Loureirin A Regulates MiR-339-5p To Promote The Differentiation Of Hair Follicle Stem Cell And Repair The Skin Wounds

ObjectiveSkin trauma is a common clinical disease.Unhealing wounds or pathological scar seriously affect the patient’s physical and mental health.Hair follicle stem cells(HFSCs)are multipotent stem cells.When the skin is injured,it can differentiate into epidermal and skin appendages(hair follicles,sebaceous glands,and sweat glands),and participate in skin wound repair.Sanguis draconis is one of the renowned traditional medicines and has been used as a “panacea of blood activation” in China for many years.It has various biological activities such as anti-bacterial,anti-fungal and anti-platelet effects.Loureirin A,as the main active ingredient of sanguis draxonis,can induce HFSCs differentiation effectively.However,the mechanism of differentiation has not been studied clearly.Micro RNAs(miRNAs)are the research hotspots in recent years.More and more studies have shown that miRNA regulate cell differentiation,biological development,and disease development.In this study,the miRNA expression of HFSCs induced by loureirin A was explored in vitro.It had been proved that DLX5 was the target gene of miR-339-5p.Moreover,it had been explored that miR-339-5p/DLX5 regulated HFSCs differentiation via classic Wnt signaling pathway.In vivo,miR-339-5p antagomir alone or combinated with loureirin A could promote the vibrissa skin wound healing.Methods1.Sprague-Dawley rats,1～3 days old,were choosed to obtain the vibrissa hair follicles.The vibrissa hair follicles were digested by “two-step enzyme”to separated the hair follicle cells.CD34 and K15,the surface markers of HFSCs,were identified by double immunofluorescence staining.2.HFSCs were devided into the control group and loureirin A-treated group.Specific expressed miRNAs were screened by miRNA microarray and verified by RT-q PCR.3.The mimic or inhibitor of miR-339-5p was transfected into HFSCs.After 48 h,Western Blot and immunofluorescence were used to detect the expression of DLX5 protein.Double luciferase reporter gene of DLX5 was constructed,and the luciferase activity of target gene was assayed.4.The miR-339-5p mimic/inhibitor/si DLX5 was transfected into HFSCs.The proteins including Involucin、K10 and Wnt3 a were examed by Western Blot in each group.5.The male SD rats,200-220 g,were choosed to build vibrisaa skin wound model.The rats were randomly divided into blank control group,negative control group and experimental group.Separate treatment groups were subcutaneously injected with saline/agomir NC/miR-339-5p agomir/antagomir NC/miR-339-5p antagomir;the combined treatment groups were treated with DMSO or loureirin A and injected with above-mentioned regents.The wound healing score、 HE staining and Masson staining were used to detect wound healing in each group on the 3 d and 6 d.Results1.The isolated and cultured cells of hair follicles are round or polygonal,like typical paving stones.Both CD34 and K15 were positive in these cells.2.A total of 92 miRNAs was changed signaficately by miRNA microarray.Compared with the control,41 miRNAs were upregulated and 51 miRNAs were downregulated in the loureirin A group.RT-q PCR results showed that miR-339-5p was reduced significantly in the loureirin A group,which was consistent with the trend of miRNA microarray.3.The expression of DLX5,compared with the corresponding NC group,was reduced in miR-339-5p mimic transfection group and increased in miR-339 inhibitor transfection group by Western Blot and immunofluorescence.The luciferase activity of p LUC-DLX5 was significantly reduced by miR-339-5p mimic,and markedly increased by the miR-339-5p inhibitor.4.The expression of involucrin、K10 and Wnt3 a was decreased in the miR-339-5p mimic and si DLX5 groups,and increased in the miR-339-5p inhibitor group.5.The skin wound of the vibrisaa was promoted by miR-339-5p antagomir.The vascularization and collagen secretion in skin wounds were promoted obviously by miR-339-5p antagomir alone and combined with loureirin A.Conclusion1.The expression of miR-339-5p in HFSCs is down-regulated by loureirin A.2.DLX5 is a potential target gene of miR-339-5p.3.Mi R-339-5p/DLX5 affects the differentiation of HFSCs through the classic Wnt signaling pathway.4.Loureirin A combined miR-339-5p antagomir promotes skin wound repair.