Cycloartenyl Ferulate Inhibits Paraquat-Induced Apoptosis in HK-2 Cells With the Involvement of ABCC1.

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.

[The protective effect of bone marrow mesenchymal stem cells carrying antioxidant gene superoxide dismutase on paraquat lung injury in mice].

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.

[Effect of PON1 overexpression on mouse diaphragmatic muscle cells injury caused by acute dichlorvos poisoning].

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.

The reversal of paraquat-induced mitochondria-mediated apoptosis by cycloartenyl ferulate, the important role of Nrf2 pathway.

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.

[An association between NF-E2-related factor 2-617 C/A gene polymorphisms and sepsis in Chinese Han population].

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.

[Protective effect of ghrelin against paraquat-induced acute lung injury in mice].

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.

[Curcumin alleviated liver oxidative stress injury of rat induced by paraquat].

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.

[Functional polymorphism of NRF2 gene promoter -617C/A in lipopolysaccharide-stimulated peripheral blood mononuclear cellular inflammatory response in patients with alcoholic liver disease].

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.

[Effects of NF-E2-related factor-2 promoter polymorphism on lipopolysaccharide-induced inflammatory responses in macrophages].

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.

Mitochondrial tRNA mutations are associated with maternally inherited hypertension in two Han Chinese pedigrees.

We report here the clinical, genetic, molecular, and biochemical evaluations in two Han Chinese families with maternally inherited hypertension. Fourteen of 20 adult matrilineal relatives of these families exhibited a wide range of severity in hypertension, while none of offspring of affected fathers had hypertension. The age-at-onset of hypertension in matrilineal relatives varied from 37 years to 83 years, with an average of 55 and 66 years, respectively. Mutational analysis of their mitochondrial genomes identified the m.4353T>C mutation in the tRNA, in conjunction with the known m.593C>T mutation in the tRNA(Phe) and m.5553C>T mutation in the tRNA(Trp). Northern analysis revealed that m.4353T>C, m.593C>T and m.5553C>T mutations caused ∼66%, 65%, and 12% reductions in the steady-state level of tRNA(Gln), tRNA(Phe) and tRNA(Trp), respectively. An in vivo protein labeling analysis showed ∼35% reduction in the rate of mitochondrial translation in cells carrying these tRNA mutations. Impaired mitochondrial translation is apparently a primary contributor to the reduced rates of overall respiratory capacity, malate/glutamate-promoted respiration, succinate/glycerol-3-phosphate-promoted respiration, or N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate-promoted respiration and the increasing level of reactive oxygen species in the cells carrying these mtDNA mutations. These data demonstrate that mitochondrial dysfunction caused by mitochondrial tRNA mutations is associated with essential hypertension in these families.

[To evaluate the therapeutic efficacy of hemoperfusion in treating intermediate syndrome following acute organophosphate poisoning].

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.

[Effects of hemoperfusion on oxidative stress, matrix metalloproteinases and their inhibitors in acute paraquat poisoning rabbits].

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.

[Effect of ulinastatin on oxidative stress and nuclear factor E2-related factor 2 expression in the lung tissues of acute hydrogen sulfide intoxicated rats].

