| ObjectiveAutoimmune hepatitis(AIH)is a chronic autoimmune disease mediated by an immune disorder that lead the immune system to attack its own hepatocytes.The pathogenesis is unclear,and the genetic susceptibility and environmental factors are the commonly recognized risk factors.As we know,intestinal and liver have a common origin in the embryo.The liver is the body's largest immune organ.The intestinal mucosal lymphocytes play the role on defensing against pathogens,and the immune cells of the liver can hold back the harmful intestinal antigens and toxin stimulation.Intestinal mucosal barrier plays a role as an important line of defense to prevent intestinal toxins from entering the intestine and circulation,which includes mechanical barrier,immune barrier,biological barrier and chemical barrier.Most AIH patients with cirrhosis may be associated with varied degrees of intestinal mucosal barrier dysfunction,portal hypertension to ischemia and hypoxia of intestinal.The inflammatory mediators and oxygen free radicals enhanced the damage of intestinal mucosal epithelial tight junction,leading to increased mucosal permeability.The aim of our study was to explore the correlation between intestinal mucosal barrier and immune injury of liver,by analyzing the changes of intestinal mechanical barrier and immune barrier in AIH patients with cirrhosis and non-cirrhosis of AIH.Further analysis was on the role of intestinal-hepatic cycle in the pathogenesis of AIH and the progression of disease is underwent.In addition,we evaluated the relationship between the degree of immune activity and the degree of hepatocyte injury by detecting the activation status of macrophages in patients with AIH,and the co-expression between TLR4 and macrophages.It is suggested that the expression of macrophages was stimulated by TLR4 pathway to affect liver immune abnormalities.MethodFirst part: Changes of Intestinal Mechanical Barrier and Immune Barrier in Patients with AIHSixty cases of AIH patients(non-cirrhosis in 35 cases,cirrhosis in 25 cases)and 15 cases of healthy controls were selected in Department of Gastroenterology and Hepatology of Tianjin Medical University General Hospital between January 2016 and December 2016.Collected the peripheral blood samples.D-Lac,DAO levels of serum were measured by enzyme linked immunosorbent assay in three groups of subjects.14 cases of AIH patients(non-cirrhosis in 6 cases,cirrhosis in 8 cases)and10 cases of healthy controls were selected between January 2016 and December 2016.Collected the terminal ileum mucosal tissue of them.The expression of ZO-1,Occludin,TLR4 and inflammatory factors were detected by Realtime-PCR.Immunohistochemical method was used to detect the macrophages in the mucosa of the ileum.Western blot was used to detect the expression of s Ig A protein,to evaluate changes in intestinal mucosal mechanical barrier and immune barrier.Second part: Study on the activation state of liver macrophages in patients with AIHThirteen cases of AIH patients(normal liver function in 5 cases,abnormal liver function in 8 cases)and 10 cases of age and gender matched healthy controls were selected in Gastroenterology Department of Tianjin Medical University General Hospital between January 2016 and December 2016.The patients of AIH underwent liver biopsy under ultrasound guidance to obtain liver pathological specimens,and the control group is the liver tissue for pancreatic cancer or cholangiocarcinoma resection of part of the normal liver specimens.Immunohistochemical staining of macrophages was performed on liver tissue of three groups to evaluate the relationship between macrophage expression and inflammatory activity.The co-expression of macrophages and Toll-like receptor 4 in liver was observed by immunofluorescence double staining,and evaluated the correlation between the degree of inflammation and the activity of liver inflammation by pathology.ResultFirst part: Changes of Intestinal Mechanical Barrier and Immune Barrier in Patients with AIH1.Part of the cirrhosis patients of AIH with ileum terminal have the gland structure damage and mucosal microvilli structure damage,and local small crypt abscess are visible.AIH patients without cirrhosis showed less regular intestinal mucosal morphology,and mild local inflammatory cell infiltration is visible compared with the control group.2.ELISA was used in serum D-Lac level.Results showed patients with AIH have increased level [non-cirrhosis:(386.1 ± 297.3)ug/L,cirrhosis:(1423.2 ± 409.4)ug/L,health control:(98.6 ± 10.2)ug/L].The D-Lac level increased significantly in AIH cirrhosis patients compared with the control group with a