ABSTRACT
Sepsis is characterized by the extensive release of cytokines and other mediators. It results in a dysregulated immune response and can lead to organ damage and death. Curcumin has anti-inflammatory properties and immunoregulation functions in various disorders such as sepsis, cancer, rheumatoid arthritis, cardiovascular diseases, lung fibrosis, gallstone formation, and diabetes. This paper investigates the effects of curcumin on immune status and inflammatory response in mice subjected to cecal ligation and puncture (CLP). Inflammatory tissue injury was evaluated by histological observation. Magnetic microbeads were used to isolate splenic CD4+CD25+regulatory T cells (Tregs), and phenotypes were then analyzed by flow cytometry. The levels of Foxp3 were detected by Western blot and real-time PCR and cytokine levels were determined by enzyme-linked immunosorbent assay. We found that the administration of curcumin significantly alleviated inflammatory injury of the lung and kidney in septic mice. The suppressive function of Treg cells was enhanced and the plasma levels of IL-10 increased after treatment with curcumin. Furthermore, the secretion of plasma TNF-α and IL-6 was notably inhibited in septic mice treated with curcumin and administration with curcumin could improve survival after CLP. These data suggest that curcumin could be used as a potential therapeutic agent for sepsis.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Curcumin/therapeutic use , Inflammation/drug therapy , Kidney/drug effects , Lung/drug effects , Sepsis/drug therapy , T-Lymphocytes, Regulatory/immunology , Animals , Cecum/surgery , Cells, Cultured , Disease Models, Animal , Forkhead Transcription Factors/metabolism , Humans , Immunomagnetic Separation , Immunosuppression Therapy , Inflammation/immunology , Interleukin-10/metabolism , Kidney/pathology , Lung/pathology , Male , Mice , Mice, Inbred BALB C , Neutrophil Infiltration , Sepsis/immunologyABSTRACT
Paraquat (PQ), as a highly effective and nonselective herbicide, induces cell apoptosis through generation of superoxide anions which forms reactive oxygen species (ROS). Mitochondria, as regulators for cellular redox signaling, have been proved to play an important role in PQ-induced cell apoptosis. This study aimed to evaluate whether and how mitochondrial fission interacts with oxidative stress in PQ-induced apoptosis in mouse alveolar type II (AT-II) cells. Firstly, we demonstrated that PQ promoted apoptosis and release of cytochrome-c (Cyt-c). Furthermore, we showed that PQ broke down mitochondrial network, enhanced the expression of fission-related proteins, increased Drp1 mitochondrial translocation while decreased the expression of fusion-related proteins in AT-II cells. Besides, inhibiting mitochondrial fission using mdivi-1, a selective inhibitor of Drp1, markedly attenuated PQ-induced apoptosis, release of Cyt-c and the generation of ROS. These results indicate that mitochondrial fission involves in PQ-induced apoptosis. Further study demonstrated that antioxidant ascorbic acid inhibited Drp1 mitochondrial translocation, mitochondrial fission and attenuated PQ-induced apoptosis. Overall, our findings suggest that mitochondrial fission interplays with ROS in PQ-induced apoptosis in mouse AT-II cells and mitochondrial fission could serve as a potential therapeutic target in PQ poisoning.
Subject(s)
Alveolar Epithelial Cells/drug effects , Apoptosis/drug effects , Mitochondrial Dynamics/physiology , Oxidative Stress/drug effects , Paraquat/toxicity , Animals , Ascorbic Acid/pharmacology , Cell Survival , Cells, Cultured , Cytochromes c/metabolism , Death-Associated Protein Kinases/metabolism , Herbicides/toxicity , Mice , Protein Transport/drug effectsABSTRACT
Silent information regulator 2-related enzyme 1 (SIRT1), a protein deacetylase, is known to strongly protect cells against oxidative stress-induced injury. The nuclear factor E2-related factor 2 (NRF2)-antioxidant response element (ARE) antioxidant pathway plays important regulatory roles in the antioxidant therapy of paraquat (PQ) poisoning. In the present study, we investigated whether the SIRT1/NRF2/ARE signaling pathway plays an important role in lung injury induced by PQ. For this purpose, mouse type II alveolar epithelial cells (AECsII) were exposed to various concentrations of PQ. The overexpression or silencing of SIRT1 was induced by transfecting the cells with a SIRT1 overexpression vector or shRNA targeting SIRT1, respectively. The protein expression levels of SIRT1 and NRF2 were measured by western blot analysis. The superoxide dismutase (SOD) and catalase (CAT) activities, as well as the glutathione (GSH) and malondialdehyde (MDA) levels were measured using respective kits. Heme oxygenase-1 (HO-1) activity was also determined by ELISA. In addition, cell apoptosis was determined by flow cytometry. The protein stability of NRF2 was analyzed using cycloheximide and its acetylation in the cells was also determined. The following findings were obtained: i) SIRT1 overexpression markedly increased NRF2 protein expression; ii) SIRT1 promoted the transcriptional activity of NRF2 and upregulated the expression of the NRF2 downstream genes, SOD, CAT, GSH and HO-1, thus inhibiting the apoptosis of AECsII; iii) the inhibition of SIRT1 activity further induced the production of malondialdehyde (MDA), which resulted in increased oxidative damage; iv) SIRT1 promoted the stability of NRF2 by regulating the deacetylation and activation of the NRF2/ARE antioxidant pathway. The findings of this study demonstrate that the protective effects of SIRT1 are associated with the activation of the NRF2/ARE antioxidant pathway in lung injury induced by PQ poisoning.
