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ABSTRACT:Objective To explore the role of HIF‐1αin the pathogenesis of hepatopulmonary syndrome (HPS) and its relationship with GRP78 .Methods The HPS model in rats was induced by multiple pathogenic factor .The samples were assessed by using Western blotting analysis for HIF‐lα, GRP78 and VEGF164 . The expressions of VEGFR‐2 and CD105 were observed by using immunohistochemical staining .Results The protein level of HIF‐1αwas significantly increased in HPS group at week 8 compared with that at week 4 and 6 groups and corresponding normal control groups .With the development of HPS ,protein level of GRP78 was gradually increased at each time point significantly and reached the highest level at week 8 ;protein level of VEGF164 showed a similar change with GRP78 ,but the peak was at week 6 .Immunohistochemical results showed that the protein expressions of VEGFR‐2 and CD105 were gradually increased in lung tissue as HPS progressed .The protein level of GRP78 was positively correlated with HIF‐1α,VEGF164 ,VEGFR‐2 and CD105 ,respectively (P<0 .05) .Conclusion HIF‐1αis most likely together with GRP78 to play a critical role in promoting pulmonary microvascular remodeling in the pathogenesis of HPS in rats .
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Objectives To explore the relativity between Alzheimer ’ s disease ( AD)-like lesions and metabolic syndrome models induced by high-sugar high-fat diet in rats.Methods Forty-eight Sprague Dawley rats were randomly divided into 2 groups.The control group (fed with normal diet, 12 rats) and high sugar and high fat group (fed with high-sucrose and high-fat diet, 12 rats) continuously for 12 months.At the end of 6, 9 and 12 months of the experiment , we observed the animal body weight and visceral fat weight .The blood lipid levels , blood glucose and MS-related biochemical parameters were determined . The brain tissues were examined by histopathology . The characteristic AD molecules hippocampus Aβand Tau were detected using ELISA and Western blotting to confirm the presence of AD lesions in the brain.Results Compared with the normal control group , the body weight and visceral fat weight of the rats in the high-sugar high-fat groups were significantly increased; the levels of TG , FPG, LDL, HOMA-IR and hippocampus Aβ,phosphorylated Tau (p-Tau) were higher, but the level of HDL was decreased (P<0.05 for all).The histopathological examination revealed inflammatory cell infiltration in the brain tissues .Conclusions Characteristic AD-like lesions may occur and accompany the rat models of metabolic syndrome , induced by high-sugar high-fat diet, and provide a new idea for the construction of Alzheimer ’ s disease animal models .
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Objective To evaluate the effect of anti-histamine treatment on intestinal endotoxemia and liver inflammation in experimental chronic hepatitis rats.Methods Thirty Wistar rats (15 males and 15 females) were randomly divided into control group (n =8),chronic hepatitis group (n =12) and hepatitis + anti-histamine group (n =10).Chronic hepatitis was induced by subcutaneous injection with 40％ of CCl4,and feeding with low protein,low choline,high cholesterol and high alcohol diet.Antihistamine treatment was given 1 week after the modeling by intragastric administration of ketotifen (1.25 mg/kg).All rats were sacrificed 4 weeks later.Plasma endotoxin,alanine aminotransferase (ALT),total bilirubin (TBil),tryptase,histamine,interferon-γ (IFNγ),iuterleukin (IL)-12,IL-10 and IL-4levels were detected,and the changes in liver histology,the morphology and ultrastructure of mast cells were observed.SPSS 13.0 software package was used