The MiRNA-122 And Metabonomics Impact Of Gandouling Tablet On The Tissue Of Copper Overloaded Rats

1.Background and purposeWilson’s disease(WD), also called hepatolenticular degeneration(HLD), is a copper metabolism disorder of autosomal recessive inheritance. As there is ceruloplasmin(CP) synthesis disorder and biliary copper excretion barrier in vivo, excessive copper accumulates in organs and tissues, such as the liver, brain, corneal, and so on, causing dysfunctions of corresponding organs with liver injury, neural/ psychiatric symptoms, cornea Kayser-Fleischer ring and kidney injury as the main manifestations,even endangering life. Researches show that the liver is one of the first involved organs of WD, with the younger age of onset, the greater possibility of liver disease as the only manifestation.Due to mutations in ATP7 B gene causing ATP7 B dysfunction, excessive copper in vivo can not be eliminated, and therefore, large deposits of copper occur in the liver, the brain, the kidney and other organs and tissues. Excessive copper in vivo induces free radical reaction and lipid peroxidation, injures hepatocytes, causing fatty degeneration and inflammation, resulting in death of hepatocytes. Copper is released into the plasma, leading to hemolysis and copper deposition in extrahepatic tissues after the death of hepatocytes, causing lesions, impaired structure and dysfunctions of involved organs and tissues, such as chronic hepatitis, hepatocirrhosis and hepatic failure.Researches show that hepatocyte apoptosis exsist in all liver diseases, and persistently regular hepatocyte apoptosis is an important reason of WD liver injury.New researches show that Mi RNA controls nearly 1/3 of human genes, regulates target gene expression in m RNA stage, and cytogenesis, cell differentiation, cell multiplication, apoptosis are regulated by mi RNA. Mi RNA-122 is specificly high expressed in the liver. Mi RNA directly and indirectly affects the process of liver cell apoptosis by targetly regulating apoptosis factors.Metabonomics develops rapidly in many fields of life science since its birth, providing new research methods for studying evaluation theories, curative effect, and other key problems of Chinese medicine.Liver is a crucial organ of metabolism, and participates in synthesis, decomposition, transformation and excretion. Researches on hepatitis, cirrhosis, liver cancer, hepatic failure have been carried out through metabonomics.However, the WD hepatic metabolome is different from other hepatopathies, so hepatic metabonomics is more important in WD researches.Previous researches on WD patients reveal that in general, patients with liver disease onset manifest no clinlcal symptom of hepatic dysfunction or hepatocirrhosis but with the onset of acute liver failure. Research group led by my tutor has proved in clinical and experimental research that compound Chinese medicinal preparation Gandouling can promote copper excretion, protect the liver, and accelerate recovery of the liver.This experiment mainly carries out the following research: establishing copper-loaded model rats, observing the effect of Gandouling on liver tissue in ALT, AST, TBIL, serum copper, liver copper and liver pathology,applying TUNEL to testing hepatocyte apoptosis, and immunohistochemical method testing NF-KB, Caspase-3, Bcl-2 and Bax, RT-PCR testing the abundance of mi RNA-122 in liver tissue, metabonomics testing the effect of Gandouling on metabolic profiling of the model rats,preliminarily exploring the molecular mechanism of Gandouling treating WD liver injury. 2. Methods 2.1 The model rats were divided into 4 groups: the control group, the model group, the penicillamine group, and the Gandouling group. 2.2 The models were established according to the references. 2.3 The Gandouling group was treated with Gandouling, and penicillamine as positive control drug. 2.4 Serum ALT 、 AST 、 TBIL were tested by automatic biochemical analyzer;serum copper and liver copper were tested by atomic absorption method. 2.5 Hepatocyte apoptosis was tested by TUNEL, and expressions of NF-KB、Caspase-3、Bcl-2 and Bax were tested by Immunohistochemical method. 2.6 mi RNA-122 expression in liver tissue was tested by RT-PRC. 2.7 Changes of small molecular metabolite profiles were tested by UPLC-MS. 3. Results 3.1 The effect of Gandouling on serum and liver copper of copper-loaded model rats: compared with those of the control group, serum and liver copper increased significantly(P<0.01)in the model group, revealing there was copper accumulation in the model rats, and the models were successfully established; compared with those of the model group, serum and liver copper decreased significantly( P<0.01) in the Gandouling and penicillamine groups;Serum and liver copper levels were lovwer than that of the Gandouling group(P<0.05). The above results reveal that there is certain copper excreation effect in Gandouling, and the effect is weaker than penicillamine. 3.2 The impact of Gandouling on liver function of copper-loaded rats.The comparison of liver function among different groups: compared with those of the control group, the levels of ALT、AST、TBIL of the model group increased significantly(P<0.01);compared with those of the model group, the levels of ALT、AST、TBIL of penicillamine and Gandouling decreased significantly(P<0.01);the levels of ALT, AST and TBIL were lower in Gandouling group than those in the penicillamine group( P<0.05, and P<0.01). 