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Objective:To investigate the changes of ROS/TXNIP/NLRP3 signaling pathway in pyroptosis of human embryonic trophoblast cells induced by high glucose.Methods:Human embryonic trophoblast cells were cultured in vitro to establish high glucose injury model, and they were randomly divided into control group, high glucose (HG) group and HG + ROS inhibitor N-acetyl-L-cysteine (HG + NAC) group. MTT assay was used to detect the cell survival rate. The level of ROS in each group was detected by dihydroethidine ROS fluorescence probe. Expression of TXNIP and NLRP3 mRNA was detected by real-time quantitative PCR (RT-qPCR). Western blot analysis was used to detect the expression levels of TXNIP, NLRP3, Caspase-1, interleukin (IL) -1β, tumor necrosis factor-α (TNF-α) and GSDMD proteins. In addition, pyroptosis was detected by flow cytometry.Results:The optimal glucose concentration for high glucose-induced injury of human embryonic trophoblast cells was 30 mmol/L. Compared with the control group (96.27±3.10) %, the survival rate of human embryonic trophoblast cells in HG group (55.44±2.15) % was significantly lower ( P<0.05), while the fluorescence intensity (ROS level) of 7 'dichlorofluorescein (DCF), the expression levels of TXNIP and NLRP3 proteins, the number of pyroptosis, expression levels of Caspase-1, GSDMD, IL-1β and TNF-α proteins were significantly higher ( P<0.05) ; Compared with HG group, the survival rate of human embryonic trophoblast cells in HG+NAC group (84.75±2.33) % was significantly higher ( P<0.05), the fluorescence intensity (ROS level) of DCF, the expression levels of TXNIP and NLRP3 proteins, the number of pyroptosis, and expression levels of Caspase-1, GSDMD, IL-1β and TNF-α proteins were significantly lower ( P<0.05) . Conclusion:Inhibition of ROS level in human embryonic trophoblast cells induced by high glucose may promote cell proliferation and reduce the occurrence of pyroptosis by inhibiting TXNIP/NLRP3 signaling pathway.
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@#Thioredoxin-interacting protein (TXNIP), which mainly regulates glucose homeostasis in pancreatic β cells, is a novel target in the treatment of diabetes.In this study, 4-hydroxybenzopyrimidine was used as the raw material, four nitrogen-containing rings (imidazole, methylpiperazine, pyrazole, morpholine) were introduced, benzopyrimidine skeleton with nitrogen-containing rings derivatives targeting TXNIP was designed and synthesized, and the protective effect of compounds on palmitic acid-stimulated islet β cells was investigated.A total of 20 benzopyrimidine derivatives were designed and synthesized, and the structures were confirmed by 1H NMR and ESI-MS.Pharmacological studies showed that most of the compounds exhibited protective effects on islet β cells, with better axtivity for compounds C-1, C-2, C-4 and D-2 (cell survival rate > 70%) compared with PA model group (38.3%), Among the four compounds, D-2 had the highest activity of 87.2%, so it could become a potential new anti-diabetic chemical entity.
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Thioredoxin peroxidase (TPx) belongs to the superfamily of peroxiredoxins, which is widely expressed in various growth and development stages of parasites and their excretory secretions. On the one hand, recombinant TPx protein can participate in host immunoregulation; on the other hand, recombinant TPx protein has high sensitivity and specificity as a diagnostic antigen, and can be used for immunodiagnosis of parasitic diseases; in addition, it can also be used as a candidate vaccine molecule for the immunoprophylaxis of parasitic diseases. This paper reviews the research progress on host immunoregulation, immunodiagnosis and immunoprophylaxis by recombinant TPx protein of important human parasites.
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Objective Drug resistance is the main cause of chemotherapy failure in hepatocellular carcinoma (HCC), and thioredoxin reductase 1 (TXNRD1), as a major influencing factor for reactive oxygen species (ROS) metabolism, has been proven to be associated with the poor prognosis of patients with HCC. This study aims to explore the role of TXNRD1 in the mechanism of multidrug resistance in HCC. Methods BEL/FU cells in BEL-7402 cell line were selected as the multidrug-resistant cell line. The siRNA was used for the intervention of TXNRD1 expression; quantitative real-time PCR and Western blotting were used to measure the expression of TXNRD1; CCK-8 assay and flow cytometry were used to evaluate the effect of TXNRD1 on hepatocyte ROS accumulation, resistance to 5-fluorouracil (5-Fu) and doxorubicin (DOX), and apoptosis in vitro; a xenograft tumor model was established to investigate the effect of auranofin (AUR) on drug resistance in vivo. The two-independent-samples t test was used for comparison of continuous data between two groups. Results As a multidrug-resistant HCC cell line, BEL/Fu showed high mRNA and protein expression levels of TXNRD1 (both P < 0.05). Compared with 5-Fu or DOX treatment alone, the TXNRD1 inhibitor AUR combined with 5-Fu or DOX had had a significant reduction in the number of colony formation ( P < 0.01) and a significant increase in apoptosis ratio ( P < 0.001). The ROS scavenger N-acetylcysteine (NAC) significantly weakened the effect of TXNRD1 knockdown by siRNA on the drug resistance of BEL/Fu cells, and the application of NAC effectively reduced the apoptosis ratio of cells after siRNA interference ( P < 0.001). Animal experiments also confirmed that compared with the nude mice treated with 5-Fu alone, the nude mice treated with 5-Fu and AUR had a significantly lower tumor mass ( P < 0.001) and a significantly smaller tumor volume ( P < 0.001). Conclusion TXNRD1 plays an important role in the drug resistance of HCC, and inhibition of its level in cells can effectively improve drug resistance. As a TXNRD1 inhibitor, AUR has great application prospects in the multimodality therapy for HCC.
