Inhibition Effect Of Ursodeoxycholic Acid On Hepatocarcinogenesis And Its Mechanism

Background and AimUrsodeoxycholic acid (UDCA) is a kind of hydrophilic dihydroxy cholic acid.Due to its functions of cholagogue, protecting cells, resistance to apoptpsis and immue adjustment, it is used in treatment of various hepatic diseases in clinical, such as primary biliary cirrhosis, cholesterol calculus, cholangitis,cholestatic liver diseases. Recently we find that UDCA can inhibit hepatocarcinogenesis, in this study we establish liver cancer model and test the level of AST、ALT、AFP in serum and the level of DNA repair gene hMTH1in liver tissue to investigate the inhibitory effect of ursodeoxycholic acid (UDCA) on hepatocarcinogenesis and explore its mechanisms.ObjectiveSPF wistar rats, male,8weeks, weight (180±10) g, bought in shandong university animal experiment center, the rats were adaptly fed a week, then they were divided into experimental group and control group.Methods1、Establishing rat hepatoma model:75wistar rats were divided into5groups randomly:normal control group, UDCA control group(UDCA30mg/kg intragastric administration per day), DEN group, UDCA low dose group(UDCA15mg/kg intragastric administration per day), UDCA high dose group(UDCA30mg/kg intragastric administration per day),15rats each group. The rats in the last three group were given an i.p. injection of diethnitrosamine(DEN) three times a week for16weeks. 2、The rats were killed at the end of16th week, body weight, took liver tissue to counting the nodules, calculating liver/weight ratio and doing pathology inspection, drew blood from the heart (2ml), used ELISA to detect the AST/ALT/AFP level in the rat serum.3、Took liver tissue in each group, extracted RNA expectively, we use real time fluorescence quantitative RT-PCR and Western Blot to detect the expression of hMTH1in the rat liver tissue.Results1、At the end of the experiment, the DEN group, UDCA low dose group and high dose group established hepatoma model successfully, and the percentage of tumorigenesis was respectively93%(14/15)、80%(12/15)、60%(9/15). There were no abnormal findings in the liver of the normal control group and UDCA control group. The liver HE pathological morphology display that the liver cell arranged radially arroud the central vein, the structure of hepatic lobules were intergrity, no degeneration and necrosis in the liver of the normal contral group(Fig.2A) and the UDCA contral group(Fig.2B). Homever the normal hepatic lobule organizational structure disappeared, liver cell cord arranged disorderly, false flocculus and cancer nests formed, serious inflammatory cell infiltration, tumor cell atypia, and visible necrosis could be found in the DEN group(Fig.2C).The pathological changes of the UDCA low dose group(Fig.2D) was similar to the DEN roup, but there were less cancer nests and necrosis.The UDCA high dose group(Fig.2E) had a better pathological manifest.2、The body weight、liver weight and the liver coefficient of the normal control group and UDCA control group had no difference, but the body weight of the DEN group and UDCA intervention group is significantly lower than the normal conrol group (P<0.05); however, compared with DEN group, the body weight of the two UDCA intervention group was apparently higher, the liver weight and the liver coefficient were obviously lower (P<0.05);. The body weight of UDCA high dose group was apparently higher than low dose group, the liver weight and the liver coefficient were obviously lower (P<0.05)3、Compared with normal control group, the level of AST、ALT、AFP in the rat serum was distinctly higher (P<0.05); however, the indicators of the UDCA intervention groups were apparently lower (P<0.05); the indicators of the UDCA high group were apparently lower than low dose group (.P<0.05)4、We take the hMTH1mRNA expression level of the normal control group as reference, the hMTH1mRNA expression level of UDCA control group、DEN group、UDCA low dose group、UDCA high dose group was respectively (1.312±0.1236)(16.226±0.7423)(9.483±0.4604)(6.125±0.3298). The expression of hMTH1mRNA in the DEN group was obviously higher than the normal control group (P<0.05), but the expression of hMTH1mRNA in UDCA intervention group were lower than DEN group (P<0.05), UDCA high dose group lower more.Conclusion1、We copied the primary hepatocarcinoma model by DEN i.p. injection in16weeks.2、UDCA could reduced the liver tumor variable ratio caused by DEN, at the same time it could reduce the level of AFP in serum, explaining that UDCA had certain preventive effect in hepatocarcinogenesis.3、UDCA could reduced the level of AST and ALT in serum, the UDCA could inhibit the development of the hepatocarcinoma by surpressing inflammatory response.4、UDCA can reduce hMTH1mRNA expression in liver during hepatocarcinogenesis,which reflect that the inhibition effect of UDCA on hepatocarcinogenesis may be relate with the inhibition of oxidative stress.5、There was no side effect on normal rats, ruling out the drug itself for the development of the primary hepatocellulur carcinoma.