Protective Effect And Mechanism Of Alcohol Dehydrogenase Inhibitor On Liver Fibrosis

Liver fibrosis results from chronic liver damage and extracellular matrix(ECM)protein accumulation,which is characteristic of most chronic liver diseases.Liver fibrosis can further develop into liver cirrhosis or even liver cancer,which cannot be reversed and there is no effective treatment.Therefore,it have great significance for the prevention and treatment of liver fibrosis to find the susceptible factors of liver fibrosis and clarify the mechanism of liver fibrosis.Alcohol dehydrogenase(ADH)is a one-phase metabolic enzyme of the body,which is most abundantly expressed in liver tissue.ADH is mainly involved in the metabolism of ethanol and retinol,in addition to the metabolism of steroids and fatty acids,and plays an important role in the body's steady state.More and more studies have shown that there is a certain relationship between the change of ADH metabolic activity and disease.For example,in liver cancer,brain cancer and kidney cancer,the serum and cancer tissues of patients showed higher ADH activity.The preliminary results of our laboratory showed that compared with normal tissues,ADH activity in liver fibrosis tissues was significantly increased;and there was a good correlation between the initial activity of ADH and liver injury indicators in the rat liver fibrosis model.The above results suggest that ADH is involved in the process of fibrosis,but the mechanism of ADH involvement in fibrosis and whether inhibition of ADH activity can improve liver fibrosis need to be further studied.Retinol and its metabolites are important for maintaining the life activity of the body.Retinol is mainly metabolized by ADH to retinal and then to retinoic acid.Normal physiological amounts of retinol and retinoic acid are necessary for the steady state of adult body tissues,however,changes in retinol metabolism may be one of the pathways of liver fibrosis process.It is reported that carbon tetrachloride induced fibrosis animal models have observed a decreased in retinol content and a marked increased of retinoic acid content.It is speculated that the increased ADH activity is related to the loss of retinol,which may be involved in the process of fibrosis by regulating the metabolism of retinol.Transforming growth factor-?1(TGF-?1)plays a vital role in the development of liver fibrosis.Studies have shown that retinoic acid,a metabolite of retinol,can induce the activation of hepatic stellate cells and the expression of TGF-?1,thereby aggravating the development of liver fibrosis.TGF-?1 signaling can up-regulate protease inhibitors such as plasminogen activator inhibitor-1(PAI-1),interfered with the production of matrix metalloproteinases(MMPs),effected on the degradation of ECM,thereby promoting liver fibrosis.The mice model of liver fibrosis induced by CCl4 is the research object,the ADH activity of mice in various stages of liver fibrosis model was detected in order to observe the change of ADH activity during liver fibrosis,explore the protective effect of ADH inhibitors(4-methylpyrazole)on liver fibrosis.In order to preliminary explore the mechanism of ADH involved in liver fibrosis,the retinol content was measured by HPLC and the expression level of TGF-?1,PAI-1 and MMP9 were determined by Real-time PCR and Western-blot.MethodsThis study was approved by the medical ethics committee of Zhengzhou University.1 Protective effect of ADH inhibitor on liver fibrosis model1.1 Liver fibrosis model95 Kunming mice were randomly divided into 4 groups,control group,model group,inhibitor group and 4-MP group.Mice in the control group were treated with olive oil(5ml/kg,i.p.)twice a week.Mice in the model group were treated with CCl4(20%CCl4 5ml/kg,i.p.)twice a week.Mice in the inhibitor group were treated with 4-MP(100 mg/kg,i.p.)three times a week outside the same treatment in the model group,The mice in the 4-MP group were given only 4-MP(100 mg/kg,i.p.)three times a week,and the model was continuously induced for 9 weeks.The weight of the mice were measured once a week.At week 3,6,and 9 after the first administration of CCl4 the animals were sacrificed,liver tissue and blood samples were harvested at this time points.Part of the liver tissue is fixed in 4%formalin,and the remaining liver tissue were divided into frozen tubes,and serum and liver tissue were stored at-80? until used for further assessment.1.2 Histological analysisAll paraffin-embedded liver tissues were cut into thick sections and stained with H&E and Masson for histological examination.Stained section were viewed with an optical microscope,and referring to the Ishak scoring standard to classify liver fibrosis