Serum sIL-2R, TNF-α and IFN-γ in alveolar echinococcosis

AIM: To approach the relationship between alveolar echinococcosis (AE) pathology and level of sIL-2R,TNF-α and IFN-γ in sera and the significance of cytokines in development of AE.METHODS: After 23 patients with AE were confirmed by ELISA and ultrasound, their sera were collected and the concentrations of sIL-2R,TNF-α and IFN-γ were detected by double antibody sandwich. Twelve healthy adults served as controls. According to the status of livers of AE patients by ultrasound scanning, they were divided into 4 groups: P2,P3, P4 groups and C group (control). Average of concentrations of sIL-2R, TNF-α and IFN-yin homologous group was statistically analyzed by both ANOV and Newman-Keuls, respectively.RESULTS: The mean of sIL-2R in P2 group was 97±29, P3:226±80, P4:194±23 and control group (111±30)×10^3 u/L(P<0.01). The mean of TNF-α in P2 group was 1.12±0.20, P3:3.67±1.96, P4:1.30±0.25 and control group 0.40±0.19 μg/L(P<0.01). The mean of IFN-γ in P2 group was 360±20, P3:486±15, P4:259±19 and control group: 16±2 ng/L (P<0.01).Judged by ANOV and Newman-Keuls, the mean concentrations of sIL-2R, TNF-α and IFN-γ had a significant difference among groups. Except for P2group, the mean sIL-2R between other groups of AE patients had a significant difference (P<0.05). The mean of TNF-α concentration in P3 group was the highest (P<0.01). The mean of IFN-γ concentration in all patients was higher than that in control group (P<0.01),but there was no difference between AE groups (P>0.05).CONCLUSION: Low sIL-2R level indicates an early stage of AE or stable status, per contra, a progression stage. Higher level of TNF-α might be related to the lesion of liver. The role of single IFN-γ is limited in immunological defense against AE and it can not fully block pathological progression.     

Expression and significance of nuclear factor κB p65 in colon tissues of rats with TNBS-induced colitis

AIM: To investigate the role of NF-κB in the pathogenesis of TNBS-induced colitis in rats. METHODS: Thirty-two healthy adult Sprague-Dawley (SD) rats were randomly divided into four groups of eight each: normal, NS, model Ⅰ, model Ⅱ groups in our study. Rat colitis model was established through 2-,4-,6-trinitrobenzene sulfonic acid (TNBS) enema. At the end of four weeks, the macroscopical and histological changes of the colon were examined and mucosa myeloperoxidase (MPO)activities assayed. NF-~B p65 expression was determined by Western blot assessment in cytoplasmic and nuclear extracts of colon tissue, and the expressions of TNF-α (and ICAM-1 protein in colon tissue were examined by immunohistochemistry. The relativities between expression of NF-κB p65 and other parameters were analyzed. RESULTS: TNBS enema resulted in pronounced pathological changes of colonic mucosa in model Ⅱ group (macroscopic and histological injury indices 6.25±1.39 and 6.24±1.04, respectively), which were in accordance with the significantly elevated MPO activity (1.69+0.11). And the nuclear level of NF-κB and expression of TNF-α, ICAM-1 in rats of model Ⅱ group were higher than that of normal control(9.7±1.96 vs 1.7±0.15, 84.09±14.52 vs 16.03±6.21,77.69±8.09 vs 13.41±4.91 P<0.01), Linear correlation analysis revealed that there were strong correlations between the nuclear level of NF-κB and the tissue positive expression of TNF-α and ICAM-1, MPO activities, macroscopical and histological indices in TNBS-induced colitis, respectively (r = 0.8235, 0.8780, 0.8572, 0.9152,0.8247; P<0.05). CONCLUSION: NF-κB plays a pivotal role in the pathogenesis of ulcerative colitis, which might account for the up-regulation the expression of TNF-α and ICAM-1.     