OBJECTIVE: To investigate the dynamic changes of oxidative stress and nuclear factor-E2 related factor 2 (Nrf2) expression in the lung tissues of acute hydrogen sulfide (H2S) intoxicated rats and intervention effects of ulinastatin (UTI). METHODS: A total of 96 SD rats of clean grade were divided randomly into four groups: normal control group (n = 8), UTI control group (n = 8), H2S -intoxicated model group (n = 40), and UTI treatment group (n = 40). The H2S-intoxicated model group and UTI treatment group were exposed to H2S (283.515 mg/m3) by inhalation for 1h, then UTI treatment group was intraperitoneally exposed to UTI at the dose of 10(5) U/kg for 2 h. H2S-intoxicated model group and UTI treatment group were sacrificed at 2, 6, 12, 24 and 48 h after exposure, respectively. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione (GSH) in the rat lung tissues were measured. The expression levels of Nrf2 mRNA in the rat lung tissues were detected. Pathological changes of rat lung tissues were observed under a light microscope and the lung injury scores were evaluated. RESULTS: Compared with control group, the pulmonary SOD, CAT and GSH levels at 2,6 and 12 h after exposure and the pulmonary GSH-Px levels at 2, 6, 12 and 24 h after exposure in H2S-intoxicated model group significantly decreased (P < 0.05 or P < 0.01). The levels of pulmonary MDA at 2, 6, 12 and 24 h after exposure in H2S-intoxicated model group were significantly higher than those in normal control group (P < 0.01). As compared with H2S -intoxicated model group, the pulmonary GSH-Px activities at 6 and 12 h after exposure, the pulmonary CAT activities at 2, 6 and 12 h after exposure, the pulmonary GSH levels at 2, 6, 12 and 24 h after exposure and the pulmonary SOD activities at 2, 6, 12, 24 and 48 h after exposure in UTI treatment group significantly increased (P < 0.05 or P < 0.01), the pulmonary MDA levels at 2, 6 and 12 h after exposure in UTI treatment group significantly decreased (P < 0.01). The expression levels of Nrf2 mRNA at 2, 6, 12, 24 h after exposure in H2S-intoxicated model group were 0.314 +/- 0.011, 0.269 +/- 0.010, 0.246 +/- 0.011 and 0.221 +/- 0.018, respectively, which were significantly higher than those (0.149 +/- 0.012) in control group (P < 0.01). As compared with H2S-intoxicated model group, the expression levels (0.383 +/- 0.017, 0.377 +/- 0.014, 0.425 +/- 0.017, 0.407 +/- 0.011 and 0.381 +/- 0.010) of Nrf2 mRNA at 2, 6, 12, 24 and 48 h after exposure in UTI treatment group significantly increased (P < 0.01). The lung injury at 24 h after exposure in H2S-intoxicated model group was higher than that in UTI treatment group. Histopathological examination showed that the scores of lung injury at 12, 24 and 48 h after exposure in UTI treatment group was significantly lower than those in H2S-intoxicated model group (P < 0.01). CONCLUSION: Oxidative stress and Nrf2 activation may be the important factors in rat lung injury induced by H2S-intoxicated, UTI may reduce the rat lung injury and protect the rat lung from damage induced by H2S by inhibiting ROS, improving the imbalance in redox and up-regulating Nrf2 mRNA expression.

[In vitro construction and confirmation of adenovirus containing a mifepristone (RU486)-inducible regulation system for NRF2 gene].

OBJECTIVE: To construct an adenovirus containing a mifepristone (RU486)-inducible regulation system for NRF2 gene, express the product in H460 cell and verify whether the mentioned system can control the gene expression and assess its efficiency. METHODS: A RU486-inducible regulation system for Nrf2 gene was introduced into an adenovirus. The confirmation was performed through the LUC and Dsred genes. And the expression pattern of Nrf2 at the viral level was examined by Western blot and RT-PCR (reverse transcription-polymerase chain reaction). RESULTS: The expressions of LUC and Dsred showed a rising trend with the incremental dose of RU486. After the transfection H460 cell with Ad-RUNrf2, the results of RT-PCR and Western blot demonstrated that the expression of Nrf2 was elevated with a rising dose of RU486. After the removal of RU486, the expression of Nrf2 was reduced. CONCLUSION: The construction of an adenovirus carrying Nrf2 gene regulated by a RU486-inducible system is successful, and RU486 can adjust the cellular expression of Nrf2 factor. The LUC and the Dsred expression assumes the dosage dependence along with RU486 to increase; after the Ad-RUNrf2 infects the H460 cell, through RTPCR and Western the Blot result demonstrated that the expression of Nrf2 increases along with the RU486 dosage increases, after removing RU486, the Nrf2 expression is weaken. Showing the construction of the adenovirus carrying Nrf2 gene regulated by the mifepristone (RU486)-inducible system is successful, and RU486 can adjust the Nrf2 factor in the cell the expression.