Subject(s)
Alveolar Epithelial Cells/pathology , Lung Injury/chemically induced , Lung Injury/pathology , NF-E2-Related Factor 2/metabolism , Paraquat , Sirtuin 1/metabolism , Acetylation , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/metabolism , Animals , Cells, Cultured , Lung Injury/genetics , Lung Injury/metabolism , Mice , Mice, Inbred ICR , NF-E2-Related Factor 2/genetics , Oxidative Stress/drug effects , Sirtuin 1/genetics , Up-RegulationABSTRACT
OBJECTIVE: To explore the possible mechanism and protective effect of BMSCs (bone mesenchymal stem cells) carrying superoxide dismutase (SOD) gene on mice with paraquat-induced acute lung injury. METHODS: To establish the cell line of BMSCs bringing SOD gene, lentiviral vector bringing SOD gene was built and co-cultured with BMSCs. A total of 100 BALB/c mice were randomly divided into five groups, namely Control group, poisoning group (PQ group) , BMSCs therapy group (BMSC group) , BMSCs-Cherry therapy group (BMSC-Cherry group) , BMSCs-SOD therapy group (BMSC-SOD group) . PQ poisoning model was produced by stomach lavaged once with 1 ml of 25 mg/kg PQ solution, and the equal volume of normal saline (NS) was given to Control group mice instead of PQ. The corresponding BMSCs therapy cell lines were delivered to mice through the tail vein of mice 4h after PQ treatment.Five mice of each group were sacrificed 3 d, 7 d, 14 d and 21 days after corresponding BMSCs therapy cell lines administration, and lung tissues of mice were taken to make sections for histological analysis. The serum levels of glutathione (GSH) , malondialdehyde (MDA) , SOD, and the levels of transforming growth factor-ß (TGF-ß) and tumor necrosis factor-α (TNF-α) in lung tissue were determined. The level of SOD was assayed by Westen-blot. RESULTS: Compared with Control group, the early (3 days) levels of SOD protein in lung tissue of PQ group obviously decreased, and the late (21 days) levels of SOD obviously increased, while in therapy groups, that was higher than that in PQ group, and the BMSCs-SOD group showed most obvious (all P<0.05) . Compared with Control group, the levels of plasma GSH and SOD of PQ group and each therapy group wae significantly lower than those in Control group, while in therapy groups, those were higher than those of PQ group, and the BMSCs-SOD group showed most obvious (all P<0.05) .Compared with Control group, the level of plasma MDA, TNF-α and TGF-ß in PQ group and therapy groups were significantly higher, while in therapy groups, that was lower than that in PQ group, and the BMSCs-SOD group showed most obvious (all P<0.05) . Lung biopsy showed that, the degree of lung tissue damage in each therapy group obviously reduced. CONCLUSION: SOD is the key factor of the removal of reactive oxygen species (ROS) in cells, that can obviously inhibit the oxidative stress damage and the apoptosis induced by PQ, thus significantly increasing alveolar epithelial cell ability to fight outside harmful environment.
Subject(s)
Acute Lung Injury/therapy , Mesenchymal Stem Cell Transplantation , Paraquat/poisoning , Superoxide Dismutase/genetics , Acute Lung Injury/chemically induced , Animals , Antioxidants/metabolism , Cell Line , Glutathione/blood , Lung/pathology , Malondialdehyde/blood , Mice , Mice, Inbred BALB C , Oxidative Stress , Superoxide Dismutase/blood , Transforming Growth Factor beta/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
OBJECTIVE: To investigate the effect of resveratrol (Res) on paraquat (PQ)-induced acute lung injury (ALI) and mortality in mice and the mechanism of nuclear factor-ΚB (NF-ΚB) inflammatory pathway. METHODS: Sixty-eight healthy male ICR mice with grade SPF were enrolled, among them 20 mice were used for mortality observation (n = 10), and other 48 were used for determination of related parameters (n = 6). The mice were randomly divided into four groups: normal saline (NS) control group, Res control group, PQ group and PQ + Res group. The mice in the latter two groups were subdivided into 6, 24, 72 hours subgroups. The PQ poisoning model of mice was reproduced by one injection of 30 mg/kg PQ intraperitoneally. The mice in PQ + Res group were given 60 mg/kg Res intraperitoneally on the contralateral side after PQ injection. The mice were sacrificed at 6, 24, 72 hours after PQ poisoning, and lung tissue was harvested. The serum levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6 and IL-1ß) were determined by enzyme linked immunosorbent assay (ELISA). The pathological changes in lung tissue were observed with electron microscopy. Apoptosis cells in the lung were identified by terminal dexynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) for the estimation of apoptosis rate. The protein expression of NF-ΚB p65 was determined by Western Blot. RESULTS: Compared with PQ group, the death number of mice at 48, 72, 96 hours in PQ + Res group was slightly decreased (0 vs. 2, 2 vs. 5, 4 vs. 6) but without statistically significant difference (all P > 0.05). Under electron microscope, the lung injury in PQ group was severer than that in NS control group, and Res was found to be able to alleviate the lung injury. Compared with NS control group [(2.45±0.61)%], the apoptosis rate at 6 hours in PQ group was significantly increased [(8.42±1.48)%], and peaked at 72 hours [(21.23±3.47)%]. Res could decrease the apoptosis rate after PQ poisoning [6 hours: (5.56±1.31)% vs. (8.42±1.48)%, 24 hours: (11.14±2.07)% vs. (16.88±2.96)%, 72 hours: (13.28±2.32)% vs. (21.23±3.47)%, all P < 0.05]. The serum levels of TNF-α, IL-6, and IL-1ß, and NF-ΚB p65 in lung tissue were all markedly increased after PQ poisoning, and they were significantly decreased after Res intervention as compared with those of PQ group [TNF-α (ng/L): 2.62±0.29 vs. 4.06±0.74 at 6 hours, 3.98±0.41 vs. 6.79±0.80 at 24 hours, 5.06±0.75 vs. 11.00±0.75 at 72 hours; IL-6 (ng/L): 14.19±1.54 vs. 16.55±1.24 at 6 hours, 13.21±1.37 vs. 19.73±0.85 at 24 hours, 13.72±0.56 vs. 22.45±0.72 at 72 hours; IL-1ß (ng/L): 8.54±1.64 vs. 12.59±0.66 at 6 hours, 10.15±0.29 vs. 16.24±1.03 at 24 hours, 16.14±0.70 vs. 19.55±0.56 at 72 hours; 6-hour NF-ΚB p65: (1.34±0.07) folds vs. (1.86±0.11) folds when the expression in NS control group was represented as 1, all P < 0.05]. CONCLUSIONS: Res cannot lower the mortality in mice with PQ poisoning, but it seems to be able to attenuate PQ-induced ALI and cell apoptosis. The mechanism responsible for the latter maybe the inhibitive effect of Res on NF-ΚB p65 translocation and cytokines production.