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This study was designed to obtain recombinant human thioredoxin (rhTXN) by gene cloning and prokaryotic expression, and evaluated its therapeutic effect in the mouse ulcerative colitis (UC) model induced by dextran sulfate sodium (DSS). The human thioredoxin gene TXN was cloned from the cDNA of Jurkat cells. The recombinant expression plasmid pCold TF-rhTXN was constructed by restriction enzyme digestion. After expression in E. coli BL21 (DE3), recombinant human thioredoxin was purified by a nickel column. Intact rhTXN recombinant protein was obtained after removal of the fusion partner-tag by enzyme digestion and the activity of disulfide reductase was detected by the insulin reduction method. The animal experiments in this study were performed in accordance with the ethical guidelines of the Laboratory Animal Welfare Ethical Review Committee of Nanjing University. Experiment ulcerative colitis was induced by providing mice with sterilized drinking water which contained 3% DSS. rhTXN was injected intraperitoneally. The therapeutic effect was studied by weight change, colon length and HE (hematoxylin and eosin) stained sections. In vivo imaging was used to study the targeting of rhTXN to DSS mice. The GSE107499 data set of GEO database was used to screen the hub genes at the lesional sites of UC and study the correlation with TXN. The experimental results showed that rhTXN was successfully expressed and purified with disulfide reductase activity. rhTXN (100 μg·kg-1) had a significant therapeutic effect on maintaining the weight change of mice (P = 0.000 5) and reducing intestinal injury (P < 0.000 1), and had a colon targeting effect on DSS mice. In GSE107499 data set, TXN in inflammatory sites of UC patients was significantly down regulated (P < 0.01) and negatively correlated with hub gene CD40 (P < 0.01) and positively correlated with hub gene fibronectin 1 (FN1) (P < 0.01). In this study, biologically active rhTXN was successfully prepared and proved to have a promising therapeutic effect on the DSS mouse model, and TXN gene was significantly correlated with the UC hub genes CD40 and FN1.
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Objective:To investigate the effects of thioredoxin reductase 1(TR1) overexpression on hippocampus in ovariectomized SD rats.Methods:Totally 54 female SD rats were divided into normal group, ovariectomized group and ovariectomized over-expressioned TR1 group (ovariectomy-TR1 group) according to the random number table method with 18 in each group. The overexpressed TR1 vector was constructed by lentivirus, and the recombinant lentivirus was injected into the hippocampus by a brain stereotactic instrument.The mRNA levels and protein levels of TR1, Bcl-2, p53 and p21 in the hippocampus of SD rats were detected by qRT-PCR and Western blot.The expression of Caspase-3 in the hippocampus of SD rats was detected by Western blot. The activity of SOD was measured by the WST-1 cell proliferation and cytotoxicity method, the content of GSH was measured by the microplate method, and the content of MDA in the hippocampus of SD rats was measured by the TBA method. The behavioral changes of SD rats were detected by the open field test and water maze test. GraphPad Prism 9.0 was used for statistical analysis.One-way analysis of variance was used for comparisons among the three groups, and the LSD test was used for further pairwise comparisons, the t-test was used to compare the mean number of samples between the two groups. Results:(1) The mRNA and protein levels of TR1 in hippocampus of ovariectomized rats were lower than those of normal rats ( t=3.125, 4.402, both P<0.05). The mRNA and protein levels of TR1 in hippocampus of ovariectomized-TR1 rats were higher than those of ovariectomized rats ( t=4.945, 4.845, both P<0.05). (2) There were significant differences among the three groups in the escape latency in water maze test, the movement distance and the stay time in central area in the open field test ( F=44.73, 33.67, 6.51, all P<0.05), the movement distance in the open field test of ovariectomized rats was more than that of the normal group ((4 700±141) mm, (3 967±163) mm, P<0.05), the stay time in the central area was longer than that of the normal group ((87.33±3.93) s, (80.83±2.48) s, P<0.05), the movement distance ((4 267±150) mm) and the stay time in the central area ((82.17±3.43) s) of ovariectomized-TR1 group were lower than that of ovariectomized group ( P<0.05). In the water maze test, the escape latency of ovariectomized rats was longer than that of the normal group ((28.67±2.50) s, (19.50±2.59) s, P<0.05), and the escape latency in the ovariectomy-TR1 group((25.00±1.67) s) was shorter than that of ovariectomized TR1 group ( P<0.05). (3)There were significant differences in the levels of MDA, SOD and GSH in the hippocampus oxidative stress injury indexes among the three groups ( F=87.41, 91.38, 28.69, all P<0.01). The level of MDA in hippocampus of ovariectomized group was higher than that of normal group, and that in the ovariectomized-TR1 group was lower than that of ovariectomized rats group ( P<0.05). And what's more the levels of SOD and GSH in ovariectomized group were lower than those of normal group ( P<0.05), and the ovariectomized-TR1 group was higher than that of ovariectomized group ( P<0.05). (4) The results of Western blot and RT-PCR showed that the levels of p21 and p53 in hippocampal tissue of ovariectomized group were higher than those of normal group ( P<0.05), while the level of aging-related protein p21 and p53 in ovariectomized-TR1 group were significantly lower than those in ovariectomized group ( P<0.05). The level of apoptotic protein Caspase-3 in the hippocampus of ovariectomized rats was higher than that in the normal group ( P<0.05), while the level of Caspase-3 in ovariectomized-TR1 group was significantly lower than that in ovariectomized rats ( P<0.05). The level of anti-apoptotic protein Bcl-2 in hippocampus of ovariectomized group was lower than that of normal rats ( P<0.05), while the level of Bcl-2 in ovariectomized-TR1 group was significantly higher than that in ovariectomized rats ( P<0.05). Conclusion:Overexpression of TR1 can reduce apoptosis of hippocampal cells by regulating oxidative damage and reducing cell senescence.
