Study Of Molecular And Metabolic Mechanisms Of Dietary Copper Regulating Hair Follicle Development In Rex Rabbits

Copper(Cu)is a redox-active metal ion,and plays an important role in antioxidant mechanism,energy homeostasis,signal network and cell metabolism in aerobic organisms ranging from bacteria to mammals.In this study,the effects of copper on hair follicle development and dermal papilla cell metabolism of Rex rabbits were studied by using metabolomics,proteomics and other molecular biological techniques.The research contents are as follows:1.Effects of dietary copper supplementation on growth performance and hair follicle development of Rex rabbitsOne hundred and sixty 90-day-old Rex rabbits were randomly divided into 4 groups,with 40 rabbits in each group.Control group: fed basal diet(measured copper co ntent 8.4mg/kg);copper groups: fed basal diet supplemented with 30、60 or 120 mg/kg Cu(measured copper content 39.1 、 67.5 and 127.7 mg/kg,respectively).The trial lasted for 42 days(including a 7-day adaptation period and a 35-day experimental period).The experiment results showed that the addition of 30 mg/kg copper to the basal diet significantly increased the average daily weight gain(P < 0.05)and the average daily feed intake(P < 0.05),but had no significant effect on the feed-to-weight ratio,total and half-cleaning rates(P > 0.05).Copper addition significantly increased the contents of plasma copper(P < 0.05)and serum ceruloplasmin(P < 0.05),but had no significant effect on the contents of serum alanine aminotransferase(P > 0.05)and aspartate aminotransferase(P > 0.05).Copper addition significantly increased hair density(P < 0.05)and the ratio of secondary hair follicles to primary hair follicles(P < 0.05)in Rex rabbits,but had no significant effect on the fur area(P > 0.05)、fur weight(P > 0.05)、fur thickness(P > 0.05)and hair length(P > 0.05).In addition,the addition of 30 mg/kg copper to the basal diet significantly increased the expression of alkaline phosphatase(ALPL),keratin associated protein 3-1(KAP3.1),keratin associated protein 6-1(KAP6.1)in the dorsal skin of Rex rabbits and significantly downregulated the expression of transforming growth factor β2(TGF-β2)and the protein levels of Phosphorylation-regulatory associated protein of m TOR(p-Raptor)and Phosphorylation-AMP-activated protein kinase(p-AMPK),and significantly increased the protein levels of Phosphorylation-Ribosomal protein S6 kinase(p-P70S6K).2.Effect of copper on proliferation of dermal papilla cells in Rex rabbitsDermal papilla cells were divided into control group and copper treatment group.Control group: basal culture medium without serum;copper treatment group: basal culture medium without serum was added with 0,0.2,0.5,1,2,5,10,100 or 1,000 nM copper.The dermal papilla cells were treated for 24 h,and the effect of copper on the proliferation of dermal papilla cells was observed.The experiment results showed that the addition of 1 nM copper to the medium significantly promoted the proliferation of dermal papilla cells(P < 0.05),and significantly increased the protein level of mitochondrially encoded cytochrome c oxidase 1(MTCO1)(P < 0.05),the production of ATP(P < 0.05),and the activity of cytochrome c oxidase(P < 0.05).The effects of copper on the metabolism of dermal papilla cells were studied based on the Ultra performance liquid chromatography/tandem mass spectrometry(UPLC-MS/MS)metabonomics.The results showed that the differential metabolites were significantly enriched with arachidonic acid metabolism,Histidine metabolism and alanine,aspartate and glutamate metabolism pathway(P < 0.05).3.Effect of copper depletion on cell metabolism of dermal papilla cells in Rex rabbitsDermal papilla cells were divided into control group and TM(Tetrathiomolybdate,a copper chelating agent)treatment group.Control group: basal culture medium;TM treatment group: basal culture medium was added with 0,0.1,1,2,5,10 or 20 μM TM.The dermal papilla cells were treated for 72 h,and the effects of TM-mediated copper depletion on the proliferation and the cellular copper contents of dermal papilla cells were observed.The experiment results showed that TM(5 μM)efficiently depleted the intracellular copper level(P < 0.05)without affecting the cell viability(P > 0.05).The results of proteomic showed that,compared to the control group,the expression levels of 256 proteins were significantly reduced and the expression levels of 145 proteins was significantly increased in the TM mediated copper depletion group.GO annotation and enrichment analysis showed that the downregulated proteins were mainly involved in oxidoreductase activity,antioxidant activity,cytochrome-c oxidase activity,and electron transport chain(P < 0.05).Several subunits of mitochondrial COX were specifically downregulated,including Mitochondrially encoded cytochrome c oxidase II(MTCO2),NDUFA4 mitochondrial complex associated(NDUFA4),and cytochrome c oxidase subunit 7A2(COX7A2)(P < 0.05),western blot analysis confirmed the downregulated protein levels of Mitochondrially encoded cytochrome c oxidase I(MTCO1),and NDUFA4(P < 0.05).KEGG enrichment analysis showed that the downregulated proteins were significantly enriched in Glut athione metabolism,Pentose phosphate,Glycolysis/Gluconeogenesis,Carbon metabolism,and Pyruvate metabolism pathway(P < 0.05).Moreover,TM mediated copper depletion significantly downregulated the expression of multiple enzymes involved in glycolysis and the tricarboxylic acid cycle(TCA cycle),including malate dehydrogenase 1(MDH1),isocitrate dehydrogenase(NADP(+))1(IDH1),aconitase 1(ACO1),aldolase,fructose-bisphosphate A(ALDOA),enolase 1(EN01),phosphoglucomutase 1(PGM1),phosphoglucomutase 2(PGM2),and glucose-6-phosphate