| Background and ObjectivesHepatic cellular cancer(HCC)is a kind of common malignant tumor in China with high degree of malignity,and high incidence of recurrence and metastasis.The death rate of HCC is the second in total malignant tumor,second only gastric cancer. Once HCC take place,the treatment is extremely difficult,and the prognosis is very bad.The survival rate of 5 years is only 2%.Therefore,preventing HCC and finding the drug which can block(or inhibit)hepatocarcinogenesis not only have significant academic value,but also can bring enormous economic benefit.Because of the multistage process of HCC development,there is a long process of hepatic precancerous lesion.Therefore,if hepatic precancerous lesion is blocked(or delayed),the genesis of HCC can be interrupted.The pathologic feature of hepatic precancerous lesion is atypical hyperplasia of hepatocyte,and abnormal proliferation and dysdifferentiation of body cells is the basic characteristic of tumor appears.It is important to find the drug which can depress abnormal proliferation of hepatocyte and induce the differentiation of hepatocyte.In recent years,some benefit studies of preventing HCC by traditional Chinese medicine have been done.Many studies have proved that some Chinese herbs may prevent liver cancer.But some factors,such as long cycle,complex influencing factor,difficulty of controlling object,cause that less studies in clinic and foundation for preventing HCC by traditional Chinese medicine have been thoroughly done.There is no effective preventive agent has been accepted generally in clinic practice.Zhengganfang(ZGF)is a compound recipe of Chinese herbs which is organized according to the HCC pathogenesis of traditional Chinese medicine.Zhengganfang composed of Radix Astragali(30g),Radix Salviae Miltiorrhizae(30g),Carapax Trionycis(15g),Fructus Ligustri Lucidi(15g),Herba Scutellariae Barbatae(15g),Herba Hedyotis Diffusae(3Og),et al,has three functions of activating blood and dissipating a mass,benefiting qi and nourishing yin,clearing away heat and toxic materials.In this study,histochemical,immunohistochemical, RT-PCR,PCR-ELISA methods and technique of computer imageanalysis were used to observe the influence of Zhengganfang on AFB1-induced precancerous lesion of rat liver,and to investigate the effect on differentiation of BEL-7402 human hepatocarcinoma cell(HHC)induced by Zhengganfang drug serum and the influence of Zhengganfang drug serum on the telomerase activity of HHC.The purposes of the study are to identify the function of Zhengganfang for preventing HCC,to find the main mechanism of action,to offer academic foundation for further clinic study,and to spread out its application.Methods1.Animal experiment in vivoOne hundred Wister rats were randomly divided into four groups:control group of model(group A,30 rats),group of Zhengganfang(ZGF)low dosage(group B,30 rats),group of ZGF high dosage(group C,30 rats)and control group of normal rats(group D,10 rats).Except the control group of normal rats,all the rats in each group were treated with AFB1 and 2-AAF successively for making model.During the period of making model,the rats in group of ZGF low dosage and group of ZGF high dosage were fed with ZGF aqua(dusts of free-decoction dissolved in water at the concentration of 0.3g/ml,0.6g/ml)according to 10ml/kg.The rats in control group of model were fed with sterile distilled water according to 10ml/kg.After eight weeks,the rats were killed,and blood and hepatic tissues of each rat were taken.After stained with HE staining and uranium- lead staining,the specimens of hepatic tissue were observed by light microscope and electron microscope,the number and size of gama-glutamyltranspeptidase-positive hyperplastic liver cell foci(GGT foci)were checked with histochemical method and technique of computer imageanalysis.The expression of GST-P,PCNA,AFP,IGF-Ⅱ,Cyclin D1,CDK4 in hepatic tissue were tested with immunohistochemistry(IHC)method.The genetic expression of Cyclin D1, CDK4,IGF-Ⅱwere detected with RT-PCR.The biochemical indicators of ALT,AST, ALP,GGT,TBIL,ALB were tested with colorimetric method.2.Experiment of serum pharmacology in vitroThe normal rats were