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OBJECTIVE@#To construct a HEK293 cell line stably overexpressing TrxR1 as a cell model for functional study of TrxR1 and screening of TrxR1-targeting drugs.@*METHODS@#TrxR1 gene was amplified by PCR and ligated with the lentivirus expression vector pLVX-Puro, which was transformed into Escherichia coli and identified by Sanger dideoxy sequencing. HEK293 cells were infected with the recombinant lentivirus vector (pLVX-Puro-TXNRD1) and screened with Puromycin for cell clones with stable TrxR1 overexpression (HEK293-TrxR1-OE cells). HEK293-TrxR1-OE cells, along with HEK293 cells infected with pLVX-Puro vector (HEK293-NC) and normal HEK293 cells, were tested for mRNA and protein expression levels of TrxR1 using RT-qPCR and Western blotting. TrxR1 enzyme activity in the cells was evaluated with insulin endpoint assay and TRFS-green probe imaging. The sensitivity of the cells to auranofin, a specific TrxR1 inhibitor, was determined with CCK8 assay.@*RESULTS@#TrxR1 gene was successfully inserted into the lentiviral vector pLVX-Puro as confirmed by DNA sequencing. The enzyme activity and mRNA and protein expression levels of TrxR1 were significantly higher in HEK293-TrxR1-OE cells than in HEK293 and HEK293-NC cells (P < 0.005). The inhibitory effects of auranofin on proliferation and cellular TrxR1 enzyme activity were significantly attenuated in HEK293-TrxR1-OE cells as compared with HEK293 and HEK293-NC cells (P < 0.005).@*CONCLUSION@#We successfully obtained a HEK293 cell line with stable TrxR1 overexpression, which shows resistance to auranofin and can be used for screening TrxR1 targeting drugs.
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Humans , Auranofin , Cell Line, Tumor , Genetic Vectors , HEK293 Cells , Lentivirus/genetics , RNA, Messenger , TransfectionABSTRACT
ObjectiveTo observe the effect of Dahuang Xiezhuo prescription on the changes in renal pathology and reactive oxygen species （ROS）/thioredoxin-interacting protein （TXNIP）/NOD-like receptor protein 3 （NLRP3） pathway expression in the kidney tissues of rats with 5/6 nephrectomy， and to explore the mechanism of Dahuang Xiezhuo prescription in protecting renal function and delaying renal interstitial fibrosis and the possibility. MethodNinety healthy male SD rats were randomly divided into a sham operation group， a model group， low， medium， and high-dose （6.825， 13.65， 27.30 g·kg-1） Dahuang Xiezhuo prescription groups， and a Niaoduqing granule group （2.60 g·kg-1）. Except the sham operation group， 5/6 nephrectomy was used to replicate the rat model of chronic renal failure （CRF）. After modeling， each administration group was given the corresponding dose of drug suspension by intragastric administration， once a day for consecutive 8 weeks. After administration， serum creatinine （SCr） and urea nitrogen （BUN） levels and 24 h urinary protein quantification （UTP） levels were detected. Western blot assay was used to detect the protein expressions of thioredoxin （TRX）， TXNIP， and NLRP3. The protein expressions of TRX， TXNIP， NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， transformation growth factor-β （TGF-β）， Collagen Ⅳ， α-smooth muscle actin （α-SMA）， and fibronectin （FN） were detected by immunohistochemistry. ResultAs compared with the sham operation group， serum levels of SCr， BUN， and UTP in the model group were increased （P<0.05）， TRX， TXNIP， NLRP3， ASC， TGF-β， Collagen Ⅳ， α-SMA， and FN proteins were increased （P<0.01）， and renal interstitial fibrosis significantly occurred. As compared with the model group， the levels of SCr， 24 h BUN， and UTP in the low， medium， and high-dose Dahuang Xiezhuo prescription groups and the Niaoduqing granule group were decreased to varying degrees （P<0.05）， TRX， TXNIP， NLRP3， ASC， TGF-β， Collagen Ⅳ， α-SMA， and FN were decreased （P<0.01）， and renal interstitial fibrosis was improved to varying degrees. ConclusionDahuang Xiezhuo prescription can protect renal function and delay renal interstitial fibrosis in rats with CRF.