to reflect the severity of liver fibrosis.1.3 Determination of mice serum ALT and ASTThe mice serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)were detected by a fully automatic biochemical analyzer.1.4 Determination of mice ADH activityTake out the serum and liver tissue in the refrigerator at-80?.The liver tissue were made into 10%liver homogenate according to the weight:volume=1:9.The protein concentration of liver homogenate were measured by BCA protein concentration determination kit.ADH activity was measured using p-nitrosodimethylaniline(NDMA)as a substrate,mixed with serum(or liver homogenate),glycine-sodium hydroxide buffer,and then mixed with n-butanol and NAD starts the reaction,and the absorbance of the substrate were detected at a wavelength of 440 nm used a multifunctional microplate reader.2 Mechanism of ADH involved in liver fibrosis2.1 Pathological staining and ADH activity determinationIn order to study the effect of ADH inhibitor use time on liver fibrosis,take another batch of mice.The mice in the control group,model group,and inhibitor-1 group were administered the same as 1.1.Three weeks after the first administration of CC14 in the inhibitor-2 group,4-MP intervention was started.The model was induced for 6 weeks,and all mice were sacrificed at 6 week.Serum and liver tissue were collected for pathological staining and determination of ADH activity.2.2 Changes of retinol in mice liver tissueRetinol contents of mice liver tissues were determined by HPLC-UV method2.3 Determination of TGF-?1,PAI-1 and MMP9The mRNA and protein expression levels of TGF-?1,PAI-1,MMP9 in mice liver tissues were detected by Real-time PCR and Western-blot.3 Statistical analysisStatistical analysis and the picture drawing of the data were performed using SPSS 20.0 and Prism 6.0.The data were analyzed using t test(two-group data)or one-way ANOVA(multi-group data).The correlation was analyzed using Pearson test(for data with normal distribution)or Spearman test(for data with non-normal distribution).Results were considered statistically significant at P<0.05.Results1 The protective effect of ADH inhibitor on liver fibrosis1.1 4-MP Toxicity assayThe serum levels of ALT and AST and the H&E and Masson staining showed that 4-MP(100 mg/kg,i.p.)had no significant liver toxicity.1.2 Changes of mice body weight and liver weightThe body weight of mice were gradually increased with time,and there was no significantly difference between the three groups(P>0.05).Compared with the control group,the liver index of the model group were significantly increased(P<0.01).The liver index of the inhibitor group was significantly lower than that of the model group at 6 weeks(P<0.01).1.3 Pathological stainingCompared with the control group(0.00 ± 0.00),the Ishak scores of the model group at week 3(3.13 ± 1.17),week 6(3.63 ± 0.70)and week 9(3.25 ± 0.43)were significantly higher(P<0.0001).The results showed that the liver fibrosis model was successfully established.At week 3(3.25 ± 0.43),there was no difference between the Ishak score of the inhibitor group and the model group(P>0.05).At week 6(1.78 ± 1.03)and week 9(2.13 ± 1.17),the Ishak score of inhibitor group was significantly lower than that of the model group(P<0.001).This indicates that with the prolongation of the use time of ADH inhibitor(4-MP),liver fibrosis has been improved,and the improvement is most significant at the sixth week.Masson staining showed that there was no obvious collagen fiber hyperplasia in the liver tissue of the control group;in the model group,there was a large number of collagen fiber hyperplasia around the portal vein and central vein in the manifold area,and bridge formation was more common.At week 6 and week 9,a small amount of collagen fiber hyperplasia was seen around the local manifold area in the inhibitor group.1.4 ALT and AST content in mice serumCompared with the control group,the serum levels of ALT and AST in the model group were significantly increased(P<0.0001).There was no difference between the inhibitor group and the model group(P>0.05),which suggested that the ADH inhibitor(4-MP)failed to improve the increased ALT and AST caused by CC141.5 ADH activity of mice during various stages of fibrosis model1.5.1 ADH activity of mice serumThere was no change in ADH activity in the control group.Compared with the serum ADH activity at week 0,the ADH activity of the model group was significantly increased at week 3,6,and 9(P<0.0001),and the ADH activity at week 3 was the highest.The results suggested that the ADH activity is the highest in the early stage of liver fibrosis.The comparison between the groups found that the ADH activity in the model group was significantly