| Fibrosis is a common chronic progressive liver disease,caused by long or repeated effects of one or more causes of diffuse liver injury,including viruses,alcohol,hepatotoxic and exogenous toxicants,autoimmune diseases,manifested as excessive deposition of intrahepatic extracellular stromal components and abnormal liver connective tissue,affecting the function of the liver.Cytochrome P450(CYP)is a superfamily metabolizing enzyme containing heme hemoglobin,which is mainly involved in a variety of endogenous substances(such as fatty acids,arachidonic acid and steroids)and exogenous substances(such as alcohols,nitrosamines,drugs,etc.)I phase biotransformation.CYP2E1 is the most important enzyme involved in the metabolism of a variety of substances.Diethylnitrosamine(DEN)is a typical carcinogen of the N-nitrosamines family,widely found in a variety of daily life things.DEN can produce hydroxyalkyl nitrosamines under the hydroxylation reaction of CYP2E1,which can bind with DNA forming DNA alkyl-adduct,eventually leading to liver damage,so diethylnitrosamine can be widely used in the rat model of liver fibrosis and cancer.Chlorimethiazole(CMZ)is a thiamine thiazoline derivative compound that has sedative,hypnotic,anxiolytic effects and is widely used in alcohol withdrawal symptoms including tremor delirium at some European countries.CMZ has a significant inhibitory effect in the human metabolism of chlorzoxazone as a CYP2E1 inhibitor.Owing to the important role of CYP2E1 in pharmacokinetics andtoxicokinetics,CMZ can be used as a specific inhibitor in vivo to elucidate the important role of CYP2E1 in the activation and metabolism of xenobiotics,and it can be further used in preventing liver from damage induced by micromolecule poison metabolised by CYP2E1.The current study was designed to investigate the inhibition of CMZ at different doses in the metabolism of DEN in normal or fibrosis rats and establish DEN-induced rat model of liver fibrosis.CMZ was used as a CYP2E1 specific inhibitor to investigate the effect of CMZ on DEN-induced pathological changes of rat fibrosis in order to provide new ideas and ways for the prevention and treatment of clinical fibrosis.Methods1 Study on the effect of CMZ on Toxicokinetics of DEN in rats1.1 Analytical MethodsThe specific and sensitive HPLC-UV method was developed for determination of DEN.Perchloric acid was used to precipitate protein for DEN.The precision,recovery,linear range,and stability were examined according to the guidance of determination of biological specimen.1.2 Study on the effect of CMZ on Toxicokinetics of DEN in normal ratsTwelve rats were used to study different doses(10mg/kg,50mg/kg,100mg/kg,i.p)of CMZ treatment on Toxicokinetics of DEN(50mg/kg,i.p).The study was a self-control design at intervals of 4 days.Blood samples were collected before and at the following time points after administration: 1,4,10,25 min and 1,2.5,4.5,7,10,24,48 h.1.3 Study on the effect of CMZ on Toxicokinetics of DEN in rats of fibrosisTen rats of fibrosis were used to study the effect of CMZ(50mg/kg,i.p)on toxicokinetics of DEN at 12 th week.2 Study on the effect of CMZ on the pathogenesis of liver fibrosis2.1 The model of liver fibrosisTen rats were used for control experiment.Ten rats were used to induce the model of liver fibrosis by DEN(50mg/kg),the rats were subjected to intraperitoneal injection of 50mg/kg DEN twice a week for 4 consecutive weeks,followed then by weekly injections for another 8 weeks.Twenty-four rats were used to study the effect of CMZ on the pathogenesis of liver fibrosis at different doses(100mg/kg,50mg/kg,10mg/kg,i.p,n=8 for each group).The rats were subjected to intraperitoneal injection of DEN(50mg/kg)combination with CMZ twice a week for 4 consecutive weeks,followed then by weekly injections for another 8 weeks.2.2 Study on the effect of CMZ on Toxicokinetics of DEN at the pathogenesis of liver fibrosisThe different inhibition effect of CMZ at different doses was studied at 1 and12 th week.Histopathological examination of all liver samples was taken at 12 th week.2.3 Histological analysis and ImmunohistochemistryThe liver index was calculated by calculating the ratio of liver weight to body weight.Tissue samples of different groups were fixed in 10% buffered formalin for72 h and stained with hematoxylin and eosin(H&E)or Masson and imaged(200x magnification).The results of pathologic staining were scored according to the Isak scoring system.Immunohistochemistry was performed using a polyclonal antibody for cell proliferation marker(Ki67),proliferating cell nuclear antigen(PCNA),Placental glutathione S-transferase(GST-P).Results were expressed as the percentage of stained cells in each field.2.4 The correlation between the activity of CYP2E1 and fibrosis stageThe correlation between toxicokinetic parameters of diethylnitrosamine after i.p administration DEN at 50mg/kg combination with CMZ at 10,50,100mg/kg at first week and liver index,Ishak score,the area of Masson positive cells.Statistics Toxicokinetic analysis of data was calculated by DAS 2.1.Statistical analysis was performed with SPSS 21.0 