Subject(s)
Acute Lung Injury , Animals , Apoptosis , Interleukin-1beta , Interleukin-6 , Lung , Male , Mice , Mice, Inbred ICR , NF-kappa B , Paraquat , Resveratrol , Stilbenes , Tumor Necrosis Factor-alphaABSTRACT
Acute kidney injury (AKI) is a serious complication of sepsis, which has a high mortality rate. Growth arrest-specific protein 6 (Gas6), the protein product of the growth arrest specific gene 6, has been shown to have an anti-apoptotic effect as well as pro-survival capability. Here, we investigated the effects of Gas6 on sepsis-associated AKI in mice subjected to cecal ligation and puncture (CLP). We found that the administration of rmGas6 significantly reduced serum urea nitrogen and creatinine and improved the survival of septic mice. Furthermore, the renal pathological damage induced by CLP was attenuated by rmGas6 treatment. Finally, rmGas6 reduced the renal tissue apoptotic index and the expression of Bax, while it upregulated the expression of Bcl-2. The data suggest that rmGas6 might be used as a potential therapeutic agent for sepsis-induced AKI.
Subject(s)
Acute Kidney Injury/drug therapy , Apoptosis/drug effects , Blood Urea Nitrogen , Creatinine/blood , Intercellular Signaling Peptides and Proteins/therapeutic use , Sepsis/pathology , Acute Kidney Injury/pathology , Animals , Cecum/surgery , Kidney/pathology , Male , Mice , Mice, Inbred BALB C , Proto-Oncogene Proteins c-bcl-2/biosynthesis , bcl-2-Associated X Protein/biosynthesisABSTRACT
Nephrotoxicity induced by chemicals such as paraquat (PQ) is a common clinical phenomenon; therefore, searching for drugs with renal protective effect is of a great practical significance. Our previous investigation found that cycloartenyl ferulate (CF) can antagonize the cytotoxic effect of PQ, and recent studies also revealed a variety of bioactivities of CF. However, specific molecular mechanisms underlying the protective effect of CF have not been explored yet. HPLC detection of PQ content indicated that CF reduced PQ accumulation in HK-2 cells and thereby improved cell survival. Western blot results showed that both PQ and CF did not affect the expression of ABCB1; however, while PQ suppressed the expression of ABCC1, CF upregulated ABCC1 expression and thereby reversed the inhibitory effect of PQ on ABCC1 expression. Meanwhile, HK-2 cells did not express ABCG2. When the expression of ABCC1 was knocked down with siRNA, the inhibitory effect of CF on intracellular PQ accumulation was blocked. Further flow cytometric analysis showed that while PQ significantly induced the appearance of sub-G1 apoptotic peak in cells, CF evidently inhibited apoptosis. TUNEL-DAPI double-staining also detected that PQ significantly induced the occurrence of DNA fragmentation in cells, whereas CF effectively inhibited the effect of PQ. Further results showed that ABCC1 siRNA effectively abolished the protective effect of CF on PQ-induced apoptosis. Taken together, these data demonstrated that in HK-2 cells, CF could antagonize PQ-induced toxicity with the involvement of regulatiion of ABCC1 protein expression, which provides a new strategy for treatments of nephrotoxicity.
Subject(s)
Coumaric Acids/pharmacology , Cytotoxins/antagonists & inhibitors , Epithelial Cells/drug effects , Paraquat/antagonists & inhibitors , Protective Agents/pharmacology , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/deficiency , ATP-Binding Cassette Transporters/genetics , Apoptosis/drug effects , Cell Line , Cytotoxins/toxicity , DNA Fragmentation/drug effects , Epithelial Cells/cytology , Epithelial Cells/metabolism , G1 Phase Cell Cycle Checkpoints/drug effects , Gene Expression Regulation , Humans , Kidney Tubules, Proximal/cytology , Kidney Tubules, Proximal/drug effects , Kidney Tubules, Proximal/metabolism , Neoplasm Proteins/deficiency , Neoplasm Proteins/genetics , Paraquat/toxicity , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Signal TransductionABSTRACT
OBJECTIVE: To investigate the protective effect of paraoxonase 1 (PON1) gene against liver oxidative stress injury in mice due to dichlorvos poisoning. METHODS: Experiment 1: 12 male Balb/c mice were randomly divided into three groups, with 4 mice in each group: control group, green fluorescent protein lentivirus control group (Lv-GFP group), and recombinant PON1 lentivirus group (Lv-PON1 group). 2 × 107 TU of Lv-GFP or Lv-PON1 was transfected via tail vein, while normal saline was given to those in control group. Blood was collected on 0, 1, 3, 5, 7, 9 days via fundus venous plexus for the assay of serum PON1 activity. PON1 mRNA and protein expression levels were respectively determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot on the 3rd post-lentivirus transfection day. Experiment 2: according to the random number table method, another 96 male Balb/c mice were divided into four groups of 24 mice in each control group, dichlorvos group, Lv-GFP intervention group, and Lv-PON1 intervention group. Lv-GFP or Lv-PON1 was transfected via tail vein followed by intraperitoneal injection of dichlorvos 9 mg/kg, while those in control group were given normal