3.3 The impact of Gandouling on hepatic tissue pathology of copper-loaded model rats.The result of HE stain revealed that there was clear structure of hepatic lobule in the liver tissues of the normal group, hepatocytes arraed orderly around the central vein and portal area, there was clear boundaries between the hepatocytes, with similar hepatocyte size; there was disorder structure of hepatic lobule in the liver tissues of the model group, blurred boundaries between the hepatocytes, the nucleus disappeared, or shrinked. The injury of liver tissues was reduced in the two treated groups than in the model group. 3.4 The impact of Gandouling on hepatocyte apoptosis of copper-loaded ratsThere was no hepatocyte expression in the normal group, abundant expression of tan hepatocyte apoptosis in the model group, expression of brown yellow and spindle hepatocyte apoptosis in the treated groups, compared with that of the normal group, the number of apoptotic cells increased significantly in the model group(P< 0.01), the number of hepatocyte apoptosis decreased significantly in the treated groups than in the model group, and there was difference between the Gandouling and penicillamine groups(P<0. 05). 3.5 The impact of Gandouling on caspase-3, bcl-2, bax and NF-KBCompared those in the normal group, there were large quantities of positive expressions of tan or brown yellow cells in the model group, there were positive expressions of tan or brown yellow cells in the treated groups, the number of positive cells in the model group was higher than that in the normal group(P< 0.01),the number of the positive cells in the treated group was lower than that in the model group(P<0.01),and there was difference between the Gandouling and penicillamine groups(P<0. 05). 3.6 The comparison of the expression of liver tissue mi RNA-122 among the groupsCompared with that in the normal group, the expression level of mi RNA-122 decreased significantly in the in the model group(P<0.01),compared with that in the model group, the expression level of mi RNA-122 increased significantly in the Gandouling and penicillamine groups(P<0.05,and P<0.01), and the expression level of mi RNA-122 in Gandouling group was higher than that in the penicillamine group(P<0.05). 3.7 The impact of Gandouling on metabonomics of copper-loaded ratsThere was decrease of lyso-phosphatidylcholine(20:5、18:2、16:0、18:3、20:1、20:3) and oleamide,increase of tryptophan, phenylalanine, stearamide, taurine chenodeoxycholic acid, glycocholic acid and cholaic acid in the model group. Gandouling increased the content of lysophosphatidylcholine(20:5、18:2、16:0、18:3、20:1、20:3) and oleamide, decreased the content of tryptophan, phenylalanine, stearamide, taurine chenodeoxycholic acid, glycocholic acid, cholaic acid. 4. Conclusion 4.1 This research adopts the copper-loaded method to establish the models.There is pathological changes of liver injury due to excessive copper in the model group, and this model can reflect pathological changes of liver injury due to excessive copper. 4.2 This research finds there is increase in ALT, AST, TBIL serum copper and liver copper in copper-loaded rats, while the above data is lower in the treated groups than in the model group.There is obvious decrease of ALT, AST and TBIL in the Gandouling group than in the model group. Serum and liver copper of the penicillamine group are lower than those of the Gandouling group, hinting that there is inhibition function on hepatic injury in Gandouling, while the copper excreation function is weaker than that of penicillamine.4.3 This research shows there is a large number of expressions of hepatocyte apoptosis in copper-loaded rats, decreased hepatocyte apoptosis in treated groups, compared with that in the model group, revealing there is inhibition function on hepatocyte apoptosis of copper-loaded rats in Gandouling. 4.4 This research finds there are positive expressions of caspase-3, bcl-2, bax, NF-KB in the model group, and there is significantly improvement in the treated groups, compared with the the model group, revealing that when hepatic injury occurs in copper-loaded rats, caspase-3, bcl-2, bax and NF-KB are activated and participate in the whole process of the occurance and development of hepatic injury in copper-loaded rats. Possibly, Gandouling plays a protective role in prohibiting caspase-3, bcl-2, bax and NF-KB. 4.5 This research finds that there is significantly lowered content of mi RNA-122 in the model group, while significant increase in the treated groups, and a significant increase in the Gandouling group, compared with that in the model group, revealing that mi RNA-122 may participate in the occurance and development of hepatic injury of copper-loaded rats, and Gandouling may protect the liver and improve the hepatic injury by increasing mi RNA-122. mi RNA-122 can be used as a specific marker of liver injury, mi RNA-122 possibly through caspase-3, bcl-2, bax, NF-KB-mediated apoptosis of liver cells. 4.6 This research applies UPLC-MS metabonomics to studying the effect of Gandouling on hepatic injury of copper-loaded rats, obtaining such metabolites as lysolecithin, oleamide, tryptophan, phenylalanine, stearamide, taurine chenodeoxycholic acid, glycocholic acid, cholaic acid, and so on. Through analyzing the metabolic pathway, the author find that copper affects lipid, amino acid and bile acid metabolisms of the liver tissue, while Gandouling protects the liver and improves hepatic injury through regulating and bettering these metabolic pathways, embodying the trait of multi way, multi level, and multi target in Chinese medicine treating diseases.A large number of researches prove that hepatic injury is reversible.So paying attention to the treatment on hepatic injury of early stage of WD is of great significance in improving hepatic function and the prognosis of WD.