| [Objectives]Liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases.Advanced liver fibrosis results in cirrhosis,liver failure,and portal hypertension and often requires liver transplantation.The cellular and molecular mechanisms of liver fibrosis are not well understood.In this thesis,the function of Fkbp5 was investigated in wide type(WT)and Fkbp5 knockout(KO)mice which liver fibrosis was induced by CCl4.[Methods]WT and Fkbp5 KO mice were separated into two groups:normal control group(WT-CON and KO-CON)and CCl4 treatment group(WT-CCl4 and KO-CCl4)(N=6 each group).1)The liver histopathology differences were observed by staining of collagen deposition by Masson staining;2)Fibrosis markers,including ?-SMA,TIMP1 and Collagen was detected by Immunofluorescence(IF)staining;3)The mRNA expression of Collagen ?,TIMP1,MMP2,CTGF,TGF-? and ?-SMA were detected using the real-time PCR;4)The expression of TGF-? signaling pathway related genes,TGF-?1?TGF-? receptor 1?TGF-? receptor 2?Smad2?Smad3?Smad4?p-Smad2 and p-Smad3 were detected by Western Blot;5)The mitochondria morphology was observed by electron microscopy,and the mitochondrial size was measured by NIH-ImageJ software;6)The expression of mitochondrial autophagy markers:Parkin,pink1 and P62 were detected by Western blotting;7)The total mRNA from livers CCl4 treated or without treated was sequenced by the next generation sequencing technique,and gene expression profiling was analyzed by DAVID?STRING and Genecard.[Results]1)Compared to WT,Fkbp5 KO significantly reduced CCl4 induced liver fibrosis.2)Fibrosis markers,?-SMA,TIMP-1 and Collagen ? were increased after CCl4 treatment in both WT and KO,but in KO-CCl4 those factors were lower than which in WT-CCl4;3)The expression of ?-SMA,TIMP-1,Collagen ?,CTGF and TGF-?1 in CCl4 treatment groups were significantly improved,while those were lower in KO-CCl4 than in WT-CCl4;4)The genes expression changes,such as TGF-?1,TGF-? R ?,TGF-?R ?,Smad2,Smad3,P-Smad2,P-Smad3,Smad4,impacted that the TGF-?/Smad signaling pathway were changed in CCl4 treatment groups;5)Furthermore,we found that the size of mitochondria was larger in KO group than WT,and the mitochondrial autophagy(mitophagy)was more sever in KO-CCl4 than in WT-CCl4.The results showed that the mitochondrial autophagy-related proteins expression of Parkin and pink1 in the KO-CON group were higher than those in WT-CON group,while the expression of autophagy negative regulator P62 was lower than that in WT-CON;6)RNA-seq analysis showed that Fkbp5 gene may affect the process of liver fibrosis in mice by multiple biological processes,such as lipid metabolism,stress process,CYP450 metabolism and redox process.[Conclusions]1)Fkbp51 knockout mice were resistant to CCl4-induced liver fibrosis;2)Fkbp51 plays role in regulating TGF-?/Smad signaling pathway;3)Fkbp51 deletion may induce Parkin mediated mitophagy;4)Fkbp51 gene may have function in liver fibrosis through multiple biological processes such as lipid metabolism,stress response,CYP450 metabolism and so on.[Objectives]Although it is known that there is a relationship between stress and metabolic regulation,the effects of stress within an obesogenic context are not very clear and molecular target at the interface remains elusive.Hypoxia is one kind of chronic stress.FKBP5(FK506-binding protein)has been identified as a target gene implicated in the development of stress-related psychiatric disorders and is a possible candidate for involvement in stress and metabolic regulation.The aim of the current study was to investigate the different mechanisms of hypoxia stimulation on primary WT and FKBP5 KO MEFs in adipogenesis and the regulation of FKBP5on adipogenesis under hypoxia.[Methods]1)Isolation and culture of primary fibroblasts:The corresponding primary fibroblasts were isolated and cultured using embryos from wild-type(WT)and FKBP5 knockout(KO)mice;2)Fat-induced differentiation experiments:The effects of hypoxia on adipogenic differentiation of WT and FKBP5 KO cells were observed under normoxia(21%02)and hypoxia(5%02).3)Oil red O staining:staining of fat-induced cells on day 6 with oil red O to observe the amount of lipid droplets accumulated;4)Western Blot was used to detected the expression of FKBP5 under hypoxia and fat-induced process;)Real-time PCR was used to detect the mRNA expression of lipid synthesis,lipid degradation and energy metabolism related genes.[Results]Herein,we found that the molecular mechanisms of WT and FKBP5 KO MEFs adipogenesis under hypoxia and normoxia were different and hypoxia could inhibit the repression of adipogenesis in FKBP5 KO MEFs.For WT MEFs differentiation groups under hypoxia,the express of adipogenic genes(CD36,Glut4,aP2)?lipolysis genes(GDPD1,Adiponectin)and energy metabolic gene(UCP1)were lower than normoxia differentiation groups.For FKBP5 KO MEFs differentiation groups under hypoxia,the adipogenic genes and UCP1 were up-regulated,however,the lipolysis genes were down-regulated compared with normoxia differentiation groups.Ultimately,hypoxia promotes the adipogenesis and lipids accumlation of WT and FKBP5 KO MEFs.These data prompt that an 02-sensitive molecular mechanism regulating adipogenesis under hypoxia and agents that regulate FKBP5 activity or 02 sensing may be used to inhibit adipogenesis and control obesity.[Conclusions]1)Hypoxia promotes the formation and accumulation of droplets of WT and FKBP5 KO MEFs2)Hypoxia affects the formation and accumulation of lipid droplets by regulating the expression of FKBP5 gene3)Hypoxia regulates lipid droplet formation and accumulation in WT and FKBP5 KO MEFs by influencing fat synthesis,lipolysis and energy metabolism... |
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