fed with with ZGF decoction(1.5g/ml),two times a day,for three days.On the forth day,the rats took full day's dose.After one hour,the blood was extracted and centrifugated to get sera.The sera were inactivated at 56℃.Serum of normal rat was taken as negative control and retinoic acid(RA)as the positive control.The drug-serum was used for Bel-7402 HHC culture.Cell proliferation was detected by MTT colorimetric assy,nucleocytoplasm ratio by image assay,α-fetoprotein(AFP)by RIA,alkaline phosphatase(ALP)activity and gama-glutamy transpeptidase(GGT)activity by dynamic colorimetric assy,and telomerase activity of Bel-7402 HHC by PCR-ELISA.Results1.Animal experiment in vivo1.1 General state of rat health in each groupAt initiation of the experiment,body weight of rats in each group had no difference in statistics(P＞0.05).In whole lab proc,body weight of rats in control group increased faster than that of model group(P＜0.01).Body weight of rats in model group(group A),group of ZGF low dosage(group B)and group of ZGF high dosage(group C)had similar condition(P＞0.05).This indicated that both low dosage and high dosage of ZGF are safe to rats.At the end of the experiment,the number of death rats in each group were 12 in group A,15 in group B,13 in group C, respectively.There was no death in group D.1.2 Appearance of hepatic tissue and hepatocyte ultrastructure of rats in each groupUnder light microscope:Sporadic,different size hyperplastic liver cell foci(or tubercle)could be seen in the histological section of rat liver in group A,B,C(HE staining).The number and size of hyperplastic liver cell foci in group A were the most, and that in group C were the least.The cells in hyperplastic foci were mainly basophilia hepatocyte,and little cells displayed cloudy swelling,disfiguration and punctiform necrosis.Some cell showed allotype,great volum,conjugate nuclei, anachromasis nuclus,megakaryocyte.Under electron microscope:There were concentrated arrangement of organelle,many mitochondrions and extending rough endoplasmic reticulurn in liver cells of hyperplastic foci.Some abnormal phenomena, such as:big anomal- nucleus,outstanding nucleolus,conjugate nuclei,conjugate nucleoli,chromatin porosity,also could be seen. 1.3 Comparison of positive GGT foci and positive GST-P foci among each groupThe areas of per positive GGT foci(mm2/entries)of group B were less than that of group A(P＜0.05).But the amount of positive GGT foci per square centimeter (entries/cm2)and the total areas of the positive GGT foci per square centimeter (mm2/cm2)had no significant deviation between tow group(P＞0.05).The amount of positive GGT foci per square centimeter,the total areas of the positive GGT foci per square centimeter and the areas of per positive GGT foci of group C were less than that of group A(P＜0.01).Comparing with group B,the amount of positive foci per square centimeter,the total areas of the positive foci per square centimeter and the areas of per positive foci of group C decreased.This indicated that there was dose-effect relationship about the function of ZGF.The amount of positive GST-P foci per square centimeter(entries/cm2),the total areas of the positive GST-P foci per square centimeter(mm2/cm2)and the areas of per positive GST-P(mm2/entries)had no significant deviation between group A and group B(P＞0.05).But the amount of positive GST-P foci per square centimeter,the total areas of the positive GST-P foci per square centimeter and the areas of per positive GST-P foci of group C were less than that of group A(P＜0.01).This indicated that ZGF could inhibite positive GST-P foci of liver precancerous lesion.1.4 Comparison of Liver function among each groupComparing with group A,ALT,GGT,TBIL of group B and groupC reduced markedly(P＜0.01 or P＜0.05).AST and ALP of group C were less than that of group A(P＜0.01 or P＜0.05).This indicated that ZGF could protect liver founction and reduce hepatic injury dueto AFB1 and 2-AAF.1.5 PCNA and AFP immunohistochemistry result of rat hepatic tissue about each groupThe numeral PU of PCNA and AFP in group C and the numeral LI of PCNA and AFP in group C were less than that of group A(P＜0.01 or P＜0.05).This indicated that ZGF