| Liver cancer is the most common liver malignancy threatening human health.The multi-stage model of the liver cancer development(hepatitis-liver cirrhosis-liver cancer)has been supported by a wide range of clinical and pathological researches.Liver fibrosis is a common pathology of various chronic liver diseases,and the necessary stage of chronic liver disease to cirrhosis.Alcohol dehydrogenases(ADH)and aldehyde dehydrogenases(ALDH)are mainly present in hepatocytes.Alcohol is metabolized to acetaldehyde by alcohol ADH.Acetaldehyde has hepatotoxicity,and can induce cancer.ADH cannot only metabolize alcohol but also metabolize retinol.Retinol and its metabolites involved in the process of hepatitis and liver fibrosis,and the recent results show that the lack of ADH can inhibit liver fibrosis.It was reported that ADHI activity was elevated in liver cancer tissues compared with normal tissues adjacent to the tumor.Serum ADH activity of liver cancer patients also increased compared with healthy people.The results of our laboratory showed that the activities of total ADH,ADHI and ADHII were significantly increased in the liver fibrosis tissues of patients with hepatocellular carcinoma compared with normal liver tissues,but the activities of total ALDH and ALDH2 did not change.The above results show that elevated ADH activity may be associated with liver fibrosis and even liver cancer,but how to change of ADH activity in serum and tissue at different stages of liver cancer development.Elevated ADH activity is the result or cause of liver injury,and whether elevated ADH activity is susceptibility to liver injury,which need to be further studied.In this study,we selected the rat model of of liver cancer induced by diethylnitrosamine(DEN)as the object of study.The activities of ADH and ALDH in liver tissue and plasma were measured at different stages of the rat liver cancer model.The relationship between basic ADH activity in plasma and the index reflecting hepatic lesions were analyzed,and the relationship between ADH activity in liver tissue and the index reflecting hepatic lesions were also analyzed,in order to explore the role of ADH in the development of liver fibrosis and even liver cancer.Methods 1 Establishment of rat liver cancer model 1.1 Animal grouping and model preparation54 male Sprague-Dawley(SD)male rats were randomly divided into 2 groups: model(n=44)and control(n=10).After feeding,Model A was sacrificed at 12 weeks.Model was injected intraperitoneally with 50mg/kg DEN twice a week for 4 weeks,then injected intraperitoneally with 50mg/kg DEN once a week to 14 weeks.Twenty rats in the model group were randomly assigned at 12 weeks,which was called Model A.The rest rats of model were fed after stopping the administration and were sacrificed at 19 weeks,which was called Model B.The rats of control were sacrificed at 19 weeks.1.2 Animal observation and sample collectionThe physiological and mental state of rats were observed every day.The death of rats was observed.The body weight of rats was recorded every week.The control group and model A and model B survived the rats’ plasma at 0 week before administration and at the 8th,12 th,16th and 19 th week after administration was taken.After the rats were sacrificed,the liver was removed and part liver tissue was used for pathological examination and the remaining liver tissue was placed in liquid nitrogen.1.3 Preparation of rat liver homogenateAppropriate amount of liver tissue(1:9 w/v)was homogenized in normal saline,centrifuged at the appropriate speed.The supernatant was used to determine enzyme activities.Determination of liver homogenate protein concentration by BCA method.1.4 Determination of ALT and AST in plasma and liver homogenateThe activity of ALT and AST in plasma and liver homogenate was determined by a microplate reader according to the instructions of kit.1.5 Histop athologyHE and masson staining was performed on liver sections of different stages.Liver specimens were graded according to the Ishak scoring system and the percentage of fibrotic area after masson staining was measured.1.6 Immu nohistochemistryThe expression levels of Ki67,PCNA and GST-p proteins in the control and model groups were determined by immunohistochemistry.2 Determination of the activity of ADH and ALDHALDH catalyzed the conversion of oxidative coenzyme I(NAD+)to reductive coenzyme I(NADH),the change of absorbance at 340 nm was measured to obtain ADH activity.NADH has absorbance at 340 nm.The ADH activity in plasma and liver homogenate was determined according to the instructions of kit.In the presence of NAD+,ALDH catalyzed the conversion of acetaldehyde and NAD+ to acetic acid and NADH,and the change of absorbance at 340 nm was measured