Study On Preventive Effect And Pharmacokinetics Of Rat Hepatic Precancerous Lesion By Paeonol

ObjectiveHepatocellular carcinoma is a tumor with high degree of malignancy and poor prognos-is,5years survival rate is only about7%, representing the fifth commonest malignancy worl-dwide and the third cause of mortality from cancer. In China, high incidence of chronic liver disease、cirrhosis、liver cancer is a result of the high infection rate of hepatitis B virus(HBV). Hepatitis-hepatic fibrosis-hepatic precancerous lesion-liver cancer is malignant development of chronic liver disease, liver fibrosis is reversible, early diagnosis and treatment can block th-e development of liver fibrosis to liver cirrhosis; five years survival rate of early hepatocellul-ar carcinoma can be improved if receiving treatment timely. Hepatic precancerous lesion is h-epatocyte dysplasia, adenomatoid hyperplasia nodules, regenerative nodules of hepatocyte, tubular metaplasia of hepatocyte and oval cell proliferation in chronic liver disease, with high probability of developing into liver cancer, especially the dysplasia or nodules and dysplastic cells in cirrhosis. In recent years, although the treatment of liver cancer has made some prog-ress, but clinical curative effect is not obviously improved, so occurrence and development of hepatic precancerous lesion is precluded or delayed, occurrence of hepatocellular carcinoma is prevented, both have important practical significance. Based on previous experiment, in this paper, we established hepatic precancerous lesion rat model, studied on prevention effect and pharmacokinetic changes of hepatic precancerous lesion by Paeonol.Methods1To establish model of rat hepatic precancerous lesionEstablished model of rat hepatic precancerous lesion induced by DEN, the dose of DEN was0,25,50,75,100mg/kg,18weeks of administration, observed pathological changes, at-ypia index, time-effect curve was regressed between atypia index and modelling processing hours[log(h)], dose-effect curve was regressed between AUCi and dose[log(dose)], discussed on time-effect relationship and dose-effect relationship of hepatic precancerous lesion induced by DEN.2Preventive effect of rat hepatic precancerous lesion by PaeonolEstablished model of rat hepatic precancerous lesion induced by DEN, and used Pae-onol to treat, compared general condition of rats and pathology of liver tissue、biochemical indexes、related indexes of morphometry, alpha-fetoprotein(AFP) expression in rat liver tissue by using immunohistochemistry and RT-PCR.3Pharmacokinetic study of Paeonol on rat hepatic precancerous lesion modelEstablished model of rat hepatic precancerous lesion induced by DEN, Paeonol was orally administered at different stages of disease, observed pharmacokinetic changes of Paeonol, detected SULT1E1expression in rat liver tissue by using immunohistochemistry and RT-PCR.Results1To establish model of rat hepatic precancerous lesionIn liver tissue lesions,0mg/kg group, pathological changes of hepatic tissue was slight-ly in each time period, appearing edema、droplet steatosis or spotty necrosis;25,50mg/kg group, metamorphic-fibrosis-cirrhosis-dysplasia were found in liver tissue;75,100mg/kg gr-oup, stage disease appeared earlier, with heavier degree. The atypia index of five doses were0.21%-0.61%、0.2%-1.17%、0.27%-1.55%、0.29%-2.24%、0.30%-2.25%respectively, increasing with time period. From atypia index of hepatocytes (corrected volume ratio of nuc-leus to cytoplasm), the half effective durations of diethylnitrosamine at0、25、50、75and100mg/kg to induce hepatic precancerous lesions were70347、1734、1536、1530and1183h, respectively. The areas under curves were0.0064,0.0084,0.0123,0.0165and0.0167[(atypia index)×log(h)], respectively. From the area under curve, the half effective dose of diethylnitr-osamine to induce hepatic precancerous lesions was48.225mg/kg. The atypia indexes in rats treated with50mg/kg diethylnitrosamine correlated positively with mitotic counts in hepatic precancerous lesions (y=0.0023x-0.0056, r=0.9217, n=10, P<0.01).2Preventive effect of rat hepatic precancerous lesion by PaeonolOn the general condition, model group began to present some pathological behaviors after giving DEN into adominal cavity for three weeks, Paeonol group had such similar condi-tion but was slighter than model group. On the gross morphology, liver