Hypoxia Induced The Effects Of Lactate Dehydrogenase On Hair-Induction In Dermal Papilla Cells Of Hair Follicles

BackgroundHair follicle is mainly composed of epithelium and dermis,and the interaction of epithelium and dermis determines the process of normal morphogenesis,hair periodic cycle,proliferation and regeneration of hair follicles.The main components of epidermis are hair follicle stem cell(HFSC),hair matrix,inner and outer root sheath,while the main components of dermis are dermal papilla cell(DPC).In the process of hair growth,epidermal cells are mainly induced by DPC to differentiate HFSC and grow downward around DPC,finally forming mature dermal papilla cells.The cells can induce the differentiation of hair matrix,inner and outer sheath,and promote the proliferation and growth of hair follicle.The studies have shown that DPC can not only regulate the development and growth of hair follicles through the signals between epithelium and dermis,but also be considered as mesenchymal stem cells with potential of differentiation and proliferation.Mesenchymal stem cells(MSCs)are multifunctional stem cells,which can proliferate and differentiate to other types of cells in a specific microenvironment.When DPC was isolated from the hair follicle microenvironment and cultured in vitro,due to the change of the culture environment,it was gradually losing proliferation ability and activity of stem cell.However,if there is corresponding signal or cytokine stimulation,DPC still have their inherent ability of inducing differentiation.DPC is very important for the growth of hair follicles,so culture DPC in vitro and make them have the ability of proliferation and differentiation has been widely concerned.Under the hypoxia environment,the proliferation and differentiation ability of MSCs can be enhanced in varying degrees.It can be seen that hypoxia plays a very important role in the activity of stem cells.In the process of cell proliferation and differentiation,glucose is mainly converted into pyruvate by glycolysis in the cytoplasm,and pyruvate oxidation(TAC)is carried out in the mitochondria.In hypoxia,pyruvate is mainly transformed into lactate by lactate dehydrogenase(LDH)in the cytoplasm,which provides energy for cell proliferation and differentiation.The study has also confirmed that LDH,as a key enzyme in glycolysis,can promote the activity of HFSC and induce the growth of hair follicles.However,we are still not clear about the proliferation of DPC under hypoxia in vitro and the expression of LDH in cell metabolism.To some extent,the hair follicle organ culture simulates the growth environment of the hair follicle and the three-dimensional structure of DPC in vivo,enhances the induction ability when cultured in vitro,so as to understand some important cytokines and metabolic substances involved in the regulation of hair growth.At the same time,we can master some important signal pathways(Wnt/?-catenin)and regulatory mechanism of hair follicle growth,which is of great significance to clarify the cell-cell interaction or cell-extracelluler matrix interaction.Bioinformatics research shows that the gene expression and the proteins level of DPC will change when cultured in vitro,leading to the change of its inherent characteristics,especially the ability of hair induction and cell aggregation.c-Myc,as the upstream regulatory gene of LDH,can regulate the expression of LDH,induce the cycle of hair follicle and promote the regeneration of hair follicle.Compared with cell culture,organ culture can better reflect the influence of the change of microenvironment in the hair cycle and cell metabolism.Although the organ culture can't be completely equal to the real environment of hair follicle growth in vivo,we can use the model to study the effect of different microenvironment and drug concentration on the proliferation and differentiation ability of DPC.In this study,we focus on microenvironment of DPC in two ways.Firstly,through the change of cell culture environment in vitro,we preliminarily understand the effect of hypoxia on the proliferation of DPC,and provide suitable microenvironment for them to retain the activity of MSCs.Meanwhile,we investigate the effect of LDH on growth of hair follicle from the changes of cell metabolism and metabolites.Secondly,through the organ culture,it is further confirmed that LDH can promote the growth of hair follicle.The results provide theoretical basis and clinical significance for improving the survival rate of transplantation of hair follicle.1.The effect of hypoxia on the proliferation capacity of DPC by LDH.Objective:Hypoxia is important to maintain the proliferation and differentiation ability of stem cells.DPC,as a special mesenchymal component of hair follicle(HF),not only regulates the development and growth of hair follicle,but also is considered as an induced pluripotent stem cells(i PS).Therefore,this study focuses on whether hypoxia could maintain the proliferation ability of DPC,and demonstrate the role of LDH in this process.Methods:DPC were obtained by microseparation and enzyme digestion.The cells density was adjusted to 1×10⁶/ml after being resuspended.DPC were cultured either in normoxia(20%O₂)or hypoxia(5%O₂),while observing the morphological and growth patterns.The cells with good growth were inoculated into 96 well plates with 4×10³cells/100?l medium per well.Cell viability assays were performed at the different time point(6h?12h?24h?48h?72h),meanwhile,LDH activity and lactate level assay in DPC were detected by ELISA.After that,DPC were cultured for 24 hours with different concentrations of LDH(1?10?g/ml).The expression of protein markers(ALP,?