| Background: Acne is a chronic inflammatory skin disease and most prevalent in adolescents.The incidence rate of acne in soldiers of China's South-East coastal areas is as high as 70%-87%,which will induce different degrees of psychological stress and affect the training of armies.Therefore,effective treatments must be taken to deal with it.Photodynamic therapy(PDT)with topical aminolevulinic acid(ALA)is effective for the treatment of acne vulgaris.However,as to patients with Fitzpatrick skin type III-IV,little is known about the optimum concentration of photosensitizer,incubation time and dosage of infrared radiation.Besides,the molecular mechanism(s)by which 5-ALA-PDT regulates the immune response need to be futher investigated.Objective:1.To investigate the efficiency and safety of 5-ALA-PDT in moderate to severe acne.2.To explore the effect of 5-ALA-PDT on the expression of Toll-like receptors,the related signaling pathway molecules and and cytokines in RAW264.7 cell line.3.To study the influence of 5-ALA-PDT on the Toll-like receptor-mediated immune response in acne model in SD rats.Methods:1.Fifty patients with moderate to severe acne were enrolled and randomly divided into two groups: the treatment group,treated with 5% 5-ALA thermosensitive gel,avoid light for 1.5 h,followed by 20 min of red light exposure once a week for 3 weeks.The control group,treated with no 5% 5-ALA thermosensitive gel,avoid light for 1.5 h,followed by 20 min of red light exposure once a week for 3 weeks.The clinical efficacies of the treatments were compared at 2 weeks,4 weeks and 6 weeks.Adverse reactions were recorded during the course of the treatment.2.Murine macrophage RAW264.7 cells were induced by Propionibacterium acnes inactivated fluid to establish an inflammatory cell model.The RAW264.7 cells were treated with different concentrations of 5-ALA combined with infrared radiation with a light dose of 16 J/cm2.The capability of macrophage phagocytosis was detected by using neutral red method.Concentration of IL-6 and TNF-? in the supernatant were measured by ELISA assay.Expression of TLR2 and MyD88 as well as phosphorylation of p38,JNK,ERK and I?B? was detected by Western Blotting in the murine macrophage cell line RAW264.7.Fluorescence intensity of cellular reactive oxygen species(ROS)was detected with fluorescence microscopy,the level of intercellular reactive oxygen species(ROS)was detected by dihydrochloride acetyl acid dichloride(DCFH-DA)3.Acne inflammation model of rat ear induced by Propionibacterium acnes were established and then treated with 5-ALA-PDT.Section was made for H-E staining.Immunohistochemical staining was used to observe the expression of CD68,CD163,TLR2,and MyD88.4.Statistical analysis was performed using SPSS 18.0 software.Variables such as age and disease duration were presented as ?±s in the clinical trial.For comparison between treatment and control groups,the independent samples t-test was used to determine differences.The numbers of rash were expressed as median(interquartile).Mann-Whitney U test was used to compare the numbers of rash between the control and treatment groups,and comparision of the effective rate between the two groups was performed by chi-squared.The quantitative data are shown as ?±s in cell experiments.Multiple comparisons among the groups were analyzed by one-way analysis of variance(ANOVA).If variables were consistent with homogeneity of variance,LSD test was used to compare the inter-group variables,and Tambane T2 test was used to evaluate the differences in presence of heterogeneity of variance.The qualitative data of animal experiments were ordinal variables which were analyzed using a nonparametric analysis.Differences of P(27)0.05 were considered statistically significant.Results:1.Data were valid in 24 patients in the treatment group and 23 patients in the control group.The numbers of comedo,papule and pustule were decreased in the treatment group compared with control group,but there was no significance in terms of numbers of cyst and nodule.The effective rates were 50.0%,75.0%,83.3% and 26.1%,43.5%,56.5% at the 2,4 and 6 weeks after treatment respectively.P was 0.092,0.028 and 0.045 respectively.The effective rates of the treatment group were significantly higher than those of the control group at 4 and 6 weeks.2.(1)The capability of macrophage phagocytosis of the model group was higher compared to that of the blank group.With the increasing concentration of 5-ALA,the capability of macrophage phagocytosis decreased in a dose-dependent manner.(2)Compared with the blank group,the concentrations of TNF-? and IL-6 were increased in the model group.The concentration of TNF-?was decreased after 5-ALA-PDT,but was not in a dose-dependent manner.With the increasing concentration of 5-ALA,the concentration of IL-6 was decreased in a dose-dependent manner.(3)Compared with the blank group,the expression levels of TLR2,MyD88,p-p38,p-I?B?,p-JNK and p-ERK were significantly higher in the model group.With the increasing concentration of 5-ALA,the expression level of the above proteins decreased in a dose-dependent manner.(4)The result of fluorescence showed,cells in the 0.12mmol/L 5-ALA group had strong red fluorescence.(5)With the increasing concentration of 5-ALA,the level of ROS was increased in a dose-dependent manner.3.(1)Acne inflammation model of rat ear was established by inoculating Propionibacterium acnes to rat ears.Diffuse infiltrations of inflammatory cell were observed by HE staining.(2)Compared with the blank group,the expression of CD68,CD163,TLR2,MyD88 were decreased in rat ears inflammatory cells,but increased after stimulated with P.acnes and increased further after Pam3CSK4 adminstration.The expression level decreased and was lower than the model group after receiving 5-ALA-PDT.Conclusion:1.Three courses of 5% 5-ALA for 1h,followed by 20-min dose of infrared radiation once a week is effective in the treatment of moderate to severe facial acne vulgaris.Moreover,short therapy course is likely to improve patient compliance.2.Propionibacterium acnes inactivated fluid can enhance macrophage phagocytosis and the expression of TLR2,leading to increased secretion of cytokines.5-ALA-PDT suppresses the expression of TLR2 and decreases cytokine secretion via TLR2 signaling pathway.A propionibacterium acnes-induced inflammatory model in RAW 264.7 cells could be used to screen and evaluate the efficacy of acne treatment.3.TLR2 expression was low in rat ear pilosebaceous unit and its surrounding tissue and the expression increased significantly after inoculating Propionibacterium acnes,which induced macrophage infitration in pilosebaceous unit.5-ALA-PDT decreased the expression of TLR2 and alleviates body's immune response to bacteria,thus relieved inflammation... |
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