| Objective: Liver failure refers to the serious liver injury caused by avariety of factors, which is characterized by variant complications such asdisturbances of blood coagulation, jaundice, hepatic encephalopathy, ascitesand so on. Now the pathogenesis of liver failure remains obscure. In recentyears, many scholars believed that immune injury, ischemia, hypoxia andendotoxemia contributed to the development of liver failure. There existedsevere endotoxemia when liver failure, endotoxin and its downstreamcytokines increased, such as tumor necrosis factor-α (TNF-α), NO,interleukin-6(IL-6) and so on, all these cytokines could lead to a large numberof hepatic cells necrosis or apoptosis, also they might influence or damage theintestinal barrier function by a certain mechanism, which accounted for theincrease of the permeability of the intestinal mucosa. Consequently, a greatnumber of macromolecular substances, bacteria and bacterial toxins mightpass through the intestinal mucosa optionally. And this had close relationshipwith spontaneous bacterial peritonitis, hepatic encephalopathy and othersevere complications. In this study, mice model of ALF can be developed byan intraperitoneal injection of D-Galactosamine. The probioticsBifidobacterium, Lactobacillus and Streptococcus Thermophilus wasapplicated to intragastric administration. In order to identify the mechanism ofthe increased permeability of the intestinal mucosa and the protection of theprobiotics more precisely, the expression of ZO-1protein and the relationshipwith the serum TNF-α and the plasma endotoxin levels were investigated.Methods:30healthy male depuratory grade BALB/c mice, six to eightweeks old, weighting18g to22g. All the mice were randomly divided into thenormal control group, the ALF group and the probiotics treatment group. Theywere housed and cared for in rooms maintained at a constant temperature of 18-22℃and humidity of60.0±10.0%. Mice were given standard feed. Themice in the normal control group and the ALF group were given anintragastric administration of normal saline (9ml/kg/d) once a day. Thetreatment group accepted an intragastric administration of probiotics(900mg/kg/d) once a day. Two weeks later, the ALF group and the treatmentgroup were given an intraperitoneal injection of D-Galactosamine (3.0g/kgbogy weight) to induce liver failure. The mice in the normal control groupaccepted an intraperitoneal injection of normal saline (equal volumD-Galactosamine/kg body weight).9h later, all the mice were sacrificed. Theserum, plasma and specimens of liver and ileum were collected respectively.The contents of alanine aminotransferase (ALT), aspartate aminotransferase(AST) were examined by Olympus AU-2700automatic biochemical analyzer.Enzyme-linked immunosorbent assay (ELISA) was used to test serum TNF-αlevel. Plasma endotoxin level of the mice was tested with Tachypleusamebocyte lysate. Some of the obtained hepatic tissue were fixed in10%formalin and embedded in paraffin. Sections of4μm thickness were obtainedand stained with Hematoxylin-eosin (HE) to observe histopathologic changes.Some hepatic tissue and ileum samples were quickly frozen in liquid nitrogen,then in-80℃fridge. The expression of TNF-α mRNA in the hepatic tissueand ZO-1mRNA in the ileum were detected by RT-qPCR. Western blot wasapplied to detect the expression of ZO-1protein in the ileum.SPSS21.0was applicated to analyse all data.Results:1The general state of mice:Mice in the normal control group were all active and had good appetitewith bright hair. Mice in the ALF group were less active and had bad appetitewith messy hair. They responded to the outside world slowly. The spirit,appetite and activity in the treatment group mice were ranged between thenormal control group and the ALF group.2Histopathological changes in the hepatic tissue:Histopathologic changes of hepatic tissue staining with HE showed that hepatic lobule was clear, hepatocytes arranged in radiation from the centralveins in the normal control group. There appeared many necrosis andinfiltrated a large number of inflammatory cells in the livers of the ALF group.Compared with the ALF group, the necrosis of hepatocytes and inflammatorycells accumulation in the treatment group was remarkably reduced.3Serum ALT, AST, TNF-α and plasma endotoxin levels in each group:The level of serum ALT (393.587±31.010), AST (475.751±41.536),TNF-α (435.971±30.415) and plasma endotoxin (3.789±0.313) were higher(all P<0.01) than those in the normal control group (38.994±9.627,55.279±7.500,51.602±8.540,0.577±0.120). All the above indexes of thetreatment group (158.271±23.637,259.216±22.492,256.289±26.221,2.716±0.214) had significantly decreased (all P<0.01) compared with the ALFgroup.4The expression of TNF-α mRNA in the hepatic tissue and ZO-1mRNAin the ileum tissue:The expression of TNF-α mRNA in the hepatic tissue and ZO-1mRNA inthe ileum tissue were detected by RT-qPCR. The expression of TNF-α mRNA(3.355±0.274) in the hepatic tissue of the ALF group was significantly higher(P<0.01) than that in the normal control group (1.00). In addition, theexpression of TNF-α mRNA (2.284±0.212) in the hepatic tissue of thetreatment group had significantly decreased compared with the ALF group(P<0.01). The expression of ZO-1mRNA (0.441±0.046) in the ileum tissue ofthe ALF group had significantly reduced (P<0.01) compared with the normalcontrol group (1.00). The expression of ZO-1mRNA (0.680±0.039) in theileum tissue of the treatment group was significantly higher than that of theALF group (P<0.01).5The expression of ZO-1protein in the ileum:The expression of ZO-1protein (0.361±0.037) in the ileum of the ALFgroup was significantly lower (P<0.01) than that of the normal control group(0.808±0.069). The expression of ZO-1protein (0.552±0.055) in the ileum ofthe treatment group had significantly increased compared with the ALF group (P<0.01).6The correlation between ZO-1protein and serum TNF-α, plasmaendotoxinPearson correlation analysis showed that the relative expression of ZO-1protein was negatively correlated with the level of the serum TNF-α, plasmaendotoxin, the r values were-0.946,-0.919respectively (both P<0.01).Conclusions:1Mice model of ALF could be successfully developed by anintraperitoneal injection of D-Galactosamine. TNF-α and LPS played animportant role in the development of ALF.2The expression of ZO-1mRNA/protein in the ileum of the mice in theALF group mice had significantly reduced compared with the normal controlgroup. And this was correlated with the level of the serum TNF-α. TNF-αcould down-regulate the expression of ZO-1protein by inhibiting theexpression of ZO-1mRNA, which damaged the tight junction betweenintestinal mucosal epithelial cells. This may be one of the probable causes ofthe increased permeability of the intestinal mucosa in mice with acute liverfailure.3The possible mechanism that probiotics improved the permeability of theintestinal mucosa was mediated through up-regulating the expression of ZO-1protein which was induced by reducing the content of endotoxin and the levelof TNF-α.4Probiotics could complement the normal gut microbiota, improve theimbalance of intestinal flora, and decrease the synthesis and release ofendotoxin and TNF-α to prevent their damage to the liver. |
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