Role Of Ldha On Hair Regeneration And Its Regulatory Mechanism In Secondary Hair Follicle Stem Cells Of Cashmere Goat

Hair regeneration has always been a research hotspot,but there are few studies on cashmere regeneration.Cashmere is a kind of downy fiber without medulla that grow from secondary hair follicles(SHFs).The characteristics of the medulla,the regular arrangement and density of hair follicles in mice are totally different from those of the coarse hair with medulla.What’s more,the growth and apoptosis of the secondary hair follicles are synchronous throughout the year.The growth of SHFs is regulated by secondary hair follicle stem cells.There is an annual cycle of initiation and termination of differentiation,which is a group of hair follicle stem cells collectively activated and quiescent synchronously.It is different from the asynchronous renewal of adult mouse hair,suggesting that SHFs at least to some extent has a specific regulatory mechanism for cyclic hair regeneration.Lactate dehydrogenase A(LDHA)is an oxidoreductase that catalyzes the conversion of pyruvate into lactate and NAD~+,and participates in the maintenance of Multipotency of stem cells.This project aims to investigate the effect and regulatory mechanism of LDHA on cashmere regeneration in the glycolytic metabolic pathway of secondary hair follicle stem cells,and reveal the new molecular mechanisms of regulating hair regeneration because of metabolic disorders.In this study,through in vivo immunofluorescence localization and in vitro cell experiments,it was found that Ldha was significantly highly expressed in the bulge where hair follicle stem cells(HFSCs)reside.The Ldha expression in secondary HFSCs was markedly elevated than that in secondary hair follicle dermal papilla cells(DPs)and matrix cells,suggesting that Ldha plays an important role in secondary HFSCs.Next,it was verified that the decrease(or increase)of Ldha expression significantly reduced(or enhanced)the migration,proliferation and differentiation potential of secondary HFSCs,indicating that the expression of Ldha in secondary HFSCs affect the activities of stem cells.Secondary HFSCs treated with different experiments were transplanted into back skin of nude mice,it revealed that the reduced Ldha expression resulted in sparse and short regenerated hair compared with the control group.Similarly,it also leaded to significant delay of hair regeneration in mice when the supernatant of secondary HFSCs were injected subcutaneously in mice.Together,these results demonstrated that Ldha regulated hair regeneration in secondary HFSCs.By performing transcriptome sequencing and expression profiling analysis of HFSCs with reduced or overexpressed Ldha,it was found that the expression of inflammatory factors related to immune response,such as Icam1,Cxcl8,Ccl20 and Il-17 were significantly increased after Ldha expression was reduced.KEGG and GO enrichment analysis showed that IL-17 and Th17signaling pathways,which play important roles in immune diseases,were markedly enriched.When overexpressed Ldha,immune-related signaling pathways such as IL-17 were significantly down-regulated,and pathways related to hair follicle growth were significantly up-regulated.This result suggested that Ldha may cause a significant immune response.Through the co-culture experiment of secondary HFSCs and immune cells,it was found that reduced Ldha would recruit Th17 and Treg cells,and the cell supernatant significantly promoted the differentiation of pro-inflammatory cell Th17 and inhibited the differentiation of anti-inflammatory Treg cell.These results suggested that the reduced expression of Ldha induced a significant immune response.It is worth noting that,in this study,through the subcutaneous injection of the inhibitor indirubin,it was found that the inhibitor can restore the phenotype of delayed hair regeneration,and can reduce the recruitment of Th17 cells and the inflammatory response.Further research showed that the inhibitor indirubin increased the expression of Wnt signaling pathway-related geneβ-catenin,Lef1 and Tcf7,and Shh signaling pathway gene Shh,Smo and Gli3.Inhibitor indirubin significantly promoted the proliferation and differentiation of HFSCs during the formation of hair follicles,revealing a new molecular mechanism of regulating hair regeneration by immune responses caused by metabolic disorders.In summary,when reduced Ldha expression,the expression of inflammation-related factors,including Icam1,Cxcl8,Ccl20 and Il-17 are significantly increased,and Th17 and Treg cells are recruited to make an immune regulatory response,Wnt and Shh signaling pathways can’t be activated normally,and inhibits the proliferation and differentiation of secondary HFSCs,significantly delaying hair regrowth.Taken together,our results uncovered a novel regulatory mechanism for hair regeneration by immune responses triggered by metabolic dysregulation.This opens up a new direction for future research on targeting specific metabolic pathways to improve cashmere production and quality,and has important reference value for future research on hair in other mammals.