for statistical analysis.ANOVA was used for the comparison of measurement data,and SNK method was used for pairwise comparison.Results Plasma endotoxin,ALT,TBil,tryptase,plasma and liver tissue histamine concentrations were (81 ± 19) pg/mL,(186 ± 140) U/L,(10.2±6.2) μmol/L,(0.75 ±0.21) mg/mL,(145 ±52) ng/mL,and (107 ±43) ng/100 mg in chronic hepatitis group,while the above parameters were significantly lower in anti-histamine group except TBil (P ＜ 0.05).Under light microscope,fatty degeneration and fibrosis were formed in liver of chronic hepatitis rats,the hepatic injury was attenuated in anti-histamine group.Toluidine blue stain showed that there was many degranulating and degranulated mast cells filled with purple granula around liver blood vessels and in fiber-interval in chronic hepatitis group,and there were few purple granula in anti-histamine group.The number of mast cells in anti-histamine group was (6.5 ± 1.5)/HP,which was significantly lower than chronic hepatitis group [(10.9 ± 1.6)/HP,P =0.000],but was still higher than that in the control group [(2.2 ± 0.9)/HP,P =0.000].Under electron microscope,the phenomenon of degranulation was severe in chronic hepatitis group and moderate in the anti-histamine group.Compared with the chronic hepatitis group,IL-4 and IL-10 in anti-histamine group were significantly decreased (P ＜0.05),IL-12 was increased (P ＜0.05),but the level of IFN-γ had no significant change (P ＞ 0.05).Conclusion Anti-histamine therapy can significantly improve liver inflammation and alleviate intestinal endotoxemia.
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Objective To investigate the changes of Th1/Th2 cytokines and its relationship with lipopolysaccharide (LPS) in pretreatment of relieving nonalcoholic steatohepatitis (NASH).Methods The 24 male Wistar rats were randomly divided into 3 groups: normal control group, liver injury group and LPS pretreatment group. The rats were given normal diet in normal control group,high-sucrose and high-fat diet both in liver injury group and in LPS pretreatment group, and the rats in LPS pretreatment group were given hypodermic injection of LPS 0. 5 mg/kg every other day. The level of plasma endotoxin (ET), activity of alanine aminotransferase (ALT), content of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) were determined. At the end of week 9, the rats were executed, and the liver tissue slices were prepared to investigate hepatic pathologic change by hematoxylin and eosin (HE) staining.Results The level of plasma ET was significantly higher in liver injury group than in normal control group. The level of plasma ALT and infiltrating lymphocytes in liver tissue were significantly lower in LPS pretreatment group than in liver injury group. The level of plasma TNF-α was significantly lower in LPS pretreatment group compared with liver injury group.In contrast, the level of plasma IL-10 was higher (P＜0. 05). Histology with HE staining showed that hepatocyte steatosis was obviously relieved with smaller lipid droplet in LPS pretreatment group than in liver injury group. Conclusions LPS pretreatment can alleviate high-sucrose and high-fat induced NASH. The disequilibrium of Th1/Th2 cytokines may be an important part of mechanism.
