The Study Of Qili San Promoting Wounds Healing By Tissue Engineering Skin And Loureirin A Regulating The Proliferation And Differentiation Of Hair Follicle Stem Cells By Activating Wnt/β-catenin Signal Pathway

ObjectiveA highly-effective and stable method for the isolation and cultivation of rat hair follicle stem cells (FSCs) and fibroblasts was established. The cultured cells were identified by immunofluorescence. Drug containing serum of Qili San was prepared and added to FSCs cultured medium in order to study its effect on FSCs proliferation.PELNAC was chosen to be the artificial dermis for tissue engineering skin, hair follicle fibroblasts were seeded on PELNAC to develop an artificial dermal substitute. They were further cultured with FSCs to construct tissue engineering skin. The substitutes above were transplanted on wound surface of nude mice. It is aimed to discuss how Qili San made positive contribution to tissue engineering skin with FSCs to repair skin defect.Qili San is a complex traditional Chinese medicine, Loureirin A is one of the main active compounds in resina draconis which is the sovereign drug in Qili San. FSCs cultured in vitro were stimulated by Loureirin A to discuss its effect on FSCs growth and cell cycle. Also the expressions of the main proteins and downstream regulated genes in Wnt/β-catenin signal pathway in FSCs were detected. Through the experiments we can know the molecular mechanism of FSCs proliferation and differentiation by Loureirin A via the Wnt/β-catenin signal pathway. The experiments also explored the action mechanism of Chinese medicine ingredients repairing skin wounds with FSCs.MethodsFSCs were isolated from SD rats’ beard using collagenase type Ⅰ and trypsin two-step digestion. Hair follicle fibroblasts were obtained by tissue block cultured method. The cultured hair follicle stem cells were identified by immunofluorescence stain to detect the expression of cytokeratin 15 (CK15), cytokeratin 19(CK19) and β1 integrin. And the expression of fibronectin (FN), laminin (LN) and Vimentin in fibroblasts were tested by the same way. Qili San drug containing serum was prepared, which was strictly followed the common principle. Cultured medium with various concentration of drug containing serum were used to stimulated FSCs for different time and then the cell viability was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay.Hair follicle fibroblasts were seeded on the PELNAC to build artificial dermis. FSCs labeled with 5-bromo-2-deoxyuridine (BrdU) were implanted on the material. The skin substitutes with two kinds of cells were transferred on the Transwell plates for air-liquid interface culture to make mature skin substitutes. Nude mice were cut off a certain area of skin on the back as animal models of skin defect. The animals were selected and randomly divided into group A and B. Animals wound of group A were coated with normal saline and group B were coated with Qili San. The constructed skin substitutes were transplanted on the surface of the wound. After the surgery, both the two groups were randomly assigned to 5 treatment groups. They were normal saline control, epidermal growth factor (EGF) positive control, Qili San for oral use, Qili San for external use, Qili San for oral and external use. The rehabilitation progress of each animal were observed and recorded every day after transplantation. The standard for evaluation was made accordingly and used to score for every animal. The skin wounds were cut off and fixed after 3 days, 5 days,7 days and 14 days postoperation. Skin specimens were tested by pathological and immunofluorescence histochemical detection.The effect of different concentration of Loureirin A on FSCs proliferation was analysed by MTT assay at different points. Cell cycle of FSCs stimulated by Loureirin A was detected by flow cytometry. Expression of protein β-catenin, glycogen synthase kinase-3β(GSK-3β) and gene transcription factor 3(Tcf3), lymphoid enhancer factor 1(Lef1), cyclin D1, c-myc were detected respectively by Western Blotting and real-time quantitative polymerase chain reaction(RT-qPCR).ResultsThe cultured follicle cells by two-step enzyme method were round and polygon. They grew with a cobblestone appearance. The cultured epithelioid cells expressed CK15, CK19 and β1 integrin in their cytoplasm. Dermal fibroblasts with spindle shape spread on the flasks and expressed FN, LN and Vimentin. 