to obtain ALDH activity.The ALDH activity in the liver homogenate was determined using the kit according to the instructions of kit.3 Statistical analysisStatistical analysis was performed with SPSS 17.0 software.The significance of difference between two groups was analyzed by independent t test.Enzyme activities among different groups were compared by One-way ANOVA.The correlation between ADH,ALDH activity,ADH/ALDH and liver injury-related indexes was analyzed by Person.The level of significance was set at 0.05.Results 1 Establishment of rat liver cancer model 1.1 Body weight,liver weight of ratsCompared with the control group,the body weight of the model group decreased from the second week(P<0.001).Compared with the control group,the liver weight of model A was not significantly changed(P> 0.05)and liver weight / body weight was significantly increased(P<0.001);the liver weight of model B was significantly increased(P<0.001)and liver weight / body weight was significantly increased(P<0.001).1.2 Mortality of ratsRats in the model group did not die in the first 12 weeks.14 of rats died in 15-19 weeks in the model group.1.3 ALT and AST activity in plasma and liver homogenateThe ALT and AST activities in plasma of model were significantly higher than those in control group at 8,12,16 and 19 weeks,and the increase rate was 2-3 times(P<0.001).The activity of ALT in liver homogenate of rats in model A was lower than that in control group(P<0.05),and the activity model B decreased more obviously(P<0.01).The activity of AST in model A and model B was higher than that in control group(P<0.05).The AST / ALT ratio model A and B was significantly higher than that in control group(P<0.001).1.4 Histopathology and incidence of liver cancerCompared with the control group,the liver tissue Ishak score and Fibrotic area% after masson staining were significantly increased in model A and B(P<0.001).Rats of model A showed varying degrees of hepatic fibrosis.In model B,5 rats had hepatoma and 9 had hepatocellular carcinoma.The incidence of liver cancer was 66.7%.1.5 immun ohistochemistryCompared with the control group,the Ki67,PCNA positive cell rate and GST-p average density of liver slices in model A and B were significantly increased(P<0.01).2 ADH,ALDH activity of rats 2.1 ADH activity in rat plasmaCompared with the control group,the ADH activity in the plasma of model was increased at 16 weeks(24.21 ± 9.86 U/ml)and 19 weeks(24.56 ± 9.65 U/ml),and there was no significant change of ADH activity in the plasma of model at 0,8 and 12 weeks(P>0.05).2.2 ADH and ALDH activity in rat liver homogenateCompared with control group(3.43 ± 1.07 U/mg prot),the ADH activity in liver homogenate of rats in model A(4.50 ± 1.05 U/mg prot)and model B(4.77 ± 2.11 U/mg prot)was significantly increased(P<0.05).Compared with control group(9.96 ± 1.52 U/g prot),the ALDH activities in the liver homogenate of rats in model A(46.02 ± 14.10 U/g prot)and model B(30.33 ± 9.80 U/g prot)was significantly increased(P<0.001).Compared with model A,the activity of ALDH in liver homogenate of model B decreased by 34%(P<0.001).3 Relationship between ADH,ALDH and index reflecting hepatic lesions 3.1 Relationship between ADH basic activity and index reflecting hepatic lesionsThe ADH basic activity at 0 week in plasma of model A was correlated with the four indexes eflecting hepatic lesions(masson area%,Ki67+%,PCNA+%,GST-p average density),and the correlation coefficients were 0.453,0.512,0.457,0.450(P<0.05).There was no correlation between ADH basic activity of model B and index reflecting hepatic lesions(P>0.05).3.2 Relationsh ip between ADH activity i n rat li ver homogenate and index reflecting hepatic lesionsThe ADH activity in rat liver homogenate of model B was correlated with the five indexes eflecting hepatic lesions(LW/BW,nodule number,largest diameter,cumulative diameter,Ki67+%),and the correlation coefficients were 0.567,0.499,0.438,0.666,0.506(P<0.05).Further analysis showed that ADH / ALDH in the liver homogenate of model B and the four indexes(LW/BW,nodule diameter,largest diameter,cumulative diameter)existed correlation,and the correlation coefficients were 0.720,0.476,0.522,0.755(P<0.01).Conclusions1.The ADH activity in the plasma of the model increased at 16 and 19 weeks compared with the control group in the same period,and the ADH activity in the liver homogenate was higher than that in the control group at 12 weeks and 19 weeks.2.The activity of ALDH in the liver homogenate of the model at 12 weeks and 19 weeks was significantly higher than that of the control group.3.Increased basic activity of ADH in rat plasma is a susceptibility factor in hepatic fibrosis... |
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