surface was smooth、 soft and pink in control group; liver surface was dark red、enlargement, tawny granular nodules formed in model group;liver surface was relatively smooth, nodules smaller and less in Paeonol group, performance of liver was between control group and model group. It was found by pathological examination that the structure of hepatic lobules was normal, hepatic cords was separated arranged radially around central vein, hepatocyte no denaturation and necrosis, hepatic sinus no stenosis in control group; central venous was departured or absence, hepatic cord arranged in disorder, hepatic sinus stenosis, a lot of connective tissue hyperplasia in portal area, collagen fibers hyperplasia obviously, false lobular formed widely; hepatocyte dysplasia lesions and dysplastic nodules were formed, the size of hepatocyte was different, nucleoli prominent, nucleus was pleomorphic in model group;Most of the structure of hepatic lobules were near normal, liver cords still arranged in disorder, degeneration and necrosis of hepatocyte, hyperplasia of connective tissue were to a lesser extent, number of hepatocyte dysplasia lesions and dysplastic nodules were decreased obviously in Paeonol group. Masson’s trichrome stain showed that the condition of liver fibrosis in each group was similar with HE’s results, but with more obvious coloring of collagen fibers. Some morphological indexes indicated that Paeonol can ameliorate the atypia of liver cell, improve false lobule volume ratio, reduce release amount of single mast cell. The result of biochemical indexes indicated that Paeonol can obviously ameliorate liver tissue inflammation, repair damaged liver cells, reduce oxidative damage of lipid peroxide. Immunohistochemistry results showed that compared with model group, Paeonol group can obviously reduce number of AFP positive foci and its area, the difference was significant(P<0.01); RT-PCR results showed that compared with model group, the relative expression of AFPmRNA in Paeonol group decreased, the difference was significant(P<0.01).3Pharmacokinetic study of paeonol on rat hepatic precancerous lesion modelBy d000-d028-d056-d112, the results of biochemical indexes indicated that the damage to hepatocyte increased gradually, antioxidant capacity was decreased gradually. The results of immunohistochemistry manifested that the positive expression of EST were observed in each group, the expression of EST in hepatocyte was decreased, the expression of EST in endothelial cell was increased by d000-d028-d056-d112-The results of RT-PCR manifested that the expression of ESTmRNA was decreased by d000-d028-d056-d112.The results of pharmacokinetic manifested that in high, middle and low doses of Paeonol,AUC and Cmax increased, V and MRT decreased,CL decreased (increased by d056-d112in middle dose) by d000-d028-d056-d112.The results of tissue distribution indicated that compared to control group, in model group, AUC and Cmax increased, CL decreased except to lung, V decreased except to stomach, MRT increased in liver、heart、kidney and stomach, MRT decreased in spleen、lung、brain and small intestine. Liver and kidney were main distribution organ of Paeonol.The results showed that there are some differences in different phases of hepatic precancerous lesion about the pharmacokinetic of Paeonol.ConclusionsThe atypia index might be used to define hepatic precancerous lesions. A rat model of hepatic precancerous lesions could be replicated by intraperitoneal injection of diethylnitrosa-mine at a dose of48.225mg/kg once a week for9wk. Paeonol can obviously ameliorate rela-ted index of hepatic precancerous lesion, decrease expression of AFP mRNA, with preventing effect on hepatic precancerous lesion. Established a method for determination of Paeonol con-centration in plasma and tissue by UPLC, through pharmacokinetic study of Paeonol in each stages of hepatic precancerous lesion, confirmed that results of pharmacokinetic in each stage were different; through tissue distribution study of Paeonol in control and model group, con-firmed that liver and kidney are main distribution organ of Paeonol, other tissues also have distribution. The main factors which influences expression of pharmacokinetics parameters are the decreased expresstion of SULT1E1in liver, the increased expresstion of SULT1E1in kidney, and the decreased efflux of Paeonol.