Expression of TNF-α and VEGF in the esophagus of portal hypertensive rats

AIM: To investigate the expression of tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF) in the development of esophageal varices in portal hypertensive rats. METHODS: Thirty male Sprague-Dawley (SD) rats in the model group in which a two-stage ligation of portal vein plus ligation of the left adrenal vein was performed, were divided into three subgroups (M7, M14, and M21) in which the rats were kiued on the seventh day, the 14^th d and the 21 d after the complete portal ligation. Thirty male SD rats, which underwent the sham operation in the control group, were also separated into three subgroups (C7, C14 and C21) corresponding to the models. The expression of TNF-α and VEGF in the esophagus of all the six subgroups of rats were measured with immunohistochemical SP technique. RESULTS: The portal pressure in the three model subgroups was significantly higher than that in the corresponding control subgroups (23.82±1.83 vs 11.61±0.86 cmH2O, 20.90±3.27 vs 11.43±1.55 cmH2O and 20.68±2.27 vs 11.87±0.79 cmH2O respectively, P<0.01), as well as the number (9.3±1.6 vs 5.1±0.8, 11.1±0.8 vs 5.4±1.3 and 11.7±1.5 vs 5.2±1.1 respectively, P<0.01) and the total vascular area (78 972.6±3 527.8 vs 12 993.5±4 994.8 um^2, 107 207.5±4 6461.4 vs 11 862.6±5 423.2 um^2 and 110 241.4±49 262.2 vs 11 973.7±3 968.5 um^2 respectively, P<0.01) of submucosal veins in esophagus. Compared to the corresponding controls, the expression of TNF-α and VEGF in M21 was significantly higher (2.23±0.30 vs 1.13±0.28 and 1.65±0.38 vs 0.56±0.30 for TNF-α and VEGF respectively, P <0.01), whereas there was no difference in M7 (1.14±0.38 vs 1.06±0.27 and 0.67±0.35 vs 0.50±0.24 for TNF-α and VEGF respectively, P>0.05) and M14 (1.20±0.25 vs 1.04±0.26 and 0.65±0.18 vs 0.53±0.25 for TNF-α and VEGF respectively, P>0.05). And the expression of TNF-α and VEGF in M21 was significantly higher than that in M7 (2.23±0.30 vs 1.14±0.38 and 1.65±0.38 vs 0.67±0.35 for TNF-α and VEGF respectively, P<0.01) and M14 (2.23±0.30 vs 1.20±0.25 and 1.65±0.38 vs 0.65±0.18 for TNF-α and VEGF respectively, P<0.01), but there was no difference between M7 and M14 (1.14±0.38 vs 1.20±0.25 and 0.67±0.35 vs 0.65±0.18 for TNF-α and VEGF respectively, P >0.05). CONCLUSION: In the development of esophageal varices in portal hypertensive rats, increased TNF-α and VEGF may be not an early event, and probably play a role in weakening the esophageal wall and the rupture of esophageal varices.     

Therapeutic effects and molecular mechanisms of anti-fibrosis herbs and selenium on rats with hepatic fibrosis

AIM:To study the therapeutic effects of anti-fibrosis herbs and selenium on hepatic fibrosis induced by carbon tetrachloride (CCl4) in rats and the underlining molecular mechanisms.METHODS:Fifty-three Wistar rats were randomly divided into:normal control group, model control group, colchicine group, anti-fibrosis herbs group (AF group) and anti-fibrosis herbs plus selenium group (AS group).The last four groups were administered with CCl4 at the beginning of experiment to induce hepatic fibrosis. Then colchicine, anti-fibrosis herbs and selenium were used to treat them. The normal control group and the model control group were given normal saline at the same time. At the end of the 6^th week, rats in each group were sacrificed. Blood and tissue specimens were taken. Serum indicators (ALT, AST, HA, LN) were determined and histopathological changes were graded. Lymphocyte CD4 and CD8 were examined by flow cytometry. Expression of TGF-β1 and NF-κB was detected by immunohistochemistry and expression of TGF-β1 mRNA was detected by semiquantified RT-PCR.RESULTS: Histological grading showed much a smaller degree of hepatic fibrogenesis in AS group and AF group than that in colchicine group and model control group.The serum content of ALT, AST, HA and LN in AF group and AS group were significantly lower than that in colchicine group (ALT:65.8±26.5, 67.3±18.4 and 96.2±20.9 in AF, AS and colchicine groups respectively; AST: 150.8±34.0, 154.6±27.3 and 215.8±24.6 respectively; HA: 228±83, 216±58 and 416±135 respectively; LN:85.9±15.0, 80.6±18.6 and 106.3±14.2 respectively) (P<0.05). The level of CD4 and CD4/CD8 ratio in AF group and AS group was significantly higher that those in cochicine group (CD4:50.8±3.8, 52.6±3.4 and 40.2±2.1 in AF, AS and colchicine groups respectively;CD4/CD8 ratio:1.45,1.46 and 1.26, respectively (P<0.05).The expression level of NF-κB and TGF-β1 in the liver tissues of AF and AS treatment groups was markedly decreased compared with that in cochicine group, and TGF-β1 mRNA was also markedly decreased (1.07±0.31 and 0.98±0.14 vs2.34±0.43, P<0.05).CONCLUSION: Anti-fibrosis herbs and selenium have beneficial effects on hepatic fibrosis in rats by enhancing immunity and inhibiting NF-κB and TGF-β1 expressions.     