[The effects of NF-E2-related factor-2 prompter polymorphism on alcoholic liver disease with Vibrio vulnificus sepsis].

OBJECTIVE: To investigate the influence of genetic polymorphism in NF-E2-related factor-2 (nrf2) gene promoter locus at 336 in alcoholic liver disease (ALD) with Vibrio vulnificus (VV) sepsis. METHODS: Through the simple random sampling method, C57B6 male mice were divided into normal feeding group (group A, 10 mice), alcoholic liver disease group (group B, 10 mice), normal feeding group infected with VV through intraperitoneal injection (group C, 8 mice), alcoholic liver disease group infected with VV (group D, 110 mice). Through gene sequencing method, nrf2 gene promoter 336 polymorphism in D group was analyzed and grouped into: non-mutation group (336T) (group D1, 7 mice) and mutation group (336C) (group D2, 10 mice). Through RT-PCR, Western-blotting and ELISA method, expressions of nrf2, tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), high mobility group protein 1 (HMGB(1)) gene and protein of liver were measured. The pathological changes in liver were recorded with light microscope. RESULTS: After infected with VV for 48 hours for A, B, C, D1, D2 group, the expression medians of nrf2 mRNA in liver were 0.115, 0.173, 0.211, 0.764, 0.352, respectively (χ(2) = 40.64, P < 0.05), the expression medians of IL-10 mRNA in liver were 0.338, 0.637, 1.002, 1.825, 1.403, respectively (χ(2) = 41.05, P < 0.05), the expression medians of TNF-α mRNA in liver were 0.140, 0.254, 0.372, 0.399, 0.699, respectively (χ(2) = 38.16, P < 0.05), the expression medians of HMGB(1) mRNA in liver were 0.230, 0.410, 0.668, 0.508, 1.021, respectively (χ(2) = 31.45, P < 0.05). After infected with VV 48 hours for mice in A, B, C, D1, D2 group, the expression medians of nrf2 protein in liver were 0.908, 1.461, 2.061, 3.982, 2.243, respectively (χ(2) = 33.72, P < 0.05), the expression medians of IL-10 protein in liver were 13.97, 22.54, 30.14, 57.98, 41.53, respectively (χ(2) = 37.31, P < 0.05), the expression medians of TNF-α protein in liver were 114.07, 142.94, 175.44, 174.60, 266.11, respectively (χ(2) = 32.29, P < 0.05), the expression medians of HMGB(1) protein in liver were 2.01, 6.05, 9.62, 6.24, 12.89, respectively (χ(2) = 36.94, P < 0.05). Compared with group A, there were large amount of fat drops, fatty changes in group B, inflammatory cell infiltration, disorder of hepatic cell in group C, and extension of hepatic duct and vein, edema of liver cells and disorder of hepatic cells in group D. CONCLUSION: The nrf2 gene promoter of T336C mutation in C57B6 mouse of ALD can significantly decrease the expression of nrf2, and intensify organ inflammation and damage when they were infected by VV.

[Effects of hemoperfusion on plasma concentration and histopathological changes in paraquat poisoning rabbits].

