The Study Of Pathogenesis And Treatment On Liver Fibrosis Induced By Pagumogonimus Skrjabini In Rats

博士研究生学位论文Background and ObjectivePagumogonimus skrjabini (P.s) is one of the three pathogenic paragonimus species in China, also is the main pathogenic paragonimus species which widely spread in Yunnan Province. If the worms parasitize in the liver for long time, it leads to liver fibrosis gradually, and develops into irreversible liver cirrhosis finally. To research the pathogenesis of liver fibrosis induced by P.s in rats, and to explore new drugs and other treatment measures,is significant and valuable for the prevention and treatment of paragonimiasis caused by P.s.P.s is one of the paragonimus species, the migration of juvenile worms mainly produce ectopic lesions. It migrates to the liver, brain, spinal cord, eye and other organs.It cause a series of specific symptoms and lesions. The liver is major damaged organs, which has attracted the attention of the clinical workers. The research on pathology, pathogenesis and diagnosis of liver disease caused by P.s has made some progress, but no systematic research report for this disease. Therefore, study the effect of P.s on liver fibrosis in rats, to provide theoretical and experimental basis for the prevention and treatment.The research shows that cytokines play an important role in liver fibrosis formation. T helper cells (Th cells) are main source of cytokines in liver. INF-y secreted by Thl cells, is anti-fibrosis cytokine. IL-4 secreted by Th2 cells, is profibrotic cytokine. The imbalanced Thl/Th2 cytokines are important factors to the formation of liver fibrosis. On the basis of this view, the experiment researchs the pathogenesis of liver fibrosis caused by P.s.At present, Triclabendazole has better insecticidal effect for P.s infection. However, the clinical dose of Triclabendazole was quite different, so the research to evaluate its therapeutic effect to the experimental rats infected with P.s by the morphology and pathological morphology, and probe into the best dosage of Triclabendazole to the juvenile worms of P.s. To provide the theoretical and experimental basis for clinical treatment is important.The recent studies have found that the process of liver fibrosis can still continue after removed the pathogenic factors in many cases. Therefore, the further anti-fibrosis therapy is necessary, besides the insecticidal treatment of Triclabendazole. Some studies found that Gentiana scabra bage plays a role on lung fibrosis and liver damage. So, this study selecting the traditional medicine- Gentiana scabra bage to treat liver fibrosis caused by P.s, and expound its pharmacological mechanism, to provide new drugs for the treatment of liver fibrosis caused by P.s.From the above, on the rats liver fibrotic model build by P.s infection, this experiment to research the pathogenesis and treatment of liver fibrosis from the overall level, cellular level and molecular level by the pathological observation, immunohistochemical and molecular biology technologies, to provide the theoretical and experimental basis for the prevention and treatment of liver fibrosis induced by P.s.MethodsThere are four parts in this research:1. The experimental study on liver fibrosis of rats infected with P.s: Using light microscope, transmission electron microscope, biochemistry and other methods to observe the pathological changes of liver tissue and dynamic changes of liver function of experimental SD rats. Evaluate the liver damage caused by P.s from different aspects, and summarized the characteristics on liver function and morphological changes after the rats infected with P.s, provide a scientific basis for the early diagnosis of the disease.2. The experimental study on role of interleukin-4, interferon-y in liver fibrosis formation of rats infected with P.s:observed the pathological changes of liver tissue in experimental SD rats by light microscope, measured the dynamic changes of expression of interleukin-4, interferon-y in the formed process of liver fibrosis in rats infected with P.s by the use of immunohistochemical, and evaluate the role of interleukin-4, interferon-y in liver fibrosis formation and regulation.3. The experimental study of Triclabendazole on the treatment of rats infected with P.s: using light microscope and transmission electron microscope, counting No.of the worms recovered, calculating worm recovered rate and worm reduction rate and measuring the sizes of the worms (length, width), weighing up wet weight of the worms, to observe the pathological changes of worms of P.s in experimental SD rats in each group.To evaluate the therapeutic effect of different doses