| Objective:To study the impact of intestinal mucosa claudin-1, Zonula Occludens-1(ZO-1)and myosin light chain kinase(MLCK) in rat models of acute liver failure(ALF) and bacterial translocation by Tumor necrosis factor-α(TNF-α) and TNF-α antagonist Methods:Healthy male Sprague Dawley(SD) rats were randomly divided into three groups,normal control(n=6) group was i.p. injected with 0.9% saline(12 ml / kg); model group(n=24) and the intervention group(n=24), were i.p. injected with a full dose of D-galactosamine(D-Gal N) 1200 mg / kg to established model of acute liver failure,but the intervention group was i.p. injected TNF-α antagonists(rh TNFR: Fc etanercept) 12.5 mg / kg and after 24 hours given D-galactosamine. Model group and the intervention group were designated as 8hr,24 hr,48hr,and 72 hr subgroups with six animals each, while the control group of animals were anesthetized and sacrificed at72 hr, homogenates of liver, spleen and mesenteric lymph nodes(MLNs) from each group were cultured in agar for bacterial outgrowth; each group was detected serum ALT, AST, Tbil and Alb concentration; enzyme-linked immunosorbent assay(ELISA)to detect level of serum TNF-α; hematoxylin-eosin(H.E.) stained sections of liver and terminal ileum were examined under an optical microscope for pathological changes; and protein expression of the terminal ileum Claudin-1, ZO-1 protein and of MLCK was determined by immunohistochemistry and Western blot.Results:1, ALT and AST were gradually increased in model group and intervention group, reached a peak at 48 hr, but the peak of the intervention group was significantly lower than the model group. During the same period of time, total bilirubin both model group and the intervention group were increased, but total bilirubin in model group was significantly faster than in intervention group. Then aminotransferase was decreased but total bilirubin continued to rise, bilirubin separation ALT. But totalVI bilirubin in intervention group 72 hr was decrease. For the 72 hr model group, normal structure of liver was lost with huge area of necrosis(more than 2/3 of the whole section) and few live hepatocytes left. The sinus hepaticus had congestion and hemorrhage, and massive inflammatory cell infiltration were seen in the portal area and necrotic area, these pathological changes were consistent with acute liver failure pathological changes and the acute liver failure model was successed.For the 72 hr intervention group, normal structure of liver was visible part of liver cellular swelling and spotty necrosis, a small amount of inflammatory cells infiltration were scattered in the hepatic lobule and portal area. Under the light microscope, the model group72 hr terminal ileum villus were visible that fell down, part of villus tip off, villus mucosa and submucosa interstitial were edema, numerous neutrophils were infiltrating in mucosal laye. The intervention group 72 hr terminal ileum kept integrity,no shedding epithelial cells necrosis, villus mucosa and submucosa interstitial were mild edema, mucosa was showing a small amount of neutrophil infiltration.2, the normal control group, and for the 8hr model group and intervention group were not bacterial grew in the organ cultures. The model group have bacterial grew in the organ cultures that were 11.1%, 38.89% and 66.67% of the rats in the 24 hr, 48 hr and 72 hr.Between control group and model group 24 hr, 48 hr and 72 hr compared the differences were statistically significant(P <0.05). However, in the intervention group24 hr, 48 hr and 72 hr organ bacterial translocation rates were 5.56%, 16.67% and27.78%. Between control group and intervention group 24 hr, 48 hr and 72 hr compared the differences were also statistically significant(P <0.05). Then, between the intervention group and the model group have statistical significance(P <0.05).3, expression of TNF-α in rats of model group 8hr(117.89 ± 13.09 pg / ml),gradually increased and peaked at 24 hr(239.83 ± 15.81 pg / ml), then began to decline, 48 hr and 72 hr were respectively decreased(149.37 ± 20.86 pg /ml and96.41±4.21pg/ml), which were significantly higher than that of the the normal control group(24.19 ± 3.57 pg / ml)(P <0.01). At the same time, expression of serum TNF-αin the intervention group 8hr( 52.06 ± 18.06 pg / ml), gradually increased and peaked at 24hr(182.71 ± 17.08 pg / ml), declined to 48h(71.78 ± 20.15 pg / ml), and 72h(57.22 ± 7.39 pg / ml), however,compared with normal control group that thesedifferences were statistically significant(the normal control group and intervention group 8h, 48 h P <0.05,24 h and 72 h P <0.01), and expression of TNF-α between the intervention group with the model group at each time point were statistical significance(P <0.01).4, western blot be used to determine relative amounts of protein, the model group at 8hr Claudin-1 protein level was 0.9932 ± 0.1849, which are significantly lower than that of the normal