| Objectives: Primary biliary cirrhosis(PBC) is a kind of autoimmune disease that is common in middle-aged women. Its pathological changes mainly include chronic non-suppurative damages of minute biliary ducts in the liver, inflammation at portal areas, chronic cholestasis and hepatic fibrosis. The pathogenesis of PBC is not fully clear but may be related to autoimmune, infection and cell lesions. Therefore, in this topic we did preliminary research about TCM syndrome types of PBC patients by the method of multivariate statistics and discussed the mechanism of Tiaogan Lipi Decotion(TGLP) in prevention and treatment actions to PBC according to the changes of CD4+CD25+Foxp3+Tregs from peripheral blood and hepatic pathology of the PBC animal model established by Polyinosinic-polycytidylic acid(poly I: C).Methods: 1. The clinical researchAccording to the clinical characteristics of PBC and the four diagnostic methods of traditional Chinese medicine(TCM), we collected the four diagnostic information such as symptoms, signs, tongue and pulse conditions in 105 patients with PBC by means of clinical epidemiological investigation, which were finally statistically analyzed through he combined method consisting of system cluster, principal component and factor analysis. 2. The experimental researchWe took 30 C57 BL / 6 mice as the normal control group by the random number table method.The remaining 150 mice were randomly divided into the model control group, the positive control group(0.1g·kg-1·d-1),the low dose TGLP group(13.5g·kg-1·d-1), the middle dose TGLP group(27.0g·kg-1·d-1), the high dose TGLP group(54.0g·kg-1·d-1), 30 in each group. They were all given PolyI:C 5mg/kg via intraperitoneal injection, twice a week until 24 th week. At the same time, the mice in the positive control group and TGLP groups were given lavage treatment with respective drugs by the volume of 20 ml·kg-1. The normal control group and the model control group were given distilled water by the equal volume once a day for 24 weeks. All mice were executed after being fasted for one day at different times(week 8, 16, 24) and their livers and serums were taken out. Anti-mitochondrial antibodies subtypeM2(AMA-M2) were detected by Enzyme-linked immunesorbentassay(ELISA).While the proportions of CD4+CD25+Foxp3+Tregs on T lymphocytes from peripheral blood in each group were analyzed through flow cytometry. Also, pathological changes in liver tissues were observed with HE and Masson staining methods, so were the changes of CK19 with immune histochemical method. Results: 1. The clinical researchBy multivariate statistical methods, the TCM syndromes of 105 patients with PBC can be divided into three types: stagnation of liver-qi with deficiency of the spleen, internal retention of fluid-dampnessin, liver-kidney Yin deficiency and qi stagnation with blood stasis.2. The experimental research(1) Apart from the normal group whose AMA-M2 is always negative, the positive rate of AMA-M2 in the other groups is gradually increasing with the modeling time going on and even reached 90% on 24 th week. Compared with the model group, the positive rate of AMA-M2 in the TGLP groups and the positive control group has decreased.(2) With the increase of the modeling time, the proportion of CD4+CD25+Foxp3+ Tregs from peripheral blood in the model group is lower than that in the normal control group in the corresponding period(P<0.05). While it tends to decline in the normal control group, the model group and the TGLP high dose group as the modeling time goes on. On the contrary, the proportion gradually increases in the TGLP middle dose group over time. And there is no statistical difference(P>0.05) when compared with the normal control group on week 24.(3) Liver histopathology shows that the hepatic lobule structure is distinct in the normal control group. Glaucous collagen fibrils are not visible around portal areas. In the model group, the hepatic lobule structure is disorder and collagen fibers has increased obviously. Furthermore, there are some streak-like hyperplasia but pseudolobuli can be seen. The pathology construction in the middle dose TGLP group, the high dose TGLP group and the positive control group have all improved compared to the model group.Immunohistochemical staining shows that CK19 was expressed on small bile ducts, around where there were no lymphocytes infiltration and bile duct proliferation.The expression of CK19 increased significantly in the model group. The number of bile canaliculus whose lumen were small and abnormal incresed significantly at portal areas and fibrous septum. And intrahepatic small bile ducts were destroyed. CK19 positive cells dispersedly distributed and lymphocytes infiltration could be seen. Compared with the model group, the expression of CK19 decreased in the high dose TGLP group,the middle dose TGLP group and the positive control group. Conclusion:1.The application of multivariate statistical methods make the research on TCM syndrome characteristic of PBC patients more standard,objective and normative.2. PolyI:C injection to C57BL/6 mice can establish PBC animal model successfully.3. The TGLP groups can maintain the state of immune tolerance through increasing the peripheral regulatory T cells to slow the disease progression of PBC.4. The TGLP groups can improve inflammation and fibrosis at the portal areas in hepatic tissues of PBC mice and reduce bile canaliculus proliferation. The curative effects are better compared to the positive control group. |
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