| Objectives:Wilson disease is an autosomal recessive disorder of copper homeostasis, the incidence of which is30/1000,000. WD typically occurs in young people. The clinical manifestations are chronic hepatitis, renal and neurological abnormalities. Research shows liver is the first organ involved, and the smaller the age is, the greater possibility of the liver disease is the only clinical manifestation. Hepatic fibrosis is the main liver pathological change almost in every WD patient. As a copper metabolic aplastic disease, the basic cause of hepatic fibrosis is the difficulty of copper to get drained through bile, which causes free copper to accumulate slowly in liver tissue.Researches prove that HSC is a key part of the happening of liver fibrosis, the activation of proliferation in HSC, phenotype change and excessive secretion in ECM play important roles in hepatic fibrosis.Among the studies on HSC signal pathway in recent years, MAPK is one of the hot points. P38mitogen-activated protein kinases pathway is an important branch in MAPK mediated signal pathway, which is involved in inflammation, proliferation, apoptosis and many kinds of other physical processes, and has become one of the key pathways in cell apoptosis.Metabolomics increasingly becomes a very powerful method in studying the functional change of life system. Since WD is a disorder of copper homeostasis,the changes of the liver metabolites spectrum may be different from other liver diseases, and studies of metabonomics in WD for pathogenesis, treatment and prognosis seem particularly important.At present, there is no specially effective means in the prevention of hepatic fibrosis especially in WD, but traditional Chinese medicine has unique curative effect with its overall adjustment in many targets and personalized treatment in the prevention of hepatic fibrosis. It is proved by my tutor’s research group through their clinical practice that catharsis and diuresis, expelling stasis and mass recipe, Gandouling, can improve the index of hepatic fibrosis, and promote the recovery of liver cirrhosis and liver function in WD.This experiment is to carry out the following research:to establish copper overloaded rats, to observe the effects of catharsis and diuresis, expelling stasis and mass recipe, Gandoutang, on p38-MAPK pathway and the apoptosis of hepatic stellate cell, to use metabonomics to inspect the effect of Gandouling on changes of metabolites of hepatic fibrosis in copper overloaded rats, to discuss the molecular mechanism of catharsis and diuresis, expelling stasis and mass recipe in treating hepatic fibrosis in WD.Methods:1.The rats were divided into5groups:control group, model group, Gandouling group, Penieillamine group,Gandoutang and Penieillamine group.2. Copper overloaded rats were modeled according to documents. 3. Copper overloaded rats in each group were intervened with catharsis and diuresis, expelling stasis and mass recipe, and Penieillamine was used as positive drugs.4. Pathological changes of hepatic fibrosis were observed.5. Expression of P38MAPK protein was observed with immunohistoche-mical and western-blot methods, and cell apoptosis of HSC was detected with TUNEL method.6. Changes of small molecules metabolites from serum and urine of hepatic fibrosis in WD were analyzed with1H-NMR techniques.Result:1.The effect of the catharsis and diuresis, expelling stasis and mass recipe on pathology of hepatic fibrosis in copper overloaded rats.Normal group had complete structure in hepatic lobule. liver cells were in order. There was no cell degeneration inside the lobule, and no infiltration of inflammatory cells. There was obvious fiber hyperplasia in portal area in model group, and the degree of hepatic fibrosis was significantly reduced in treatment groups. There were green collagen fibers under light microscopy in Masson dyeing. There were only a few green collagen fiber distributions in central venous wall in normal group. There were thick green collagen fiber distributing in portal area and central venous wall in model group, which was of significant meaning compared with normal group (P<0.01).The degree of hepatic fibrosis was significantly reduced in treatment groups, which was of significant meaning compared with normal group (P<0.01).There was a significant decrease of pat-hogenic score in the integrated treatment of Gandoutang and Penieillamine group (P<0.01).Pathogenic score decreased obviously in Gandoutang and Penieillamine groups(P<0.05).2.The effect of the catharsis and diuresis, expelling stasis and mass recipe on HSC in copper overloaded rats Cell apoptosis detected with TUNEL method,there were visible expressions of hepatic stellate cells in model group, and there were visible expressions of apoptotic cells in treatment groups.The apoptosis index of treatment groups was significantly higher than that of model group(P<0.05). The apoptosis index of Gandoutang and Penieillamine group was significantly higher(P<0.01).3.The