saline. Six mice in each group were sacrificed respectively at 6, 12, 24, 48 hours, and liver tissue was collected. PON1 mRNA and nuclear factor E2-related factor 2 (Nrf2) mRNA expression levels were determined by RT-PCR, and PON1 protein level was determined by Western Blot. The content of malondialdehyde (MDA) and glutathione (GSH) in the liver tissue were determined by chemical colorimetry. The activity of superoxide dismutase (SOD) and catalase (CAT) were measured by double antibody sandwich enzyme linked immunosorbent assay (ELISA). RESULTS: Experiment 1: after Lv-PON1 was transfected to normal mice, PON1 activity in serum gradually increased and maintained a high level on 3rd day, while that of the control group and Lv-GFP group showed a normal low level. On the 3rd post-lentivirus transfection day, PON1 mRNA and PON1 protein expressions in liver were significantly higher than those of control group and Lv-GFP group. Experiment 2: compared with control group, the mice in dichlorvos group showed significant decreases in PON1 mRNA, PON1 protein, Nrf2 mRNA as well as GSH, SOD, CAT levels at 6 hours [PON1 mRNA (gray value): 0.237 ± 0.075 vs. 0.674 ± 0.011, PON1 protein (gray value): 0.602 ± 0.086 vs. 0.998 ± 0.124, Nrf2 mRNA (gray value): 0.089 ± 0.012 vs. 0.126 ± 0.010, GSH (mg/g): 3.84 ± 0.33 vs. 5.52 ± 0.40, SOD (µg/g): 0.383 ± 0.040 vs. 0.564 ± 0.052, CAT (ng/g): 7.32 ± 1.28 vs. 12.46 ± 1.54, all P < 0.05 ], and remarkable increase in MDA content (nmol/g: 7.78 ± 0.41 vs. 2.34 ± 0.25, P < 0.05). With the extension of time, PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels gradually increased, MDA content gradually decreased, Nrf2 mRNA expression level had risen to the level of control group at 24 hours (0.133 ± 0.019 vs. 0.126 ± 0.009, P > 0.05), and it was higher than that of the control group at 48 hours ( 0.206 ± 0.028 vs. 0.124 ± 0.010, P < 0.05). Compared with that of the dichlorvos group, Lv-PON1 intervention group showed a significant increase in PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels [PON1 mRNA (gray value): 0.726 ± 0.021 vs. 0.237 ± 0.075, PON1 protein (gray value): 0.739 ± 0.050 vs. 0.602 ± 0.086, Nrf2 mRNA (gray value): 0.158 ± 0.007 vs. 0.089 ± 0.012, GSH (mg/g): 4.30 ± 0.22 vs. 3.84 ± 0.33, SOD ( µg/g): 0.454 ± 0.062 vs. 0.383 ± 0.040, CAT (ng/g): 8.98 ± 1.02 vs. 7.32 ± 1.28, all P < 0.05 ], and a decrease in MDA content (nmol/g: 6.56 ± 0.44 vs. 7.78 ± 0.41, P < 0.05). CONCLUSIONS: Regulation of PON1 gene can reduce MDA content, enhance SOD and CAT activities, increase GSH content, and it may also up-regulate Nrf2 mRNA expression to play a protective effect against oxidative stress of liver injury induced by dichlorvos poisoning.
Subject(s)
Dichlorvos/toxicity , Liver/injuries , Oxidative Stress , Animals , Aryldialkylphosphatase , Male , Malondialdehyde , Mice , Mice, Inbred BALB C , NF-E2-Related Factor 2 , RNA, Messenger , Superoxide DismutaseABSTRACT
OBJECTIVE: To investigate the effect of paraoxonase1 (PON1) overexpression on mouse diaphragmatic muscle cells injury caused by acute dichlorvos poisoning. METHODS: Mouse diaphragmatic muscle cells were cultured routinely and infected with overexpression lentivirus. Cells were divided into normal control group, DDVP group, LV-GFP + DDVP group, LV-PON1 + DDVP group. Cell viability was determined by CCK-8 assay. Flow cytometry was used to detect cell apoptosis. The mRNA and protein expression of PON1 and Nrf2 in mouse diaphragmatic muscle cells was measured by RT-PCR and Western blot. Enzyme-linked immunosorbent assay was used to determine levels of acetyl cholinesterase (AchE), heme oxygenase 1 (HO-1) and quinone oxidoreductase-1 (NQO-1) in mouse diaphragmatic muscle cells. The activity of superoxide dismutase (SOD) and catalase (CAT) as well as malondialdehyde (MDA) content in cells was measured by chemical colorimetry. RESULTS: After induced by 0, 80, 160, 320, 640 µmol/L DDVP for 24 hours, the viability of mouse diaphragmatic muscle cells was (100 ± 3.82)%, (82.13 ± 2.60)%, (53.57 ± 5.05)%, (30.77 ± 3.30)%, (14.20 ± 2.19)% respectively, changing in a concentration-dependent manner (P < 0.05). After induced by 160 µmol/L DDVP for 0, 6, 12, 24 hours, the viability of mouse diaphragmatic muscle cells was (100.17 ± 2.74)%, (76.13 ± 6.01)%, (66.53 ± 3.55)%, (53.57 ± 5.05)%, changing in a time-dependent manner (P < 0.05). The PON1 protein level in LV-PON1 group was higher than that of blank control group (0.370 ± 0.015 vs 0.232 ± 0.004, 0.197 ± 0.015 vs 0.037 ± 0.003, P < 0.05). The cell viability of LV-PON1 group is higher than that of DDVP group at different time point after induction of DDVP (P < 0.05). After induced by DDVP for 24 hours, the cell apoptosis rate and MDA content in LV-PON1 group were lower than those of DDVP group (P < 0.05). While levels of AchE, PON1 and Nrf2 protein expression, SOD and CAT, HO-1 and NQO-1 were higher than those of DDVP group (P < 0.05). CONCLUSIONS: The overexpression of PON1 could effectively alleviate AchE inhibition by DDVP and induce Nrf2 expression to exert antioxidant effect, thus protected the mouse diaphragmatic muscle cells.