of triclabendazole in rats infected with the juvenile worms of P.s. To investigate regularity of the dose-effect relationship.4. The experimental study on therapeutic effects of GSB on hepatic fibrosis induced by P.s in rats:using light microscope, immunohistochemical staining, RT-PCR to observed the pathological changes of liver tissue, the expression of collagen I, III and TIMP-1 protein and mRNA in liver samples in experimental SD rats. To evaluate whether it is necessary to make further anti-fibrosis treatment after Triclabendazole therapy on rat liver fibrosis caused by P.s, evaluate the therapeutic effects of GSB on rat liver fibrosis caused by P.s.Results1. The experimental study on liver fibrosis of rats infected with P.s:①Light microscopy observation to pathological changes of liver tissue:one week after infection, liver cells became swelling, some liver cells became fatty degeneration, no obvious necrosis in liver cells, no fiber proliferation in hepatic portal canal area; two weeks after infection,liver showed acute suppurative inflammation, small abscess could be seen in liver tissues; eight weeks after infection, fibroplastic proliferation could be seen in liver tissues; fifty-two weeks after infection, the hepatic portal canal area expanded significantly, proliferated small bile duct and fibrous tissues were all obvious, and formed clumps or rope-like structure.liver tissues fibrosis was obvious, a lot of false hepatic lobules formed.②Under transmission electron microscope:one week after infection, liver cells became swelling, some liver cells became fatty degeneration; the smooth endoplasmic reticulum became dilatation in some hepatocytic cytoplasm; congestion and fat storing cells could be seen in hepatic sinusoids, the liver damage relatively mild in this period; two weeks after infection, liver cells became more swelling, inflammatory cell infiltration in the local liver tissue, in this period, the most prominent change is the increased fat storing cells, and appeared collagen fibers between the liver cells, the liver Fibrosis appeared in this period; four weeks after infection, liver cells became balloon-degeneration and necrosis, even patchy necrosis,the inflammatory cells infiltration was more obvious than that after two weeks; six weeks after infection,the structure of hepatic lobules was still complete, but the small abscess formed and the patchy necrosis appeared in liver tissue, the seosinophil, neutrophils, lymphocytic infiltration and a little of proliferated fibrous tissues in hepatic portal canal area. eight weeks after infection, the liver cells showed severe necrosis and hemorrhage, local liver tissues fibrosis was rather obvious; fifty-two weeks after infection, a lot of false hepatic lobules formed.③Dynamic detection of liver function:four, six and eight weeks after infection, serum levels of albumin were decreased, and globulin were increased; six and eight weeks after infection, serum levels of ALT and AST were increased.2. The experimental study on role of interleukin-4, interferon-γin liver fibrosis formation of rats infected with P.s:①Light microscopy observation to pathological changes of liver tissue:As the infection time, the collagen fiber area ratio (S/T) and liver fibrosis rating increased, the degree of liver fibrosis gradually increased.②Immunohistochemical staining:The expression of interferon-γbegan to rise at one to eight weeks after infection, and reached the peak at eight weeks, then decline from twelve to sixteen weeks.Cytokines interleukin-4 gradually increased at one to sixteen weeks after infection, increased rapidly at the end of twelve weeks.3. The experimental study of Triclabendazole on the treatment of rats infected with P.s:①According to worm recovered rate,worm reduction rate:on the first day after finishing the treatment,there were no significant differences without exception between each other of rats in control group,low,middle and high-dose group (P>0.05); on the fifteenth,twenty-two day after finishing the treatment,there were significant differences between each other of rats in control group,low,middle and high-dose group (P<0.01), but there were no significant differences between rats in low-dose group and control group on the fifteenth day after finishing the treatment (P>0.05).②Wet weight mensurating of the worms:on the first day after finishing the treatment,there were no significant differences without exception between each other of rats in control group,low,middle and high-dose group (P>0.05); on the fifteenth,,twenty- two day after finishing the treatment,there were significant differences between each other of rats in control group, low, middle and high-dose group (P<0.01), but there were no significant differences between rats in middle and high-dose group on the fifteenth day after finishing the treatment (P>0.05).