could reduce the PCNA and AFP expression of rat liver precancerous lesion, and inhibit the hepatocellular paraplasm of rat liver precancerous lesion.1.6 Cyclin D1 and CDK4 expression of rat hepatic tissue in each group Both numeral PU and numeral LI of Cyclin D1 in group B and group C were less than that in group A(P＜0.01).The numeral PU of Cyclin D1 in group C were also less than that in group B.This presented dose-effect relationship.The numeral PU of CDK4 in group B and group C were less than that in group A(P＜0.01),and the numeral PU of CDK4 in group C were also less than that in group B.Comparing with group A,Cyclin D1 and CDK4 mRNA expression of both group B and group C reduced markedly(P＜0.01).This results suggested that ZGF could depress the abnomal expression of Cyclin D1 and CDK4 in hepatic tissue of rat hepatic precancerous lesion at protein and mRNA level.1.7 IGF-Ⅱexpression of rat hepatic tissue about each groupThe numeral PU of IGF-Ⅱin group B and group C were less than that in group A(P＜0.01 or P＜0.05),and the numeral LI of CDK4 in d group C were also less than that in group A(P＜0.05).Among four group,IGF-ⅡmRNA expression of group D was the lowest.Comparing with group A,IGF-ⅡmRNA expression of group B and group C reduced markedly(P＜0.01).Comparing with group B,IGF-ⅡmRNA expression of group C decreased markedly(P＜0.01),which showed dose-effect relationship.This results suggested that ZGF could depress the abnomal expression of IGF-Ⅱin hepatic tissue of rat hepatic precancerous lesion at protein and mRNA level.2.Experiment of serum pharmacology in vitro2.1 The effect of ZGF drug serum on the proliferation of Bel-7402 cell(MTT colorimetric method)The numeral A of the group of negative control,ZGF drug-serum and RA were 1.704±0.102,1.483±0.077,1.510±0.104,respectively.Comparing with the group of negative control,numeral A of the group of ZGF drug-serum reduced markedly(P＜0.01).This meanted ZGF could inhibit the proliferation of Bel-7402 cell.2.2 The effect of ZGF drug serum on the nucleocytoplasm ratio of Bel-7402 cellThe nucleocytoplasm ratio of the group of negative control,ZGF drug-serum and RA were 1.04±0.09,0.71±0.07,0.69±0.08,respectively.Comparing with the group of negative control,nucleocytoplasm ratio of the group of ZGF drug serum decreased markedly(P＜0.01).This indicated that ZGF could reduce nucleocytoplasm ratio of Bel-7402 cell.2.3 The effect of ZGF drug serum on the AFP secretion of Bel-7402 cellThe secretory volume of AFP(ng/106)in the group of negative control,ZGF drug-serum and RA were 22.58±2.68,14.60±1.85,13.50±2.50,respectively.The secretory volume of AFP in the group of ZGF drug serum and RA were markedly lower than that in the group of negative control,which indicated that both ZGF and RA could inhibit the secretion of AFP of Bel-7402 cell.2.4 The effect of ZGF drug serum on GGT and ALP of Bel-7402 cellThe GGT of the group of ZGF drug serum and RA were markedly lower than that of the group of negative control(P＜0.05),while the ALP of the groups were markedly higher than that of the group of negative control.(P＜0.01).This indicated that ZGF could decrease GGT of Bel-7402 cell and raise ALP of Bel-7402 cell.2.5 The effect of ZGF drug serum on telomerase activity of Bel-7402 cellThe numeral A of the group of Bel-7402 cell control and ZGF drug-serum were 0.929±0.066,0.360±0.105,respectively.The telomerase activity of ZGF drug serum was markedly lower than that of the group of Bel-7402 cell control,which indicated ZGF could inhibit the telomerase activity of Bel-7402 cell.ConclusionZGF has inhibitive effect on the process of hepatic precancerous lesion induced by chemical carcinogen.The mechanism of inhibitive effect of ZGF may involve that the agent can regulate abnormal expression of IGF-Ⅱoncogenes and oncoproceins, then affect cell-cycle process by endogenous regulation and(or)exogenous regulation, inhibit proliferative activity of hepatocytes and retard the malignant proliferation and cancerization of hepatocytes.In addition,ZGF can induce the differentiation of Bel-7402 HHC,the main mechanism maybe through the supression of the telomerase activity.
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