Subject(s)
Diaphragm , Muscle Cells , Animals , Antioxidants , Apoptosis , Aryldialkylphosphatase , Catalase , Cells, Cultured , Dichlorvos , Heme Oxygenase-1 , Malondialdehyde , Membrane Proteins , Mice , Superoxide DismutaseABSTRACT
OBJECTIVE: To discuss the protective effect of bone marrow mesenchymal stem cell (BMSC) on lung injury induced by vibrio vulnificus sepsis and its mechanism. METHODS: BMSCs were isolated by whole bone marrow adherent culture from mouse. Male ICR mice were randomly divided into normal saline control group (NS group), normal saline + BMSC control group (NSB group), vibrio vulnificus sepsis group (VV group), vibrio vulnificus sepsis + BMSC group (VVB group) according to random number table, with 40 mice in each group. Sepsis mouse model was reproduced by injecting vibrio vulnificus (1 × 107 cfu/mL) 5 mL/kg through the left side peritoneal cavity, and caudal intravenous injection of BMSC (4 × 105 cfu/mL) 5 mL/kg for intervention after model reproduction. Ten mice in each group were sacrificed at 6, 12, 24 or 48 hours after injecting vibiro vulnificus, and their lung tissues were harvested. The lung wet/dry (W/D) ratio was calculated. The expression of nuclear factor-ΚBp65 (NF-ΚBp65) in nucleus was measured by Western Blot. The levels of tumor necrosis factor-α (TNF-α) and interleukins (IL-1ß, IL-6) in lung tissue were detected by enzyme-linked immunosorbent assay (ELISA). The pathological changes in lung tissue were observed after hematoxylin-eosin (HE) staining and uranyl acetate-lead citrate staining. RESULTS: After vibrio vulnificus injection, lung W/D ratio, the expression of NF-ΚBp65 in nucleus, and the levels of TNF-α, IL-1ß, IL-6 in the lung tissues were significantly increased in VV group compared with those in NS group at all the time points, and peaked at 12 hours. Compared with the VV group, the VVB group had significantly decreased levels of lung W/D ratio, NF-ΚBp65 expression, and the levels of TNF-α, IL-1ß, IL-6, with significant differences at all the time points [VV group vs. NS group at 12 hours: lung W/D ratio 7.22 ± 0.03 vs. 5.21 ± 0.02, NF-ΚBp65 expression (glay scale) 1.86 ± 0.74 vs. 0.75 ± 0.07, TNF-α (ng/L) 433.24 ± 3.23 vs. 106.57 ± 1.21, IL-1ß (ng/L) 35.64 ± 0.15 vs. 10.64 ± 0.48, IL-6 (ng/L) 58.84 ± 0.55 vs. 17.69 ± 1.35, all P<0.05; VVB group vs. VV group at 12 hours: lung W/D ratio 6.49 ± 0.06 vs. 7.22 ± 0.03, NF-ΚBp65 expression (A value) 1.16 ± 0.08 vs. 1.86 ± 0.74, TNF-α (ng/L) 357.22 ± 3.25 vs. 433.24 ± 3.23, IL-1ß (ng/L) 27.77 ± 0.59 vs. 35.64 ± 0.15, IL-6 (ng/L) 38.6 8 ± 1.29 vs. 58.84 ± 0.55, all P<0.05]. There were no significant differences in above indexes between NS group and NSB group. In the NS and NSB groups pathological changes were not obvious under light microscopy, in the VV group lung tissue hyperemia and edema was significant, the edema fluid, red blood cells and inflammatory cells also could be seen, and in the VVB group lung damage that mentioned above could be alleviated. In the NS and NSB groups epithelial cell structure of type I and type II was completed, and the changes were not obvious under the transmission electron microscopy. In the VV group the alveolar walls were damaged significantly, with type I epithelial cell cytoplasm swelling, bubbling and rupture, with type II epithelial cells visible cytoplasm decrease, cavitation, addiction to osmium lamellar corpuscle emptying, lysosome hyperplasia, microvilli reduction, and in the VVB group the above damage was alleviated. CONCLUSIONS: Vibrio vulnificus sepsis can cause acute lung damage and edema, and BMSC can down regulate inflammatory cytokines, reduce lung injury caused by vibrio vulnificus sepsis.
Subject(s)
Bone Marrow Cells/cytology , Lung Injury/prevention & control , Mesenchymal Stem Cells/cytology , Sepsis/complications , Vibrio Infections/complications , Vibrio vulnificus , Animals , Cytokines , Disease Models, Animal , Interleukin-1beta , Interleukin-6 , Lung Injury/etiology , Male , Mice , Mice, Inbred ICR , Random Allocation , Tumor Necrosis Factor-alphaABSTRACT
OBJECTIVE: To investigate the association between the single nucleotide polymorphisms (SNPs) in NF-E2-related factor 2-617 (NRF2-617) promoter region with the susceptibility to the risk of sepsis. METHODS: In this case-control association study, 203 healthy controls and 174 patients with sepsis in Wenzhou Han population were enrolled and genotyped by DNA direct sequencing. RESULTS: (1) The (CA+AA) genotype frequency was significantly higher in the sepsis group than in the control group (59.2% vs 46.3%, P = 0.012). (2) Compared with the general sepsis group, higher (CA+AA) genotype frequency was found in the severe sepsis group (47.5% vs 65.5%, P = 0.033) . However, no significant difference was shown in the (CA+AA) genotype frequency between the shock group and the non-shock group as well as between the death group and the non-death group (61.8% vs 57.1%, P = 0.221; 56.8% vs 66.7%, P = 0.258) . (3) The unconditional logistic regression analysis showed that the mutation of C to A at the gene promoter locus of Nrf2-617 was associated with the increased onset risk of sepsis (OR = 1.584, 95%CI 1.025-2.447, P = 0.038) and the severity of sepsis (OR = 0.453, 95%CI 0.233-0.878, P = 0.019). CONCLUSION: The mutation of C to A at the gene promoter locus of Nrf2-617 may increase the onset risk of sepsis and organ failure in sepsis patients, while not associated with the incidence of shock and the prognosis of sepsis.
