Study On The Preventive Effects And Mechanism Of Erzhu Jiedu Decoction On DEN-induced Liver Precancerous Lesions In Rats

Objective: Hepatocellular carcinoma(HCC)is the fifth most common cancer among men worldwide,and the seventh among women,and an important cause of malignant tumor-related death.Precancerous lesions of liver cancer are the intermediate stage of the development of liver cancer from cirrhosis.Therefore,some scholars believe that this process is reversible.Early intervention measures can effectively prevent the occurrence of liver cancer and prolong the life of patients,thus bringing greater economic effects.Erzhu Jiedu Decoction is Professor Jianjie Chen,s empirical formula in the treatment of cirrhosis,liver precancerous lesions and liver cancer according to the academic philosophy of “Preventive treatment of disease”.Erzhu Jiedu Decoction has the following effects: invigorating spleen and eliminating dampness,clearing heat and detoxifying,softening hardeness and dissipating nodulation”,and has achieved good curative effect in actual clinical practice and has high research value.The purpose of this study was to evaluate the preventive effect of Erzhu Jiedu Decoction on liver precancerous lesions induced by diethylnitrosamine(DEN)in rats and to explore the molecular mechanism.Methods:Sixty-two SPF male Wistar rats were adaptively fed for one week,then randomly divided into 6 groups according to their body weight: normal group(n=8),model group(n=14),and Cinobufotalin group?Erzhu Jiedu Decoction low?medium high-dose group(n=10).Model group,Cinobufotalin group,Erzhu Jiedu Decoction low,medium and high dose groups were injected intraperitoneally with DEN for liver precancerous lesion model.The intragastric dosages of Erzhu Jiedu decoction in the low,medium and high dose groups were 0.525g/Kg,1.05g/Kg and 2.1g/Kg per day.The intragastric dosages of Cinobufotalin group was 0.15g/Kg per day.The rats in the normal group and the model group received equal doses of normal saline by gavage,and the volume of gavage in each group was 5ml/Kg.At the end of week 16,the rats fasted for 12 hours after the end of intragastric administration.The rats were anesthetized with pentobarbital sodium.Blood was taken from the abdominal aorta,and the liver and spleen were weighed.Test indexes: body weight,liver and spleen indexes;ALT,AST,GGT;Liver HE staining;Immunohistochemistry was used to detect the expressions of GST-Pi,AFP,CK19,c-Myc and Ki67 in tumor tissues.RT-PCR was used to detect the expressions of AFP m RNA,CK19 m RNA,c-Myc m RNA,YAP m RNA and TAZ m RNA in tumor tissues.Results:(1)Compared with the model group,the body weight of Cinobufotalin group and Erzhu Jiedu Decoction increased significantly(P<0.01);compared with the lowdose group of Erzhu Jiedu Decoction,The body weight of Erzhu Jiedu Decoction medium-dose group t was significantly increased(P<0.05).(2)From week 4,rats in the model group,Cinobufotalin group and Erzhu Jiedu Decoction in low,medium and high doses began to eat significantly less than those in the normal group,with vertical dorsal hair,slow movement,listlessness,slow or gradual weight loss and other manifestations.However,the levels of Cinobufotalin group and Erzhu Jiedu Decoction the low,medium and high dose groups were better than those in the model group..(3)Compared with the model group,the liver index and spleen index of Cinobufotalin group and the low,medium and high dose groups of Erzhu Jiedu Decoction were significantly lower(P<0.01).(4)Rat liver morphology: the liver tissue of the model group was dull,rough and hard with a dark red color,and the liver surface was changed by single or overspread white tumor nodules with different sizes,nodules with different diameters(0.5 ? 1.5cm),or with different degrees of cirrhosis,but no ascites were observed.The liver tissues of the rats in the Cinobufotalin group and Erzhu Jiedu Decoction low,medium and high dose groups had dark luster and hard texture with dark red color and fewer nodules on the surface of the liver.Liver HE staining,the model group rats liver cells have different degree of degeneration,necrosis,disordered arrangement is multifocal,nests,beam pattern,cellular pleomorphism,big nucleus,a dual-or multicore,dyeing is deeper,increased density,increased nuclear/cytoplasmic ratios,and liver cell dysplasia,accompanied by fibrous tissue rights,false flocculus to form.Compared with the model group,the hepatocytes of rats in the medium dose group of Erzhu Jiedu Decoction were degenerated and necrotic,with nests in disorder,and the degree of connective tissue hyperplasia was relatively low.The hepatocytes of rats in the Cinobufotalin group and Erzhu Jiedu Decoction low,high dose groups were between the model group and the Erzhu Jiedu Decoction medium dose group.(5)Compared with the model group,Cinobufotalin group and the low,medium and high doses of Erzhu Jiedu Decoction could significantly down-regulate serum ALT,AST and GGT levels(P<0.01).(6)Compared with the model group,the expressions of GST-Pi,AFP,CK19,c-Myc and Ki67 in the low,medium and high dose groups of Erzhu Jiedu Decoction and Cinobufotalin group were significantly decreased.(7)Compared with the model group,the expressions of AFP m RNA,CK19 m RNA,c-Myc m RNA,YAP m RNA and TAZ m RNA in the low,middle and high dose groups of Ershu Jiedu Decoction and Cinobufotalin group were significantly decreased.Conclusions:(1)Erzhu Jiedu Decoction has a significant inhibitory effect on DENinduced liver precancerous lesions in rats.(2)Erzhu Jiedu Decoction can inhibit the expression of YAP and TAZ,but whether it is related to the activation of Hippo pathway,further research is needed.