software.The significance of difference between groups was analyzed by One-way ANOVA.The level of significance was set at 0.05.Results1 Study on the effect of CMZ on Toxicokinetics of DEN in rats1.1 Study on the effect of CMZ on Toxicokinetics of DEN in normal ratsCompared with the single DEN treatment,the CL decreased by(44.49±10.16)%,(58.35±9.14)% and(69.35±5.03)%,whereas t1/2 increased by(89.82±121.70)%,(317.64±128.80)%,(438.32±127.51)%,AUC0-t increased by(80.12±32.85)%,(117.83±45.71)%,(192.37±47.77)%,AUC0-?increased by(88.97±44.88)%,(127.58±43.27)%,(218.21±51.27)% in different doses(10,50 and 100mg/kg)of CMZ treated rats,respectively(P<0.05).It suggested that the metabolism of DEN was significantly inhibited by CMZ.1.2 Study on the effect of CMZ on Toxicokinetics of DEN in rats of fibrosisCompared with fibrosis period of rats,the CL decreased by(64.32±10.02)%,whereas t1/2 increased by(189.00±173.95)%,AUC0-t increased by(168.46±35.96)%,AUC0-? increased by(385.60±174.39)%,Cmax increased by(38.17± 16.50)% of CMZ treated(P<0.05).It suggested that the metabolism of DEN was significantly inhibited by CMZ in rats of fibrosis.2 Study the effect of CMZ on the pathogenesis of liver fibrosis2.1 Effect of CMZ in pathological indicators of liver in DEN-induced fibrosisCompared with control group,the liver index,the positive area of Masson,the positive cell of PCNA,the average density of GST-P and the Isak score showed a significant increase(P<0.05),but the body weight of rats showed significant decreased in model and CMZ groups(P<0.05),and there was no significant difference of weight among CMZ groups and model group(P>0.05).Compared with model group,the positive area of Masson,the positive cell of PCNA and the positive cell of Ki-67 showed significant decreased in CMZ groups(P<0.05).Compared with low CMZ group,the Isak score,the liver lidex and the positive area of Masson showed significant decreased in high CMZ group(P<0.05),but the positive cell of PCNA showed significant increased in medium CMZ group(P<0.05).Compared with medium CMZ group,the Isak score and the positive area of Masson showed significant decreased in high CMZ group(P<0.05).The liver index and the Isak score showed no significant difference in the low and medium CMZ group(P>0.05).Proliferative activity studied by Ki67 immunostaining showed no significant difference among CMZ groups(P>0.05).The average density of GST-P showed no significant difference between CMZ groups and the model group(P>0.05).2.2 Effect of CMZ on Toxicokinetics of DEN in DEN-induced ratsCompared with the single DEN treatment,the CL decreased by(57.79±6.83)%,whereas t1/2 increased by(260.95±121.88)%,AUC0-t increased by(139.90±40.77)%,AUC0-? increased by(142.81±42.13)% in low group(10mg/kg)of CMZ treated rats(P<0.05);the CL decreased by(72.57±3.72)%,whereas t1/2 increased by(349.22±121.25)%,AUC0-t increased by(269.63±50.40)%,AUC0-? increased by(270.21±50.38)% in medium group(50mg/kg)of CMZ treated rats(P<0.05);the CL decreased by(81.69±2.25)%,whereas t1/2 increased by(522.32±443.48)%,AUC0-t increased by(428.89±56.72)%,AUC0-? increased by(453.43±68.26)% in high group(100mg/kg)of CMZ treated rats(P<0.05).Compared with the first CMZ combination with DEN at normal period of rats,the CL decreased by(29.83±28.38)%,whereas t1/2 increased by(63.29±82.17)%,AUC0-t increased by(45.63±38.58)%,AUC0-? increased by(60.50±54.83)% in low group(10mg/kg)of CMZ treated rats(P<0.05);the CL decreased by(45.90±16.06)%, whereas t1/2 increased by(136.85±130.47)%,AUC0-t increased by(61.60±24.46)%,AUC0-? increased by(100.18±60.74)% in medium group(50mg/kg)of CMZ treated rats(P<0.05);the CL decreased by(25.54±34.50)%,whereas t1/2 increased by(218.50±250.04)%,AUC0-t increased by(61.62±19.73)%,AUC0-? increased by(62.77±76.86)% in high group(100mg/kg)of CMZ treated rats(P<0.05).2.3 The correlation between the activity of CYP2E1 and fibrosis stageThe toxicokinetic parameters(AUC0-t,AUC0-?)of diethylnitrosamine after i.p.administration DEN combination with CMZ at first week were significantly negatively correlated with Ishak score and Masson staining in CMZ groups(P<0.05),but the CL was positively correlated with the above three indexes(P<0.05).With the increase of CMZ dose,the inhibitory effect of CMZ on the metabolism of DEN was more obvious.Lower the clearance rate of DEN in rats,lighter the liver fibrosis induced by DEN.It was further suggested that DEN induced the hepatic fibrosis in rats by the metabolism of CYP2E1 and CMZ significantly reduced the degree of liver fibrosis induced by DEN by inhibiting the activity of CYP2E1.Conclusions1.CMZ significantly reduces the area of liver fibrosis at different doses and the protective effect shows dose-dependent.2.CMZ has significantly inhibition about the metabolism of DEN in normal and fibrotic rats at different doses.3.The prevention of CMZ on DEN-induced rat liver fibrosis is significantly correlated with inhibitory effect of CMZ on CYP2E1 activity.CMZ significantly reduces the degree of liver fibrosis induced by DEN by inhibiting the activity of CYP2E1. |
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