OBJECTIVE: To observe the effects of hemoperfusion on plasma concentration and histopathological changes in paraquat (PQ) poisoning rabbits. METHODS: Sixteen rabbits were randomly divided into exposure group (PQ group, n = 8) and hemoperfusion plus PQ exposure group (HPQ group, n = 8). HPQ group were given hemoperfusion in 45 min after exposure to PQ. The plasma PQ concentrations at 0.5, 1.0, 1.5, 2.0, 3.0, 6.0, 12.0, 24.0, 48.0 and 72.0 hours after exposure were measure in 2 groups. The histopathological changes of lung, liver and kidney were examined, the behavior changes and the survival number of 7 days were observed. RESULTS: The poisoning symptoms of HPQ group were generally better than those of PQ group, in each group six animals survived for 7d. The plasma PQ concentrations at 1.0, 1.5, 2.0, 3.0, 6.0, 12.0, 24.0, 48.0, 72.0 h after exposure in HPQ group were significantly lower than those in PQ group (P < 0.05 or P < 0.01). In HPQ group, the plasma PQ peak concentration [(5.01 ± 0.15] µg/L], area under the curve [(54.03 ± 5.31) mg×h(-1)×L(-1)] and PQ half-life time [(16.29 ± 3.26) h] after treatment of HP were significantly lower than those [(11.97 ± 0.75) µg/L, (141.40 ± 10.10) mg×h(-1)×L(-1) and (31.16 ± 9.85) h] in PQ group (P < 0.05). The apparent volume of distribution and PQ clearance rate in HPQ group were significantly higher than those in PQ group (P < 0.05). Congestion, edema, cell infiltration and other pathological changes were found in lung, liver and kidney in PQ group under the light microscope, which were significantly more severe than those in HPQ group. The pathologic scores of lung tissue, liver and renal tubular damage on the 1st, 3rd, 7th days after exposure in HPQ group were significantly lower than those in PQ group (P < 0.05). CONCLUSION: When acute PQ poising, rabbits appeared the quick absorption, high toxicity and long half-life time of PQ. The early hemoperfusion can effectively remove the toxicant in plasma and reduce the pathological injury in major organs, which may be beneficial for further treatment.

[Intervention effect of dimercaptopropansulfonate sodium on central toxic induced by bromoxynil in vivo].

OBJECTIVE: to investigate the changes of γ-aminobutyric acid (GABA) and glutamate (Glu) in the cerebral cortex following acute bromoxynil intoxication in mice and the protective effect of sodium dimercaptopropane sulfonate (Na-DMPS). METHODS: 30 ICR mice were randomly divided into blank control group (10), exposure group (10) and Na-DMPS protection group (10). The levels of GABA and Glu in the cerebral cortex were measured by RP-HPLC. The glutamine (Gln) level and the glutamine synthetase (GS), glutamate decarboxylation enzyme (GAD), γ-aminobutyric acid transaminase (GABA-T) activity in the cerebral cortex were determined by UV colorimetric. RESULTS: compared with the control group [GABA: (3.41 ± 0.12) micromol/g, Glu (14.00 ± 0.16) micromol/g, Gln (1.25 ± 0.19) micromol/g, GAD (13.50 ± 0.25) micromol × g(-1) × h(-1), GABA-T (25.51 ± 0.21) micromol × g(-1) × h(-1), GS(142.19 ± 1.31) U/mg pro], the level of GABA [(3.14 ± 0.14) micromol/g] was decreased (P < 0.05), whereas the level of Glu [(17.54 ± 0.40) micromol/g] and Gln [(3.35 ± 0.27) micromol/g] were increased (P < 0.05), the activity of GAD [(11.93 ± 0.15 micromol × g(-1) × h(-1)], GABA-T [(24.15 ± 0.22) micromol × g(-1) × h(-1)], GS [(140.75 ± 1.01) U/mg pro] was decreased (P < 0.05) in acute intoxication group; Compared with the acute intoxication group, the level of GABA [(3.52 ± 0.30) micromol/g] was increased (P < 0.05), whereas the level of Glu [(14.20 ± 0.32) micromol/g] and Gln [(1.32 ± 0.17) micromol/g] were decreased (P < 0.05), the activity of GAD [(13.01 ± 0.45 micromol × g(-1) × h(-1)], GABA-T [(25.19 ± 0.26) micromol × g(-1) × h(-1), GS [(142.35 ± 1.20) U/mg pro] was increased (P < 0.05); In contrast, the levels of GABA, Glu, Gln and the activity of GAD, GABA-T, and GS in Na-DMPS protection group were not significantly different in comparison with control group (P > 0.05). CONCLUSION: the central toxic effects of mice with acute bromoxynil intoxication may be related to the changes of GABA and Glu content in the cerebral cortex;Na-DMPS can protect mice from bromoxynil-induced central toxic effects and GABA and Glu abnormal change in the cerebral cortex.

