Functional And Mechanism Research Of Retinol Dehydrogenase 13 In Mouse Acute Liver Injury And Chronic Liver Fibrosis Induced By Carbon Tetrachloride

Part I FUNCTIONAL AND MECHANISM STUDY OF RETINOL DEHYDROGENASE 13 IN ACUTE LIVER INJURY INDUCED BY CCL₄Short chain dehydrogenase/reductase（SDRs）is a superfamily which shares analogous sequence and similar function.Up to now,there are 140kinds of proteinases having been found that belong to this superfamily.Retinol dehydrogenase 13 is a recently identified short-chain dehydrogenase/reductase that shares the similar sequence with retinol dehydrogenase 11,retinol dehydrogenase 12,retinol dehydrogenase 5 and retinol dehydrogenase 14 belonging to dual-substrate specificity short chain retinol dehydrogenases.It has been reported that the other members of RDH family play an important role in the visual cycle.In our work,we found that Rdh13 played an important role in retinal light damage which is consistent with Rdh11 and Rdh12 in mice.In the previous report,Rdh13 is situated on the outer side of the inner mitochondrial membrane while Rdh11 and Rdh12 are pitched in the endoplasmic reticulum.The localization of Rdh13 suggests that its function may be different from Rdh11,Rdh12 and Rdh14.RDH13 protein has 80%homology between human and mice,indicating the conservation in the species evolution.Our purpose is to research the function of Rdh13 from mice to human.Our study showed that Rdh13 m RNA mostly expressed in the liver of mice,which was 3-5 times higher than that of other tissues.To further research the Rdh13 function,we generate Rdh13 knockout mice,and Rdh13 homozygous mice could be born and grew to adulthood,and the offspring of heterozygete mating accorded with Mendel’s law of inheritance,and there was no gross difference among the body weight and body length among wildtype,heterozygete and homozygous mice.On physiological condition,there is no obvious difference about the serum biochemistry and the liver section H&E staining between wildtype and homozygous mice.The harm of liver diseases is very huge,and the animal model of acute liver injury induced by carbon tetrachloride had been commonly used to study the development and progress of liver diseases.In our study,we observed slight injury and low level of hepatocyte proliferation following carbon tetrachloride in Rdh13knockout mice.The possible mechanism was that Rdh13 regulated the level and stability of Cyp2E1 which determined the extent and degree of CCl₄-induced liver injury via affecting the metabolism of thyroxine and hnfα.These results for the first time define that Rdh13 is an important regulator associated with the injury and repair process induced by CC1₄in mice liver.Part II FUNCTIONAL AND MECHANISM STUDY OF RETINOL DEHYDROGENASE 13 IN LIVER FIBROSIS INDUCED BY CCL4The mitochondrion-localized retinol dehydrogenase has been recently identified to be highly expressed in liver and acts as short-chain dehydrogenase/reductase participating in vitamin A metabolism of both human and mouse.In our first part,we observed slight injury and low level of hepatocyte proliferation following in Rdh13 knockout mice.To further investigate the function of Rdh13 in liver,we generated animal models of chronic liver fibrosis induced by carbon tetrachloride,and observed that Rdh13 knockout mice displayed weaker phenotype of liver fibrosis compared with wildtype mouse under the treatment of carbon tetrachloride.Observations of masson’s trichrome staining indicated that knockout livers displayed smaller fibrosis region compared with the wildtype.Consistently,after the exposure to carbon tetrachloride,the liver of Retinol dehydrogenase 13 knockout mice had less obvious increase of the expression of liver fibrosis marker genes encoding collagen type I,α-smooth muscle actin（α-SMA）,tissue inhibitor of metalloproteinase 1（TIMP-1）,transforming growth factor beta 1（TGF-β1）and platelet-derived growth factor（PDGF）than those of the wildtype,suggesting that Rdh13-mediated biochemical pathway is involved in CCl4-induced liver fibrosis.These data indicate that Rdh13 plays a significant role in the progress of liver fibrosis giving us some clues on the therapy of liver fibrosis.Part III FURTHER PHENOTYPIC RESEARCH OF RETINOL DEHYDROGENASE 13 KNOCKOUT MICEExperiments in vitro showed that Rdh13 had the function of oxidation retinol andreduction retinaldehyde.In vivo,retinol was metabolized into retinoic acid through an intermediate of retinaldehyde.Retinoic acid is the active form of retinol in vivo,and involves in the organism development,cell differentiation,apoptosis and regeneration.Retinaldehyde is toxic to organism body,so we guess Rdh13 may maintain the balance of retinol,retinaldehyde and retinoic acid.It had been reported Rdh13 was situated on the outer side of the inner mitochondrial membrane,so our working key was to research the function of Rdh13 in the mitochondrial.We measured the expression level of genes about oxidative phosphorylation,and genes associated with the number and synthesis of mitochondria,and some index of mitochondrial antioxidant function including superoxide dismutase,malonaldehyde,and total antioxidant capacity and so on.