Study On The Mechanism Of Primary Biliary Cirrhosis And The Evaluation Of The Effect Of Yugan Prescription Combined With Ursodeoxycholic Acid

Objective:Primary biliary cirrhosis (PBC) is a chronic and progressive autoimmune disease of liver cholestasis, marked by non-purulent inflammation of small bile duct and high titer AMA (antimitochondrial antibody) in the blood. Immunological factors are generally considered to play a very important role since the exact mechanism of the disease is still not clear. Interleukin-33 (IL-33) is a cytokines participating widely in the process of inherent immunity and acquired immunity. IL-33 proinflammatory cytokines(IL-8、IL-1β) and histamine. Furthermore, IL-33 directly acts on the neutrophil by up-regulating chemokine receptor CXCR2 to strengthen the neutrophil migration.However, it is rare reported of IL-33 functions in PBC patients, and the expression character, the action on neutrophil, and the correlation with PBC severity of disease as well as the histopathology are still unclear. This study primarily monitored IL-33 distributed in peripheral blood plasma and liver tissue, and explored the correlation between liver IL-33 mRNA expression and MPO+liver infiltration in PBC patients (Child-Pugh A=21, Child-Pugh B=29, Child-Pugh C=18). Furthermore, the neutrophil chemotaxis induced by blood IL-33 was also studied in vitro. Finally, the possible mechanism of the interaction between IL-33 and neutrophil in the progression of PBC was discussed.Methods:1. Serum IL-33 in PBC patients (Child-Pugh A, Child-Pugh B and Child-Pugh C) was examined by ELISA to explore the correlation with ALT, AST, ALP and GGT. The relationship between IL-33 expression and the disease severity was discussed.2. Liver IL-33 and ST2 mRNA in PBC patients were examined by in situ immunohistochemical analysis and RT-PCR. Cells of IL-33 positive in different pathological stages were observed. The correlation of the amount of MPO+ and IL-33 mRNA expression in the liver tissue was explored.3.The function of blood IL-33 to the neutrophil in PBC was clarified by neutrophil chemotaxis test.Results:1. Serum IL-33 elevated in PBCSerum IL-33 elevated significantly in PBC patients (69.44±10.70 pg/ml) in superior to healthy controls (32.24±8.37 pg/ml) (P< 0.001). IL-33 in Child-Pugh B (74.93±6.56) and C (75.61±7.95 pg/ml) increased significantly higher than Child-Pugh A (57.24±6.55 pg/ml) (P< 0.001), while there was not statistically significance between Child-Pugh B and　C (P>0.05)2. Serum sST2 elevated in PBCSerum sST2 elevated significantly in PBC (70.47±25.81 pg/ml) in superior to healthy controls (144.94±224.60 pg/ml) (P< 0.001), while there were no significant differences among the subgroups of Child-Pugh A(1343.84±583.28 pg/ml), B(1438±485.42 pg/ml), and C(1439.37±503.73 pg/ml)(P> 0.05).3. Serum IL-33 correlated with ALP in PBCSerum IL-33 in PBC significantly correlated with ALP (R= 0.421, P< 0.001), but this correlation has not been found with ALT, AST and GGT (R=-0.023、R=-0.071、R= 0.158,P>0.05)4. The increment of the amount of IL-33+ cells correlated with the Child-Pugh stagesThe amount of IL-33+ cells within the liver tissue, measured by in situ immunohistochemical analysis, significantly increased in PBC (P< 0.001), especially more significantly in PBC Ⅲ and IV in superior to PBC Ⅰ and Ⅱ (P< 0.001). Simultaneously, IL-33 mRNA was found more expressed in PBC III or IV than the healthy controls, PBC I and II (P< 0.001)5. Significant migration of the neutrophil in the liver tissue of PBC closely correlated with IL-33 mRNA expression.The migration of the neutrophil into the liver tissue of PBC was also studied by in situ immunohistochemical analysis to find that the myeloperoxidase (MPO) positive cells significantly increased than the healthy controls (P< 0.001), which was more markedly in PBC III and IV than PBC I and II (P< 0.001). Similarly, the amount of MPO+cells significantly correlated with IL-33 mRNA (R= 0.81, P< 0.001)6. The neutrophil chemotaxis was facilitated by IL-33In order to further investigate the function of IL-33 to the neutrophil, we explore the neutrophil chemotaxis and found plasma of PBC, in superior to the healthy controls, attracted more neutrophils to migrate (P< 0.001).furthermore, in comparison of the neutrophil cultured in PBC plasma, the neutrophil chemotaxis index significantly increased when adding IL-33 back to the plasma of PBC (P< 0.001)ConclusionSerum IL-33, IL-33+ cells and IL-33 mRNA expression in PBC significantly elevated, which correlated with the disease progression. Along the PBC progression, the neutrophil migration to liver tissue was marked and correlated with IL-33 mRNA expression, which suggested that the neutrophil played an important role in the progression of PBC. Plasma sST2 in PBC also significantly increased. Taking account of the neutrophil cheomotaxis induced by IL-33 in vitro, it was suggested that the PBC progression might be the result of the neutrophil migration towards to the liver tissue, which was attracted by IL-33/ST2. It was also suggested that the neutrophil migration induced by IL-33 might be the future treatment target.Objective:This study aims to investigate the efficacy of ursodeoxycholic acid capsule combined with Yuganfang to improve liver function in patients with primary biliary cirrhosis (PBC), and to clarify the clinical efficacy of combined application of Chinese and Western medicine for PBC.Methods:Subjects were selected from patients diagnosed with primary biliary cirrhosis by our department from September 2011 to September 2012. Sixty of these patients that met the inclusion criteria were randomly split into a test group that received ursodeoxycholic acid capsule plus Yuganfang, and a control group that received ursodeoxycholic acid capsule only. Indicators of liver function were assessed before and after 4 and 12 weeks of treatment, which included alkaline phosphatase, total bilirubin, alanine aminotransferase, aspartate aminotransferase, y-glutamyl transferase. Statistical analysis was performed with SPSS17.0, count data was compared using paired t test, rates were compared by x2 test, and P<0.05 was deemed statistically significant, so as to evaluate the clinical efficacy of ursodeoxycholic acid capsule combined with Yuganfang to treat PBC.Results:1.Analysis of efficacy (Table 1):after 4 weeks of treatment, total effective rate was 83.3% for the test group and 63.3% for the control group, the difference between the two was significant (P<0.05); after 12 weeks of treatment, total effective rate was 90.0% for the test group and 66.7% for the control group, and the difference was more obvious (P<0.001).2. Analysis of indicators of liver function (Figure 1、2、3):before of treatment, no significant differences were detected in indicators of liver function between the two Groups (P>0.05). ALP, GGT, ALT and AST of both groups showed obvious reduction after 4 weeks of treatment (**P<0.05,***P<0.001),but the reduction of TBil was not obvious in the two groups (P>0.05).the reduction of ALT、AST、GGT and ALP was more obvious in the test group than in control group, and the difference was significant (**P<0.05,***P<0.001),but the reduction of TBil was no significant difference (P>0.05). After 12 weeks of treatment, still significant differences were detected in indicators of liver function between the two groups (**P< 0.05,***P< 0.001). Reduction of TBil、ALP、ALT、ASTand GGT was more obvious in the test group than in the control group, and the differences were significant (**P<0.05,***P< 0.001)Conclusion:Ursodeoxycholic acid capsule combined with Yuganfang can effectively improve liver function in patients with primary biliary cirrhosis. It’s safe and free of adverse side effects, thus worthy of clinical promotion.