Research On Zi Shui Qing Gan Recipe Adittion And Substraction Decoction In Preventing And Treating Hepatocellular Carcinoma Induced By Hydrodynamics-based AKT/N-Ras Plasmid Administration Via Tail Vein

PartⅠEstablishment of Primary Liver Cancer ModelObjective:Construction of primary liver cancer model by injection of AKT/N-Ras plasmid by hydrodynamics-based administration via tail vein.Method:In this experiment,the animal model of primary liver cancer was constructed by hydrodynamics-based administration.The AKT/N-Ras plasmid was rapidly injected into the experimental animals through the tail vein.The experimental animals were FVB mice,C57BL/6J mice and C57BL/6J Ang-1^（-/-）mice,24 animals each,half male and half female,were randomly divided into normal group and model group.The mice in the model group were injected with AKT/N-Ras plasmid to induce the primary liver cancer,after injection,observe the general condition of the mice,after dissection,the color,size,texure and lesion of the liver were observed,the liver weight was weighed,the liver organ index was calculated,and the diseased liver tissue was fixed by formalin,embedded with paraffin,made into paraffin sections,which were stained with HE,and the liver histopathology changes were observed under the microscope.Immunohistochemistry was used to detect the expression of AKT,P-AKT,ANG and CD133 in liver tissues.Results:1)In the FVB mouse model:the mice in the model group began to have abdominal augmentation in the third week,and the liver was significantly enlarged compared with the normal group.The longest survival time after modeling was five weeks.In the normal group,the liver was reddish,the surface was smooth,and the texture was soft.The liver of the model group was pink at the first week,and white lesions were visible on the surface.At the third week,the appearance was significantly changed compared with the normal group,and the surface of the liver was full of grayish white granules which are unequal in reddish brown.At the fourth week,the liver is yellowish white overall,with fatty lesions and hard texture.The liver coefficient of the model group was larger than that of the normal group,with significant difference（p<0.001）.HE sections showed that the liver of the model group had mixed liver cancer at the third week of modeling.The type of cancer was mainly hepatocellular carcinoma,and the liver nucleus became large,deep stained,and nuclear abnormalities appeared.The positive rates of AKT,P-AKT and ANG in the first,third and fourth week of model group were significantly higher than those in the normal group,with significant difference（p<0.001）.The positive rates of AKT,P-AKT and ANG in the third week of the model group was higher compared with the first week,with significant differences（p<0.001）,increased by fourth week compared with the third week,with a significant difference（p<0.001）.2)In the C57BL/6J mouse model:the mice in the model group began to have abdominal augmentation in the fourth week,and the mice survived for a maximum of six weeks after modeling.In the model group,the liver was yellow in the fourth week,the texture was soft and the surface was smooth.On the sixth week,the liver was pink,soft in texture,rough in surface and erosive.The hepatic coefficient was larger than that of the normal group,with a significant difference（p<0.001）.HE sections showed that at the fourth week,the liver had mixed liver cancer,and in the fourth week,cholangiocarcinoma was the main disease,and in the sixth week,hepatocellular carcinoma was the main.The positive rates of AKT,P-AKT,ANG and CD133 of model group were significantly higher than those in the normal group（p<0.001）.The positive rates of AKT,P-AKT,ANG and CD133 in sixth week increased compared with the fourth week,with significant difference（p<0.001）.3)In the C57BL/6J Ang-1^（-/-）mouse model:the abdominal bulge began to increase in the fifth week of the model group,and the longest survival time of the mice after modeling was seven weeks.At five weeks of model mice,the liver surface was uneven in color and soft in texture.At the seventh week,the liver was erosive and soft.There was a significant difference（p<0.001）in the model liver organ coefficient compared with normal group.HE slices showed that the liver produced mixed liver cancer in the fifth week,most of cholangiocarcinoma in the fifth week,and most of hepatocellular carcinoma in the seventh week.The positive rates of AKT,P-AKT,ANG and CD133of model group at the fifth and seventh weeks were significantly different from those in the normal group（p<0.001）.The positive rate of AKT,P-AKT,ANG and CD133 in the seventh week of the model group was higher than that in the fifth week,with a significant difference（p<0.001）.Conclusion:The primary liver cancer model was induced in all three animals successfully,and all three models were mixed liver cancer.In FVB mouse mixed liver cancer,hepatocellular carcinoma accounts for the majority,and mice have a short survival time,most of which is around four weeks;C57Bl6/J and C57Bl6/J Ang-1^（-/-）mouse liver cancer models,In the fourth week,the type of liver cancer was mainly cholangiocarcinoma.After five weeks,hepatocellular carcinoma was the main type.The survival time of the mice was slightly longer,but not more than seven weeks.In addition,C57Bl6/J and C57Bl6/J Ang-1^（-/-）liver cancer model mice had different