Subject(s)
NF-E2 Transcription Factor, p45 Subunit/genetics , Polymorphism, Single Nucleotide , Sepsis/genetics , Arthropod Proteins , Asian People , Case-Control Studies , Enzyme Precursors , Genetic Predisposition to Disease , Genotype , Humans , Mutation , Polymorphism, Genetic , Prognosis , Promoter Regions, Genetic , Serine EndopeptidasesABSTRACT
OBJECTIVE: To investigate the effect of curcumin on liver injury in rats induced by paraquat-mediated oxidative stress and the mechanism underlying its effect. METHODS: Sixty rats were randomly divided into 4 groups: control group, curcumin control group (curcumin 50 mg/kg), paraquat group (2% paraquat solution 100 mg/kg), and curcumin intervention group (curcumin 50 mg/kg at 15 min, 24 h, or 48 h after paraquat exposure). On days 1, 3, or 7 after paraquat administration, and liver tissue was collected thereafter. The content of malonaldehyde (MDA) and the activities of superoxide dismutase activity (SOD) and catalase (CAT) in the liver tissue were determined by chemical colorimetry. The activities of heme oxygenase 1 (HO-1) and quinone oxidoreductase 1 (NQO-1) in the liver tissue were determined by ELISA. The mRNA and protein levels of NF-E2-related factor 2 (Nrf2) were determined by RT-PCR and Western blot, respectively. The pathological changes of liver tissue were examined by optical microscopy. RESULTS: No significant change was observed between the control group and the curcumin control group in any examination of this study (P > 0.05). Both paraquat group and curcumin intervention group showed increase in MDA content, decreases in SOD and CAT activities, increases in HO-1 and NQO-1 activities, and increases in the protein and mRNA levels of Nrf2, in comparison with the control group (P < 0.05 for all except HO-1 activity in paraquat group on day 7). In comparison with the parquet group on the same day, the curcumin intervention group showed decrease in MDA content, increases in the activities of SOD, CAT, HO-1, and NQO-1, and increases in the mRNA and protein levels of Nrf2 on days 1, 3, and 7 (P < 0.05). The pathological examination revealed that the damage of liver tissue in the paraquat group was the most serious on the 3rd day after paraquat exposure, and the damage was consistently alleviated by curcumin intervention on days 1, 3, and 7, as compared with the paraquat group. CONCLUSION: Oxidative stress plays an important role in paraquat-induced acute liver damage in rats, and curcumin can exert a hepatoprotective effect against oxidative stress by increasing the expression of Nrf2 and the activities of HO-1, NQO-1, SOD, and CAT and reducing the content of MDA.
Subject(s)
Curcumin/pharmacology , Liver/pathology , Oxidative Stress/drug effects , Paraquat/poisoning , Animals , Catalase/metabolism , Disease Models, Animal , Heme Oxygenase (Decyclizing)/metabolism , Liver/drug effects , Liver/metabolism , Male , Malondialdehyde/metabolism , NAD(P)H Dehydrogenase (Quinone)/metabolism , Rats , Rats, Sprague-Dawley , Superoxide Dismutase/metabolismABSTRACT
OBJECTIVE: To measure the levels of ghrelin-induced expression or activation of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) in the PQ-injured lungs of mice and to evaluate the protective effect of ghrelin against paraquat (PQ)-induced acute lung injury in mice. METHODS: According to the random number table method, 50 ICR mice of clean grade were assigned to 5 groups: normal control group (n = 10), PQ group (n = 10), and ghrelin intervention groups (n = 30). For PQ group, mice were injected with a single dose of PQ (20 mg/kg, i.p.); for ghrelin intervention groups, mice were injected with a single dose of PQ (20 mg/kg, i.p.), and then ghrelin was injected at three concentrations (16.58, 33.15, and 49.73 µg/kg). Lung tissues were collected and proceeded to the following studies. HE staining was used for histopathological examination under a light microscope, and the changes in nuclear expression of Nrf2 were evaluated by Western blot. The activities of HO-1 and NQO1 were measured by ELISA. Malondialdehyde (MDA) content and MPO activity were measured by colorimetry. Another 40 mice were divided into PQ group (n = 10) and 16.58, 33.15, and 49.73 µg/kg ghrelin intervention groups (n = 10 for each); mortality and clinical manifestations were recorded within 72 h. RESULTS: Compared with the normal control group, the PQ group showed significant increases in nuclear protein level of Nrf2, content of MDA, and activities of HO-1, NQO1, and MPO (P < 0.05 for all). Compared with the PQ group, ghrelin treatment significantly increased the expression of Nrf2 and activities of HO-1 and NQO1 and significantly reduced the content of MDA and activity of MPO (P < 0.01 for all). Histopathological studies indicated that ghrelin showed an antioxidant property that reduced the histological changes induced by PQ in the lungs. The ghrelin intervention groups had a significantly lower mortality than the PQ group, and there was a significant difference between the high-dose ghrelin intervention group and PQ group (P < 0.05). CONCLUSION: Ghrelin can up-regulate nuclear expression of Nrf2, increase the activities of HO-1 and NQO1, and reduce the activity of MPO and content of MDA, thus protecting PQ-exposed mice from acute lung injury.
Subject(s)
Acute Lung Injury/metabolism , Ghrelin/pharmacology , Lung/metabolism , Paraquat/poisoning , Acute Lung Injury/chemically induced , Animals , Heme Oxygenase-1/metabolism , Lung/drug effects , Male , Malondialdehyde/metabolism , Membrane Proteins/metabolism , Mice , Mice, Inbred ICR , NAD(P)H Dehydrogenase (Quinone)/metabolism , NF-E2-Related Factor 2/metabolism , Oxidative Stress , Peroxidase/metabolismABSTRACT
OBJECTIVE: To investigate the influence of NRF2 gene polymorphism at locus -617 on inflammatory response of lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) in patients with alcoholic liver disease (ALD). METHODS: Venous blood samples from 82 patients with ALD were collected and PBMCs were separated using Ficoll density gradient centrifugation. T cell subgroup was detected by flow cytometry. The polymorphisms in NRF2 gene promoter -617C/A was determined by gene sequencing. According to the results of gene sequencing, patients were divided into non-mutation group (genotype CA and AA) and mutation group (genotype CC). After stimulation with LPS, the expression levels of NRF2, tumor necrosis factor (TNF)α, interleukin (IL)-1ß and IL-10 were measured by reverse transcription-PCR (RT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. RESULTS: Among the 82 patients with ALD, 32 were homozygous for the C allele (CC), 44 heterozygous (CA), and 6 AA. The frequencies of allele C and A were 65.9% and 34.1%, respectively. There were no differences in clinical data, such as liver function and distribution of T cell subsets between the two groups (all P values >0.05) .Under LPS stimulation, the NRF2 mRNA expression in the non-mutation group was significantly higher than that in the mutation group (P < 0.05). The TNFα, IL-1ß mRNA and protein expression in the mutation group were significantly higher than those in the non-mutation group (P < 0.05) and IL-10 mRNA and protein expression of the mutation group was higher than that in the non-mutation group without statistical significance (P > 0.05). CONCLUSION: The gene promoter NRF2-617C mutated to A in LPS-stimulated PBMC of patients with ALD significantly decreases the expression of NRF2 and releases early proinflammatory cytokines.