2.5% volume fraction of drug containing serum from SD rats given Qili San for 3 days worked best in FSCs proliferation for 24 hours. Hair follicle fibroblasts were seeded on PELNAC and they were cultured for 5 days. The cultured artificial dermis was free of contamination and the hair follicle fibroblasts grew well. FSCs were seeded on the above dermis to culture for 5 days in vitro and then the whole compound artificial skin was cultured by air-liquid interface culture for another 5 days. The substitutes were soft and of moderate flexibility and toughness.After the transplantation of FSC tissue engineering skin, each animal lived in a good condition. There were no bleeding and infection on the surface of the wound. The substitutes were attached on the wound. All the animals had not obvious immune rejection.3 days after operation, the wound was gradually recovery and the scab was formed.5 days after operation, the scab became harder and thinker.7 days after operation, the scab area turned smaller. Renascent skin could be seen at the peripheral area of the scab.14 days after operation, scab of most nude mice was sloughed. The scab of Qili San for external use, Qili San for oral and external use group came off earlier than that of other groups and had already epithelized. The renascent skin was soft and thinker. The color was white or pink and it could differ from normal skin.Semi-quantitative standard for evaluation was made by observing the wound healing. Results showed that 3 days after operation, there was significant difference between Group A and B, which demonstrated that wound pretreatment of Qili San played a positive role in inhibiting wound bleeding, reducing the inflammatory reaction, promoting scab to cover the wound earlier at wound healing period; also there was significant difference among all the groups in both Group A and B, which indicated that at early stage after transplantation, different kinds of therapeutic methods were beneficial to wound healing.5 days after operation, as the wound was at reparation stage, there was no difference between the two groups.7 and 14 days after operation, there was significant difference between Group A and B, which meant wound pretreatment of Qili San was significant to tissue engineering skin with FSCs repairing skin wound. Among all the groups, Qili San for external use, Qili San for oral and external use of Group B got the highest score, which showed that external use, oral and external use were the best use of pharmaceuticals.Pathology results showed that 3 days after operation, animals of all the groups had obvious inflammatory reaction. Scab covered the engineered skin tissue. Undegraded PELNAC and a majority of lymphocyte could be seen. The cells of Qili San for external use group increased and concentrated.5 days after operation, the inflammatory reaction was reduced. Epidermis cells of each animal began to proliferate but they had not covered the wound yet. The connection between new epithelia and demis was not tight and they were easy to break apart. Granulation tissue of the animals in Group B proliferated distinctly.7 days after operation, inflammatory reaction significantly reduced and collagen fibers increased. The newly grown epithelium could be seen around the wound.14 days after operation, no obvious scaffold material could be seen. There were still small areas to heal in both normal saline control group and Qili San for oral use group. Animals of the other groups were all recovered. Skin lamination was fine and both the epidermis and dermis were well differentiated.After operation, the results of immunofluorescence histochemistry showed that BrdU-labeled FSCs in each animal expressed CK15、CK19 and β1 integrin. Also, as time went by, the expression of cytokeratin 14(CK14)in each specimen increased and strengthened.MTT results showed that Loureirin A at 20μ g/mL worked best on FSCs proliferation. The results of flow cytometry indicated that the quantity of FSCs at S phase and G2 phase increased stimulated by Loureirin A. Loureirin A could changed the cell cycle of FSCs. After stimulated by Loureirin A, GSK-3 β expression in FSCs was down-regulated while β-catenin was up-regulated in Wnt/β-catenin signaling pathway. Gene Tcf3, Lefl, cyclin D1 and c-myc in nucleus were all up-regulated.ConelusionDrug containing serum of Qili San could promote FSCs proliferation. The constructed tissue engineering skin with FSCs was of fine repairing effect to skin defect. Most of the implanted FSCs got involved in the differentiation of the epidermis. Qili San was beneficial for the improvement of wound microenvironment and tissue engineering skin repairing full-thickness skin defect by resolving stagnation and promoting regeneration therapy. Loureirin A activated the Wnt/β-catenin pathway, changing the cell cycle and contributing to FSCs proliferation and differentiation.