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ObjectiveTo investigate the effect of Gegen Qinliantang （GGQLT）-medicated serum on free fatty acid （FFA）-induced nonalcoholic steatohepatitis （NASH） in vitro model of human hepatoma cells HepG2. MethodNASH model of HepG2 cells was established in vitro， and the cells were intervened with different volume fractions of GGQLT-medicated serum and resveratrol. Intracellular lipid deposition in each group was detected by oil red O staining， the level of reactive oxygen species （ROS） in each group were detected by flow cytometry， the levels of glutathione peroxidase （GSH-Px）， superoxide dismutase （SOD）， triglyceride （TG） and malondialdehyde （MDA） in each group were detected by kits. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure the mRNA expression levels of nuclear transcription factor （NF）E2-related factor 2 （Nrf2）， heme oxygenase-1 （HO-1）， quinone oxidoreductase 1 （NQO1）， Kelch-like epichlorohydrin-associated protein-1 （Keap1）， NF-κB， thioredoxin interacting protein （TXNIP）， interleukin-1β （IL-1β） in HepG2 cells of each group. The protein expression of Nrf2， TXNIP in cells of each group was detected by Western blot. ResultFFA induced large accumulation of intracellular lipids. Compared with the normal group， the activities of GSH-Px and SOD were significantly decreased （P<0.01） and the contents of TG， ROS and MDA were significantly increased （P<0.05， P<0.01） in the model group. Compared with the model group， all GGQLT groups and resveratrol group could elevate intracellular SOD activity to different degrees （P<0.05， P<0.01） and significantly reduce the levels of intracellular ROS and MDA （P<0.05， P<0.01）， GGQLD high- and medium-dose groups and resveratrol group significantly elevated GSH-Px activity （P<0.01）， GGQLD medium- and low-dose groups and resveratrol group significantly decreased TG content （P<0.05， P<0.01）. Compared with the model group， GGQLT high- and medium-dose groups and resveratrol group could significantly upregulate the mRNA expression levels of Nrf2， HO-1 and NQO1 （P<0.01）， all GGQLT groups and resveratrol group could significantly downregulate the TXNIP protein expression level， as well as significantly downregulate the mRNA expression levels of Keap1， NF-κB （P<0.05， P<0.01）. Nrf2-siRNA transfection of cells revealed that Nrf2 expression was significantly downregulated （P<0.01） in the Nrf2-siRNA group of cells by comparing with NC-siRNA group at the corresponding dose of drugs， and the inhibitory effects of GGQLT and resveratrol on TXNIP， IL-1β were attenuated. ConclusionFFA induces the production of ROS and inflammatory factors in HepG2 cells， and GGQLT can improve the anti-inflammatory and antioxidant capacities of cells， and its mechanism may be related to the regulation of Nrf2/TXNIP signaling pathway， so as to improve NASH.
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ObjectiveTo observe the effect of Danggui Buxuetang on podocyte pyroptosis in diabetic kidney disease （DKD） rats and to explore the possible mechanism of its prevention and treatment of DKD and podocyte pyroptosis. MethodEight of the 50 male SD rats were randomly classified into a normal group， and the remaining 42 were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 35 mg·kg-1 streptozotocin （STZ） for inducing type 2 diabetes. After successful modeling， they were randomized into the model group， low- （0.72 g·kg-1） and high-dose （1.44 g·kg-1） Danggui Buxuetang group， and irbesartan （0.017 g·kg-1） group and gavaged with the corresponding drugs， while those in the normal group and model group with an equal volume of normal saline， once per day， for 20 weeks. During the medication， the fasting blood glucose （FBG） and 24 h urine protein （24 h-UTP） were measured regularly. After administration， the pathological changes in renal tissues were observed by periodic acid-silver metheramine （PASM） staining， followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope （TEM）. Serum levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） were determined by enzyme-linked immunosorbent assay （ELISA）. The DNA damage in renal tissue cells of rats was detected by in situ nick end-labeling （TUNEL） assay. The protein expression levels of thioredoxin interacting protein （TXNIP）， cysteine-dependent aspartate-directed protease-1 （Caspase-1）， and gasdermin D （GSDMD） in renal tissues of rats were detected by immunohistochemistry （IHC）， the expression levels of nucleotide binding domain like receptor protein 3 （NLRP3） and Wilms tumor protein-1 （WT-1） in podocytes by immunofluorescent （IF） staining， and the expression levels of TXNIP/NLRP3/Caspase-1/GSDMD pathway proteins and Synaptopodin in renal podocytes by Western blot. ResultCompared with the normal group， the model group exhibited increased FBG and 24 h UTP， glomerular hypertrophy， mesangial hyperplasia， increased extracellular matrix， thickened basement membrane， K-W nodules， vacuolar degeneration in renal tubular epithelial cells， foot process fusion or loss， elevated serum IL-1β and IL-18 levels and TUNEL-positive cells in renal tissue， enhanced NLRP3 but diminished WT-1 expression in podocytes， down-regulated Synaptopodin protein expression， and up-regulated TXNIP/NLRP3/Caspase-1/GSDMD protein expression （P<0.01）. Compared with the model group， Danggui Buxuetang high-dose group remarkably lowered FBG， 24-h UTP， and TUNEL-positive cells in renal tissue， improved renal histopathology and podocyte injury and loss， down-regulated NLRP3 expression in podocytes and TXNIP/NLRP3/Caspase-1/GSDMD protein expression levels， and up-regulated WT-1 expression in podocytes and Synaptopodin protein expression （P<0.05， P<0.01）. ConclusionDanggui Buxuetang inhibits podocyte pyroptosis to reduce proteinuria and delays the development of DKD possibly by regulating the TXNIP/NLRP3/GSDMD signaling pathway.