[Expression of angiotensin converting enzyme and angiotensin converting enzyme 2 gene in lung of paraquat poisoning rats and protection of sodium dimercaptopropane sulfonate].

OBJECTIVE: To investigate the expression of angiotensin converting enzyme (ACE) and ACE2 Gene in lung of paraquat poisoning rats and the protection of sodium dimercaptopropane sulfonate (Na-DMPS). METHODS: One hundred SD male rats were randomly equally divided into 4 groups:normal control group (10 rats), drug control group (40 rats), paraquat poisoning group (40 rats) and drug intervention group(40 rats). The paraquat poisoning and drug intervention group rats were injected intraperitoneally by paraquat (20 mg/kg). The rats in drug intervention group rats were protected by intraperitoneal injection with Na-DMPS (200 mg/kg) 15 min before exposure of paraquat. Behavioral changes of the rats and histological changes of lung tissues under light microscope were observed. And the expression of ACE and ACE2 mRNA in lung tissues of rats both in paraquat poisoned group and drug intervention group were measured by RT-PCR at different time of 6 h, 24 h, 3 and 7 d after poisoning. RESULTS: The poisoning symptoms of shortness of breath, cramps appeared and deteriorated progressively in rats after paraquat exposure and the protection of NA-DMPS could delay and reduce these symptoms significantly. Histological appearance of disorganization of pulmonary capillary and alveolus, exudation in alveolar space, pulmonary edema, severe bleeding, and inflammatory cells infiltration were obvious in lungs of rats after paraquat poisoning, whereas the histological changes were extenuated by protection of NA-DMPS. As compared with normal control group (NC group), the expressions of ACE, ACE2 mRNA in lung tissue decreased, and the lowest level of ACE mRNA expressions appeared at 24 h (0.457 +/- 0.262), on 3 d (0.385 +/- 0.179) after Paraquat exposure (P < 0.05), while lowest level of ACE2 mRNA expressions appeared on 3 d (0.415 +/- 0.247), 7 d (0.365 +/- 0.215) (P < 0.05). As compared with paraquat poisoned group, the expressions of ACE mRNA in lung tissue of rats in NA-DMPS protected group increased significantly at 24 h (0.739 +/- 0.558) and 3 d (0.749 +/- 0.414) (P < 0.05), while the expressions of ACE2 mRNA increased markedly on 3 d (0.584 +/- 0.345) and 7 d (0.493 +/- 0.292) (P < 0.05). But the expression of ACEmRNA and ACE2 mRNA in lungs had no statistical significance between normal control group and drug intervention group (P > 0.05). CONCLUSION: The expressions of ACE and ACE2 mRNA in lung tissue of the rats with paraquat poisoning are decreased. Na-DMPS can effectively improve the balance of RAS in local lung tissue and reduce the pathological changes of lung tissue, delay the poisoning symptoms and show protective effects for acute lung injury induced by paraquat.

Dynamic expressions of liver tissue apoptosis-related genes of Vibrio vulnificus sepsis rats and the effects of antibacterial agents.

