| Objective:1.To observe the ratio of Th17/Treg cells and the expression of IL-17,IL-23,IL-6,and TGF-?1 in peripheral blood of patients with acne vulgaris.2.Establishing the rabbit ear acne model by vitro experiment to observe the expression of IL-17,IL-6,TGF-?1 in the skin lesions of acne.And preliminarily explore the effect mechanism of super molecular salicylic acid at different concentrations in the treatment of rabbit ear acne model,to provide a new target and theoretical basis for the non-antibiotic treatment of acne vulgaris.Methods:1.A total of 125 subjects were studied,including 30 healthy controls and 95 patients with acne vulgaris?30 mild,32 moderate and 33severe?.The ratio of Th17/Treg cells in peripheral blood was determined by FCM,and the concentration of IL-17,IL-23,IL-6,TGF-?1 in peripheral blood serum was determined by ELISA.2.Thirty 12-week-old healthy and clean male New Zealand white rabbits with large ears were selected as the experimental objects.24 rabbits?48 ears?were randomly selected as the model group to be constructed the rabbit ear acne model by the Kligman method.The other 6rabbits?12 ears?were used as the normal control group without any treatment.The crude coal tar oil was applied to the opening of the ear canal on both sides of the model rabbit ears,with an area of 2cm×2cm,once a day,about 0.5ml/ear each time for 14 days.P.acnes bacterial suspension was injected subcutaneously at multiple points on the 7th day of the model,at a concentration of 1×108CFU/ml,about 50ul/ear.Twenty-four rabbits were randomly divided into the model control group,positive control group,2%super molecular salicylic acid group and 30%super molecular salicylic acid group.Each group had 6 rabbits?12 ears?.The model control group was smeared with normal saline,once a day for 28 consecutive days.The positive control group was smeared with Fusidic Acid Cream,once a day for 28 consecutive days.2%super molecular salicylic acid treatment group was smeared with 2%super molecular salicylic acid acne removing and conditioning gel,once a day for 28consecutive days.30%super molecular salicylic acid treatment group was smeared with 30%super molecular salicylic acid facial mask,once a week for 4weeks.Before treatment?immediately after modeling?,on the 7th,14th,21th and 28th day after treatment,the curative effect of each group was observed by naked eye and dermatoscope.Before the treatment?to verify whether the model is successful?,on the 14th and 28th day after the treatment,skin biopsy was performed on the ear lesions of the experimental groups.HE staining,light microscope was used to observe the histomorphological changes of the specimens.The thickness of epidermis and cuticle,the diameter of sebaceous gland,the area and keratosis degree of hair follicle,the degree of true dermatitis infiltration were measured and recorded by morphological image analysis system.At the same time,the expression of IL-17,IL-6 and TGF-?1 were detected by immunohistochemistry.Results:1The expression of Th17/Treg cells in peripheral blood:?1?The proportion of Th17 cells in patients with moderate and severe acne was positively correlated with the severity of acne,which was significantly higher than that in healthy people and mild acne patients?P<0.05?;the difference between patients with moderate and severe acne was statistically significant?P<0.05?;there was no statistical difference between patients with mild acne and healthy people?P>0.05?.?2?The proportion of Treg cells in patients with mild to moderate to severe acne was negatively correlated with the severity of acne,which was significantly lower than that in healthy people?P<0.05?;the differences among patients with mild,moderate and severe acne were statistically significant?P<0.05?.?3?There was a positive correlation between Th17/Treg ratio and the severity of acne,which was significantly higher than that in healthy people?P<0.05?;the differences among patients with mild,moderate and severe acne were statistically significant?P<0.05?.2.The expression levels of IL-17,IL-23,IL-6 and TGF-?1 in the peripheral blood serum:The expression levels of IL-17,IL-23,IL-6,TGF-?1 in patients with moderate and severe acne were positively correlated with the severity of acne,and significantly higher than that in healthy and mild acne patients,with statistical significance?P<0.05?;the difference between patients with moderate and severe acne was statistically significant?P<0.05?;there was no statistical difference between patients with mild acne and healthy people?P>0.05?.3.The results of acne rabbit ear modeling:?1?Macroscopic observation:the rabbit ears are thickened and hardened,the hair follicle mouths are further enlarged and locally uplifted,showing a red inflammatory papule of the size of rice grain to soybean.There are obvious yellow white or black hair follicle angle thrombus,acne,and even pustules,etc.?2?Dermoscopic observation:Under the red background,there are obviously thick pores with diffuse distribution,a large number of white and black acne like structures,scattered inflammatory papules with greasy scales,and pleomorphic vascular shadows between