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Objective:To study the effect of thioredoxin domain containing protein 5 (TXNDC5)-peroxiredoxin 2 (Prx2) on the drug resistance of prostate cancer cells.Methods:Prostate cancer PC3 cells were cultured in vitro, treated with the chemotherapy drug cyclophosphamide (5, 10, 15 μmol/L) for 24 hours, and PC3 cells without any treatment was served as the control group. The expression levels of TXNDC5 in PC3 cells were detected by real-time fluorescent quantitative PCR (RT-qPCR) and Western blotting. PC3 cells with TXNDC5 knocking down were exposed by cyclophosphamide and CCK-8 was used to detect the cell viability of siTXNDC5 group and siNC group. The content of reactive oxygen free radicals was determined by reactive oxygen detection kit. PC3 cells and its parental cyclophosphamide-resistant ones with TXNDC5 knocking down were treated by 10 μmol/L cyclophosphamide and subjected for CCK8 assay. The expression of Prx2 in PC3 cells was detected by Western blotting after TXNDC5 was silenced. Prx2 expression was silenced in PC3 cells overexpressing TXNDC5, and cell viability and reactive oxygen free radical content were detected in Vec-Ctrl group, pcTXNDC5 group, siNC group, siPrx2 group and pcTXNDC5+ siPrx2 group. Results:Compared with the control group, cyclophosphamide treatment significantly increased the expression of TXNDC5 at mRNA and protein levels in PC3 cells. After PC3 cells were treated with cyclophosphamide (10, 15 μmol/L) for 12 h, compared with the siNC group, the cell viability in the siTXNDC5 group was significantly suppressed (0.44±0.08 vs. 0.74±0.10, t=3.647, P=0.031; 0.30±0.04 vs. 0.53±0.06, t=6.115, P=0.006). When PC3 cells were treated with 10 μmol/L cyclophosphamide for 6 and 12 h, compared with the siNC group, the production of reactive oxygen free radicals in the siTXNDC5 group was significantly increased (2.68±0.19 vs. 1.58±0.26, t=-6.027, P=0.005; 4.56±0.37 vs. 2.73±0.26, t=-6.995, P=0.003). When PC3 cells and its cyclophosphamide-resistant ones were treated with 10 μmol/L cyclophosphamide for 12 h, compared with the siNC group, the cell viability was significantly inhibited in the siTXNDC5 group. Western blotting analysis showed that the expression of Prx2 was significantly reduced when TXNDC5 was silenced. Silencing Prx2 could significantly attenuate the increase of cell viability and the decrease of reactive oxygen content resulting from TXNDC5 overexpression. PC3 cells were treated with 10 μmol/L cyclophosphamide for 12 h, and the cell viabilities of the Vec-Ctrl group, pcTXNDC5 group, siNC group, siPrx2 group and pcTXNDC5+ siPrx2 group were 0.52±0.07, 0.69±0.03, 0.56±0.05, 0.43±0.05, 0.58±0.07, respectively, and there was a statistically significant difference ( F=8.868, P=0.003). Furthermore, the cell viability in the pcTXNDC5+ siPrx2 group decreased significantly when compared to that of the pcTXNDC5 group ( P=0.045). The contents of reactive oxygen free radicals in the above 5 groups were 3.26±0.46, 2.09±0.49, 3.16±0.38, 4.62±0.26, 2.87±0.36, respectively, and there was a statistically significant difference ( F=16.037, P<0.001). The content of reactive oxygen radicals in the pcTXNDC5+ siPrx2 group was higher than that of the pcTXNDC5 group ( P=0.036). Conclusion:TXNDC5 can reduce the level of reactive oxygen free radicals in prostate cancer cells by regulating the expression of Prx2, so as to promote the drug resistance of prostate cancer cells.
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The thioredoxin system is composed of thioredoxin (Trx), thioredoxin reductase (TR) and reduced nicotinamide adenine dinucleotide phosphate. Trx is an important antioxidant molecule that can resist cell death caused by various stresses and plays a prominent role in redox reactions. TR is a protein containing selenium (selenocysteine), mainly in three forms, i.e. TR1, TR2 and TR3. TR1 mainly distributed in the cytoplasm, TR2 mainly distributed in the mitochondria, and TR3 mainly distributed in the testes. TR can regulate cell growth and apoptosis. After the cell becomes cancerous, the expression of TR increases to promote cell growth and metastasis. Trx system is closely related to neurodegenerative diseases, parasitic infections, acquired immunodeficiency syndrome, rheumatoid arthritis, hypertension, myocarditis and so on. The Trx system can remove the reactive oxygen species (ROS) in the body, keep the inside and outside of the cell in a balanced state, and it interacts with the thioredoxin interacting protein (TXNIP), which plays an important role in the regulation of glucose metabolism and tumor treatment. The Trx system is an important target for drug treatment of many diseases. In this paper, the research progress of the thioredoxin system was reviewed.
