Mechanism Of Vitamin A Regulating Hair Follicle Development In Heat-stressed Rex Rabbits

Rex rabbit is an important fur-bearing rabbit.Heat stress severely reduces the fur quality of Rex rabbit.The purpose of this study was to investigate the effect of vitamin A（VA）addition in diet on hair follicle development and related signaling pathways in heat-stress Rex rabbits.The main elements of the study were as follows.（1）Research of VA effect on hair follicle development in heat-stressed Rex rabbitsIn the experiment,120 Rex rabbits were randomly divided into four groups:control group（20-25℃,fed basal diet）,heat stress group（30-34℃,fed basal diet）,and heat stress+VA group（30-34℃,fed 6,000 and 12,000 IU/kg VA on top of basal diet）.The experiment results showed that the addition of 12,000 IU/kg VA to the basal diet significantly increased the average daily weight gain（P<0.01）and significantly reduced the feed-to-weight ratio（P<0.01）of Rex rabbits under heat stress,but had no significant effect on the average daily feed intake,total and half-cleaning rates（P>0.05）,and serum biochemical indexes（P>0.05）.VA addition significantly increased hair follicle density（P<0.01）,hair length（P<0.05）and the ratio of secondary hair follicles to primary hair follicles（P<0.05）in heat-stressed Rex rabbits.In addition,dietary VA addition significantly inhibited the expression of bone morphogenetic protein 2（BMP2）,bone morphogenetic protein 4（BMP4）,fibroblast growth factor 5（FGF5）,transforming growth factorβ1（TGF-β1）and micro RNA-214（miR-214）in heat-stressed Rex rabbits and significantly upregulated the expression of noggin,insulin-like growth factor（IGF1）,insulin-like growth factor receptor（IGF1R）,Wnt10b,β-catenin,hedgehog factor（SHH）and micro RNA-203（miR-203）expression and the levels of Wnt10b and p-β-catenin,but did not affect significantly the expression of epidermal growth factor（EGF）,hepatocyte growth factor（HGF）and Micro RNA-205（miR-205）expression.（2）Effect of VA on proliferation and apoptosis of dermal papilla cells in the condition of heat stressedCells were divided into control group（37℃,5%CO₂）,heat treatment group（every 7h45℃for 1h,5%CO₂）,heat stress+VA（0.05,0.1,0.2,0.4 or 0.8 mg/m L）groups.After 48hours of treatment,the cell proliferation,apoptosis,cycle and related gene protein expression were detected.The results of cell proliferation assay kit（CCK8）showed that the addition of0.4 mg/m L VA to mediam in the condition of heat stress could significantly accelerate cell proliferation（P<0.01）and inhibit the early and late apoptosis of dermal papilla cells（P<0.01）.Heat stress can lead to the arrest of dermal papilla cells in G1 phase,and VA can slow down the arrest of G1 phase（P<0.05）.VA had no significant effect on S phase and G2 phase of dermal papilla cells under heat stress（P>0.05）.Heat stress significantly increased the expression of Caspase3 and HSP70 protein（P<0.05）,and VA addition in heat stress could inhibit the expression of Caspase3 and HSP70 protein compared with heat stress group（P<0.05）.Compared with heat stress group,VA significantly inhibited the expression of BMP4,FGF5,SHH,miR-214,miR-205 and miR-195 in heat-stressed dermal papilla cells（P<0.05）,and significantly up-regulated the gene expression of IGF1,IGF1R,Wnt10b andβ-catenin（P<0.05）.BMP2,Notch1,SHH in heat-stressed dermal papilla cells were not significanlty affected by VA（P>0.05）.VA significantly increased the levels of Wnt10b and p-β-catenin protein in heat-stressed dermal papilla cells compared with heat stress group（P<0.05）,and significantly inhibited the expression of p-GSK-3βprotein in heat-stressed dermal papilla cells（P<0.05）,but had no significant effect on the expression of EGF,HGF and miR-203（P>0.05）.（3）Function of ocu-miRNA-195/IGF1 signaling pathway in VA regulation of heat-stressed dermal papilla cell developmentmiRNAs and target genes with different results in animal and cellular assays were predicted to have a target relationship.ocu-miRNA-195 and IGF1 were found to have binding sites,and a double luciferase gene reporter was performed to verify their targeting relationship.The results showed that the luciferase activity of ocu-miR-195-5p was highly significantly decreased after overexpression of ocu-miR-195-5p when transfected with wild-type vector plasmid compared with the control group（P<0.01）.And the luciferase activity of ocu-miR-195-5p was highly significantly increased after overexpression of ocu-miR-195-5p when transfected with mutant vector plasmid compared with the wild-type vector plasmid group（P<0.01）.This result indicates that miR-195-5p has a targeting relationship with IGF1.Then miR-195-5p was transfected into dermal papilla cells for overexpression assay.Cells were divided into normal group（37℃）,heat stress group（45℃）,heat stress+VA（0.4mg/m L）group,and heat stress+VA（0.4 mg/m L）+miR-195 group.After transfection of miR-195 into heat-stressed cells,the cells were collected in culture for 48h,and cell proliferation viability,cell cycle and apoptosis were detected,together with the expression of related genes.The results showed that after miR-195 overexpression,cell viability was significantly decreased（P<0.05）,apoptosis was significantly increased（P<0.05）,cell cycle was not significantly affected（P>0.05）,and IGF1 and IGF1R expression was significantly decreased（P<0.05）compared with the heat-stressed+VA group.（4）Role of Wnt/β-catenin signaling in VA regulating heat-stressed dermal papilla cell developmentThe results of animal and cellular experiments have revealed that VA could activate Wnt/β-catenin signaling.To further investigate the role of Wnt/β-catenin signaling in VA regulating heat-stressed dermal papilla cell development,the normal group（37°C）,heat stress group（45°C）,heat stress+VA（0.4 mg/m L）group,heat stress+VA+IWP-2（Wnt signaling inhibitor）group,heat stress+VA+XAV-939（β-catenin signaling inhibitor）group.Cells were collected for a period of time in culture,and cell proliferation viability and apoptosis were detected,along with the expression of related genes and proteins.The results showed that the cell viability was significantly decreased compared with the heat-stressed+VA group（P<0.05）.And apoptosis was significantly increased（P<0.05）after the addition of IWP-2compared with the heat-stressed+VA group.There was no significant effect on the expression ofβ-catenin,noggin,Notch1,BMP2 and BMP4 genes（P>0.05）compared with the heat-stressed+VA group;the expression of Wnt10b and p-β-catenin protein was significantly decreased compared with the heat-stressed+VA group（P<0.05）.And Caspase3protein expression significantly increased compared with the heat-stressed+VA group（P<0.05）.The addition of XAV-939 significantly decreased cell viability（P<0.05）,significantly increased apoptosis and p-β-catenin and Caspase3 protein expression（P<0.05）,decreased BMP4 gene expression and had no significant effect onβ-catenin,noggin,Notch1,and BMP2gene expression compared with the heat-stressed+VA group（P>0.05）.In conclusion,the aim of this study was to elucidate the alleviating effect of VA on hair follicle development in heat-stressed Rex rabbits and its mechanism.Our result demonstrated:（1）heat stress reduces hair length and hair follicle density in Rex rabbits,and the addition of VA in diet can alleviate the effect;（2）heat stress can reduce hair papilla cell viability and proliferation,leading to apoptosis,and VA addition in culture medium can alleviate this phenomenon;（3）VA can regulate the development of heat-stressed dermal papilla cells through miR-195/IGF1 and Wnt10b/β-catenin signaling.