| BackgroundCutibacterium acnes(C.acnes)infection is one of the important factors that promote the development and progress of acne.To date,antibiotics and retinoic acid have been used as a fisrt-line treatment for severe acne.However,with the increase of bacterial resistance and tretinoid-related adverse reactions,new and safer therapeutic strategy is needed in clinical practice.5-aminolevulinic acid photodynamic therapy(ALA-PDT)is an emerging tool combing the drug and devices that has been widely used in many medical disciplines,especially for the treatment of moderate to severe acne.However,the mechanism of its therapeutic effect on acne has not been very clarified.The aim of this study was to determine the inhibitory effect of ALA-PDT on C.acnes in vitro and explore the regulatory effect on the expression of IL-17 in the lesions of rabbit ear acne modelObjectives1.To determine the optimal photosensitizer concentration and incubation time of ALA with C.acnes,and the antibacterial effect of different ALA-PDT illunination doses on C.acnes,establishing the best parameters for ALA-PDT treatment of bacterial infection;2.To investigate the therapeutic effect of ALA-PDT on the acne and the expression change of IL-17 in the lesions before and after ALA-PDT treatment by developing a rabbit ear acne model Methods1.Determine the relationship between PpIX fluorescence intensity and photosensitizer concentration and incubation durationThe experiment was divided into control group and experimental group.The control group was C.acnes suspension.In the experimental group,C.acnes suspension was incubated with ALA at different concentrations(0.0625 mg/mL,0.125 mg/mL,0.25 mg/mL,0.5 mg/mL,1 mg/mL,2 mg/mL,4 mg/mL)and each group was incubatedat 37? for 0h,1h,2h,4h,8h,12h,24h,48h,72h,96h respectively.After incubation,the Multi-function microplate reader was used to detect the fluorescence intensity of PpIX at each time point.The samples after treatment in the experimental group were cultured on the BHI solid medium.The optimal experimental therapeutic parameters(the photosensitizer ALA concentration and duration of application)of ALA-PDT on C.acnes were determined by measuring the fluorescence intensity of PpIX.2.Determine the antibacterial effect on C.acnes of different illumination doses of ALA-PDT Based on the results from the first step,the optimal ALA concentration and incubation time were determined.The experiment in this step was divided into four groups:blank control,single drug group,single light group,and ALA-PDT group.The third fourth group were irradiated with red light(632±7 nm)at different doses(0,20,40,80,100 J/cm2)5 and the first second group were absence of illumination.The inhibitory effects in vitro on C.acnes of ALA-PDT with different illumination doses were evaluated by colony-forming units(CFU),live death staining and transmission electron microscopy.3.Establish a rabbit ear acne modelNew Zealand rabbits were selected in this step.Freshly prepared C.acnes bacteria solution(3×108 cells/mL)was intradermal injected into the inner side of rabbit ears every other day for 21 days to induce acne-like lesions and ultimately establishing a rabbit ear acne model to facilitate.4.To detect the changes of skin lesions and the expression of IL-17 in rabbit ear acne model before and after ALA-PDT treatment,and to explore the mechanism of ALA-PDT influencing the IL-17 expression in acne lesionsVisual observation and histopathological evaluation were used to evaluate the effect of model construction,and the developed rabbit ear acne model were randomly divided into two group of ALA-PDT treatment group and non-intervention group.The inflammation in acne lesions was evaluated by observing the general performance and histopathological changes before and after treatment at 4h,24h,4d,and 7d.At the same time,Real-time PCR was used to detect the expression of IL-17 and the two upstream key transcription factors RORa and ROR?t before and after treatment with ALA-PDT at 4h,24h.4d and 7d.Results1.Relationship between PpIX fluorescence intensity and drug concentration and incubation durationThe fluorescence intensity in the experimental group showed no significant change from 0h,1 h,2h,4h,8h,to 12h,but increased slightly at 12h and 24h,and the fluorescence intensity increased significantly after 48h.Therefore,48h was identified as the optimal incubation time.In the control group,the fluorescence intensity in the C.acnes suspension group increased with the incubation time but with no significance.At the same time,C.acnes was incubated for 48 h at different concentrations of ALA.The fluorescence intensity was higher than that of other concentrations(p<0.05)when the ALA concentration was 0.5 mg/mL.2.Antibacterial effect of different red light doses of ALA-PDT on C.acnesThe results of CFU showed that the single-light with doses of 20 J/cm2,40 J/cm2,and 60 J/cm2 had no significant inhibitory effect on C.acnes.However when the irradiation dose reached 80 J/cm2,the number of survived C.acnes survival reduced significantly(p<0.05).In the ALA-PDT group,the C.acnes was significantly inhibited at a doses from 20-100 J/cm2 the bactericidal effect was significantly correalated with the increase of illumination dose(p<0.01).Live death staining showed that the survival rate of C.acnes gradually decreased with the increase of the dose of ALA-PDT.Transmission electron microscopy showed that the cell wall,cell membrane and cytoplasm of the C.acnes were damaged after ALA-PDT treatment.3.Establishment of rabbit ear acne modelIntradermal injection of C.acnes in rabbit ears can induce acne-like lesions.There are papules,enlarged hair follicles with lipid plugs and partial abscesses in the injection area.The rabbit ear acne model developed in this study simulates the acne-like skin lesions and based on this good model,further study of ALA-PDT treatment on acne and tissue inflammatory factors expression can be performed successfully.4.The effect of ALA-PDT on the lesions of rabbit ear acne model and the tissues expression change of IL-17 before and after treatment and its possible regulatory mechanismIn general,the inflammation in the lesion area aggravated gradually from 4h to 24h after treatment compared with that before treatment in the ALA-PDT group.The lymphocytic infiltration was relieved at after 4d of treatment.After 7d of treatment the lesions improved obviously and were better than that of pre-treatment and non-intervention groups.The results of Real-time PCR showed that the expression levels of IL-17,RORa and ROR?t in ALA-PDT group increased after 4h of treatment(p=0.0819;p=0.0296;p=0.0426)and a further increase was observed at 24h(p=0.0018;p=0.0006;p=0.0027).After 4d of treatment,their expression started to decrease(p=0.2066;p=0.0286;p=0.1945)and reduced significantly on the 7th day after treatment(p=0.0224;p=0.0027;p=0.0233).Conclusions1.C.acnes can absorb ALA and transform it as PpIX.ALA-PDT has a significant inhibitory effect on C.acnes in vitro.When incubating with ALA(concentration:0.5mg/mL)for 48h,the antibacterial effect of ALA-PDT will enhance with the increase of irradiation dose.ALA-PDT may play a role in disrupting the structure of the bacterial.2.The in vitro antibacterial parameters of ALA-PDT were significantly different from the parameters used in clinical practise(5%ALA concentration and 3 h medication),suggesting that the curative effect of ALA-PDT on acne may not be dependent on direct eradication of C.acnes.3.Intradermal injection of C.acnes suspension can develop a rabbit ear acne model which can simulate well the clinical acne lesions and can be used as a research model for treatment effect of ALA-PDT treatment on acne.4.ALA-PDT has a significant improvement effect on acne lesions.It can up-regulate IL-17 to amplify the inflammatory response,then down-regulate IL-17,and finally effectively cure acne.This regulation effect may be achieved by two key transcription factors ROR? and ROR?t. |
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