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Objective:To study the protective effect of essential oil from Alpiniae Zerumbet Fructus （EOAZF） against high glucose （HG）-induced injury of human umbilical vein endothelial cells （HUVECs） <italic>in vitro</italic>， so as to provide experimental evidence for the treatment of diabetes-induced cardiovascular diseases with EOAZF. Method:The cells were divided into the normal group， model group （25 mmol·L<sup>-1</sup> glucose）， positive control group （100 mg·L<sup>-1</sup> vitamin C）， and the low- （0.25 μg·L<sup>-1</sup>）， medium- （1 μg·L<sup>-1</sup>）， and high-dose （4 μg·L<sup>-1</sup>） EOAZF groups. The HUVECs were damaged by HG. The secretion amounts of malondialdehyde （MDA）， nitric oxide （NO）， and endothelin-1 （ET-1） in HUVECs of different groups were measured to assess the protective effect of EOAZF against HG-induced injury. The effects of EOAZF on the apoptosis and reactive oxygen species （ROS） generation of HUVECs damaged by HG were detected by Annexin V-fluorescein isothiocyanate/propidium iodide （Annexin V-FITC/PI） staining and dichloro-dihydro-fluorescein diacetate （DCFH-DA） assay. The protein and mRNA expression levels of thioredoxin interacting protein （TXNIP） and thioredoxin 1 （Trx-1） were determined by Western blot and Real-time polymerase chain reaction （Real-time PCR）， followed by the measurement of total intracellular Trx-1 activity with insulin disulfide reduction method. Result:The comparison with the control group revealed that the proliferation of HUVECs in the model group was significantly inhibited and their shape was damaged. Compared with the model group， EOAZF protected HUVECs against HG-induced injury in a concentration-dependent manner. The secretion amounts of MDA and ET-1 （<italic>P</italic><0.05） in the model group were increased in contrast to those in the control group， while the NO level was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at all the three concentrations， especially at 4 μg·L<sup>-1</sup>， obviously reduced the secretion of MDA and ET-1 （<italic>P</italic><0.05）， but elevated NO after HG induction （<italic>P</italic><0.05）. The cell apoptosis assay and ROS detection results demonstrated that the apoptosis and ROS level in the model group were higher than those in the control group （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L<sup>-1 </sup>significantly lowered the ROS level and apoptosis （<italic>P</italic><0.05） of HUVECs damaged by HG. The Western blot assay and Trx-1 activity detection uncovered that the protein and mRNA expression levels of TXNIP in the model group were significantly up-regulated as compared with those in the control group （<italic>P</italic><0.05）， whereas the Trx-1 activity was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L<sup>-1 </sup>significantly down-regulated the mRNA and protein （<italic>P</italic><0.05） expression levels of TXNIP and enhanced the total Trx-1 activity （<italic>P</italic><0.05） in HUVECs， thus suppressing the oxidative stress. Conclusion:EOAZF exerts the protective effects against HG-induced injury in HUVECs by improving the endothelial function and reducing intracellular ROS and apoptosis. Its efficacy in anti-oxidative stress may be related to the down-regulation of mRNA and protein expression levels of TXNIP and the enhancement of Trx-1 activity.
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Thioredoxin reductase (TrxR) is one class of the most important antioxidant selenoproteins and is involved in regulating tumor genesis and progression. It has been reported that naphthoquinones can target and inhibit TrxR1 activity therefore produce reactive oxygen species (ROS) mediated by TrxR1, resulting into cellular redox imbalance and making the naphthoquinone compounds to become potential antitumor chemotherapy drugs. The purpose of this work is to explore the interaction between TrxR1 and menadione using biochemical and mass-spectrometric (MS) analyses, to further reveal the detailed mechanisms of TrxR1-mediated naphthoquinone reduction and inhibition of TrxR1 by naphthoquinone compounds. Using the site-directed mutagenesis and recombinantly expressed TrxR1 variants, we measured the steady-state kinetic parameters of menadione reduction mediated by TrxR1 and its variants, performed the inhibition analysis of menadione on TrxR1 activity, and eventually identified the interaction between menadione and TrxR1 through MS analysis. We found that Sec-to-Cys mutation at residue of 498 significantly enhanced the efficiency of TrxR1-mediated menadione reduction, though the Sec⁴⁹⁸ is capable to catalyze the menadione reduction, indicating that TrxR1-mediated menadione reduction is dominantly in a Se-independent manner. Mutation experiments showed that Cys⁴⁹⁸ is mainly responsible for menadione catalysis in comparison to Cys⁴⁹⁷, while the N-terminal Cys⁶⁴ is slightly stronger than Cys⁵⁹ regarding the menadione reduction. LC-MS results detected that TrxR1 was arylated with one molecule of menadione, suggesting that menadione irreversibly modified the hyper-reactive Sec residue at the C-terminus of selenoprotein TrxR1. This study revealed that TrxR1 catalyzes the reduction of menadione in a Se-independent manner meanwhile its activity is irreversibly inhibited by menadione. Hereby it will be useful for the research and development of naphthoquinone anticancer drugs targeting TrxR1.
Subject(s)
Catalysis , Drug Development , Oxidation-Reduction , Thioredoxin Reductase 1/metabolism , Vitamin K 3/metabolismABSTRACT
Objective To find the target of auranofin with the antibacterial activity against gramnegative bacteria and to investigate the effect of the combination of auranofin and vorinostat on the antibacterial activity against gram-negative bacteria.Methods The strains of E.coli lacking thioredoxin reductase (TrxR)was used to find the target gene.The potential synergies of the combination of auranofin and vorinostat for E.coli strain,A.baumannii strain,P.aeruginosa strain,K.pneumonia strain and muhidrug-resistant (MDR)A.baumannii strain were evaluated using susceptibility tests,micro-dilution checkerboard tests and time-kill studies.The genes related to Trx (trxA,trxB,trxC) and the gene expressed glutathione (gor) of E.coli BW25113 strains (WT) were separately knocked out to observe the effect of auranofin on minimum inhibitory concentration (MIC) and the time-kill kinetics of △trxC and △gor.Furthermore,the complemented strains (C-trxA,C-trxB,C-trxC,C-gor) were used to verify and define the genetic targets.Results According to the results of susceptibility tests,MICs of auranofin were 64 mg/L for E.coli strain BW25113 and K.pneumonia strain ATCC 43816,128 mg/L for P.aeruginosa strain PA14 and 32 mg/L for both A.baumannii strain ATCC 17978 and A.baumannii strain AB5075.However,MICs of vorinostat are 512 mg/L for all isolates.The fractional inhibitory concentration indexes (FICIs) of the combination of auranofin and vorinostat for E.coli strain BW25113,A.baumannii strain ATCC 17978,MDR A.baumannii strain AB5075,K.pneumonia starin ATCC 43816 and P.aeruginosa strain PAl4 were 0.313,0.375,0.375,0.375,and 0.375,respectively,with all values ＜ 0.5,which showed synergy.In susceptibility tests of knockout strains,MICs of auranofin for △trxC increased from 64 mg/L to 256 mg/L,decreased to 16 mg/L for △gor,and no changes for △trxA and △trxB.Auranofin showed the same antibacterial activities against the complemented strains (C-trxC,C-gor) and E.col BW25113,which decreased by about 1.8 lg colong formins units (CFU)/mL of bacterial counts.However,the antibacterial activity of auranofin was significantly reduced for △trxC,and decreased by ＜ 1 lg CFU/mL of bacterial counts.For Agor,bacterial counts decreased 4.6 lg CFU/mL,and the antibacterial activity markedly increased.Conclusions The potential target gene of auranofin against gram-negative bacteria could be trxC,which provides new ideas and methods for the clinical treatment of multidrug-resistant gram-negative bacteria.