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Objective To investigate the relationship between dendritic cell (DC)and intestinal endotoxemia in patients with chronic hepatitis B (CHB).Methods Peripheral blood were collected from CHB patients (n = 80)and healthy controls (n = 21 ).Plasma endotoxin (ET)levels,liver function (alanine transaminase,total bilirubin)were detected.According to plasma ET concentration,all CHB patients were divided into two groups:ET positive and ET negative.The peripheral blood mononuclear cells (PBMCs)were isolated and then cultured with recombinant human granulocyte-macrophage colony-stimulating factor ( rhGM-CSF),recombinant human interleukin-4 ( rhIL-4 ),FMS-related tyrosine kinase 3 ligand (Flt3L)and tumor necrosis factor-alpha (TNF-α)to derive DC.The phenotypic patterns were characterized by flow cytometry.The proliferation of T lymphocytes was evaluated with mixed leukocytes reaction (MLR)and the levels of IL-12 and interferon-γ (IFN-γ)produced by DC were analyzed with enzyme-linked immunosorbent assay (ELISA).Comparisons among the two groups and healthy control group were done by single factor analysis of variance.Results Compared to healthy controls,the expressions of CD83,CD80,CD86,human leucocyte antigen (HLA)-DR and the proliferation of allogeneic T lymphocytes by DC were all significantly reduced in CHB patient groups.The expressions of CD83,CD80,CD86,HLA-DR and the activation of proliferation in ET positive subjects were lower than those in ET negative subjects [CD83 (8.25±3.63)% vs(11.39±4.35)% ,CD80 (10.63±4.52)% vs (13.56±5.13)%,CD86 (36.61±16.16)% vs (45.90±15.35)%,HLA-DR (61.65±14.33)% vs (70.35±18.89)%,the activation of proliferation0.812±0.311 vs 1.153±0.324; F=5.123,4.213,3.714,3.323 and 3.125,respectively; all P＜0.05].After cultured for 9 days,the secretions of IL-12 and IFN-γ by DC were significantly lower in CHB patients than in healthy controls [IL-12 (16.99± 6.74)pg/mL vs (44.51±14.56)pg/mL,IFN-γ (10.52±4.19)pg/mL vs (17.94±5.86)pg/mL].The level of IL-12 in the ET positive group was significantly lower than that ET negative group [( 13.14 ±5.71)pg/mL vs (20.98 ± 9.03)pg/mL; F= 3.225,P = 0.016].The level of IFN-γ was not different between two groups [(9.46 ± 3.24)pg/mL vs (11.54 ± 5.20)pg/mL; F = 2.003,P =0.076].Conclusion The intestinal endotoxemia may play a role in DC dysfunction in CHB patients.
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AIM: To investigate that nicotine inhibits HMGB1 expression and release in RAW264.7 cells.METHODS: (1) RAW264.7 cells were cultured in 6 wells plate, treated with 250 μg/L LPS and 1 μmol/L or 10 μmol/L nicotine, in which the cells treated with or without 250 μg/L LPS were regarded as nicotine 1 group (N1), nicotine 2 group (N2), LPS group (LPS) and control group (C), respectively. HMGB1 protein in the cell culture media and in cell nuclear was examined by Western blotting and the cellular HMGB1 mRNA level was detected by RT-PCR. (2) Transfected with antisense RNA or sense RNA of α~7 subunit-containing nicotinic receptor (α~7nAChR), RAW264.7 cells were treated with 250 μg/L LPS and 10 μmol/L nicotine, HMGB1 protein in the culture media was also tested by Western blotting.RESULTS: (1) HMGB1 mRNA level in C group was low (1 659.20±121.05) and no significant statistical difference among groups of N1, N2 and LPS was observed (P>0.05). (2) Higher HMGB1 accumulation in the cell culture media was detected in LPS group (445.34±28.52) than that in C group. Compared to LPS group, both N1 and N2 groups distinctly attenuated HMGB1 accumulation in culture media (P<0.05). (3) Nuclear HMGB1 accumulation was lower in LPS group than that in C group, and two different nicotine concentrations markedly increased the nuclear HMGB1 accumulation compared to LPS group (P<0.05). (4) No significant difference of HMGB1 levels in culture media between antisense RNA group and LPS group was observed (P>0.05). In sense RNA group, however, HMGB1 level was observably reduced compared to antisense group (P<0.05).CONCLUSION: The present results suggest that nicotine dramatically inhibits RAW264.7 cell nuclear HMGB1 translocation and extracellular release, and this effect relies on α~7nAch receptor expression.