Hepatic preconditioning of doxorubicin in stop-flow chemotherapy：NF-kB／IkB-α pathway and expression of HSP72

AIM: To provide hepatic protection through administration of doxorubicin before stop-flow chemotherapy (SFC) and to investigate the expression of heat shock protein 72(HSP72) and role of nuclear factor kappa B (NF-kB) in this effect.METHODS: The hepatic preconditioning of doxorubicin was established in a porcine model by injection of doxorubidn(1 mg/kg) before SFC. The experimental animals were randomized into two groups: groups receiving doxorubicin(DOX) and normal saline (NS). Serial serum and tissue samples were taken from both groups to evaluate the protection of doxorubicin. Western blot and immunoprecipitation were applied to detect the expression of HSP72, NF-kB p65 protein, inhibitor kB-α (IkB-α) and phosphorylated IkB-α as well. The expression of tumor necrosis factor α (TNF-α) was estimated by semiquantitative RT-PCR. And the extent of the hepatic injury was estimated with the level of serum aminotransferases.RESULTS: An abundance production of HSP72 in porcine liver was observed after 24 h of intravenous administration of doxorubicin, but without any change in the expression of NF-kB p65 subunit in cytoplasm. NF-kB p65 subunit accumulated in nuclei at the end of SFC and reached its highest level at 30 rain after the restoration of the abdominal circulation and decreased gradually during the 6 h after SFC in NS group, while there was little change in DOX group. There was also a slight decrease of IkB-α at 30 min after the restoration of the abdominal circulation in NS group accompanying with the appearance of phosphorylated IkB-α. The expression of TNF-α was significantly higher in NS group than that in DOX group (average 1.40±0.17 vs 0.62±0.22, P<0.01) at serial time points after SFC. Serum ALT and AST levels of NS group were higher after 24 h than those of DOX group (93.2±7.8 IU/L vs 53.34±13.9 IU/L,217.0±29.4 IU/L vs 155.0±15.6 IU/L for ALT and AST respectively, P<0.05) and alter 48 h than those of DOX group (66.6±18.1 IU/L vs 43.3±16.7 IU/L, 174.4±21.3 IU/Lvs 125.7±10.5 IU/L for ALT and AST respectively, P<0.05).CONCLUSION: Doxorubicin renders the liver to be tolerant to the hepatic influence in SFC in a porcine model through the NF-kB/IkB-α pathway with the expression of HSP72.     

Effects of cholesterol on proliferation and functional protein expression in rabbit bile duct fibroblasts

AIM:To investigate the effect of cholesterol (Ch) on the growth and functional protein expression of rabbit bile duct fibroblasts.METHODS:The cultured bile duct fibroblasts were divided randomly into two groups:the control group and the experiment group (fibroblasts were incubated respectively with 0.6g/L Ch for 12, 24, 36 and 48 h).The growth and DNA synthesis of bile duct fibroblasts were measured by the means of ^3H-TdR incorporation.The total protein content of fibroblast was measured by BSA protein assay reagent kit,then the expression of α-actin was analyzed semiquantitatively by Western blot.RESULTS:After treatment with 0.6g/L Ch for 12, 24,36 and 48h, the values of ^3H-TdR incorporation of bile duct fibroblasts were respectively 3.1±0.39, 3.8±0.37,4.6±0.48 and 5.2±0.56mBq/cell, and the values of the corresponding control groups were 3.0±0.33, 3.2±0.39,3.7±0.49 and 4.3±0.43mBq/cell. After comparing the values of experiment groups and their corresponding control groups,it was found that the 3H-TdR incorporation of bile duct fibroblasts after treatment with 0.6g/L Ch for 24, 36 and 48h were significantly increased (P<0.05, P<0.01, P<0.01),while the ^3H-TdR incorporation of 12-h group was not different statistically from its control group.Ch had no obvious effect on the total protein content of fibroblasts.After incubated with 0.6g/L Ch for 12, 24, 36 and 48h,the total protein content of each experiment group was not altered markedly compared with its corresponding control group.The values of experiment groups were 0.246±0.051,0.280±0.049, 0.263±0.044 and 0.275±0.056ng/cell, and those of corresponding control groups were 0.253±0.048,0.270±0.042,0.258±0.050 and 0.270±0.045 ng/cell.Western blot analysis revealed that the α-actin expression in fibroblasts affected by Ch for 12 and 24 h was not markedly changed compared with their corresponding control groups (P>0.05),the values of total gray scale of 12- and 24-h groups were 1 748±185 and 1 756±173,respectively. But after stimulation with Ch for 36 h, the total gray scale of fibroblasts (1923±204) was significantly higher than that of control group (1734±197).When the time of Ch treatment was lengthened to 48h, the α-actin expression was markedly elevated, the total gray scale was 2189±231 (P<0.01 vs control group).CONCLUSION:Moderately concentrated Ch can promote the proliferation of bile duct fibroblasts at early stage, With the prolongation of Ch treatment, the α-actin expression of fibroblasts was also increased,but the hypertrophy of fibroblasts was not observed,     