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Objective:To explore the mechanism of modified Tianwang Buxindan on oxidative damage of Trx system in parachlorophenylalanine (PCPA) insomnia model rats. Method:Sixty male SD rats were randomly divided into blank group and model group. Insomnia model was prepared through intraperitoneal injection with PCPA (150 mg·kg-1). The discontinuous injection lasted for 7 d. After successful modeling, the rats were divided into model group (the same volume of normal saline), low, medium, high-dose Tianwang Buxindan groups (8.8, 17.6, 35.2 g·kg-1·d-1) and estazolam group (0.1mg·kg-1·d-1), with 10 in each group. Autonomous activity video was used to detect circadian activity rhythm. Transmission electron microscopy (TEM) was used to observe supra chiasmatic nucleus (SCN) morphology and Organelle integrity. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expressions of superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) in serum. Expressions of Trx2, TrxR2 in SCN cells were detected by immunofluorescence (IF) and Western blot. Result:Compared with blank group, the activity rhythm of model group was irregular, the activity time increased (PPPPPPPPConclusion:The effect of modified Tianwang Buxindan on insomnia model rats is related to the regulation of Trx2 and TrxR2 protein expressions in Trx system.
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Objective@#Ischemia-reperfusion (IR) injury is a leading cause of flap compromise and organ dysfunction during free-tissue transfer, and remains a great challenge for plastic surgeons. Thioredoxin-1 (Trx-1) was proved to protect the IR flap by mitigating the oxidative stress, and inhibiting the activation of apoptosis signal-regulating kinase-1 (ASK-1) and mitogen-activated protein kinase (MAPK) pathway. The aim of this study is to investigate the distinction of Trx-1 expression, apoptosis indices in different layers of IR flaps, and the feasibility of tissue-layer-specific administration of Trx-1.@*Methods@#Ten patients′ specimens of IR flaps for DIEP breast reconstruction were collected and assessed for apoptosis and Trx-1 expression. Twenty mice were used to establish the IR flap model. The mice were sacrificed twenty-four hours after reperfusion. The flap tissues were harvested and tested by immunohistochemistry staining and TUNEL assay. The tissue-layer-specific dermoprotective effect of Trx-1 and the molecular mechanisms were assessed by an in vitro epithelial skin cell hypoxia-reoxygenation model. The statistics were conducted by t test and ANOVA using SPSS 20.0.@*Results@#Trx-1 expression and apoptotic cells were observed mainly located in the basal layer of epidermis and the papillary layer of dermis in human IR flaps and mice models. Trx-1 depletion was 24.19 %± 2.23% in the basal layer of epidermis and the papillary layer of dermis of patient IR flaps, decreasing significantly compared with 70.71% ± 6.38% in control group (t = 27.54, P< 0.001). Similar tissue-layer-specific down regulation of Trx-1 also displayed in mice IR flap models (19.83% ± 2.34% vs. 76.59% ± 4.88%; t = 34.71, P<0.001). The apoptotic index in human samples significantly increased from 1.32% ± 1.52% in control group to 43.71 %± 3.17% in IR group (t =38.23, P<0.001); while it was proved to be dramatically raised in mice models from 0.86% ± 1.15% in control group to 41.14 %± 4.21% in IR group (t= 36.96, P < 0.001). Western Blot analysis revealed Trx-1 down regulation and a significant increase in ASK-1, p-p38, and c-PARP abundance in the hypoxia-reoxygenation-treated HaCaT cells (P < 0.01). Supplementation of recombinant human Trx-1 significantly reduced the apoptosis-related protein expression.@*Conclusions@#The basal layer of epidermis and the papillary layer of dermis are the main damaged tissue layers in the early stage of skin flap ischemia-reperfusion injury. The IR flap can be protected by precisely replenishing the vulnerable layers with Trx-1.
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Objective@#To investigate the diagnostic and predictable value of the levels of serum Trx (S-Trx), urinary Trx (U-Trx) and S-Trx/U-Trx ratio in acute pyelonephritis of children.@*Methods@#A total of 120 children with urinary tract infection were divided into APN group (67 cases) and non-APN group (53 cases).In addition, 67 children with APN were assigned to severe group (23 cases)and non-severe group (44 cases). The leves of serum C-reactive protein(CRP), precalcitonin(PCT), cystatin C (CysC), Trx (S-Trx) and urinary β2-microglobulin(β2-MG), neutrophill gelatinase-related apolipoprotein (NGAL), Trx (U-Trx) were collected. Besides, the ratio of S-Trx/U-Trx was also counted. The diagnostic and predictable value of each index were determined by receiver operating characteristic (ROC).@*Results@#Serum PCT, S-Trx, urinary β2-MG, NGAL, U-Trx was markedly increased and S-Trx/U-Trx ratio was obviously decreased in the APN group compared to that of the non-APN group (P<0.05). Moreover, S-Trx, urinary NGAL, U-Trx was significantly increased and S-Trx/U-Trx ratio was significantly decreased in the severe group compared to that of the non-severe group (P<0.05). Both in APN group or severe APN group, S-Trx and U-Trx displayed significantly positive correlation (P<0.05). The results from ROC demonstrated that AUC were 0.960, and optimal cut-off value were 16.17 μg/L of U-Trx diagnostic APN in children, with a specificity and sensitivity of 94.34% and 92.54%, respectively. The optimal cut-off value was 1.29.AUC of S-Trx/U-Trx 0.960 predicted the severe of APN in children, with a specificity and sensitivity of 81.82% and 82.61%, respectively.@*Conclusions@#U-Trx is useful for diagnostic APN in children, S-Trx/U-Trx is an independent predictor of the severe APN.