degrees of canceration in the group,and the degree was inconsistent.The degree of canceration in the FVB mice group was consistent,which was more suitable for drug evaluation.With the prolongation of modeling time,the degree of canceration is aggravated,the expression of AKT,P-AKT and ANG in liver tissue is significantly enhanced,so the expression of AKT,P-AKT and ANG can be used as an indicator to measure the degree of cancer.According to CD133 staining results,it is shown that the reason why FVB is inconsistent with C57Bl6/J and C57Bl6/J Ang-1^（-/-）model mouse liver cancer types may be due to different tumor cell origin.Part Ⅱ Effect and Mechanism of Zi Shui Qing Gan Recipe Adittion and Substraction Decoction on Prevention and Treatment of Primary Liver Cancer in FVB Mice Induced by AKT/N-Ras PlasmidObjective: This part aims to investigate the effect and mechanism of Zi Shui Qing Gan Recipe Adittion and Substraction Decoction on preventing and treating primary liver cancer Induced by AKT/N-Ras Plasmid.Method: 36 healthy FVB mice were randomly divided into control group,model group,Zi Shui Qing Gan Recipe Adittion and Substraction Decoction 5g.kg-1.d-1,10 g.kg-1.d-1,15 g.kg-1.d-1,20 g.kg-1.d-1 dose groups,there were 6 mice in each group,half male and half female,in addition to the normal control group,all the mice were injected AKT/N-Ras plasmid through hydrodynamics-based administration via tail vein to establish primary liver cancer,then all mice were observed every day,after one week of injection,the normal group and the model group were given normal saline,and mice of Zi Shui Qing Gan Recipe Decoction group were administered with 5g.kg-1.d-1,10 g.Kg-1.d-1,15 g.kg-1.d-1 and 20 g.kg-1.d-1 of Zi Shui Qing Gan Recipe Adittion and Substraction Decoction,continue administration for 21 days,the body weight of the mice were recorded every three days.On the 21 st day after administration of Zi Shui Qing Gan Recipe Adittion and Substraction Decoction,blood was taken from eyes of the mice which will be sacrificed.Blood routine and serum biochemical indexes were measured.The liver appearance was observed after dissection,the weight of liver and spleen was weighed,and liver organs and spleens indexes were calculated.The organ coefficient was taken from the diseased liver tissue for fixation,paraffin embedding,sectioning and HE staining.The histopathology of the liver was observed under microscope.The expression of AKT,P-AKT,ANG and Ki67 in tissues was detected by immunohistochemistry,and apoptosis was detected by TUNEL method.Results: Compared with the model group,the mice in the Zi Shui Qing Gan Recipe Decoction groups had improved in general status,but there was no significant difference in body weight.In the normal group,the surface of the liver was smooth and the color was red.The whole liver of the model group was grayish white,with white lumps on the surface,and the texture was soft.The surface of mice in Zi Shui Qing Gan Recipe Decoction groups was red,and some of them were more normal than model tissues.Among them,the pathological changes of liver tissue of mice in the 15 g.kg-1d-1 dose group of Zi Shui Qing Gan Recipe Decoction were significantly improved,and the liver organ coefficient and spleen organ coefficient were decreased.In blood routine examination,the number of white blood cells and platelets was significantly higher in the model group than in the normal group（p<0.001）.Compared with the model group,the number of white blood cells in the ZSQGY-10 and ZSQGY-15 dose groups decreased（p<0.05）,the number of white blood cells in the ZSQGY-20 dose group was significantly lower（p<0.01）,and the number of platelets in the ZSQGY-15 dose group was significantly lower（p<0.01）.In the serum biochemical index,ALT,AST,TB,TC of the model group was significantly higher than that of the normal group（p<0.001）.Compared with the model group,the ALT of ZSQGY-10 dose group was lower（p<0.05）,and the ZSQGY-15 dose group was significantly lower（p<0.01）;the AST of ZSQGY-5 and ZSQGY-10 dose group decreased（p<0.05）,ZSQGY-15 dose group decreased significantly（p<0.01）;the TB of ZSQGY-5 dose group decreased（p<0.05）,ZSQGY-10 dose group decreased significantly（p<0.01）,ZSQGY-15 dose group（p<0.001）;the TC of ZSQGY-10 dose group was lower（p<0.05）,and the ZSQGY-15 dose group was significantly lower（p<0.01）.AKT,P-AKT,ANG and Ki67 expression was not observed in the liver of normal mice,and AKT,P-AKT,ANG and Ki67 were highly expressed in the liver tissues of the model mice.The expression of AKT,P-AKT,ANG and Ki67 of Zi Shui Qing Gan Recipe Decoction groups was significantly decreased compared with the model group（p<0.001）;the number of TUNEL staining positive cells of the model group was significantly increased compared with the normal group（p<0.001）,Compared with the model group,the number of positive cells in the ZSQGY-5 dose group increased（p<0.05）,There was a significant increase in the number of positive cells with the ZSQGY-10,ZSQGY-15 and ZSQGY-20 dose group（p<0.001）.Conclusion: Zi Shui Qing Gan Recipe Adittion and Substraction Decoction in this experiment has a certain preventive effect on the primary liver cancer of FVB mice induced by AKT/N-Ras plasmid.The mechanism may be related to inhibiting PI3K/AKT pathway,inhibiting tumor cell proliferation,inhibiting angiogenesis,promoting tumor cell apoptosis and regulating immunity.