To Study The Change Mechanism Of Intestinal SIgA In Acute Liver Necrosis

AimIntestinal barriers prevent bacterium and toxin from getting gut lumen through intestinal wall. Intestinal barriers consist of mechanical barrier, microorganism barrier, immunology barrier , chemical barrier. Intestinal M cells, iIEL, lymphocyte within the lamina propria, secretory IgA(SIgA) make up intestinal immunology barrier. SIgA may inhibit bacterium or virus to adhere on intestinal epithelial, so it prevents them from get through intestinal barrier. Therefore, SIgA in gut influences intestinal immunology barrier. In our study intestinal SIgA of hepatitis gravis was detected, and we built the model of acute liver necrosis as well as cultured intestinal epithelial cells. The change of IgA, SIgA and SC were detected as well as the change of SC in intestinal epithelial cells stimulating by TNF - a and NO were detected. These were to study the change of intestinal immunology barrier in acute liver necrosis so that we may study the machine - processed of SBP.Materials and methods1. Objective1. 1 Clinical patients: in 2004 - 2005 hepatitis gravis patients were 30 from China Medical University NO. 2 Clinical Hospital. The intestine and liver of hepatitis gravis patients were 20 from YUOAN Hospital in Beijing and XINAN Hospital of NO. 3 Army Medical University.1.2 The model of acute liver necrosis: Male Balb/C mice were 390 andwere divided into eight groups at random. Groupl: saline control group;Group2: LPS control group;Group 3: D - GalN control group;Group 4: LPS acuteliver necrosis group ( LPS/GalN);Group 5: TNF - a acute liver necrosis group(TNF-a/GalN);Group6: anti - TNFa group (anti-TNFa+ LPS + GalN);Group 7: TNF - a control group;Group 8: anti - TNF - Rl group (anti - TNF-Rl + LPS + GalN). There were 5 time point (2h, 6h, 9h, 12h, 24h) ineach group except group 6 and group 8. In group 6 and group 8 time point was9h. Intestinal and live tissues, serum, fecal, were got to remained.2. Method2. 1 IgA and SIgA in serum and fecal of hepatitis gravis were examined by immunity rate nephelometry and radioimmunoassay.2. 2 IgA and SC ( SIgA) of intestinal tissues and liver in hepatitis gravis were detected by immunohistochemistry.2. 3 SIgA in the cytoplasm and on the membranes of intestinal epithelial cells in hepatitis gravis were detected by double immunofluorescence.2.4 ELISA was established to check up IgA in the serum, fecal in acute liver necrosis.2. 5 SIgA in fecal of acute liver necrosis were examined by radioimmunoassay.2. 6 IgA of intestinal tissues in acute liver necrosis were detected by immunohistochemistry .2.7 Secretory component mRNA of mouse intestine in acute liver necrosis were determined by Real - time PCR.2. 8 The change of SC in intestinal epithelial cells stimulated by TNF - a and NO were determined immunohistochemistry, Weastern blot, Real - time PCR.Result1 . IgA and SIgA in serum and fecal of hepatitis gravis were markely increased compared with the control (P <0.01).2. SC and IgA in the cytoplasm and on the membranes of intestinal epithelial cells in hepatitis gravis were remarkably decreased. We analysed that the OD of SC and IgA was notably decreased compared with normal control (P < 0?. 01).3 . Double immunofluorescence showed normal intestinal epithelial cytoplasm was tangerine, and membranes were yellow, and plasma cells within the lamina propria were tangerine and yellow, but intestinal epithelial cytoplasm, membranes and plasma cells within the lamina propria in hepatitis gravis were remarkably decreased.4 . IgA and SIgA of serum and fecal in acute liver necrosis induced by LPS/ GalN were notably elevated compared with the control (P <0.01).5 . IgA in the cytoplasm and on the membranes of intestinal epithelial cells as well as plasma cells within the lamina propria in acute liver necrosis induced by LPS/GalN were remarkably decreased. We analysed that the OD of IgA was notably decreased at 2h to 12h compared with NS control (P <0.01).6 . SC mRNA of intestinal tissues in acute liver necrosis induced by LPS/ GalN: In acute liver necrosis intestinal tissues SC mRNA was notably fall at 2h points to 9h compared with NS group and at 9h was the lowest (P < 0.01).7 . The mortality and the change of intestine and liver in acute liver necrosis induced by TNF - a/GalN were consistency with those of LPS/GalN. This showed that TNF - a was a important factor in acute liver necrosis.8 . IgA and SC (SIgA) in the cytoplasm and on the membranes of intestinal epithelial cells