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Objective@#To analyze the changes of protein expression in aqueous humor of patients with primary open angle glaucoma (POAG), and to explore the biological markers and potential therapeutic targets associated with disease occurrence.@*Methods@#A retrospective case-control study was adopted.Ten patients with age-related cataract and 10 patients with POAG combined with cataract.were collected at the Tianjin Medical University Eye Hospital from October 2016 to December 2017.In the process of surgery, 100 μl of aqueous humor were collected with 1 ml syringe and No.1 needle through the surgical access.Those proteins extracted from aqueous humor were analyzed by quantitative proteomic mass spectrometry.The differential significance test was performed by Maxquant significances A. The differential proteins of the two groups were screened and determined with the conditions of P<0.05 and difference multiple>2.The biological big data was annotated by the function of GO and KEGG.The study was approved by the Ethics Committee of the Tianjin Medical University Eye Hospital (No.2013KY[L]-10). Patients and their guardians all signed the informed consent.@*Results@#Ninty-seven differential proteins were detected in this proteomic analysis, including 48 up-regulated proteins and 49 down-regulated proteins.GO analysis significantly differs in protein function involved in many aspects, some different proteins including lipopolysaccharide-binding protein (LBP), cluster of Differentiation 163(CD163), C-reactive protein (CRP), annexin A1 (ANXA1) were involved in inflammation; redox-related proteins were glutathione S-transferase P (GSTP1), thioredoxin (TXN), some different proteins related with cell adhesion movement were cartilage oligomeric matrix protein (COMP), desmocollin-2 (DSC2) and laminin subunit beta-2 (LAMB2); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1), solid protein (tenascin, TNC) are associated with fibrosis; some different proteins related to nerve growth were reelin (RELN), semaphorin-3F(SEMA3F), semaphorin-4B(SEMA4B). Metabolism-related proteins included pyruvate kinase (PKM), carb oxypeptidase N subunit 2 (CPN2) and so on.KEGG analysis indicated that the expression of NrF2/ERK signaling pathway and TGF-β/NF-κB signaling pathway were different between the two groups.@*Conclusions@#The expression of GSTP1 and TXN in the aqueous humor of POAG is significantly decreased, which may regulate cell adhesion and activity and expression of extracellular matrix by regulating NrF2/ERK1/2 and TGF-β/NF-κB signaling pathways.GSTP1 and TXN may serve as a potential biomarker and therapeutic target of POAG.
ABSTRACT
Objective To analyze the changes of protein expression in aqueous humor of patients with primary open angle glaucoma ( POAG ) , and to explore the biological markers and potential therapeutic targets associated with disease occurrence. Methods A retrospective case-control study was adopted. Ten patients with age-related cataract and 10 patients with POAG combined with cataract. were collected at the Tianjin Medical University Eye Hospital from October 2016 to December 2017. In the process of surgery,100 μl of aqueous humor were collected with 1 ml syringe and No. 1 needle through the surgical access. Those proteins extracted from aqueous humor were analyzed by quantitative proteomic mass spectrometry. The differential significance test was performed by Maxquant significances A. The differential proteins of the two groups were screened and determined with the conditions of P<0. 05 and difference multiple>2. The biological big data was annotated by the function of GO and KEGG. The study was approved by the Ethics Committee of the Tianjin Medical University Eye Hospital (No. 2013KY[L]-10). Patients and their guardians all signed the informed consent. Results Ninty-seven differential proteins were detected in this proteomic analysis,including 48 up-regulated proteins and 49 down-regulated proteins. GO analysis significantly differs in protein function involved in many aspects,some different proteins including lipopolysaccharide-binding protein (LBP),cluster of Differentiation 163(CD163),C-reactive protein (CRP),annexin A1 (ANXA1) were involved in inflammation;redox-related proteins were glutathione S-transferase P (GSTP1),thioredoxin (TXN), some different proteins related with cell adhesion movement were cartilage oligomeric matrix protein ( COMP ) , desmocollin-2 ( DSC2 ) and laminin subunit beta-2 ( LAMB2 );procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1),solid protein (tenascin,TNC) are associated with fibrosis;some different proteins related to nerve growth were reelin (RELN),semaphorin-3F(SEMA3F),semaphorin-4B(SEMA4B). Metabolism-related proteins included pyruvate kinase ( PKM ) , carb oxypeptidase N subunit 2 ( CPN2 ) and so on. KEGG analysis indicated that the expression of NrF2/ERK signaling pathway and TGF-β/NF-κB signaling pathway were different between the two groups. Conclusions The expression of GSTP1 and TXN in the aqueous humor of POAG is significantly decreased,which may regulate cell adhesion and activity and expression of extracellular matrix by regulating NrF2/ERK1/2 and TGF-β/NF-κB signaling pathways. GSTP1 and TXN may serve as a potential biomarker and therapeutic target of POAG.