Changes of gastric and intestinal blood flow, serum phospholipase A2 and interleukin-1β in rats with acute necrotizing pancreatitis

AIM: To explore the relationship between gastric and intestinal microcirculatory impairment and inflammatory mediators released in rats with acute necrotizing pancreatitis (ANP). METHODS: A total of 64 rats were randomized into control group and ANP group. ANP model was induced by injection of 5% sodium taurocholate under the pancreatic membrane. Radioactive biomicrosphere technique was used to measure the gastric and intestinal tissue blood flow at 2 and 12 h after the induction of ANP, meanwhile serum phospholipase A2 (PLA2) activities and interleukin-lβ levels were determined. Pathologic changes in pancreas, gastric and intestinal mucosae were studied. RESULTS: The gastric blood flow in ANP group (0.62±0.06and 0.35±0.05) mL/(min·g) was significantly lower than that in control group (0.86±0.11 and 0.85±0.06) mL/(min·g) (P<0.01) at 2 and 12 h after induction of ANP. The intestinal blood flow in ANP group (0.80±0.07 and 0.50±0.06) mL/(min·g) was significantly lower than that in control group (1.56±0.18 and 1.61±0.11) mL/(min·g) (P<0.01). Serum PLA2 activities (94.29±9.96 and 103.71±14.40) U/L and IL-Iβ levels (0.78±0.13 and 0.83±0.20) μg/Lin ANP group were higher than those in control group (65.27±10.52 and 66.63±9.81) U/L, (0.32±0.06 and 0.33±0.07)μg/L (P<0.01). At 2 and 12 h after introduction of the model, typical pathologic changes were found in ANP. Compared with control group, the gastric and intestinal mucosal pathologic changes were aggravated significantly (P<0.01) at 12 h after induction of ANP. Gastric and intestinal mucosal necrosis, multiple ulcer and hemorrhage occurred. CONCLUSION: Decrease of gastric and intestinal blood flow and increase of inflammatory mediators occur simultaneously early in ANP, both of them are importantpat hogenic factors for gastric and intestinal mucosal injury in ANP.     

Effects of Yigan Decoction on proliferation and apoptosis of hepatic stellate cells