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Objective To investigate the effects of Shuangligan and Glycine on Thl/Th2 balancing on severe acute pancreatitis ( SAP) in rats. Methods Thirty-two Wistar rats weighing (260 ± 20) g were randomly divided into sham operation (SO) group, SAP group, SAP + Slg (with the treatment by Shuangligan) group and SAP + Gly (with the treatment by Glycine ) group. Each group included 8 rats, which accepted different treatment according to the experimental design. Changes of plasma level of endotoxin ( ET) and serum amylase (AMY) and the effects of Shuangligan and Glycine on Thl/Th2 ratio at the 24th hour after operation were observed respectively. Results The plasma endotoxin (ET) level ( (0. 67 ±0. 11) EU/ml),proinflammatory cytokine (INF-γ:(8.43 ± 0.86) ng/L, IL-12: (8.26 ± 1.97) ng/L) and Thl/Th2 ratio (0.36 ± 0.07) in SAP group were significantly higher than those in SO group( ET: (0. 44 ±0.07) EU/ml, INF-γ: (3. 80 ±0. 55) ng/L, IL-12: (3. 34 ± 1. 34)ng/L,Thl/Th2 ratio (0. 24 ±0. 05) ) (P <0. 05). Compared with SAP group, SAP + Slg and SAP + Gly group had remarkably decreased plasma ET level ( (0. 57 ± 0. 08,0. 52 ± 0. 04) EU/ml) (P < 0. 05) and the Thl/Th2 ratio reached equilibrium ( SAP + Slg group; (0. 29 ± 0. 04 ), SAP + Gly group: (0. 25 ± 0. 06 )) . Conclusions In the earlier stage of SAP, the rising plasma ET level may cause the overreaction of the cell mediated immune response, which leads to the aggravated damages in tissue cells. Our data indicates that Shuangligan and Glycine can restrain the formation of intestinal endotoxemia and alleviate or prevent the tissue injuries.
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<p><b>OBJECTIVE</b>To observe the effect of glycine on the expression of CD(14) mRNA and protein of hepatic tissue in the course of developing cirrhosis of rats.</p><p><b>METHODS</b>The cirrhotic model of Wistar rats was established by complex pathogens, who were respectively fed with control diets and control diets adding glycine (1g/d, giving by intragastric infusion) or 5% glycine containing diets at the same time. The rats were sacrificed at 2, 4, and 8 weeks, respectively. Hepatic tissues were collected to measure the expression of CD(14) mRNA and CD(14) protein by the reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot analysis.</p><p><b>RESULTS</b>The expression of CD(14) mRNA and CD(14) protein in the hepatic tissue of fatty liver and cirrhotic rats fed with diets containing glycine was weaker than their control groups, and the expression of CD(14) mRNA and protein was the weakest in 8 weeks cirrhotic rats fed with the diets.</p><p><b>CONCLUSIONS</b>Glycine can markedly downregulate the expression of CD(14) mRNA and CD(14) protein in hepatic tissues of cirrhotic rats.</p>
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Animals , Male , Rats , Disease Models, Animal , Gene Expression , Glycine , Pharmacology , Lipopolysaccharide Receptors , Genetics , Liver Cirrhosis , Genetics , Metabolism , RNA, Messenger , Rats, Wistar , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Objective To investigate the effect of hepatic microcirculatory disturbance induced by intestinal endotoxemia in liver injury. Methods The model of rats with intestinal endotoxemia induced by thioacetamide(TAA) was established. 25 Male Wistar rats were divided into 4 groups as normal control group (N), Heparin control group(H), TAA treated group(T), and TAA + heparin treated group(T +H). The changes of plasma biochemistry and hepatic histopathology were measured. Results The plasma endotoxin level, alanine transaminase (ALT) activity and malondialdehyde (MDA) content in TAA treated rats were markedly higher than that in the control ones (P < 0.05), while plasma endotoxin level was lower ( P > 0.05) and ALT as well as MDA were decreased significantly ( P < 0.05) in TAA + heparin group than in TAA group. Conclusion Intestinal endotoxemia could induce disturbance of hepatic microcirculation, which results in ischemia and hypoxia of liver cell. Heparin could not only correct disturbance of hepatic microcirculation induced by intestinal endotoxemia, but also reduce liver injury induced by ischemia and hypoxia.