statistically significant difference(t=13.049,P<0.01).Serum DAO levels were elevated in both groups of patients with AIH [non-cirrhosis:(6.1±2.3)U/m L,cirrhosis:(11.7±2.7)U/m L,health control:(3.7±1.1)U/m L].The DAO level increased significantly in AIH cirrhosis patients,and the difference was statistically significant(t=10.983,P<0.01).In addition,the levels of serum D-Lac,DAO were significantly negatively correlated with WBC,Hgb,PLT(P<0.01).And,D-Lac was significantly associated with elevated levels of DAO(r=0.722,P<0.01).3.Real time-PCR results showed that the relative expression of ZO-1 in ileal mucosa of patients with AIH was significantly lower than that in control group[cirrhosis vs health control:(0.20±0.14)vs(1.67±0.51),P<0.01;non-cirrhosis vs health control:(0.99 ± 0.37)vs(1.67 ± 0.51),P<0.05].The relative expression of Occludin in patients with AIH was significantly lower,and the decrease of Occludin expression was not statistically significant in non-cirrhosis patients [cirrhosis vs health control:(0.12±0.09)vs(0.90±0.21),P<0.01;non-cirrhosis vs health control:(0.64±0.30)vs(0.90±0.21),P=0.057].4.CD68 immunohistochemical staining showed that the number of terminal mucosal macrophages increased in patients with AIH.Compared with the control group,cirrhosis patients in AIH with high magnification of the absolute number of visual field expression significantly increased(P<0.05),and the expression of patients without cirrhosis in AIH also increased but the difference was not statistically significant(P=0.057).5.Realtime-PCR results showed a relatively high expression of proinflammatory cytokines in intestinal mucosa of patients with AIH.The relative expression of IL-2and IFN-? in intestinal mucosa of patients with AIH cirrhosis was significantly higher than that in control group[IL-2:(1.11±0.43)vs(0.24±0.16),IFN-?:(3.50±1.92)vs(0.32±0.30),P<0.01].The expression of IL-2 and IFN-? in AIH patients without cirrhosis increased,but the difference was not statistically significant(P=0.1628,0.0622).Inhibited inflammatory cytokines were relatively low in expression.The relative expression of IL-10 in ileal mucosa of both AIH patients in cirrhosis and non-cirrhosis were lower than that in control group [cirrhosis vs health control:(0.30± 0.19)vs(0.74 ± 0.23),P<0.05;non-cirrhosis vs health control:(0.42 ± 0.24)vs(0.74 ± 0.23),P<0.05].The relative expression of IL-4 decreased,and the difference was not statistically significant [cirrhosis vs health control:(1.01 ± 0.58)vs(1.45 ±0.31),P=0.0584;non-cirrhosis vs health control:(1.13 ± 0.54)vs(1.45 ± 0.31),P=0.1548].6.Realtime-PCR showed that the relative expression of TLR4 in ileal mucosa was elevated in AIH patients.The level of TLR in AIH patients with cirrhosis was significantly different from that in the control group [(6.74±5.13)vs(0.89±0.70),t=3.596,P<0.01].The expression of TLR4 in patients without cirrhosis increased and the difference was statistically significant [(3.74±3.65)vs(0.89±0.70),t=2.455,P=0.028].7.Western blot was used to detect the expression of s Ig A protein in the mucosa of the ileum: the level of s Ig A protein in AIH patients with cirrhosis was significantly decreased(P<0.05).The expression of s Ig A in patients without cirrhosis decreased,but the difference was not statistically significant(P=0.238).Second part: Study on the activation state of liver macrophages in patients with AIH1.The absolute number of CD68 positive cells in AIH patients was significantly increased.The macrophage expression was more obvious in AIH patients with elevated liver function(P=0.032).The macrophage expression in the patients with AIH who have normal liver function also increased(P=0.041).The expression of CD68 was more obvious in inflammatory cell aggregation area.2.Immunofluorescence double staining method was used to detect the expression of CD68 and TLR4.Compared with the control group,the TLR4 located in CD68 positive cell counted per high magnification visual field was significantly increased in each group of patients with AIH.With the increase of liver function,there was an increasing trend in expression of cells(normal liver function: P=0.037,elevated liver function: P=0.028).Conclusion1.Early non-cirrhosis of AIH patients have intestinal mucosal morphology abnormalities,and the mucosal mechanical barrier and immune barrier are destroyed.Such damage in AIH patients with cirrhosis is more significant than non-cirrhosis patients.2.Macrophages are more expressed in patients with AIH.TLR4 was co-expressed with macrophages,and has association with the degree of inflammation in the liver. |
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