③The results measured on the sizes of the worms:on the first day after finishing the treatment,there were no significant differences without exception on the sizes of the worms(length,width) between each other of rats in control group,low,middle and high-dose group respectly (P>0.05); on the fifteenth,twenty-two day after finishing the treatment,there were significant differences without exception on the sizes of the worms(length,width) between each other of rats in control group, low, middle and high-dose group (P<0.01), but there were no significant differences on the sizes of the worms(length, width) between rats in low,middle and high-dose group on the fifteenth day after finishing the treatment (P>0.05).④Light microscope observation: low-dose group,body walls and intestinal walls were dropsical; middle-dose group,the external tegument dropped,smooth muscle exposed,parts of cortex cells degenerated, necrosised and dissolved,the cell bodies of gut epithelia dropped and decreased obviously; high-dose group,only muscle layer of body walls could be seen,cortex cells necrosised and dissolved,altitudinal swelling and looseness could be seen among parenchymal cells,gut epithelia dropped nearly outright, basal membranes of the intestinal walls could be seen only.⑤Transmission electron microscope observation(dissected on the twenty-two day after finishing the treatment):Triclabendazole had different extent breakage on ultrastructure of body walls and intestinal walls of the juvenile worms of P.s, the effect of low-dose group on the worms was slighter, the destructive effect of middle-dose group on the worms was stronger than that of low-dose group, but the destructive effect was not deadly, the destructive effect of high-dose group on the worms was deadly.The worms recovered of rats in control group showed no pathological changes.4. The experimental study on Gentiana scabra bages role against hepatic fibrosis induced by P.s in rats.①Light microscopy observation to pathological changes of liver tissue:Triclabendazole insecticidal treatment to improve the degree of liver fibrosis was not obvious, the expression of collagenⅠ,Ⅲand TIMP-1 protein in liver were not markedly reduced, has no difference when compared with the model group (P>0.05); compared with the fibrotic model group, the GSB could obviously improve the pathological changes in liver and enhance the degradation of collagen in the rat fibrotic liver, Liver fibrosis score significantly lower(P<0.05).②Immunohistochemical staining and mRNA expression all showed:Compared with model group, the expression of positive area of COL-Ⅰ, COL-Ⅲ, TIMP-1 in liver were markedly reduced in Gentiana scabra bage treated group, there are significant difference between them (P<0.05)Conclusions1. P.s can cause the injury of liver function in rats, the most prominent changes in liver function are the decrease of albumin, the increase of globulin and the increased activity of AST and ALT2. P.s infection can induce liver fibrosis in rats. The ultrastructural changes of liver tissue were for cells and organelles.Two weeks after infection. fat storing cells and collagenous fiber could be seen among liver cells, TEM observation to liver tissue is valuable for the early diagnosis of liver fibrosis caused by P.s.3. The mechanical damage of P. s to liver and the imbalanced Th1/Th2 are important factors to the formation of liver fibrosis in rats.4. The expression of IL-4, INF-y were increased in the formation of liver fibrosis in rats infected with P.s, the grade of liver fibrosis was related to infection time and the expression of cytokine IL-4, INF-y.5. Triclabendazole has remarkable therapeutic effect on rats infected with the juvenile worms of P.s.The therapeutic effect was improved obviously with the extension of time and increase of doses. The best dose is about 200 mg/kg.d.6. The mechanism of Triclabendazole to kill P.s is that the Triclabendazole damage the body walls and intestines tissue,which result in handicap of both pHysiological function and biochemical metabolize of assimilation, absorb, exudation and excretion etc.7. The liver fibrosis caused by P.s still continued after the treatment of Triclabendazole in. rats, so the further anti-fibrosis therapy is necessary.8. GSB plays an anti-fibrotic role by inhibiting liver collagen synthesis, promoting collagen degradation, and downregulating the expression of TIMP-1.9. TIMP-1 may be a potential therapeutic target in the GSB treatment on liver fibrosis caused by P. s.