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AIM:To investigate LPS(lipopolysaccharide)stimulated cytokine secretion from normal rat kupffer cells in vitro. METHODS: Kupffer cells were isolated from wistar rats liver and cultured. Tumor necrosis factor - α (TNF- α) and endothelin- 1 (ET- 1 ) secreted by LPS stimulated kupffer cells were detected. RESULTS: LPS had an stimulative effect on kupffer cell activity. LPS in definite concentrations promoted kupffer cell secretion. CONCLUSION: LPS promotes kupffer cell secretion, which may be associated with liver injury induced by LPS.
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AIM: The goal of the present study was to evaluate the possibility about enterogenous endotoxemia in pathogenesis of hepatopulmonary syndrome. METHODS: The rat model of cirrhosis was prepared with compound factors. A small dose of lipopolysaccharide (LPS) was administered intraperitoneally once to aggravate endotoxemia of animal with cirrhosis. The normal rats injected with LPS or injected with LPS combined with glycine (LPS antagonist) were designed as controls. RESULTS: Hepatopulmonary syndrome of rats with cirrhosis had occurred in the end of eighth weeks. Pulmonary pathological changes of cirrhosis rats were exacerbated after administration of a given dose of LPS. Glycine sharply antagonized the biological effect of LPS in vivo and in vitro, inhibited the production of TNF-? by LPS and alleviated various pathological changes of hepatopulmonary syndrome. CONCLUSIONS: Enterogenous endotoxemia in cirrhosis rats might be an important mechanism in the development of hepatopulmonary syndrome. Endotoxin and its mediating effect by way of cytokines (TNF-?) may play a role in the pathogenesis of hepatopulmonary syndrome.
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AIM: To examine the effects of inhibition of Kupffer cell and splenectomy on intestinal endotoxemia and hepatic injury. METHODS: The hepatic injury model was established by treatment with thioacetamide (TAA). At the same time, inhibition of Kupffer cells by intravenous GdCl_3 and splenectomy were performed. Serum alanine aminotransferase (ALT), TNF-?, endotoxin content and phagocytic index were observed. RESULTS: In the TAA+GdCl_3 group, and TAA+splenectomy group, the endotoxin content was significently higher than that in normal and TAA group (P
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AIM: To investigate the effects of nitric oxide (NO) on hepatic encephalopathy in cirrhotic rats induced by LPS. METHODS: The cirrhotic model of rats was established by complex pathogeny. Since the end of the 8 th week, the rats were intragastrically-infused with 0.9% salt, L-arginine(L-arg) and LNNA respectively for 2 weeks.The hepatic encephalopathy in cirrhotic rats were induced by 3 mg/kg LPS (ip) 4 hours before the rats were sacrificed. RESULTS: The normal behaviors and electroencephalograph were appeared in L-arg group. LNNA group showed hepatic encephalopathy. The content of NO-_2/NO-_3 of brain tissue was markedly higher in L-arg group than LNNA group(P
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AIM:To investigate LPS(lipopolysaccharide)stimulated cytokine secretion from normal rat kupffer cells in vitro. METHODS: Kupffer cells were isolated from wistar rats liver and cultured.Tumor necrosis factor -? (TNF-?) and endothelin-1(ET-1) secreted by LPS stimulated kupffer cells were detected. RESULTS: LPS had an stimulative effect on kupffer cell activity. LPS in definite concentrations promoted kupffer cell secretion.CONCLUSION: LPS promotes kupffer cell secretion, which may be associated with liver injury induced by LPS.
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0.05).Both HMGB1 mRNA expression and HMGB1 protein level were remarkably higher in LPS treatment group than that in control group(P0.05).CONCLUSION:RPM inhibits HMGB1 expression not only by directly suppressing STAT3 activation,but also by indirectly reducing TNF-? level.
