Biological Effects Of Low-lever Laser (LLL) On Mesenchymal Stem Cells (MSCs)

Photobiomodulation(PBM)affected the activity of endogenous photoreceptors by light to alter cell proliferation and metabolism through mitochondrial retrograde signaling pathways.Low-level lasers(LLL)was a common light source used in photobiostimulation therapy.Light in this region of the spectrum can penetrate tissue and lacks the carcinogenic properties.Low level lasers(LLL)has been considered to be a non-invasive physiotherapy in clinical application,for example osteoporosis,with changes of the state of bone mesenchymal stem cells.Since light therapies are widely used in medicine,combination of stem cell therapy and light therapy appears to be a wise choice.The effect mechanism of LLL on MSCs from human(hMSCs)remained to be discovered.One possible mechanism is photon absorption by cytochrome C oxidase in the mitochondrial respiratory chain.Microarray technology discovered a significant upregulation of gene expression in pathways involved in energy production and cellular antioxidant protectionafter irradiation.In first part of this article,cell viability,migration,anti-apoptisis ability and pertinent mechanism were assessed.cell proliflation was measuredusing MTS assays and cell cycle was evaluated by FACS.The influence of LLL on mitochondrial biogenesis(fission or fusion)and function(ATP,ROS,NO)were evaluated by TEM,FACS,QRT-PCR and immunocytochemistry.Cell migration and cytoskeleton alteration(actin and tubulin)were evaluated using transwell assay,immunocytochemistry,ELISA and Western blotting.Cell apoptosis was evaluated using FACS,immunocytochemistry,and Western blotting.We investigated that certain influence of low level lasers(LLL)on MSCs in vitro 6 hours or 24 hours after 1 hour of LLL irradiation.The mechanism of the effects included proliferation rate increasing mediated by increased S phase proportion;mitochondria biogenesis and function altering mediated by fusion(Mfn1,Mfn2 and Opa-1)and fission(Fis1,Drp-1 and MTP18)related proteins,NRF1,TFAM,PGC-1a and upregulated intracellular ROS and NO concentration;migration acceleration via ERK1/2 and FAK pathway and upregulation of growth factors such as HGF and PDGF;resistant to apoptosis with increased Bcl-2 and decreased Bax,or through tunneling nanotubes(TNTs)formation between LLL treated MSCs and 5-fluorouracil induced apoptotic MSCs.Therapeutic use of MSCs for regenerate medicine showed promising results in patients.The mechanisms of MSCs therapy include:paracrine activity like secretion of proteins;transfer of organelles by tunneling nanotubes;transfer molecules through exosomes.It is known that MSCs are recruited to sites of stress or inflammation to fulfill their repair function.MSCs are not spontaneously immunosuppressive but require ’licensing’ or activation to exert their immunosuppressive effects.MSCs are among the cells that express TLR proteins,TLR signaling has been implicated in the licensing of MSCs.With TLR4 priming inducing a pro-inflammatory phenotype and secretion of IL-6,IL-8 and TGF-b,in contrast,TLR3 priming induced anti-inflammatory MSCs(producing IDO,PGE-2,IL-4 and IL-1RA).In the second part of this article,we investigate the anti-inflammatory effect of LLL on human adipose-derived mesenchymal stem cells(hADSCs)in which the inflammatory response was induced by LPS treatment.The production and expression of pro-inflammatory cytokines were evaluated by ELISA kits and RT-qPCR,ROS generation was measured with CM-H2DCFDA and NO secretion was determined by DAF-FM.LLL reduced the secretion of IL-1β,IL-6,IL8,ROS and NO in LPS treated MSCs.Immunofluorescent assay demonstrated the nuclear translocation decrease of NF-κB in LLL treated LPS induced MSCs.Western blot analysis also suggested that LLL suppressed NF-κB activation via regulating the phosphorylation of p65 and IκBα.We studied surface expression of lymphocyte activation markers when purified peripheral blood mononuclear cell(PBMC)were activated by phytohaemagglutinin(PHA)in the presence of 3 types of treated MSCs.MSC significantly reduced the expression of activation markers CD25 and CD69 on PHA-stimulated lymphocytes.In conclusion,we used a fluence of 11-16 J/cm2 LLL to enhance MSCs survival by promote proliferation,migration and anti-apoptosis ability,and therapeutic function by good anti-inflammatory effect.LLL can be applied as MSCs preconditioning prior to transplantation towards the safe long-term efficacy of stem cell based therapy.