control group 1.640 ± 0.1879(t = 4.908, p <0.01),gradually decreased, at 24 hr drop to the lowest 0.3546 ± 0.068, significantly lower than that of the control group(t = 12.87, p <0.01), 72 hr for 0.8322 ± 0.1169, lower than the control group(t = 7.301, p <0.01); the intervention group at 8hr Claudin-1protein was 1.387 ± 0.2163, that compared with the control group was no difference(t = 1.770, p > 0.05), but the intervention group at 8hr Claudin-1 protein was relatively higher than that of the model group at 8hr, the difference was statistically(t= 2.765, p <0.05); at 24 hr plunged to 1.051 ± 0.3702, significantly lower than the control group(t = 2.838, p <0.05), but higher than the model group at 24hr(t = 3.700,p < 0.05); that of 72 hr was 1.18103 ± 0.1501 that significantly lower than that of the control group(t = 3.818, p <0.01), higher than the model group at 72hr(t = 3.668, p<0.05). For the 8hr model group, ZO-1 protein level was 0.7256 ± 0.09153, and the normal control group 1.015 ± 0.1503, the difference was statistically significant(t =3.293, p <0.05), gradually decreased, at 24 hr minimize 0.3874 ± 0.09148,significantly lower than the control group(t = 7.137, p <0.01), at 72 hr for 0.5867 ±0.1780, significantly lower than the control group(t = 4.534, p <0.01); intervention group at 8hr ZO-1 protein was 0.8179 ± 0.1190, that compared with the control group was no difference(t = 2.060, p > 0.05), and compared with the model group at 8h was relatively higher, but the difference was not statistically(t = 1.229, p> 0.05), at 24 hr plunged to 0.6423 ± 0.08182, significantly lower than the control group(t = 4.36, p<0.05), but higher than the model group at 24 hr, the difference was statistically significant(t = 4.153, p < 0.01), that of 72 hr was 0.7622 ± 0.09943, and compared with the control group that the difference was statistically significant(t = 2.653, p<0.05), still higher than the model group at 72hr(t = 2.824, p <0.05). For the 8hr model group, MLCK protein levels was 0.6959 ± 0.1295, and the normal controlgroup was 0.4597 ± 0.06882, that difference was statistically significant(t = 3.221, p<0.05), gradually increased, peaked at 24 hr was 1.298 ± 0.1935, significantly higher than the control group(t = 8.164, p <0.01), at 72 hr was 0.9255 ± 0.1575, was significantly higher than that of the control group(t = 5.420, p <0.01); in the intervention group at 8hr MLCK protein was 0.5716 ± 0.1566, that compared with the control group was no difference(t = 1.308, p > 0.05), and compared with the model group at 8h was relatively lower, but the difference was not statistically(t = 1.224,p> 0.05), then gradually increased, at 24 hr was 1.033 ± 0.07282, was significantly higher than the control group(t = 11.44, p <0.01), but lower than the model group at24 hr, the difference was statistically significant(t = 2.564, p <0.05), at 72 hr recovery to 0.6338 ± 0.09899, that compared with the control group, the difference was statistically significant(t = 2.889, p = 0.0277, p <0.05), and compared with the model group at 72 hr that the difference was statistically significant(t = 3.136, p <0.05).Immunohistochemistry showed normal control group Claudin-1 and ZO-1 protein were a large of brown stained positive signal in the top of the intestinal cell membrane, MLCK protein was a large of brown stained positive signal in the cytoplasm. In the 72 hr model group, Claudin-1 and ZO-1 protein had a weak positive staining, have an irregular shape with a serrated border, but MLCK protein had a strong positive brown staining signal. Claudin-1 protein in the intervention group within 72 hr had a weakened slightly positive signal, compared with the control group positive staining signal declined, but the model group increased significantly positive signal; ZO-1 protein in the intervention group within 72 hr had the brown staining positive signal, and compared with the normal control group had been weakened, but have stonger positive staining signal than the model group; MLCK protein in the intervention group 72 hr had strong positive staining signals in the ileal mucosal epithelial cells, slightly stronger than the normal control group, but weaker than that of model group at 72 hr.Conclusions:Expression of TNF-α in rats model of acute liver failure were increased,activating MLCK by TNF-α that shrunk eptithelial cells and made cell gap increased,while TNF-α made expression of Claudin-1 and ZO-1 protein were decreased,leading to tight junctions fracture along with increased intestinal bacterial translocation. However, it had used of TNF-α antagonists(etanercept rh TNFR: Fc)that significantly improved liver function, reduced inflammation, inhibited the levels of MLCK increased, prevent expression of Claudin-1 and ZO-1 protein from decreasing and prevent intestinal permeability from increasing to reduce the incidence of bacterial translocation. |
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