AIM：To investigate the effects of Chinese herb Yigan Decoction on proliferation and apoptosis of the hepatic stellate cells(HSC) in vitro .METHODS:The study in vitro was carried out in the Culture of HSC lines.Various comcentration of Yigan Decoction were added and incubated .Cell proliferation was detected with MTT colorimetric assay.Cell apoptosis was detected by electron microscopy,flow cytometry and TUNEL.RESULTS:The proliferation of HSC was inhibited by Yigan Decoction,which depending on dose and time significantly.The HSC proliferation rates of groups at the end concentrations 144 and 72(g.L^-1) were 21.62%and 40.54% respectively,significantly lower than that of normal control group(P<0.01),The HSC proliferation rates of groups at the and concentrations 36,18 and 9(g.L^-1) were 54.05%,45.95%and 51\35% respectively,lower than that of control group(P<0.05).When the end concentration was 4.5g.L^-1,the proliferation rate was 83.78%,which appeared no significant differences compared with control group.At the same concentrations of 18 g.L^-1,the inhibitory effects of Yigan Decoction at 24h,48h and 72h time point were observed,the effects were time-dependent and reached a peak at 72h.Meanwhile,it was showed that the inducing effects of Yigan Decoction on HSC apoptosis were dose dependent and time-dependent,The apoptosis index(AL)was detected by TUNEL.After Yigan Decoction had been incubated for 48h at the end concentration of 18g.L^1,the Al(14.5±31.%),was significantly higher than that of control group(4.3±1.3)^(P<0.01).When visualized under transmission electron microscopy,some apoptotic stellate cells were found ,i.e.dilated endoplasmic reticulum,irregular nuclei,chromatin condensation and heterochromatin ranked along inside of nuclear membrane.By flow cytometry detection,after HSC was treated with Yigan Decoction at different concentrations of 36,18and 9(g.L^-1)for48h,Al(%)were 13.3±3.2,10.7±2.7and 10.1±2.5respectively,which were significantly higher than that of control group(4.1±1.9)(P<0.01),At the same concentration of 18g.L^-1 or 24h,48h and 72h,Al(%)were9.3±1.8,10.7±2.7and 14.6±4.3repectively,which were significantly higher than that of control group(P<0.01).CONCLUSION:Yigan Decoction could significantly inhibit HSC proliferation and increase the apoptosis index of HSC dose-dependently and time-dependently,which may be related to its mechanism of antifibrosis.     

Ameliorative effects of bombesin and neurotensin on trinitrobenzene sulphonic acid-induced colitis, oxidative damage and apoptosis in rats

AIM： TO investigate the effects of bombesin （BBS） and neurotensin （NTS） on apoptosis and colitis in an ulcerative colitis model.
METHODS： In this study, a total of 50 rats were divided equally into 5 groups. In the control group, no colitis induction or drug administration was performed. Colitis was induced in all other groups. Following the induction of colitis, BBS, NTS or both were applied to three groups of rats. The remaining group （colitis group） received no treatment. On the 11th d after induction of colitis and drug treatment, blood samples were collected for TNF-α and IL-6 level studies. Malondialdehyde （MDA）, carbonyl, myeloperoxidase （MPO） and caspase-3 activities, as well as histopathological findings, evaluated in colonic tissues.
RESULTS： According to the macroscopic and microscopic findings, the study groups treated with BBS, NTS and BBS ＋ NTS showed significantly lower damage and inflammation compared with the colitis group （macroscopic score, 2.1 ± 0.87, 3.7 ± 0.94 and 2.1 ± 0.87 vs 7.3 ± 0.94; microscopic score, 2.0 ± 0.66, 3.3 ± 0.82 and 1.8 ± 0.63 vs 5.2 ± 0.78, P 〈 0.01）. TNF-α and IL-6 levels were increased significantly in all groups
compared with the control group. These increases were significantly smaller in the BBS, NTS and BBS ＋ NTS groups compared with the colitis group （TNF-α levels, 169.69 ± 53.56, 245.86 ± 64.85 and 175.54 ± 42.19 vs 556.44 ± 49.82; IL-6 levels, 443.30 ± 53.99, 612.80 ± 70.39 and 396.80 ± 78.43 vs 1505.90 ± 222.23, P 〈 0.05）. The colonic MPO and MDA levels were significantly lower in control, BBS, NTS and BBS ＋ NTS groups than in the colitis group （MPO levels, 24.36 ± 8.10, 40.51 ± 8.67 and 25.83 ± 6.43 vs 161.47 ± 38.24; MDA levels, 4.70 ± 1.41, 6.55 ± 1.12 and 4.51 ± 0.54 vs 15.60 ± 1.88, P 〈 0.05）. Carbonyl content and caspase-3 levels were higher in the colitis and NTS groups than in control, BBS and BBS ＋ NTS groups （carbonyl levels, 553.99 ± 59.58 and 336.26 ± 35.72 vs 209.7     

Preventative effects of a probiotic, Lactobacillus salivarius ssp. salivarius, in the TNBS model of rat colitis

