Experimental Study On The Effect And Mechanism Of Low-level Laser On Hair Follicle Stem Cells In Skin Wound Healing

BackgroundIt has been more than half a century since the biological effect of low-level laser was first discovered,and it has been used in many aspects of medical practice by various clinical departments.There are also household low-level laser devices on the market to treat hair loss,relieve pain and promote recovery.However,there is still controversy in academic circles on the optimal frequency and energy density of low-level laser treatment in different application scenarios,the mechanism of low-level laser’s therapeutic effect,effector cells and even whether they really work on some diseases.Delayed healing of wounds will not only bring great physical and mental pain to patients,but also increase the burden on the social health care system.At present,promoting wound healing is one of the most common applications of low-level laser treatment,and skin wound healing is the result of multicellular synergism,and many kinds of skin stem cells,including hair follicle stem cells,play an important role in this process.However,there is no study on the effect and mechanism of low-energy laser on hair follicle stem cells in skin wound healing at present.Objectives1.To explore the effect of low-level laser on hair follicle stem cells in full-thickness skin wound healing in mice and its possible mechanism.2.To explore the effect of low-level laser on the biological behavior of mouse CK 15+hair follicle stem cells in vitro and its mechanism.Methods1.C57/BL6N mice with back hair removal were made into full-thickness skin trauma animal model with a diameter 5 mm punch,and were randomly divided into low-dose laser treatment group(LLLT-Low),high-dose laser treatment group(LLLT-High)and control group(Control).From the day of establishment of the model to the day before the skin was taken,the wound and wound margin skin of LLLT-Low group and LLLT-High group were irradiated with laser comb every 24 hours,and the energy density was 1 J/cm2(irradiation time=38 s)and 10 J/cm2(irradiation time=380 s),respectively.The control group was irradiated with ordinary fluorescent lamp.The wound closure ratio was analyzed at 0,3,5,10 and 14 days,and hematoxylin eosin staining(hematoxylinandeosin,HE)and immunofluorescence staining were performed at 3,5,10 and 14 days after modeling.Ten days after low dose laser treatment,the samples were sequenced by Whole transcriptome sequencing(RNA-Seq),gene ontology analysis(GeneOntologyanalysis,GO analysis)and verified by protein immunoblotting(WesternBlot,WB)and enzyme-linked immunosorbent assay(ELISA).2.Hair follicle stem cells in vitro were obtained by isolation,culture,purification and immunofluorescence identification,and were divided into low-level laser treatment group(LLLT)and control group(Control).The LLLT group was irradiated with low-level laser with energy density of 1 J/cm2 every 6 hours,and the control group was irradiated with ordinary fluorescent lamp.After irradiation,the ability of cell proliferation was detected by CCK-8 test and BrdU fluorescence staining test,the cell migration ability was detected by cell scratch test and Transwell chamber test,and the expression of IL 34 cytokines was detected by ELISA,RT-PCR and WB Western blotting test.Results1.The analysis of the percent of wound closure showed that healing was accelerated(significantly from 5 d to 10 d)in the LLLT-Low group,but there was no clear change in the LLLT-High group.HE staining showed that the LLLT-Low group had an increasing number of hair follicles and a tendency to migrate to the center of the wound.There was no significant increase in the number of hair follicles and no obvious migration in the LLLTHigh group.Immunofluorescence staining showed that the total number of CK 15+hair follicle stem cells in the LLLT-Low group was higher than that in the Control group and LLLT-High group at all time points.The number and farthest migration distance of CK 15+hair follicle stem cells increased significantly with time,and after 5 d,they were significantly higher than those in the control group and LLLT-high group.RNA-Seq and Western blot analysis showed that the expression of related genes in hair follicle stem cells,including CK 15,in the LLLT-Low group was upregulated.GO analysis and ELISA showed that the expression of many cytokines,represented by IL 34,in the LLLT-Low group was upregulated.2.CCK-8 test and BrdU fluorescence staining test showed that low-level laser treatment could promote the proliferation of CK 15+hair follicle stem cells in vitro.Cell scratch test showed that the migration ability of CK 15+ hair follicle stem cells in 24 h and 48 h LLLT group was better than that in the control group,and the promoting effect was more significant in 48 h.Transwell chamber test showed that the migration of hair follicle stem cells in the LLLT group was significantly higher than that in the control group at 12 h.ELISA and RT-PCR experiments showed that low-level laser treatment could promote the expression of cytokines such as IL 34 from different levels,and WB Western blotting confirmed the existence of this up-regulation.Conclusions1.Low-level laser treatment can promote the proliferation,differentiation and migration of CK 15+hair follicle stem cells by upregulating the cytokine IL 34,thereby promoting skin wound healing in mice.2.1J/cm2 low-level laser can promote the proliferation and migration of CK 15+hair follicle stem cells in vitro and promote the expression of IL 34.