Study On Dose-effect Relationship And Biological Effects Of Photobiomodulation In The Treatment Of Diabetic Ulcer

Diabetic foot ulcers(DFUs)is one of the most serious and costly chronic complications of diabetes,and in severe cases can lead to amputation and death,severely affecting the quality of life of patients.Photobiomodulation therapy(PBM)is considered one of the effective physical therapies to promote the healing of DFUs.However,there are problems of confusion of light parameters and insufficient theoretical basis in the treatment.AIMS:To address the problem of inadequate theoretical foundation and confusion of optical parameters in PBM treatment of DFU,a full-spectrum range of LED was used to conduct studies on comparison effects of different optical parameters on DFU healing-related cells and studies in vitro and in vivo on the biological mechanism of PBM treatment in DFUs.It provides the theoretical basis and reliable evidence for optical parameter compatibility in PBM treatment for DFU,so as to promote its clinical applicationMethods:(1).Diabetic cell models were constructed including skin fibroblasts(WS1),human umbilical vein endothelial cell(HUVEC),macrophages(U937)and keratinocyte(HACAT),which were DFUs healing-related key cells.The cells cultured in normal glucose setting(NG)or high glucose setting(HG)were inoculated into 96-well cell culture plates at the appropriate cell concentration.Each cell received a total 168 irradiation scheme with 4 multiple wells respectively at the bottom of the 96-well plate.The optical parameters contained 12 wavelengths(405nm,425nm,455nm,495nm,510nm,530nm,560nm,630nm,660nm,730nm,805nm,and 850nm),2 power densities(10 and 40mW/cm2),and 7 energy densities(0.5,1,2,4,6,8,and 10 J/cm2).The cell prolifelation ratio were detected by MTT analysis at 24h after PBM to high-throughput screen for optimal irradiation parameters.(?).Intracellular ATP levels were detected at 6h after PBM with the optimal irradiation parameters as screened above.IL-1?p,IL-6,and ICAM-1 in the supernatant of the cells were measured at 12h after PBM by Elisa.Cell migration ability were evaluated by scratching experiments.(?).T2DM rat model was obtained by high-fat diet in combination with STZ induction.Then DFU rat model was constructed by clipping the whole skin within 6 X 8 mm of the dorsum of the hind limb.Rats were divided into three groups:health control group(C0),T2DM control group(CDM)and PBM groups.Rats in PBM groups were randomized to receive diverse PBM irradiation scheme.They were divided into six subgroups,including five single-wavelength treatment groups(PBM425,PBM510,PBM630,PBM730,PBM850)and a multi-wavelength compatibility group(PBMcom).Irradiation was practiced by our homemade portable LEDs,which power density is 10mW/cm2 and energy density is 4J/cm2,once a day,continuous irradiation for 21 days or until the wound completely healed,each irradiation for 5 days,rest for 2 days.Percents of wound closure were calculated every two days.Skin blood flow perfusion were measured by laser scattering at 1,3,7 days after wound.The whole skin of rats containing the wound and surrounding normal skin was cut on day 3,7 and 15 after the wound for tissue histopathology staining,including HE staining,MASSON staining,CD34,PCNA,CD 11B,MMP-9 immunohistochemical staining.Results:(?).PBM promotes proliferation of HACAT cells in high glucose culture conditions with an optimal effect parameter of 10 mW/cm2,0.5 J/cm2.In which 425,510,660 nm PBM made the diabetic HACAT cells proliferate over 10%.PBM promotes proliferation of HUVEC cells in high glucose culture conditions with an optimal effect parameter of 10 mW/cm2,0.5 J/cm2.In which 425,495 nm PBM made the diabetic HUVEC cells proliferate over 10%.PBM promotes proliferation of WS1 cells in high glucose culture conditions with an optimal effect parameter of 10 mW/cm2,2 J/cm2.In which 495,510,530 nm PBM made the diabetic WS1 cells proliferate over 10%.PBM promotes proliferation of U937 cells in high glucose culture conditions with an optimal effect parameter of 10 mW/cm2,1 J/cm2.In which405,425,455,495,510,530,560,630,660,805,850 nm PBM made the diabetic WS1 cells proliferate over 10%.(?).PBM(495nm,510nm,805nm,850nm)increased the ATP level in diabetic WS1 cells(P<0.05).PBM(495nm,510nm,530nm,560nm,630nm,660nm,730nm,805nm and 850nm)increased ATP levels in diabetic HUVEC cells(P<0.05).PBM had no effect on ATP in diabetic U937 cells(P>0.05).PBM promoted diabetic WS1 cells to secrete IL-1beta(405nm,455nm,495nm,510nm,530nm,560nm,630nm,660nm,805nm and 850nm,P<0.05),but IL-6 and ICAM-1(all P>0.05).PBM(530 nm)promoted diabetic U937 cells to secrete IL-1beta(P<0.05).PBM(560 nm,630 nm,660 nm,and 730 nm inhibited diabetic U937 cells to secrete IL-6(all P<0.05).PBM(405 nm and 455nm)promoted the secretion of ICAM-1 by diabetic U937 cells(P<0.05).PBM promoted the secretion of IL-1beta(495nm and 730nm,both P<0.05)by diabetic HUVEC cells,but ICAM-1(all P>0.05).PBM promoted the migration of diabetic WS1 cells(405nm and 530nm,both P<0.05)and diabetic HACAT cells(405nm,510nm,530nm and 560nm,all P<0.05),but diabetic HUVEC cells.(?).Compared to CDM group,PBM(425nm,630nm,730nm,850nm and PBMcom)directly promote the wound closure of T2DM rats and shorten the healing time.Laser scattering showed PBM at 630nm,730nm and 850nm increased the wound blood flow in T2DM rats.HE staining showed PBM at 425 nm,510 nm,630 nm and 730 nm promoted the re-epithelialization of wound in T2DM rats.MASSON staining showed the collagen volume fraction(CVF)of wound skin were increased in all PBM groups(all P<0.05).Immunohistochemical staining showed increased proliferating cells in PBM425,PBM510,PBM730,PBM850 and PBMcom groups,increased wound neovascularization in PBM730 and PBMcom groups,increased early wound inflammatory cell aggregation in PBM425,PBM630,PBM730 and PBMcom groups,improved late inflammation persistence,decreased MMP-9 expression in all PBM treatment groups.Conslusions:The biological response of different cells involved in the healing of DFU to PBM is complex,and the optimal parameters of PBM varies from cell to cell.It makes more sense to treat with multiple wavelength combinations.PBM promote wound healing in T2DM rats by promoting re-epithelialization,promoting wound cell proliferation,correcting angiogenesis delay,improving wound blood circulation,promoting inflammatory cell aggregation,improving inflammation persistence,promoting collagen deposition,and reducing matrix metalloproteinase activity.