isomerase(GPI)(P < 0.05).Nutrient analysis in DPCs showed that copper depletion significantly increased pyruvate consumption and led to an accumulation of citric acid,succinate and α-ketoglutarate(P < 0.05)in DPCs,but had no significant effect on glucose consumption(P > 0.05).Moreover,copper depletion significantly increased the activities of Pyruvate carboxylase(PC),Pyruvate dehydrogenase(PDH),Citrate(Si)-synthase(CS),and α-ketoglutarate dehydrogenase(α-KGDH)(P < 0.05),all of which are enzymes that regulate carbon entry into the TCA cycle.Western blot analysis confirmed copper depletion significantly increased the protein levels of Dihydrolipoamide S-acetyltransferase(DLAT),Dihydrolipoamide succinyltransferase(DLST),and lipoylation(P < 0.05).By using 2-DeoxyD-glucose(2-DG)and oligomycin,the experiment results showed that copper depletion significantly inhibited mitochondrial ATP production(P < 0.05)rather than glycolysis(P >0.05).Moreover,we observed that the removal of TM or the addition of copper to TM-treated DPCs significantly increased the Mitochondrial membrane potential(MMP)and the ATP production,and decreased the total cellular Reactive oxide species(ROS)levels in DPCs.4.Effect of copper depletion on ALP activity of dermal papilla cells in Rex rabbitsDermal papilla cells were divided into control group and TM treatment group.Control group: basal culture medium;TM treatment group: basal culture medium was added with 0,0.1,1,2 or 5 μM TM.The dermal papilla cells were treated for 72 h,and the TM-mediated copper depletion on the proliferation,ALP activity and ALPL expression of dermal papilla cells were observed.The results showed that TM(5 μM)-and BCS(1,000 μM)-mediated copper depletion reduced the ALP activity(P < 0.05)and the ALP m RNA levels(P < 0.05).To further investigate the mechanisms of copper depletion compromised the ALP activity in DPCs,multiple concentrations of glycolysis inhibitor(2-DG),ANT inhibitors(bongkrekic acid and ibipinabant),and several known mitochondrial inhibitors(CCCP,rotenone,and oligomycin)was added to the basic medium,and the effects of these on the cell proliferation and ALP activity were observed after 24 h.The experiment results showed that oligomycin(P< 0.05),but not 2-DG(P > 0.05)treatment,significantly inhibited ALP activity;neither of the ANT inhibitors affected ALP activity(P > 0.05).Both rotenone and CCCP reduced ALP activity to the same extent as oligomycin(P < 0.05),despite their opposing effects on the MMP(P < 0.05).Rosup,a ROS inducer,significantly reduced ALP activity(P < 0.05);NAC and AA,two ROS scavengers,partially prevented the copper depeltion-and Rosup-mediated inhibition of ALP and reduced the generation of cellular ROS(P < 0.05).5.Effect of copper depletion on ferroptosis of dermal papilla cells in Rex rabbitsDermal papilla cells were divided into control group and BCS treatment group.Control group: basal culture medium;BCS treatment group: basal culture medium was added with 0,100,200,500,1,000 or 2,000 μM BCS.The dermal papilla cells were treated for 72 h,and the effects of BCS-mediated copper depletion on the proliferation and oxidative stress of dermal papilla cells were observed.The results showed that BCS treatment(1,000 μM)mediated copper depletion significantly reduced the protein levels of MTCO1 and NDUFA4(P < 0.05).BCS mediated copper depletion did not affect the protein level of cytochrome c(P > 0.05)but resulted in a significant reduction in the oxidation of cytochrome c(P < 0.05).BCS mediated copper depletion significantly depolarized the mitochondrial membrane potential(P < 0.05)and increased the total cellular ROS levels(P < 0.05).BCS mediated copper depletion led to a significant increase in the contents of protein carbonylation(PCO),lipid peroxide(LPO)and malondialdehyde(MDA),a significant reduction in the activity of SOD1 and GSH-Px,a significant decrease in the glutathione/oxidized glutathione(GSH/GSSH)ratio and a significant decrease protein level of glutathione peroxidase 4(GPX4)(P < 0.05).The result of transmission electron microscopy revealed that the DPCs treated with BCS exhibited shrunken mitochondria with a decreased number of cristae morphology.The results of Metabonomics showed that BCS-mediated copper depletion significantly reduced the contents of glycerol phospholipids,which constitute various biomembranes(P < 0.05),and increased the contents of arachidonic acid,adrenic acid,glutamic,and gl utamine(P < 0.05).Moreover,the combination of BCS and Erastin significantly enhanced the erastin induced cell death and ferroptosis events,including increased MDA and total cellular ROS levels,GSH and cysteine depletion,and GSSG generation(P < 0.05).The ferroptosis inhibitor(Ferrostatin-1)partially prevented BCS-mediated cell death and inhibited the generation of the total cellular ROS and LPO(P < 0.05).In brief,copper depletion enhances ferroptosis via mitochondrial perturbation and reduction in antioxidative mechanisms.In conclusion,the aim of this study was to elucidate the effect of copper on hair follicle development in Rex rabbits and its molecular and metabolic mechanisms.Our results suggested that:(1)the addition of copper in diet can increase the density of hair follicle in Rex rabbits.(2)copper addition in culture medium can promote the proliferation and increase the ALP activity of dermal papilla cells.(3)copper depletion mediated inactivation of copperdependent mitochondrial cytochrome c oxidase as the primary metabolic defect in dermal papilla cells,copper depletion decreases the activity of alkaline phosphatase in dermal papilla cells by increasing mitochondrial ROS production.(4)copper depletion enhances ferroptosis via mitochondrial perturbation and reduction in antioxidative mechanisms.