Subject(s)
Leukocytes, Mononuclear/metabolism , Liver Diseases, Alcoholic/genetics , Liver Diseases, Alcoholic/metabolism , NF-E2-Related Factor 2/genetics , Polymorphism, Single Nucleotide , Adult , Aged , Cells, Cultured , Female , Genotype , Humans , Inflammation/chemically induced , Inflammation/metabolism , Male , Middle Aged , Promoter Regions, Genetic , RNA, Messenger/geneticsABSTRACT
OBJECTIVE: To explore the effects of NF-E2-related factor-2 (NRF2)-617C/A promoter polymorphism on NRF2 expression as well as lipopolysaccharide-induced inflammatory responses in macrophages. METHODS: NRF2-617C/A promoter fragments were synthesized by chemical method and cloned into a pUC57 vector. The dul-luciferase reporter assay was employed to determine the activity of promoters. Then recombinant adenoviral vectors were constructed and transfected into macrophages. The expression of Nrf2 was examined by Western blotting and reverse transcription (RT)-PCR. The expressions of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-10 (IL-10) in macrophages after the stimulation of LPS were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The activity of NRF2-617C promoter-luciferase reporter (FLuc/RLuc activity ratio) was significantly higher than that of NRF2-617A group (0.584 ± 0.016 vs 0.258 ± 0.018, P < 0.05).The NRF2 protein and mRNA levels in -617C group were much higher than those of 617A group (1.123 ± 0.080 vs 0.951 ± 0.057,1.889 ± 0.031 vs 1.647 ± 0.323, both P < 0.05). After the stimulation of LPS, the NRF2 protein expression in macrophages significantly increased (0.584 ± 0.016 vs 0.258 ± 0.018, P < 0.05). Compared with -617A group, there was a significantly higher expression of NRF2 in -617C group (0.671 ± 0.033 vs 0.751 ± 0.014, P < 0.05). Additionally, the productions of IL-6 and IL-10 in -617C group were markedly lower than those in -617A group as well as IL-6/IL-10 (both P < 0.05). However, no significant difference existed in the levels of TNF-α between -617C and -617A groups (P > 0.05). CONCLUSIONS: The -617C/A promoter polymorphism of NRF2 may influence the NRF2 expression. And it appears to be associated with the LPS-induced inflammatory responses in macrophages.
Subject(s)
Inflammation , Macrophages/pathology , NF-E2-Related Factor 2/genetics , Cells, Cultured , Humans , Interleukin-10/metabolism , Interleukin-6/metabolism , Lipopolysaccharides , Macrophages/metabolism , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Tumor Necrosis Factor-alpha/metabolismABSTRACT
In this study, we demonstrate the protective effects of Cycloartenyl ferulate (CF) against Paraquat (PQ)-induced cytotoxicity and elucidate the underlying molecular mechanisms. The results show that, CF could reverse the PQ-induced growth inhibition and release of lactate dehydrogenase in HK-2 human proximal tubular cells. Treatment with PQ induced apoptosis in HK-2 cells, as evidenced by accumulation of sub-G1 cell population, chromatin condensation, DNA fragmentation, and translocation of phosphatidylserine, which were significantly attenuated by co-incubation with CF. Mitochondria-mediated apoptosis pathway contributed importantly to PQ-induced apoptosis, as revealed by the activation of caspase-3/-9, cleavage of PARP, depletion of mitochondrial membrane potential regulated by Bcl-2 family members, and overproduction of reactive oxygen species, which were also effectively blocked by CF. Moreover, treatments of PQ strongly inhibited the expression of Nrf2 and the downstream effectors, HO1 and NQO1. However, co-treatment with CF effectively reversed this action of PQ. Furthermore, silencing of Nrf2 by the siRNA technique significantly blocked the cytoprotective effects of CF against PQ-induced apoptosis, which suggest the important role of Nrf2 signaling pathway an cell apoptosis induced by PQ. Taken together, this study provides a novel strategy for molecular intervention against PQ-induced nephrotoxicity by using phytochemicals.
Subject(s)
Apoptosis/drug effects , Coumaric Acids/pharmacology , Mitochondria/drug effects , NF-E2-Related Factor 2/metabolism , Paraquat/toxicity , Analgesics/pharmacology , Blotting, Western , Cell Line , Flow Cytometry , Humans , NF-E2-Related Factor 2/genetics , Reactive Oxygen Species/metabolism , Signal TransductionABSTRACT
OBJECTIVE: To compare the sedative effects of propofol and midazolam, or combination of them on emergency critically ill patients on mechanical ventilation. METHODS: Medical records of 68 patients treated in emergency intensive care unit (EICU) receiving mechanical ventilation and sedation care from August 2007 to July 2011 were reviewed retrospectively. According to the type of sedatives used, patients were assigned to propofol group (n=28), midazolam group (n=20), combination of propofol and midazolam group (combination group, n=20). Patients in the former two groups were given a loading dose of propofol or midazolam and followed by continuous infusion of the same drugs. Those in the combination group were given a loading dose of propofol and followed by continuous infusion of propofol together with midazolam. In this study, Ramsay anesthesia score was used to evaluate the effectiveness of sedation. The patients in three groups were maintained at depth of sedation level 2-4 according to the Ramsay score, and reassessed every 1-2 hours after the initiation. The change in vital signs and respirator related parameters were observed before and after administration in three groups, and the treatment information of sedative and mechanical ventilation were recorded. RESULTS: Heart rate (HR), respiratory rate (RR), systolic blood pressure (SBP), diastolic blood pressure (DBP), tidal volume (VT) were decreased at 1 hour after treatment compared with those before treatment in all the three groups, while the blood oxygen saturation (SpO2) was increased. There were no significant differences in RR and SpO2 at 1 hour after treatment among three groups. HR, SBP, DBP at 1 hour after treatment in propofol group were significantly decreased compared with those in midazolam group and combination group (HR: 20.43 ± 13.52 bpm vs. 15.27 ± 13.71 bpm, 18.54 ± 10.07 bpm; SBP: 39.26 ± 16.64 mm Hg vs. 25.80 ± 21.09 mm Hg, 31.50 ± 28.20 mm Hg; DBP: 21.35 ± 12.91 mm Hg vs. 14.07 ± 10.53 mm Hg, 16.42 ± 13.55 mm Hg, P<0.05 or P<0.01). VT at 1 hour after beginning of the treatment in combination group was decreased significantly compared with propofol group and midazolam group (121.06 ± 96.50 ml vs. 33.36 ± 28.49 ml, 39.94 ± 33.24 ml, both P<0.01). The drug dosage in combination group was decreased significantly compared with propofol group and midazolam group (total dosage of propofol: 25.21 ± 15.33 mg/kg vs. 90.83 ± 17.42 mg/kg, total dosage of midazolam: 2.37 ± 1.87 mg/kg vs. 4.02 ± 3.62 mg/kg, both P<0.01), but there was no significant difference in sedation time among groups. EICU stay days in combination group was shortened significantly compared with propofol group and midazolam group (7.75 ± 5.20 days vs. 12.53 ± 8.24 days, 15.20 ± 8.33 days, both P<0.05), but there was no significant difference in mechanical ventilation duration among groups. CONCLUSIONS: A combination of propofol with midazolam for emergency critically ill patients on mechanical ventilation not only can achieve a good sedative effect, reduce total amount of the drug, but also alleviate the inhibitory effect of propofol on the circulation, improve the symptoms of asynchronous ventilation, and reduce stay time in EICU.