higher(P<0.0001)compared with the corresponding control group.The results showed that ADH activity significantly increased during fibrosis.At the same time,the serum ADH activity of the inhibitor group was significantly lower(P<0.0001)than in the model group,and the ADH activity was no difference from the control group.The results showed that ADH inhibitors can inhibit the increase of ADH activity during liver fibrosis and reduce their activity to normal levels.1.5.2 ADH activity of mice liver homogenateAt week 3,week 6 and week 9 of the induction model,the ADH activity in mice liver homogenate of the control group(27.27 ± 19.51 nmol/min/mg protein,21.79 ±10.65 nmol/min/mg protein,33.97 ± 8.74 nmol/min/mg protein),the ADH activity in the liver homogenate of the model group was significantly increased,respectively 111.81 ± 31.59nmol/min/mg protein,76.96 ± 27.92 nmol/min/mg protein and 80.41 ±28.34 nmol/min/mg protein(P<0.0001).Compared with the model group,the ADH activity in the liver homogenate of the inhibitor group was significantly reduced(P<0.001),which were 58.58±30.87 nmol/min/mg protein,33.68±10.76 nmol/min/mg protein and 29.90±16.19 nmol/min/mg protein,respectively.1.5.3 Correlation between the ADH activity in serum and liver homogenateThere was a significant correlation between ADH activity of serum and liver homogenate at week 3,6 and 9(P<0.0001),with correlation coefficients of 0.6307,0.5578 and 0.7035.1.6 Correlation between the ADH activity and Ishak scoreADH activity of serum and liver homogenate were positively correlated with Ishak score at week 3,week 6 and week 9,and the correlation coeff-icient was between 0.4693-0.7243(P<0.0001).The results suggested that the higher the ADH activity,the worse the liver fibrosis.2 Mechanism of ADH involved in liver fibrosis2.1 Histopathology of mice liverConsistent with the results of 1.3,Ishak score of the inhibitor-1 group was significantly lower compared with the model group(P<0.01).Compared with the model group,the Ishak score of the inhibitor-2 group showed a decreasing trend but no statistical significant difference(P>0.05).These results showed that early use of ADH inhibitors improved liver fibrosis more obviously.2.2 Changes of retinol contentThe retinol measurement method had a high specificity,the standard curve is C=0.0152A-0.0777(R2=0.9998),the linear range is 0.39?9.38 ?g/ml.The intra-day and inter-day precision,the absolute recovery rate and the relative recovery rate are in line with biological sample measurement requirements.Compared with the retinol content in the control group(13.26 ± 4.52ng/mg liver),the retinol content in the model group(8.30 ± 1.23 ng/mg liver)was significantly reduced(P<0.01).In the inhibitor-1 group,the retinol content(10.99±2.92 ng/mg liver)was significantly higher than in the model group(P<0.05).In the inhibitor-2 group,the retinol content(10.02 ± 4.23 ng/mg liver)of the model group tended to increase compared with the model group,but there was no statistical significant difference(P>0.05).The results suggested that the development of liver fibrosis is accompanied by the loss of retinol,ADH inhibitors inhibited the reduction of retinol.2.3 Correlations between ADH activity,retinol content and Ishak scoreADH activity in mouse serum and liver homogenate was positively correlated with liver fibrosis score(P<0.0001),and negatively correlated with retinol content in liver tissue(P<0.05).The above results showed that the higher the ADH activity,the more severe the fibrosis,and the higher ADH activity,the lower retinol content,suggested that the increased ADH activity may be involved in liver fibrosis by participating in the metabolism of retinol.2.4 Changes of TGF-?1,PAI-1 and MMP9The mRNA and protein expressions of TGF-?1,PAI-1,and MMP9 in the liver tissues of the model group were higher than control group(P<0.001).The expression levels of TGF-?1,PAI-1,and MMP9 in the inhibitor-1 group were significantly reduced compared with the model group(P<0.01).The expressions of PAI-1 and MMP9 in the inhibitor-2 group were significantly lower than those in the model group(P<0.01),and there was no significant change in TGF-?1(P>0.05).The results suggested that inhibition of ADH may protected liver fibrosis in mice by affected the expression of TGF-?1,PAI-1 and MMP9.Conclusion1.The activity of ADH was significantly increased during liver fibrosis,and its activity is highest in the early stage of liver fibrosis.2.ADH activity in serum and liver tissue are positively correlated with the severity of liver fibrosis.3.Inhibition of ADH can improve liver fibrosis in mice,and its mechanism was related to inhibition of retinol loss and regulation of TGF-?1,PAI-1,and MMP9 factors.