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 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.

[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.

[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.

[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.

[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.

[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.

[Expression of thrombomodulin, endothelial protein C receptor in lung tissue of acute paraquat poisoned rats and intervention of sodium dimercaptopropane sulfonate].

OBJECTIVE: To observe the expression and effect of thrombomodulin (TM) mRNA and endothelial protein C receptor (EPCR) mRNA in lung tissue of acute paraquat poisoned rats, and intervention of sodium dimercaptopropane sulfonate (Na-DMPS). METHODS: Eighty male SD rats were randomizedly divided into four groups: the normal control group (n=8), the Na-DMPS control group (n=8, administered with 200 mg/kg Na-DMPS intraperitoneally), the PQ group (n=32, administered with 20 mg/kg 1% PQ intraperitoneally), the NA-DMPS protected group (n=32, administered with 200 mg/kg Na-DMPS intraperitoneally before with 20 mg/kg 1% PQ). The expression of TM mRNA and EPCR mRNA in the PQ group and the Na-DMPS protected group was evaluated at the six hour, on the first, third and seventh day. RESULTS: The expression of TM mRNA and EPCR mRNA in lung tissue of poisoned rats, was significantly increased and reached the peak at the six hour, was decreased slowly on the first day, and returned to normal level on the seventh day. In the Na-DMPS protected group, at the six hour and on the first day, the expression of TM mRNA (1.071 +/- 0.097, 1.055 +/- 0.051) was less than that in the PQ group (P<0.05 or P<0.01). EPCR mRNA (0.678 +/- 0.005), (0.650 +/- 0.007) at the six hour and on the first day in the Na-DMPS protected group was less than that in the PQ group (P<0.05 or P<0.01). CONCLUSION: The expression of TM mRNA and EPCR mRNA of rats after PQ intoxication is increased, and can significantly be decreased after administered with Na-DMPS.

[Effect of bromoxynil on membrane potential and respiratory control rate in isolated mitochondria from mice liver and intervention effect of NAC].

OBJECTIVE: To demonstrate the effect of bromoxynil on membrane potential and respiratory control rate (RCR) in isolate mitochondria from mice liver tissue in vitro and the intervention of NAC. METHODS: The mitochondrial was randomized to control group, bromoxynil-poisoned group and NAC-protected group. S3, S4 and RCR of the mitochondria in each sample was detected by the method of oxygen electrode. Each sample was stained by JC-1 and the changes of membrane potential of mitochondria were observed under fluorescence microscope. RESULTS: The S3 [(0.031 +/- 0.008) nano atoms oxygen x mg(-1) x min(-1)], RCR (1.820 +/- 0.181) of bromoxynil-poisoned group and RCR (4.253 +/- 0.210) of NAC-protected group were significantly lower than those of control group (P<0.01); the S4 [(0.017 +/- 0.004) nano atoms oxygen x mg(-1) x min(-1)] of NAC-protected group was significantly higher than control group (P<0.01). The S3 [(0.046 +/- 0.005) nano atoms oxygen x mg(-1) x min(-1)] and RCR of NAC-protected group were significantly higher than group B (P<0.01), S4 [(0.011 +/- 0.001) nano atoms oxygen x mg(-1) x min(-1)] of NAC-protected group was significantly lower than bromoxynil-poisoned group (P< 0.01). Observation under fluorescence microscope: the red fluorescence of mitochondria was dim or disappeared in bromoxynil-poisoned group while brightened in NAC-protected group but still dimmer than control group. CONCLUSION: In vitro, the mitochondrial RCR and the mitochondrial membrane potential are decreased after the mitochondria is incubated with bromoxynil, and NAC could improve it.

[Oxidative stress of acute paraquat poisoned rats and sodium dimercaptopropane sulfonate intervention].

OBJECTIVE: to study the oxidative stress of rats with acute paraquat poisoning and the intervention of Sodium Dimercaptopropane Sulfonate (NA-DMPS). METHODS: Eighty male SD rats were randomizedly divided into: the normal control group (n=8), NA-DMPS control group (n=8), the PQ group (n=32, the rats were intraperitoneally injected with 1% PQ solution at the dosage of 20 mg/kg) and the NA-DMPS protected group (n=32). The rats in the groups of normal and NA-DMPS control were sacrificed 1d after administration of NS or NA-DMPS. And the rats in the PQ group and the NA-DMPS protected group were sacrificed at 6h, 1, 3, 7d after poisoning. Samples of serum, bronchoalveolar lavage fluid (BALF) and lung tissue were gathered. The MDA and CAT in serum, BALF and lung homogenate, the glutathione (GSH) in serum and BALF were measured. And the expression of Nuclear factor E2-related factor 2 (Nrf2) mRNA in lung was tested with RT-PCR. RESULTS: Compared with the normal control group, the activities of MDA and CAT in serum, BALF and lung homogenate are higher in both groups of PQ and NA-DMPS protected. And compared with the PQ group, the activities of MDA in serum, BALF and lung homogenate of the NA-DMPS protected group decreased significantly at 6h, 1d after poisoning, whereas the activities of CAT are higher at 6h, 1, 3d in serum and 1, 3d in BALF and lung homogenate (P<0.05 or P<0.001). The serum GSH at 6h, 3d of the NA-DMPS protected group [(730.07 +/- 16.23), (793.66 +/- 7.40)] were higher than those in the PQ group. And the BALF GSH at 1, 3d of the NA-DMPS protected group [(609.75 +/- 6.74), (631.83 +/- 12.03)] were also markedly higher than the PQ group (P<0.05 or P<0.001). The expression of NRF2 mRNA of the lung at 1, 3, 7d in the PQ group [(0.71 +/- 0.061), (1.023 +/- 0.158), (0.969 +/- 0.046)] and the NA-DMPS protected group [(1.005 +/- 0.06), (1.464 +/- 0.166), (1.066 +/- 0.191)] were significantly higher than those in the control groups. Compared with the PQ group, the expression of NRF2 mRNA of the lung increased markedly in the NA-DMPS protected group at 1, 3d (P<0.01). CONCLUSION: Na-DMPS decreases the activity of MDA and increases the activity of CAT, GSH and the expression of Nrf2 mRNA. NA-DMPS can protected rats from PQ intoxication by improving the balance of redox reaction.

[Expression of inflammatory factors in lung tissue of acute paraquat poisoned rats].

OBJECTIVE: To investigate the expression of inflammatory factors in lung tissue of acute paraquat (PQ) poisoned rats. METHODS: Fifty male SD rats were randomized divided into two groups: the normal control group (NC group, n = 10) and the PQ group (n = 40). The 1 ml saline was administered once in normal control group. The PQ group was administered with 20 mg/kg 1% PQ by intraperitoneal injection to establish the model of PQ induced lung injury. At six hours, at the first, the third and the seventh day the PQ group were sacrificed, while at the first day the normal control group was sacrificed. The level of tumor necrosis factor alpha (TNF-alpha) mRNA, interleukin 10 (IL-10) mRNA, high mobility group box 1 (HMGB-1) mRNA in lung of rats were detected. Meanwhile, pathological changes of the lung were examined under optical microscope. RESULTS: Compared with that in normal control group, TNF-alpha mRNA expression in lung tissue of PQ group reached the peak at the six hour and decreased slowly at the first day [(0.740 +/- 0.100) and (0.584 +/- 0.049) respectively]. At the six hour and the first day in PQ group it was significantly higher than that in normal control group (P < 0.05 or P < 0.01). IL-10 mRNA expression in lung tissue of PQ group was elevated at the six hour, reached the peak at the first day, at the third day [(0.551 +/- 0.016) and (0.524 +/- 0.010) respectively] and the seventh day also higher than that in normal control group. At the first and the seventh day in the PQ group it was significantly higher than that in normal control group (P < 0.01). Meanwhile, HMGB-1 mRNA expression in lung tissue of PQ group was also elevated at the six hour, reached the peak at the first day, at the third [(0.695 +/- 0.060), (0.871 +/- 0.154) and (0.819 +/- 0.188) respectively] and the seventh day also higher than that in normal control group. At six hour, the first and the third day in the PQ group it was significantly higher than that in normal control group (P < 0.01). The histological changes such as alveolar edema, hemorrhage and inflammatory cell infiltration in the PQ group were more than those in the normal control group. CONCLUSION: In rats after PQ intoxication the levels of the inflammatory factors TNF-alpha, IL-10 and HMGB-1 are higher than normal rats, and inflammatory could play an important role in lung injury of poisoned rats.

[Changes of toll-like receptor, TNF-alpha and IL-10 in liver tissue of rats before and after intragastric infusion with alcohol with vibrio vulnificus sepsis].

OBJECTIVE: To detect the effects of antimicrobial agents on the toll-like receptor (TLR) and so on in liver tissue of rats after intragastric infusion with alcohol with vibrio vulnificus (VV) sepsis. METHODS: Sprague-Dawley rats were randomly divided into normal control group (N group, n = 6), rats after intragastric infusion with alcohol control group (group A, n = 6), drug intervention on rats after intragastric infusion with alcohol control group (group AA, n = 6), rats after intragastric infusion with alcohol with VV sepsis group (group AV, n = 24, killed at 2, 6, 12, 24 hours after injecting VV respectively, six rats per group), as well as drug intervention on rats after intragastric infusion with alcohol with vibrio vulnificus sepsis group (group AVA, n = 30, killed at 6, 12, 24 hours and one week after injecting VV respectively, six rats per group). The expressions and dynamic changes of TLR4 mRNA and so on by RT-PCR in liver tissue of each group were measured. RESULTS: The expressions of TLR4 mRNA in AV-6 hours group was 0.775 +/- 0.101, the expressions of TLR4 mRNA in AVA-6 hours group was 0.600 +/- 0.064; the expressions of TLR4 mRNA in AV-12 hours group was 0.918 +/- 0.133, the expressions of TLR4 mRNA in AVA-12 hours group was 0.583 +/- 0.112; the expressions of TLR4 mRNA in AV-24 hours group was 0.732 +/- 0.110, the expressions of TLR4 mRNA in AVA-24 hours group was 0.512 +/- 0.118. Compared with AV group, the expressions of TLR4 mRNA in liver diminished greatly in AVA group at 6, 12 and 24 hours after being injected with VV (AVA-6 hours group compare with AV-6 hours group, t = -3.573, P < 0.01; AVA-12 hours group compared with AV-12 hours group, t = - 4.722, P < 0.01; AVA-24 hours group compare with AV-24 hours group, t = - 3.340, P < 0.01). CONCLUSION: The treatment with antibacterial agents may reduced the expression of TLR and so on in liver of rats after intragastric infusion with alcohol with VV sepsis. The treatment with antibacterial agents may regulate the balance of the inflammatory response in VV sepsis and generate the visible therapeutical effect for VV sepsis.

[A protocol for diagnosis and treatment of Vibrio vulnificus sepsis].

OBJECTIVE: Vibrio vulnificus sepsis is one of the most fatal disease with a high mortality which exceeds 50%. But at present there is no evidence-based guidelines for diagnosis and therapy of Vibrio vulnificus sepsis because of its dispersion in occurrence and low incidence. METHODS: Based on our ten-year research and review of literature, we try to draft a protocol to improve the diagnostic criteria and treatment of Vibrio vulnificus sepsis. Animal experiments and clinical research were undertaken and the related literature from CINK and PUBMED were reviewed. RESULTS: (1)A criterion for early clinical diagnosis of Vibrio vulnificus sepsis was proposed, including an abrupt onset with fever during the months from April to November, characteristic cutaneous lesions (most commonly haemorrhagic bullae on the extremities) or even extensive necrosis of skin and muscular tissue, progressive hypotension or shock accompanied by multiple organ dysfunction syndrome (MODS), preexisting liver disease or chronic abuse use of alcohol, and consumption of raw seafood or contact with seawater within 1-2 weeks. (2)The best antibiotic therapy is early administration of a combination of the third-generation cephalosporins and the quinolones in full dosage. (3)Aggressive wound debridement, appropriate dermoplasty and supportive care contribute to a better outcome. CONCLUSION: This protocol will help improve the outcome of patients with Vibrio vulnificus sepsis. But it is a crude guideline and needs to be updated when some important new knowledge becomes available.

[Experimental study on detoxication effect of sulfhydryl compounds in acute poisoning of dimethylformamide].

OBJECTIVE: To study the change in oxidase and anti-oxidase in liver of the mice poisoned by dimethylformamide (DMF), and the effects of the treatment with sulfhydryl compounds in acute poisoning of DMF. METHODS: The sulfhydryl compounds included sodium dimercaptopropane (Na-DMPS), N-acetylcysteine (NAC), glutathione (GSH) and dimercaptosuccinic acid (DMSA). The model of acute poisoning with DMF in mice was reproduced, and the left hepatic lobes were harvested at 6, 12, 24, 48 and 72 hours after DMF to detect the dynamic changes in the activities of superoxide dismutase (SOD) and xanthine oxidase (XOD) in liver homogenate. Treatment groups included intraperitoneal injection of Na-DMPS, NAC, GSH, DMSA respectively. In the control groups, the activities of XOD and SOD in liver were determined 24 hours after intragastric administration of DMF. RESULTS: The activities of XOD, SOD in liver were elevated at 24 hours after intragastric administration of DMF (both P<0.01), and returned to the normal levels at 48-72 hours. Compared to the poisoning group, the activities of XOD, SOD in liver homogenate were significantly lowered after the treatment of Na-DMPS, NAC and DMSA (P<0.05 or P<0.01). The activity of XOD in liver homogenate was reduced 24 hours after treating with GSH (P<0.05), and no obvious change was observed in SOD (P>0.05). As far as the activity of SOD was concentrated, Na-DMPS, NAC, DMSA showed better effects than GSH (all P<0.05), and Na-DMPS was the best. There was no significant differences in XOD among the four sulfhydryl compounds. CONCLUSION: The balance of oxidase and anti-oxidase is interrupted by DMF, which might be one of the mechanisms of damage to the liver. Na-DMPS, NAC and DMSA could protect liver function by restoring the balance.