Subject(s)
Hypnotics and Sedatives/therapeutic use , Midazolam/therapeutic use , Propofol/therapeutic use , Respiration, Artificial , Adolescent , Adult , Aged , Aged, 80 and over , Critical Care , Critical Illness , Female , Humans , Male , Midazolam/administration & dosage , Middle Aged , Propofol/administration & dosage , Retrospective Studies , Young AdultABSTRACT
OBJECTIVE: To evaluate the therapeutic efficacy of hemoperfusion in the treatment of intermediate myasthenia syndrome (IMS) following acute organophosphate poisoning (AOPP). METHODS: Eighty cases of IMS following AOPP, who were admitted to the Emergency Department of our hospital from 2006 to 2011 and had complete clinical records, were divided into HP treatment group (n = 36) and non-HP (NHP) treatment group (n = 44). The therapeutic efficacy of HP was evaluated by comparing the clinical data of the two groups. RESULTS: The HP treatment group showed significantly increased serum cholinesterase activity at 24h and 72 h after admission (P < 0.05), while the NHP treatment group showed significantly increased serum cholinesterase activity at 72 h after admission (P < 0.05). The serum cholinesterase activity in the HP treatment group was significantly higher than that in the NHP treatment group at 24 h after admission (P < 0.05). Compared with the NHP treatment group, the HP treatment group had significantly decreased total atropine dose, time of ventilatory assistance, length of ICU stay, recovery time from coma, incidence of pulmonary infection, and mortality due to respiratory failure (P < 0.05). There were no significant differences in the incidence of upper gastrointestinal hemorrhage and total mortality between the two groups (P > 0.05). CONCLUSION: Hemoperfusion is an effective therapy for improving clinical symptoms, shorten the course of disease, reducing complications, and decreasing the mortality due to respiratory failure in the patients with IMS following AOPP.
Subject(s)
Hemoperfusion , Muscle Weakness/therapy , Organophosphate Poisoning/therapy , Cholinesterases/blood , Female , Humans , Male , Muscle Weakness/etiology , Syndrome , Treatment OutcomeABSTRACT
OBJECTIVE: To observe the effects of hemoperfusion on oxidative stress status and the levels of matrix metallo proteinase (MMP-2, MMP-9), tissue inhibitor of metalloproteinase (TIMP-1) in lungs, livers and kidneys in paraquat poisoning rabbits, and to explore the mechanism of therapeutic effects induced by HP on acute paraquat poisoning. METHODS: Seventy eight rabbits were randomly divided into normal control group (N group, n=6), exposure groups (PQ group, n=24), hemoperfusion treatment group (HP treatment group, n= 24) and blank control group (HP group, n=24). The PQ, HPQ and HP groups were divided into 4 observation time groups (1, 3, 7 and 21 d). N group was exposed to 5 ml normal saline and PQ group was exposed to 50 mg/kg PQ by oral gavage. In 1 h after PQ exposure, HPQ group was exposed to the activated carbon hemoperfusion for 2 h. The content or activity of MDA, SOD and GSH-Px in lungs, livers and kidneys were detected, the expression levels of MMP-2, MMP-9 and TIMP-1 were measured with immunohistochemical SP method for all groups. RESULTS: The contents of MDA in lungs, livers and kidneys of PQ and HPQ groups decreased and the activities of SOD and GSH-Px in lungs, livers and kidneys of PQ and HPQ groups increased with observation time. The expression levels of MMP-2, MMP-9 and TIMP-1 in PQ and HPQ groups enhanced on the first day, PQ group was most obvious. Along with the observation time extended, all kinds of positive expression were still high. Compared with normal control group, the activities of serum SOD and GSH-Px in PQ and HPQ groups declined significantly, but the contents of serum MDA increased; the expression levels of MMP-2, MMP-9 and TIMP-1 in lung, liver and kidney tissues increased obviously, the ration between MMP-9 and TIMP-1 significantly increased (P < 0.05). Compared with PQ group, the activities of SOD and GSH-Px in HPQ group significantly increased, the content of MDA declined, the expression levels of MMP-2, MMP-9 and TIMP-1 in lung, liver and kidney tissues declined obviously, the ration between MMP-9 and TIMP-1 significantly declined, but higher than N group, the differences were statistically significant (P < 0.05). CONCLUSION: The oxidative stress and MMPs may be involved in the pathogenesis of tissue injuries induced by paraquat. The treatment with HP could obviously reduce oxidative stress and the expression levels of MMP-2, MMP-9 and TIMP-1, enhance the ration between MMP-9 and TIMP-1. So HP treatment could play a role in rescuing the PQ poisoning and protecting the organs function.