Dynamic changes in mRNA expressions of liver tissue apoptosis-promoting genes Fas and Bax and apoptosis-inhibiting gene Bcl-2 of vibrio vulnificus sepsis rats were detected and the effects of antibacterial agents were examined. The rat model with Vibrio vulnificus sepsis (VV group) was established and some of the Vibrio vulnificus sepsis rats were treated with antibacterial agents (AA group). The mRNA expressions of Fas, Bax and Bcl-2 were measured by reverse transcription polymerase chain reaction (RT-PCR). As compared with normal control group (NC group), the expressions of Fas and Bax mRNA in liver tissue at all different time points in VV group were increased significantly (P<0.05), and the highest levels of Fas and Bax mRNA expressions were 6 and 12 h after the infection, respectively. At the same time, the expression of Bcl-2 mRNA in liver tissue at all different time points in VV group were decreased significantly (P<0.05), and the lowest level of Bcl-2 mRNA expression appeared 2 h after the infection. The mRNA expressions of Bcl-2 in liver tissue 9 and 12 h after the infection in AA group were increased significantly (P<0.05) compared with NC group, while the expressions of Fas and Bax mRNA were not significantly different from those of NC group. Compared with VV group, the expression of Fas mRNA in AA group was decreased (P<0.05) and Bax mRNA was decreased significantly 12 and 16 h after the infection (P<0.05), while the expressions of Bcl-2 mRNA were increased significantly 9, 12 and 16 h after the infection (P<0.05). It is concluded that the mRNA expressions of liver tissue apoptosis-promoting genes Fas and Bax were increased remarkably in vibrio vulnificus sepsis rats, whereas the expression of apoptosis-inhibiting gene Bcl-2 mRNA was decreased obviously in sepsis rats in early stage. The treatment with cefoperazone sodium and levofloxacin lactate could inhibit the expression of Fas mRNA and Bax mRNA and enhance the expression of Bcl-2 mRNA at the same time.

[Effects of antimicrobial agents on the Toll like receptors and myeloid differentiation protein-2 in liver tissue of alcohol-induced liver disease: experiment with rats].

OBJECTIVE: To investigate the effects of antimicrobial agents on the Toll like receptors (TLRs) and myeloid differentiation protein (MD)-2 in liver tissue of alcohol-induced liver disease with Vibrio vulnificus (VV) sepsis. METHODS: Eighty SD rats were randomly divided into 2 groups: alcohol gastric lavage group (n = 74) undergoing alcohol gastric perfusion once a day for 10 weeks and normal control group (Group N, n = 6). 66 surviving rats in the gastric perfusion group were randomly divided into 6 equal subgroups: Subgroup A was alcohol-induced liver disease control subgroup. Subgroup AA, alcohol-induced liver disease and antibacterial drug control subgroup, underwent intraperitoneal injection of cefoperazone sodium and levofloxacin (LVFX). The other 9 subgroups underwent subcutaneous injection of VV to establish animal model of VV sepsis, the rats of 4 of which were killed 2, 6, 12, and 24 h later respectively with their livers taken out (Subgroups AV), and the rats of 5 of which underwent intraperitoneal injection of cefoperazone sodium and LVFX 4 h after VV injection twice a day (Subgroups AVA) and were killed 6, 12 , 24 , 36 h, and 1 week later with their livers taken out. The behavioral changed were observed. PCR was used to detect the mRNA expression of TLR2, TLR4, and MD-2. RESULTS: The mRNA expression levels of TLR2, TLR4, and MD-2 in liver 2, 6, 12, and 24 h after in Subgroups AV were all significantly higher than those of Group N (P < 0.05 or P < 0.01) with the maximum level in the AV-12 h subgroup. The mRNA expression levels of TLR2, TLR4, and MD-2 in liver 6 h, 12 h, and 24 h after the VV injection of Subgroup AVA were all significantly lower than those of Group AV (P < 0.05 or P < 0.01). CONCLUSION: The mRNA expression levels of TLR2, TLR4, and MD-2 in liver tissue of alcohol-induced liver disease with VV sepsis, which may be reduced by treatment of cefoperazone sodium and LVFX, are associated with the development of VV sepsis. This treatment is effective on this disease. Dynamic monitoring of the mRNA expression levels of TLR2, TLR4, and MD-2 in liver tissue benefits observation of the VV sepsis progress and treatment.