AIM: To investigate the intestinal anti-inflammatory effect and mechanism of a probiotic Lactobacillus salivarius ssp. salivarius CECT5713 in the TNBS model of rat colitis. METHODS: Female Wistar rats (180-200 g) were used in this study. A group of rats were administered orally the probiotic L. salivarius ssp. salivarius(5×108 CFU suspended in 0.5 mL of skimmed milk) daily for 3 wk. Two additional groups were used for reference, a non-colitic and a control colitic without probiotic treatment, which received orally the vehicle used to administer the probiotic. Two weeks after starting the experiment, the rats were rendered colitic by intracolonic administration of 10 mg of TNBS dissolved in 0.25 mL of 500 mL/L ethanol. One week after colitis induction, all animals were killed and colonic damage was evaluated both histologically and biochemically. The biochemical studies performed in colonic homogenates include determination of myeloperoxidase (MPO) activity, glutathione (GSH) content, leukotriene B4 (LTB4) and tumor necrosis factor a (TNF-α) levels, as well as inducible nitric oxide synthase (iNOS) expression. In addition, the luminal contents obtained from colonic samples were used for microbiological studies, in order to determine Lactobacilli and Bifidobacteria counts. RESULTS: Treatment of colitic rats with L salivarius ssp. salivarius resulted in amelioration of the inflammatory response in colitic rats, when compared with the corresponding control group without probiotic treatment. This anti-inflammatory effect was evidenced macroscopically by a significant reduction in the extent of colonic necrosis and/or inflammation induced by the administration of TNBS/ethanol (2.3±0.4 cm vs 53.4±0.3 cm in control group, P<0.01) and histologically by improvement of the colonic architecture associated with a reduction in the neutrophil infiltrate in comparison with non-treated colitic rats. The latter was confirmed biochemically by a significant reduction of colonic MPO activity (105.3±26.0 U/g vs 180.6±21.9 U/g, P<0.05) a marker of neutrophil infiltration. The beneficial effect was associated with an increase of the colonic GSH content (1 252±42 nmol/g vs 1 087±51 nmol/g,P<0.05), which is depleted in colitic rats, as a consequence of the oxidative stress induced by the inflammatory process. In addition, the treatment of colitic rats with L. salivarius resulted in a significant reduction of colonic TNF-α levels (509.4±68.2 pg/g vs 782.9±60.1 pg/g, P<0.01) and in a lower colonic iNOS expression, when compared to TNBS control animals without probiotic administration. Finally, treated colitic rats showed higher counts of Lactobacilli species in colonic contents than control colitic rats, whereas no differences were observed in Bifidobacteria counts. CONCLUSION: Administration of the probiotic L. salivarius ssp. salivarius CECT5713 facilitates the recovery of the inflamed tissue in the TNBS model of rat colitis, an effect associated with amelioration of the production of some of the mediators involved in the inflammatory response in the intestine, such as cytokines, including TNF-α and NO. This beneficial effect could be ascribed to its effect on the altered immune response that occurs in this inflammatory condition.     

p38丝裂原活化蛋白激酶抑制剂SB203580对溃疡性结肠炎肠黏膜肿瘤坏死因子α表达的影响

目的探讨溃疡性结肠炎（UC）患者肠黏膜组织中磷酸化p38丝裂原活化蛋白激酶（p38 MAPK）的表达及p38 MAPK抑制剂SB203580对活动性UC患者肠黏膜组织TNFα表达的影响。方法30例活动性UC患者被纳入本研究,以15例结肠癌患者的癌旁正常组织作为对照。免疫组化法检测UC患者肠黏膜活检组织中磷酸化p38 MAPK的表达。体外组织培养条件下观察SB203580对UC患者肠黏膜组织TNFα表达的影响,ELISA法检测培养上清液中TNFα含量。结果（1）UC患者肠黏膜磷酸化p38MAPK的表达明显高于正常肠黏膜,A值分别为549．22±32．54、143．52±11．89,阳染面积分别为[（1680．61±115．30）×10^-5、（351．68±12．73）×10^-5]μm^2,P值均〈0．01。（2）与未用SB203580处理的UC组比较,处理后UC肠黏膜组织分泌TNFα的水平明显较低,分别为（549．96±107．63、72．07±20．30）ng／L（P〈0．01）。（3）与未用SB203580处理的UC组比较,处理后UC肠黏膜组织p38 MAPK下游分子活化转录因子-2（ATF2）的活性表达明显减少,A值分别为688．32±47．37、265．82±40．25,阳染面积分别为[（2489．02±193．63）×10^-5、（1213．76±204．77）×10^-5]μm^2,P值均〈0．01,但对磷酸化p38 MAPK的表达无影响,A值分别为480．34±38．87、465．64±38．69,阳染面积分别为[（1536．68±182．16）×10^-5、（1486．26±165．49）×10^-5]μm62,P值均〉0．05。结论p38 MAPK信号传导通路在UC的发病中起着重要作用,阻断该通路可减少炎性细胞因子的释放。提示p38 MAPK信号传导通路可以为UC的治疗提供一个新的靶标,SB203580有可能成为UC治疗的新药物。     