-catenin and LEF-1)in DPC was evaluated by immunofluorescence staining and Western blotting.Results:Hypoxia did show positive effect on proliferation of DPC.The cell morphology showed that DPC adhered to the wall earlier and proliferated faster after 24 hours later.The CCK-8 results showed that DPC proliferated under the two different oxygen concentrations(20%O₂and 5%O₂)in the first 12 hours,but the cells proliferated faster under hypoxic condition(5%O₂).At 24 hours after culture,the proliferation rate of cells in hypoxia was significantly higher than that in normal oxygen group(P<0.05).The LDH activity of DPC cultured under hypoxic condition was significantly higher than that of cultured under normoxic condition within 24 hours(P<0.05).The level of lactate increased gradually,but there was no significant difference between the two groups(P>0.05).After treatment with different concentrations of LDH(5?10?g/ml),the proteins(ALP,?-catenin and LEF-1)expression levels of DPC increased and were significantly higher than that in the control group(P<0.05).Conclusion:Our findings show that the proliferation activity of DPC could be maintained under hypoxia.Meanwhile,LDH plays an important role in maintaining the activity of DPC in hypoxic condition.Further research is needed to confirm how the characteristics of DPC could be maintained in hypoxia and mechanism of LDH promoting the growth of HF.If possible,this will provide new ideas and techniques for the culture of DPC maintaining the activity of stem cell in vitro.2.The expression of LDH in mouse hair follicles and its effect on hair follicle growth in vitro.Objective: Lactate dehydrogenase(LDH),an important rate-limiting enzyme in the glycolysis metabolism,plays a critical role in the growth of hair follicle stem cells(HFSCs).However,the exact expression of LDH during hair follicle growth and the relevant signalling pathways involved are not yet determined.In this study,we further confirmed whether LDH can promote the growth of hair follicle and explored the biological characteristics of DPC through Wnt/?-catenin signal pathway,using vibrissae follicle(VF)organ culture model.Methods: First,Ten female wild-type C57BL/6J mice of 4 weeks were selected.The expression of LDH in DPC and hair matrix(HM)regions in the different anagen phase of the back skin was assessed by immunochemistry.The anagen stage vibrissae follicles were carefully dissected from the upper lip pad of mice and were cultured in 24-well plates.VFs were randomly divided into four groups,each group was cultured for 3 days with different concentrations of LDH(1¹0?g/ml).The experiment was repeated three times.The length and cycle of hair follicles were observed.Then,the proliferation of intracellular keratinocytes in hair follicles was assessed by Ki-67 and TUNEL double immunofluorescence staining.In addition,DPC were cultured with 5¹0?g/ml LDH for 24 hours and the expression of the associated proteins in the Wnt/?-catenin signalling pathway were measured via western blot analysis.Finally,10 littermate mice were depilated to induce a fully synchronised anagen and were randomly divided into vehicle-treated group(without LDH in DMEM medium)and LDH-treated group(contain 5?g/ml LDH in DMEM medium).The effect of LDH on hair growth in vivo was observed by injecting it into the mice subcutaneously.Images were taken at 0?7?14 and 28 days.Meanwhile,the samples of mice back skin were taken and stained with hematoxylin and eosin(HE).Results: By immunohistochemistry,we observed that the expression sites and intensity of LDH were different in the growth cycles of hair follicles.LDH was mainly expressed in DPC and HM regions during the anagen stage.5?g/ml LDH significantly promoted hair shaft elongation in cultured VFs,and increased the percentage of VFs in the anagen stage of hair cycle after 3 days of organ culture(P<0.05).After cultured with 5 and 10?g/ml LDH for 72 h,Ki-67 and TUNEL double fluorescence staining experiments showed that the keratinocyte proliferation in HM was significantly increased compared with the control group(P<0.05).Western blotting was used to detect the proteins expression of DPC in different groups.It was showed that 5?g/ml LDH significantly increased expression levels of ALP and LEF-1 in DPC(P<0.05).Meanwhile,the channel proteins of Wnt5 a and ?-catenin were also highly expressed in the Wnt/?-catenin signaling pathway(P<0.05).Finally,LDH noticeably accelerated the transition of the hair cycle from the telogen to anagen phase in vivo.Compared with the control group,the thickness of back skin in the 5?g /ml LDH-treated group was significantly increased at 7 and 14 days(P<0.05).There was no statistically significant difference in the bulb diameter of the hair follicles between the two groups(P>0.05).Conclusion: In this study,we have demonstrated that LDH has the potential to stimulate mouse hair growth using the hair follicle organ culture.LDH can induce the biological characteristics of DPC through Wnt/?-catenin signalling pathway,and stimulate hair follicles to advance into the anagen phase.At present,LDH induced the proliferation of hair follicle has only been verified in animal experiments,and its specific signal pathway and mechanism need to be further confirmed.At the same time,this study provides experimental basis for further research to confirm that LDH has the same activation on human dermal papilla cells.