (gamma-GCS) and heme oxygenase 1 (HO-1) in the myocardial tissue were determined by Western blot method. Results Compared with the blank control group, the quantity of drinking water, submandibular gland index, spleen index, heart rate (HR), cardiac index (HI), left ventricular systolic pressure (LVSP), left ventricular diastolic pressure (LVEDP), MDA, ROS, TNF-alpha, ROS protein expression, RNS protein expression, Keap 1 mRNA expression, Maf mRNA expression, Nfr2 mRNA expression, and HO-1 protein expression, and gamma-GCS protein expression significantly increased (P <0.01); body mass, +/-dp/dtmax, SOD, TAC, IL-35, GSH, and TRX significantly decreased (P <0.01) in the model group. Compared with the model group, the quantity of drinking water, submandibular gland index, spleen index, LVEDP, MDA, ROS, TNF-alpha, ROS protein expression, RNS protein expression, Keap1 mRNA expression, Maf mRNA expression, Nfr2 mRNA expression, and HO-1 protein expression, and gamma-GCS protein expression significantly decreased (P<0.05); body mass, +/-dp/dtmax, SOD, TAC, IL-35, GSH protein expression, and TRX protein expression significantly increased (P < 0.05, P <0.01) in the AR group and the hydroxychloroquine group. In the hydroxychloroquine group HR increased (P <0.05). In the AR group HR and LVSP decreased (P <0. 05, P <0. 01). Compared with the hydroxychloroquine group, HR, LVEDP, - dp/dtmax, y-GCS protein expression significantly decreased (P <0. 05, P <0. 01); SOD, TAC, GSH, TRX, HO-1 protein expression increased (P <0.01 )in the AR group. HI was positively correlated with ROS (P <0. 05). LVSP and LVEDP were positively correlated with Keap1 -Nrf2/ARE signaling pathways (P <0. 01) , and negatively correlated with TAC (P <0. 05, P <0. 01 ). +/-dp/dtmax was negatively correlated with Keap1-Nrf2/ARE signaling pathways(P <0.05), and positively correlated with TNF alpha (P <0. 05).

CONCLUSIONSDeclined heart function exists in SS rats. The mechamechanism of APS for improving the heart function might be closely correlated with activating Keap1-Nrf2/ARE signaling pathway.

Animals ; Astragalus Plant ; Blotting, Western ; Carboxylic Ester Hydrolases ; metabolism ; Heme Oxygenase-1 ; metabolism ; Hydroxychloroquine ; Male ; Malondialdehyde ; metabolism ; Myocardium ; enzymology ; NF-E2-Related Factor 2 ; metabolism ; Plant Extracts ; therapeutic use ; Polysaccharides ; metabolism ; Rats ; Rats, Wistar ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Sjogren's Syndrome ; Submandibular Gland ; Superoxide Dismutase ; metabolism ; Thioredoxins ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism