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Endotoxemia is a pathophysiological manifestation caused by the release of large amounts of endotoxin from bacteria in the blood or in the lesion. It can cause multiple organ failure, irreversible shock, and even death. The mortality rate of endotoxemiaIt is high. Bacterial endotoxin is the main cause of endotoxemia. At present, there is no safe and effective drug to treat endotoxemia in clinic. Research shows that blood purification can effectively reduce endotoxin level in the blood, then achieve the goal of treatment of endotoxemia. In this paper, the pathogenesis of endotoxemia, the development of hemoperfusion therapy technology, the mechanism and research status of endotoxin adsorption by different hemoperfusion resin were discussed, and the performance and safety requirements of hemoperfusion adsorbent materials for endotoxemia treatment were studied, so as to provide theoretical support for the synthesis of new hemoperfusion adsorption materials for the treatment of endotoxemia.
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Endotoxemia is a pathophysiological manifestation caused by the release of large amounts of endotoxin from bacteria in the blood or in the lesion. It can cause multiple organ failure, irreversible shock, and even death. The mortality rate of endotoxemiaIt is high. Bacterial endotoxin is the main cause of endotoxemia. At present, there is no safe and effective drug to treat endotoxemia in clinic. Research shows that blood purification can effectively reduce endotoxin level in the blood, then achieve the goal of treatment of endotoxemia. In this paper, the pathogenesis of endotoxemia, the development of hemoperfusion therapy technology, the mechanism and research status of endotoxin adsorption by different hemoperfusion resin were discussed, and the performance and safety requirements of hemoperfusion adsorbent materials for endotoxemia treatment were studied, so as to provide theoretical support for the synthesis of new hemoperfusion adsorption materials for the treatment of endotoxemia.
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<p><b>OBJECTIVE</b>To observe the effect of crocetin on autophagy in rat hepatocytes exposed to lipopolysaccharide (LPS) and D-galactosamine (D-gal) and explore the mechanism.</p><p><b>METHODS</b>Cultured rat hepatocytes were exposed to LPS (1 mg/L) and Dgal (60 mg/L) to induce cell injury and treated with crocetin, 3MA, or crocetin+3MA. Twelve hours after the treatments, the cells were examined for levels of ALT, AST and LDH in the supernatant using ELISA. LC3 fluorescence in the cells following immunofluorescence staining was observed using fluorescence microscopy. Autophagosomes in the cells were observed by transmission electron microscopy, and the cellular expressions of LC3, p62 and SIRT1 were detected using Western blotting.</p><p><b>RESULTS</b>The levels of ALT, AST and LDH in the hepatocytes were elevated after LPS- and D-gal-induced injury, reached the highest levels after 3MA treatment, but were decreased significantly by crocetin treatment. LC3 fluorescence increased obviously in the injured hepatoctyes, and the increment was the most obvious in crocetin-treated cells; LC3 fluorescence was decreased significantly after 3MA treatment. Cell injury induced obvious increase in autophagy in the hepatocytes, and the number of autophagosomes increased significantly after crocetin treatment but was reduced significantly after 3MA treatment. The cell injury caused an obvious up-regulation of LC3 and SIRT1 expression and down-regulated p62 expression. LC3 and SIRT1 expression levels were the highest and the expression of p62 was the lowest in cells with crocetin treatment. 3MA treatment significantly reduced the expression of LC3 and SIRT1 and increased the expression of p62 in the injured cells.</p><p><b>CONCLUSIONS</b>Autophagy is increased in injured rat hepatocytes, and crocetin can promote autophagy in the injured cells to reduce further cell injury.</p>
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Objective To study the liver protection of crocetin against paraquat (PQ) poisoning induced acute liver injury in rats. Methods Fifty-four male Wistar rats were randomly divided into control group, exposure group and treatment group, and the rats in each group were subdivided into the 0.5th, 2nd, and 6th day after exposure subgroups (n = 6). The model of acute liver failure induced by PQ poisoning was reproduced by intraperitoneal injection of 20 mg/kg of 20% PQ, and the rats in control group was injected with the same amount of normal saline. The rats in treatment group were given with intraperitoneal injection of 50 mg/kg crocetin after 0.5 day, once a day until they were sacrificed; the other two groups were injected with the same amount of normal saline. The rats in all groups were sacrificed at the corresponding time points, and blood was collected from inferior vena cava and hepatic tissue was harvested. Hematoxylin and eosin (HE) staining was used to observe the pathological changes in liver tissue on the 6th day under light microscope. Enzyme linked immunosorbent assay (ELISA) was used to detect the serum tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA expressions of inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB). The activities of apoptosis related factors, including caspase-8, -9, -12, in hepatic tissue were determined on the 6th day with chromogenic substrate method. Results In the liver tissue of exposed group, extensive infiltration of the inflammatory cells and the diffuse fragments necrosis were visible, and the regeneration of the liver cells was not obvious, and severity of the injury in a time dependent way. In the treatment group, the structure of hepatic artery was visible, and the infiltration of necrosis, congestion and inflammatory cells were not obvious. On the 0.5th, 2nd, and 6th day, serum levels of IL-6 and TNF-α, the mRNA expressions of iNOS and NF-κB in liver tissue, and the caspase-8, -9, -12 activities on the 6th day in the exposure group and treatment group were significantly higher than those in the control group. And the parameters in treatment group were significantly lower than those of the exposure group [IL-6 (ng/L): 188.37±64.21 vs. 376.61±82.42 on the 0.5th day, 287.18±58.69 vs. 432.77±96.28 on the 2nd day, 234.24±10.17 vs. 375.41±37.59 on the 6th day; TNF-α (ng/L): 472.36±76.43 vs. 688.33±102.19 on the 0.5th day, 189.32±87.54 vs. 296.21±89.77 on the 2nd day, 99.28±16.13 vs. 168.41±66.78 on the 6th day; iNOS mRNA (gray value): 2.998±0.801 vs. 3.453±0.026 on the 0.5th day, 3.126±0.306 vs. 5.259±0.153 on the 2nd day, 0.841±0.135 vs. 1.225±0.057 on the 6th day; NF-κB mRNA (gray value): 1.569±0.818 vs. 2.361±0.063 on the 0.5th day, 2.345±0.489 vs. 4.668±0.368 on the 2nd day, 2.348±0.316 vs. 3.972±0.449 on the 6th day; caspase-8 (pmol/mg): 126.77±9.97 vs. 199.18±66.48 on the 6th day; caspase-9 (pmol/mg): 213.12±69.06 vs. 321.62±89.39 on the 6th day; caspase-12 (pmol/mg): 183.46±70.52 vs. 219.68±53.93 on the 6th day, all P < 0.05]. Conclusion Crocetin has protective effect on liver in rats with PQ poisoning, which role is related with reducing the blood level of inflammatory factors, inhibiting the hepatic caspase-8, -9, -12 activities and gene expressions of iNOS and NF-κB.
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Objective To observe hepatocellular apoptosis and inflammatory cytokines expression and their mechanisms after paraquat poisoning in rat.Methods Forty Wistar rats were divided into control group (n =8) and model group (n =32) by random number table.Rats in model group were intraperitoneally injected with 30 mg/kg 20％ paraquat concentrate,while those in control group were injected with normal saline.0.5,1,3,7 days after reproduction of the model,8 rats were sacrificed,and blood was collected from inferior vena cava and hepatic tissue was harvested.The serum levels of interleukin-1β (IL-1 β) and tumor necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA).The mRNA expressions of IL-1β,TNF-α,inducible nitric oxide synthase (iNOS) and p53 were determined by reverse transcription-polymerase chain reaction (RT-PCR).Cysteine-containing aspartate-specific proteases (caspase-3,-8,-9,-12) activity in hepatic tissue was determined on the 3rd day with chromogenic substrate method.The liver histopathological changes were observed after hematoxylin-eosin (HE) staining.Results In model group,hepatic tissue showed extensive necrosis with inflammatory cell infiltration in time dependant manner.Serum IL-1β and TNF-α levels were significantly higher in model group half a day after reproduction than those in control group [IL-1β (ng/L):220.13 ± 69.74 vs.0.14 ± 0.03,TNF-α (ng/L):102.66 ± 26.43 vs.0.16 ± 0.02,P＜ 0.01 and P＜0.05],and peaked on the 3rd day and 1st day [IL-1β:(423.72 ± 153.11) ng/L,TNF-α:(690.35 ± 229.64) ng/L].They then decreased gradually,but were still significantly higher than those in control group on the 7th day [IL-1 β:(357.47 ± 87.28) ng/L,TNF-α:(12.39 ± 5.06) ng/L,both P＜0.05].The contents of IL-1β,TNF-α and iNOS mRNA expressions in hepatic tissue were significantly higher than those in control group,and the highest values were seen on the 1st day,the 1st day,and the 3rd day [IL-1β mRNA (gray value):1.569 ± 0.057 vs.0.123 ± 0.016,TNF-α mRNA (gray value):0.683 ± 0.077 vs.0.261 ± 0.025,iNOS mRNA (gray value):3.259 ± 0.135 vs.0.002 ±0.001,P＜0.05 or P＜0.01].There was no difference in p53 mRNA expression between model group and control group at early stage,and both of them showed low expression,and p53 mRNA expression was significantly higher in model group on the 7th day (gray value:2.959 ± 0.086 vs.0.263 ± 0.032,P＜0.01).In model group,caspase activity (pmol/mg) in liver tissue were significantly higher on the 3rd day than those in control group (caspase-3:857.25 ± 309.26 vs.169.73 ± 48.21,caspase-8:199.18 ± 61.41 vs.32.26 ± 11.09,caspase-9:321.62 ± 80.73 vs.90.38 ± 29.76,caspase-12:413.13 ± 89.77 vs.26.73 ± 9.86,all P＜0.01).Conclusion Paraquat can cause acute liver injury in rats,with caspase-3,-8,-9,-12 activities markedly enhanced,and liver injury may be associated with an early high expression of TNF-α,iNOS and p53 gene.
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Objective The aim of this article was to deeply study the effects of different molecular weight of proteins and different structures of chemical substances on the adsorption specificity of HB-H-6 resin.Methods HB-H-6 resin was adopted to adsorb 5 different molecular weight proteins and different structural chemical substances including proteins,saccharides,human serum albumin (HAS),dextran and lipid,and then underwent static adsorption experiments in vitro.The adsorption rates of different structural chemical substances were analyzed from two experiments and the results were compared.Results The experiment results of HB-H-6 resin adsorption showed that the average adsorption rates of 5 different molecular weight proteins,myoglobin (Myo,16 700),ovalbumin (OVA,44 000),HAS (66 200),β-gal (130 000) and IgG (150 000),were significantly different:(0.00±0.33)％,(8.02± 1.23)％,(43.19±2.31)％,(34.25±1.07)％ and (0.00±0.69)％.In the studies on adsorption of different structural chemical substances,the average adsorption rates of different structural chemical substances proteins,saccharides,lipid were significantly different:the absorption rates of plasma total protein,albumin,globulin,glucose,triglyceride and cholesterol groups were:(11.18±0.72)％,(10.74±0.66)％,(11.74± 1.22)％,(7.17±0.12)％,(1.06± 1.04)％,(3.05± 0.65)％.The absorption rates of HAS and dextran groups were:(43.19±2.31)％ and (5.44±1.46)％.Conclusion In conclusion,the proteins' molecular weight of best adsorption condition is from 66 Ku to 130 Ku.The average adsorption rates of different structural chemical substances proteins,saccharides,lipid are significantly different.The average adsorption rates of proteins are higher than that of saccharides and lipid.It shows that HB-H-6 resin has adsorption specificity on different molecular weight proteins and different structural chemical substances.
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Several studies have demonstrated the role of 4-1BBL in T cell activation. Furthermore, enhanced 4-1BB/4-1BBL interaction has been shown to amplify T-cell-mediated antitumor immunity in several mouse models. However, when applied in humans, it was difficult to generate sufficient T cells ex vivo and whole cell vaccines to transfer back into patients. To overcome this difficulty, we have focused on producing the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein. In this report, PCR and overlap PCR were used to construct the human 4-1BBL extracellular domain/anti-CD20 Fab' expression vector. DNA sequence was analyzed by the Terminus of Dideoxy Nucleotide. The product was purified by affinity chromatography and analyzed by SDS-PAGE and HPLC; its antigen binding activity was examined by rosetting assay. The data of DNA sequence showed that the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein was corrected. The fusion protein was recovered in high yield (up to 200 microg/mL) after E-taq purification. The fusion protein was capable of simultaneous binding to stimulated Jurkat cells and Raji cells as shown by cellular rosetting. In conclusion, the human 4-1BBL extracellular domain/anti-CD20 Fab' fusion protein was induced to express in E. coli 16C9. The results of some biological activity experiments indicated that the fusion protein could bind to stimulated Jurkat cells and Raji cells. Furthermore, 4-1BBL-negative tumors can be converted into 4-1BBL-positive tumors by the fusion protein without the need for 4-1BBL gene transfer to the malignant cells.
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Humanos , Ligante 4-1BB , Genética , Anticorpos Biespecíficos , Alergia e Imunologia , Antígenos CD20 , Alergia e Imunologia , Fragmentos Fab das Imunoglobulinas , Genética , Imunoterapia , Métodos , Linfoma não Hodgkin , Terapêutica , Proteínas Recombinantes de Fusão , Genética , Alergia e ImunologiaRESUMO
Objective To observe hepatocellular apoptosis and inflammatory cytokines expression and their mechanisms for lipopolysaeeharide (LPS)-induced acute liver failure in D-ga|actosamine (D-GalN)-sensitized rats. Methods Fifty-six rats were randomly divided into following groups: 0, 1, 2, 4, 6, 24 and 48 hours. 0 hour group served as control group and the rest did as treatment groups. The rats in the treatment groups received intraperitoneal injections of LPS (50 ng/g) and D-GaIN (300 μg/g) dissolved in 1 mL sterile 0.9% sodium chloride solution, while the rats in control group were treated with 1 mL sterile 0.9% sodium chloride solution only. The rats were sacrificed in the corresponding time points and their sera and liver tissues were collected. Liver tissues were fixed and stained with hematoxylin and eosin for optical microscopy examination. The serum cytokine expressions were detected by enzyme-linked immunosorbent assay (ELISA). The expressions of tumor necrosing factor (TNF)-α, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS) and p53 gene were detected by reverse transcriptase-polymerase chain reaction, and the 24 hours treated rats liver Caspase-3,8,9,12 activity were detected by chromogenie substrate method. Data for the experiments were expressed as x±s, and differences among means were compared using the analysis of variance. Results After drug treatment, liver tissues showed piecemeal necrosis and inflammatory cell infiltration, which significantly increased from 6 hours, 24 hours to 48 hours. The 1 hour treatment group with the highest concentration of TNF-α (727. 8 ± 261. 3) ng/L were significantly higher than the control group and other treatment groups(F= 49.82, P<0.01), 2 hours treatment group (156.4 ± 52.2) ng/L was significantly lower than the 1 hour group, but significantly higher than the control group (F = 30. 23, P< 0.01 ). But serum concentrations of IL-1β gradually increased, reaching the highest level in 24 hours group (360.5±121.6)ng/L (F= 18. 61, P<0. 01). Liver Caspase-3,8,9, 12 activity in 24 hours treatment group was significantly higher than in the control group (F= 84.96, P<0.01). The mRNA expression of iNOS gene, which were not detected in normal controls, reached the peak at 6 hours group after drug treatment and notably dropped in 24 hours and 48 hours groups(F=34.07,P<0.01), p53 gene expression significantly upregulated at 24 hours and 48 hours groups(F=37.43,P<0.01). TNF-α and IL-1β gene expression in the treatment group were higher than in the control group(F=2.94,P<0.05), and both reached the peak at 1 hour treatment group. Conclusions Acute liver failure can be induced by low dose LPS in D-GaiN-sensitized rats. One of the features changes is that Caspase-3,8,9,12 activities are markedly enhanced, and the occurrence of liver injury may be associated with the early high expression of TNF-α, iNOS and p53 gene.
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Wilms tumor(WT) is one of the most common renal malignancies in children.Although several genetic loci such as the WT1,WT2,p53 and ?-catenin genes have been considered to be associated with WT,the causes of the tumor are still unknown.Recently the US researchers have identified a new tumor suppressor gene that is mutated in WT.The biological function of the protein encoded by WTX is yet unknown,however,the gene's location in the X chromosome is of particular interest.This review highlights the current study of the gene mutated in Wilms tumor.