The Regulation Of Loureirin A To Promote Proliferation And Differentiation Of Follicle Stem Cells And Loureirin A Repair Vibrissa Skin Wound Of Rats

ObjectiveTrauma caused by skin defect is a common clinical problem, large skin defect cause scar formation, without skin and hair follicle, sweat gland, and other skin adnexal, affect the patients’ physical and mental health seriously. Hair Follicle Stem Cells (Follicle Stem Cells, FSCs) is one kind of adult Stem Cells that has high proliferation potential and multi-directional differentiation potential, under some special conditions can be divided for the hair follicles, sebaceous glands, and other skin adnexal suit for treating skin defect with significant clinical superiority. However, FSCs in vitro proliferation is not ideal, it is an urgently needed to solve that to give full use to the FSCs proliferation potential and effective induction of the directional differentiation for better applied to tissue trauma repair.According to the Compendium of Materia Medica records, Sanguis Draconis with warm, sweet and salty, non-toxic characteristics, the main effect of quicken the blood and transform stasis, astringency and hemostasis, engender flesh and close wounds, soften hardness and dissipate binds, disperse swelling and relieve the pain. Modern pharmacological studies have shown that Sanguis Draconis has a variety of bioactivities such as anti-inflammatory, anti-nociceptive, anti-oxidation, intensifying immunizing power, stimulate the circulation of blood and regulate body metabolism, is an ideal trauma drugs. In this study, the tentacle skin defect model of rat was built to explore the effect on skin wound repair of Loureirin A and the role of FSCs directional differentiation in vivo and in vitro; Wnt/β-catenin classic signaling pathways involved in cell proliferation and differentiation and so on, this study discussed the way Loureirin A effects on FSCs Wnt/β-catenin classic signaling pathway in vitro cultivation, and built lentivirus LEF1 over expression plasmid, transfected FSCs in vitro culture, for further explore of regulating changes of Loureirin A of FSCs Wnt/β-catenin classic signaling pathways; used the miRNA gene chip technology, predicted the targets of Loureirin A controls FSCs in the wound healing in related pathways.Methods1. Used 200 g adult male SD rats to build vibrissa skin wound model, according to the different treatment methods, animals can be divided into experimental group (Loureirin A), positive control group (Epidermal growth factor, EGF), blank control group (saline), observing each group wound healing conditions, statisticaling wound areas, healing rates, using the HE dyeing to observe pathological changes during skin wound repair process, frozen section immunofluorescence showed the epidermal cell differentiation of different groups skin of rats in the process of wound repair.2. Used 1-3 d SD rats, took the vibrissa skin hair follicle, isolated, cultured, identificated FSCs in vitro. Set up the experimental group (including Loureirin A K-SFM medium), positive control group (including the lithium chloride K-SFM medium), blank control group (K-SFM medium),compare Loureirin A, lithium chloride of FSCs promote proliferation role by MTT method; immunofluorescence observated differentiation related proteins (CK15, CK14, CK19, Involuvcrin, CK10) expression to explore the function of Loureirin A, lithium chloride, in FSCs differentiation detection.3. Tested before and after the protein change ofβ-catenin, GSK-3β, detected purpose gene LEF1, TCF3, downstream c-myc, cyclin Dl to explore the role of Loureirin A in FSCs Wnt/β-catenin signaling pathway. Used lentivirus LEF1 over expression plasmid and transfected FSCs, further explore the role of Loureirin A in FSCs Wnt/β-catenin signaling pathway.4. Used the miRNA gene chip technology high-throughput screened FSCs of before and after Loureirin A role of microRNA, and proved by qRT-PCR experiments for effective preliminary explore Loureirin A regulation of microRNA in FSCs and on the basis to forecast the related signaling pathway.Resuls1. Loureirin A can stop vibrissa skin wound bleeding of rat effectively, promote shrinkage, scabby, inhibit inflammation and promote fibroblasts proliferation, the secretion of extracellular matrix, restoration of skin and hair follicles.2. Solated, cultured FSCs in vitro, CK15 and CK19 were positive expression, while CK14, Involuvcrin, CK10 were positive expression, and Loureirin A can lead FSCs to the epidermal cell differentiation.3. After Loureirin A roled FSCs, GSK-3β expression decreased, β-catenin expression, LEF1, TCF3, c-myc, cyclin Dl gene expression are raised, and Loureirin A activates FSCs Wnt/β-catenin signaling pathway. Lentivirus LEF1 over expression plasmid transfected FSCs with the efficiency up to 37%, LEF1, cyclin Dl gene expression raised in transfection cells.4. After Loureirin A roled FSCs, screened by gene chip to predict the miRNA, the expression of miR-21, miR-299 raised and miR-296, miR-335 were lower, prompt Loureirin A might through the TGFβ-Smad pathway, BMP, and/or MAPK pathways to regulate the proliferation and differentiation of FSCs.Conclusion1. Loureirin A can promote the restoration of skin wounds in rats.2. Loureirin A can promote the proliferation and differentiation of FSCs in vitro.3. Loureirin A can through regulating the Wnt/β-catenin signaling pathway, affect the proliferation and differentiation of FSCs.4. Loureirin A possible through regulating the TGF β-Smad pathway BMP and/or MAPK pathways regulating affect FSCs proliferation and differentiation.