Nuclear factor-kappaB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-kappaB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-kappaB decoy ODNs or scrambled ODNs. NF-kappaB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-kappaB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-alpha, IFN-gamma and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-alpha and IFN-gamma were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. RESULTS: NF-kappaB activation in liver graft was induced in a time-dependent manner, and NF-kappaB remained activated for 16 h after graft reperfusion. NF-kappaB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-kappaB decoy ODNs significantly suppressed NF-kappaB activation as well as mRNA expression of TNF-alpha, IFN-gamma and ICAM-1 in the liver graft. The hepatic NF-kappaB DNA binding activity [presented as integral optical density (IOD) value] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16+/-0.78 vs 36.78+/-6.35 and 3.06+/-0.84 vs 47.62+/- 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-alpha, IFN-gamma and ICAM-1 [presented as percent of beta-actin mRNA (%)] in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31+/-3.48 vs 46.37+/-10.65 and 7.46+/- 3.72 vs 74.82+/-12.25 for hepatic TNF-alpha mRNA, 5.58+/-2.16 vs 50.46+/-9.35 and 6.47+/-2.53 vs 69.72+/-13.41 for hepatic IFN-gamma mRNA, 6.79+/-2.83 vs 46.23+/-8.74 and 5.28+/-2.46 vs 67.44+/-10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-kappaB decoy ODNs almost completely abolished the increase of serum level of TNF-alpha and IFN-gamma induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-alpha and IFN-gamma in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7+/-13.6 vs 176.7+/-15.8 and 48.4+/-15.1 vs 216.8+/-17.6 for TNF-alpha level, 31.5+/-12.1 vs 102.1+/-14.5 and 40.2+/-13.5 vs 118.6+/-16.7 for IFN-gamma level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-kappaB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-kappaB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17+/-0.07 vs 1.12+/-0.25 and 0.46+/-0.17 vs 1.46+/-0.32 for hepatic MPO content, 71.7+/-33.2 vs 286.1+/-49.6 and 84.3+/-39.7 vs 467.8+/-62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P<0.001). CONCLUSION: The data suggest that NF-kappaB decoy ODNs protects against I/R injury in liver graft by suppressing NF-kappaB activation and subsequent expression of proinflammatory mediators.     

Ameliorative effects of sodium ferulate on experimental colitis and their mechanisms in rats

AIM: To investigate the ameliorative effects of sodium ferulate (SF) on acetic acid-induced colitis and their mechanisms in rats.METHODS: The colitis model of Sprague-Dawley rats was induced by intracolon enema with 8 % (WV) of acetic acid.The experimental animals were randomly divided into model control, 5-aminosalicylic acid therapy group and three dose of SF therapy groups. The 5 groups were treated intracolonically and daily (8:00 am) for 7 days 24 h following the induction of colitis. A normal control group of rats clystered with normal saline instead of acetic acid was also included in the study.Pathological changes of the colonic mucosa were evaluated by the colon mucosa damage index (CMDI) and the histopathological score (HS). The insulted colonic mucosa was sampled for a variety of determinations at the end of experiment when the animals were sacrificed by decapitation.Colonic activities of myeloperoxidase (MPO) and superoxide dismutase (SOD), and levels of malondialdehyde (MDA)and nitric oxide (NO) were assayed with ultraviolet spectrophotometry. Colonic contents of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2)were determined by radioimmunoassay. The expressions of inducible nitric oxide synthase (iNOS), cyclo-oxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB) p65 proteins in the colonic tissue were detected with immunohistochemistry.RESULTS: Enhanced colonic mucosal injury, inflammatory response and oxidative stress were observed in the animals clystered with acetic acid, which manifested as the significant increase of CMDI, HS, MPO activities, MDA and NO levels,PGE2 and TXB2 contents, as well as the expressions of iNOS,COX-2 and NF-κB p65 proteins in the colonic mucosa,although the colonic SOD activity was significantly decreased compared with the normal control (CMDI: 2.9±0.6 vs0.0±0.0;HS: 4.3±0.9 vs0.7±1.1; MPO: 98.1±26.9 vs24.8±11.5; MDA:57.53±12.36 vs9.21±3.85; NO: 0.331±0.092 vs0.176±0.045;PGE2: 186.2±96.2 vs 42.8±32.8; TXB2: 34.26±13.51 vs 8.83±3.75; iNOS: 0.365±0.026 vs0.053±0.015; COX-2:0.296±0.028 vs0.034±0.013; NF-κB p65:0.314±0.026 vs 0.039±0.012; SOD: 28.33±1.17 vs36.14±1.91; P<0.01).However, these parameters were found to be significantly ameliorated in rats treated locally with SF at the given dose (CMDI: 1.8±0.8, 1.6±0.9; HS: 3.3±0.9, 3.1±1.0; MPO:63.8±30.5, 36.2±14.2; MDA: 41.84±10.62, 37.34±8.58; NO:0.247±0.042; 0.216±0.033; PGE2: 77.2±26.9, 58.4±23.9;TXB2:18.07±14.83; 15.52±8.62; iNOS:0.175±0.018, 0.106±0.019;COX-2: 0.064±0.018, 0.056±0.014; NF-κBp65: 0.215±0.019,0.189±0.016; SOD: 32.15±4.26, 33.24±3.69; P<0.05-0.01).amelioration of colonic mucosal injury as evaluated by CMDI and HS.CONCLUSION: Administration of SF intracolonically may have significant therapeutic effects on the rat model of colitis induced by acetic acid enema, which was probably due to the mechanism of antioxidation, inhibition of arachidonic acid metabolism and NF-κB expression.     