influence of the the catharsis and diuresis, expelling stasis and mass recipe on copper overloaded rats in p38MAPK signal pathway.3.1The expression of p38MAPK signaling pathway was detected with immunohistochemical methodThe positive signal of P38MAPK protein was tan-yellow in color, and there were visible positive expression in model group and treatment group. The data of model group ware significantly enhanced than those of normal group (P<0.001).The positive signal of P38MAPK protein of treatment groups decreased more than that of the model group(P<0.05), and there were no significant differences between the groups.3.2The expression of p38MAPK signaling pathways was detected with western-Blot method.There were significant differences in P38MAPK protein levels between the groups.Compared with the normal group, there were visible specific protein strip expressions of p38MAPK in model group and treatment groups, and positive protein expression increased remarkably in model group than in normal group(P<0.001).Positive protein expression decreased more obviously in treatment groups than in model group,and was of statistics significance(P<0.05), and there was no significant difference between the groups.4.The metabolomic effect of the catharsis and diuresis, expelling stasis and mass recipe on copper overloaded rats4.1The influence of the characteristic metabolites in serum Model group contained more lactate,glycoprotein,lipid, taurine and pyruvate than normal group.Model group contained less glucose,choline, betaine,alanine,glycine,leucine, isoleucine and myoinositol than normal group.The catharsis and diuresis,expelling stasis and mass recipe could obviously increase the content of leucine, isoleucine, choline, glucose and3-hydroxybutyrate in serum of copper overloaded rats, and could reduce the content of glycoprotein, lipid, lactate and pyruvate. The catharsis and diuresis, expelling stasis and mass recipe had obvious adjustment effect on small molecules metabolites in copper overloaded rats, and played a repair role in metabolic abnormalities of hepatic fibrosis in copper overloaded rats.4.2The influence of the characteristic metabolites of urineModel group contained more lactate, lipid, taurine, TMA, creatine and creatinine than control group. Model group contained less TMAO and hippuratethan than control group. The catharsis and diuresis, expelling stasis and mass recipe could obviously increase the content of TMAO and hippuratethan in urine of copper overloaded rats, and could reduce the content of lactate, lipid, taurine, TMA, creatine and creatinine. The change of the characteristic metabolites in urine can not only reflect the liver damage itself, but also indicate the damage of kidney and other organs in copper overloaded rats, and are in accordance with the pathological characteristics of multiple organs damage of WD.Conelusion:1.In the study,the model of copper overloaded rat was esta blished by copper excess load in the rat. There were visible pathological changes of hepatic fibrosis in model group, and this model can reflect the pathogenesis of liver damage caused by sedimentation of excessive copper.2.This study found visible hyperplasia expression of HSC in copper overloaded rats. The apoptosis index increased signi ficantly in treatment groups than in model group, indicating that the catharsis and diuresis, expelling stasis and mass recipe played the role of inducing apoptosis of HSC, and the effect was better when it was used with Penieillamine.3.The study found there was positive expression of protein p38MAPK signal pathway in model group. The expression was obviously improved in treatment groups than in model group, but in normal group there were no corresponding change. The above results indicated the p38MAPK of hepatic fibrosis was activated in copper overloaded rats, and was possibly involved in the occurrence and development process of hepatic fibrosis in copper overloaded rats. The catharsis and diuresis, expelling stasis and mass recipe maybe play a role of inhibiting fibrosis in improving liver function through p38MAPK signaling pathway.4.This study got some characteristics metabolites such as lactate,taurine and choline by exploring the effect of catha rsis and diuresis, expelling stasis and mass recipe on serum and urine in copper overloaded rats with ’H-NMR metabolomicmethod. Through analyzing the metabolic pathway of the chara cteristics metabolites,we found copper not only affected the metabolism of glucose, lipids and proteins,but also caused the disorders of amino acids and other metabolic system. We also found the catharsis and diuresis, expelling stasis and mass recipe adjusted small molecules metabolites of overloaded rats in many ways through many targets and metabolic network, had obvious adjustment effect on small molecules metabolites,and played a repair role in abnormal metabolism of hepatic fibrosis of WD. |
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