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AIM:To study the effect of intestinal endotoxemia(IETM) on hepatic energy metabolism in acute liver failure. METHODS:Intoxication by thioacetamide (TAA) was used to establish rat model of acute liver injury.Ketone body(acetoacetate and ?-hydroxybutyrate) in arterial blood and ATP content of hepatocellular mitochondria were determined by using enzymatic fluorimetric micromethod.Colectomy was adopted in observing the changes in plasma endotoxin content and serum alanine aminotransferase (ALT) activity. RESULTS:In the TAA group,plasma endotoxin content and serum ALT activity were all significantly higher than those in the control group(P
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AIM: To investigate the effect of LPS on phagocytosis of Kupffer cells in vitro. METHODS: Isolated Kupffer cells were treated with LPS in vitro . The phagocytosis, microfilament, microtubules and apoptosis of Kupffer cells were determined by the beads phagocytosis test, fluorescence staining, fluorometry and flow cytometric analysis. RESULTS: LPS enhances the phagocytosis, actin content, microtubules fluorescence density of Kupffer cells in vitro , while at a large dose or for a long time, it lessened the phagocytosis increasing or phagocytosis, inhibites the microfilament and microtubules expression, and induced apoptosis. CONCLUSION: LPS enhances the phagocytosis of Kupffer cells in vitro , but in large amount, it inhibites the phagocytosis of Kupffer cells, which is probably related to LPS -induced microfilament, microtubules expression changes and apoptosis in Kupffer cells.
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AIM:The objective of the study was to explore whether intestinal endotoxemia participate in the development of Alzheimer disease.METHODS:Adult Wistar rats were subjected to 90 days intraperitoneal injection with D-galactose and aluminum trichloride(AlCl3) to establish the model of Alzheimer disease.After the administration,the study and memory ability in the rats were observed by Morris water maze.The level of lipopolysaccharide(LPS) in the sera of Alzheimer disease's rats was determined by tachypleus amebocyte lysate method.The level of tumor necrosis factor-?(TNF-?) and interleukin-1(IL-1) in the sera were determined by radioimmunoassay.The expressions of amyloid ?-protein precursor(APP),presenilin 1(PS1) and ?-site APP-cleaving enzyme(BACE) in hippocampus were detected by RT-PCR.RESULTS:Compared with the normal control,the level of LPS in the sera and the expressions of APP,PSI,BACE mRNA in the hippocampus were markedly increased(P
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AIM: To investigate the correlation of intestinal endotoxemia (IETM), histaminemia and cellular immune function in the patients with hepatitis B. METHODS: Peripheral blood was collected from patients with chronic hepatitis B (n=80) and healthy individuals (n=18). According to plasma endotoxin concentration, total patients were divided into two groups: ET positive and ET negative. Serum IL-10, IL-12, IFN-?, IL-2, IL-4 concentrations were detected. In addition, the serum histamine (HA), tryptase (TS) and AP50 levels were studied. RESULTS: Compared to control group, the concentrations of IL-4 and IL-10 were increased, but IL-12 and IFN-? were decreased obviously in total patients (P
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AIM:To investigate the effect of lipopolysaccharides(LPS)preconditioning on CCl4-induced liver injury and the change of LPS signal transduction.METHODS:The male Wistar rats were divided randomly into liver-injury group,which were injected with CCl4 5 mL/kg first,three days later were injected 0.3 mL 40% CCl4 and 60% olive oil. Animals in LPS preconditioning group were injected with LPS 0.5 mg/kg before the day CCl4 was given. Rats received high fat diet were as liver injury group,and normal control group received normal diet. The lymphocytes infiltrated in the liver tissue were counted. The endotoxin and ALT level in rat plasma,TNF-? content and expressions of TLR4,p38,p-p38,I??,NF-?? in the rat livers were also determined.RESULTS:The lymphocytes in liver slice and ALT level of the plasma in LPS preconditioning group were lower significantly than those in the liver injury group,and the expressions of TLR4,p-p38,NF-?? in the liver were the same. In contrast,the expression of I?? was higher.CONCLUSION:LPS preconditioning relieves obviously CCl4-induced chronic liver injury. The mechanism may be associated with change of signal transduction of LPS,which results in decrease of pre-inflammatory cytokines.