as well as plasma cells within the lamina propria in acute liver necrosis induced by TNF - ot/GalN were remarkably decreased. We analysed that the OD of IgA was notably decreased at 2h to 12h compared with NS control (P < 0.01). IgA and SC (SIgA) in the cytoplasm and on the membranes of intestinal epithelial cells as well as plasma cells within the lamina propria of anti -TNFa group and anti - TNF - Rl group didn' t change. This fully proved that TNF - a was a important factor in acute liver necrosis.9 . SC mRNA of intestinal tissues in acute liver necrosis induced by TNF -a/GalN: In acute liver necrosis intestinal tissues SC mRNA was notably fall at 2h points to 9h compared with NS group and at 9h was the lowest (P <0.01).SC mRNA of anti - TNFa group and anti - TNF - Rl group didn' t change. This fully proved that TNF - a was a important factor in acute liver necrosis from another view.10 . Immunocytochemical analysis of effect of TNF - a and NO on SC -positive cells;Immunocytochemistry demonstrated that SC - positive cells accounted for about 0.5 -0.6% of Caco -2 cells cultured in the absence of TNF-a . Treatment of Caco -2 with TNF - a (50ng/ml, lOOng/ml, 200ng/ml, 400ng/ml) increased the proportions of SC - positive cells to approximately 0.5-1% , 1.5 -2% , 2-3% , 3.0-3.5% , respectively. The SC was localized mainly in the cytoplasm and on membranes of Caco - 2 cells. SC - positive cells accounted for about 0. 5 - 0. 6% of Caco - 2 cells cultured in the absence of Sinl. Treatment of Caco-2 with Sinl (125fjuM, 250/xM, 500jaM, IOOOjxM) decreased the proportions of SC - positive cells to approximately 0.46 -0. 50% , 0.26 -0.30% , 0.12 -0.15% , 0.18 -0.22%.11 . SC protein of Caco - 2 detected by Westeren blot: SC molecular was 80 KD. Our result showed that there was a remarkable strap in situation of 80 KD. SC protein was increased in Caco - 2 stimulated by TNF - a treatment compared with untreatment (P < 0. 01). SC protein was notably decreased in Caco -2 stimulated by Sinl treatment compared with untreatment (P <0.01).12 . Real - time PCR analysis of SC mRNA of Caco - 2 cells by TNF - a treatment: mRNA levels for these target genes were normalized to the mRNA levels for the housekeeping gene GAPDH. Expression of SC genes increased significantly by TNF - a treatment in a dose - dependent manner compared with untreatment (P<0.01).13. Real - time PCR analysis of SC mRNA of Caco -2 cells by Sinl teat-ment;mRNA levels for these target genes were normalized to the mRNA levels for the housekeeping gene GAPDH. Expression of SC genes decreased significantly in response to various concentrations of Sinl treatment in a dose - dependent manner compared with untreatment (P <0.01).Conclusion1. IgA and SIgA in serum and fecal of hepatitis gravis were remarkably increased compared with the control. But IgA and SC (SIgA) in the cytoplasm and on the membranes of intestinal epithelial cells in hepatitis gravis were remarkably decreased.2. The decrement of IgA and SC (SIgA) in the cytoplasm and on the membranes of intestinal epithelial cells in hepatitis gravis induced falling of intestinal immunology barrier and the attenauation of intestinal immunology defence. This may be a factor of SBP in hepatitis gravis.3. IgA and SIgA in serum and fecal in acute liver necrosis were notably elevated compared with the control. But IgA and SC(SIgA) in the cytoplasm and on the membranes of intestinal epithelial cells as well as plasma cells within the lamina propria in acute liver necrosis were remarkably decreased. This was consistency with that of hepatitis gravis. Hence acute liver necrosis was a good model to study intestinal immunology barrier in hepatitis gravis.4. In acute liver necrosis the notably falling of intestinal epithelial cells SC mRNA was the reason for the decrement of SIgA on the intestinal epithelial cells membranes.5. The mortality and the change of intestinal SC and SIgA in acute liver necrosis induced by TNF - a/GalN were consistency with those of LPS/GalN. This showed that TNF - a was a important factor in acute liver necrosis.6. TNF - a - induced increased the proportion of SC - posistive cells and SC protein and SC mRNA in Caco - 2 cells. This showed that TNF - a wasn' t a direct factor to give rise to the decrement of SIgA, SC in acute liver necrosis.7. NO - induced decreased the proportion of SC - posistive cells and SC protein and SC mRNA in Caco - 2 cells. This showed that NO was a direct factor to give rise to the decrement of SIgA, SC in acute liver necrosis.