Effect of traditional Chinese medicinal enemas on ulcerative colitis of rats

AIM: To investigate the effects of traditional Chine semedicinal enema (TCME) on inflammatory and immune response of colonic mucosa of rats with ulcerative colitis(UC), and to observe the pathogenic mechanism.METHODS: Thirty UC rats, induced by intestinal enema together with 2.4-dinitrochlorobenzene (DNCB) and acetic acid, were randomly divided into 3 groups, i.e., GⅠ, GⅡ and GⅢ. Groups GⅠ and GⅡ were administered with TCME and salazosulfapyridine enema (SASPE), respectively. Group GⅢ was clystered with only normal saline (NSE), served as control. Group GIV was taken from normal rats as reference,once daily, from the 7th day after the establishment of UC for total 28 d. Interleukin-6 (IL-6) in the colonic mucosa was assayed by ^3H-TdR incorporation assay. Colonic mucosal lymphocyte subpopulation adhesive molecules, CD4^+CD11a^+,CD4^+CD18^+, CDs^+CD11a^+, CDs^+CD18^+ (LSAM), tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ), were detected by enzyme linked immunosorbent assay (ELISA). Moreover, the expression of TNF-α mRNA and IFN-γ mRNA in colonicmuc osa were detected by polymerase chain reaction (RT-PCR).RESULTS: Before therapies, in model groups, GⅠ, GⅡ and GⅢ,levels of IL-6, TNF-α, IFN-γ, CDs^+CD11a^+ and CD8^+CD18^+ were significantly different (38.29+2.61 U/mL, 16.54+1.23 ng/L,8.61+0.89 ng/L, 13.51+2.31% and 12.22+1.13% ,respectively) compared to those in GIV group (31.56+2.47 U/mL, 12.81+1.38 ng/L, 5.28+0.56 ng/L, 16.68+1.41% and 16.79+1.11%, respectively). After therapeutic enemas,in GI group, the contents of IL-6 (32.48&#177;2.53 U/m), TNF-α(13.42&#177;1.57 ng/L) and IFN-γ (5.87+0.84 ng/L) were reduced; then, the contents of CD8^+CD11a^+ (16.01+1.05 %)and CD8^+CD18^+ (16.28&#177;0.19%) were raised. There was no significant difference between groups GⅠ and GIV, but the difference between groups GⅠ and GⅡ was quite obvious (P&lt;0.05). The expressions of TNF-α mRNA and IFN-γ mRNAin group GⅢ were much higher than those of group GIV,but those in group GI were significantly suppressed by TCME therapy.CONCLUSION: Ulcerative colitis is related to colonic regional mucosal inflammatory factors and immune imbalance. TCME can effectively inhibit regional mucosal inflammatory factors and improve their disorder of immunity.