them.?3?Histological observation:the cells in each layer are arranged in disorder,the epidermis and the hair follicle epithelium are irregularly thickened,the keratosis of the cuticle is excessive,and some granular layer and spine layer are correspondingly thickened;the hair follicle mouth is severely expanded,and the keratosis is obvious,the hair follicle cavity and the opening are filled with red-stained and dense keratosis material,the funnel part is enlarged,in the shape of a kettle,and there is a huge horn plug;the diameter of the sebaceous gland of a single gland is increased,and the diameter of the gland is increased The area of hair follicle was enlarged,and some of the adjacent expanded hair follicles could be seen to fuse.The upper dermis capillaries were expanded,and inflammatory infiltration could be seen around the hair follicles and capillaries.?4?The normal control group and the model group were graded according to the histological criteria of experimental acne.The pathological changes?Z=-3.140,P=0.002?and pathological scores?Z=-3.140,P=0.002?were statistically significant.The pathological score of the model group was higher than that of the normal control group.?5?The measurement indexes of HE staining:a.the thickness of epidermis and cuticle,the sebaceous glands diameter and hair follicle area in the ear tissue of the model rabbits were significantly increased compared with the normal control group?t=-9.65,P<0.001;t=-7.324,P<0.001;t=-10.874,P<0.001;t=-6.494,P=0.001?.b.There were significant differences in the keratosis degree of hair follicle?Z=-3.146,P=0.002?and the score of keratosis?Z=-3.146,P=0.002?between the normal control group and the model group.The keratosis degree of hair follicle in the model group was higher than that in the normal control group.c.There were significant differences in the inflammatory infiltration degree of dermis?Z=-3.108,P=0.002?and the degree of infiltration?Z=-3.108,P=0.002?between the normal control group and the model group.The inflammatory infiltration degree of dermis of the model group was higher than that of the normal control group.?6?Immunohistochemical staining results:in the ear tissue of acne model rabbits,the mod values of IL-17,IL-6 and TGF-?1 were 0.23±0.01,0.23±0.02 and0.22±0.01 respectively;in the skin tissue of normal control group,the mod values of the three were 0.16±0.01,0.12±0.02 and 0.16±0.03 respectively.There was significant difference between the two groups?t=-10.305,P<0.001;t=-8.833,P<0.001;t=-4.615,P=0.005?.4.The therapeutic effects of four groups of acne rabbit ear modes:?1?Macroscopic and dermoscopic observation:on the7th,14th,21th and 28th day after treatment,the acnes,and inflammatory papules in each group gradually decreased,the degree of pore expansion and hair follicle angle thrombus decreased in different degrees,the greasy scales and skin roughness decreased in different degrees,The therapeutic effect of 2%supermolecular salicylic acid treatment group?once a day?is equivalent to that of 30%supermolecular salicylic acid treatment group?once a week?,which is better than that of the positive control group,followed by the model control group.?2?Histological observation:on the 14th and 28th day after treatment,the thickening of epidermis and hair follicle epithelium gradually decreased,the size of hair follicle decreased,the keratinization decreased,the size of sebaceous gland decreased and the lobulation decreased,the inflammatory cell infiltration decreased in different degrees.The curative effect of 2%supermolecular salicylic acid treatment group?once a day?and 30%supermolecular salicylic acid treatment group?once a week?was equal and better than that of the positive control group,followed by the model control group.?3?The histopathological changes of the four groups were classified according to the criteria of experimental acne histology:a.Comparison among groups:On the 14th day??2=2.738,P=0.434?and the 28th day??2=7.156,P=0.067?,there were no statistical differences in histopathological grading among the four groups b.Comparison within the group:The difference of histopathologic grades between the positive control group on the 14th and 28th day of treatment was statistically significant?Z=-2.000,P=0.046?.There were no statistical differences between the 14th and 28th day after treatment of the model control group,the 2%super molecular salicylic acid treatment group and the 30%super molecular salicylic acid treatment group?Z=-1.000,P=0.317;Z=-1.732,P=0.083;Z=-1.732,P=0.083?.?4?The measurement indexes of HE staining:?1?Thickness of epidermis:a.Comparison among groups:On the 14th day of treatment?F=3.983,P=0.022?,on the 28th day of treatment?F=24.429,P<0.001?,the differences in thickness of epidermis among the four groups were all statistically significant;b.Comparison within the group:The average thickness of epidermis in the positive control group,2%super molecular salicylic acid treatment group and 30%super molecular salicylic acid treatment group on the28th day was thinner than that on the 14th day,and the differences were all statistically significant?t=6.919,P=0.001;t=4.011,P=0.010;t=5.865,P=0.002?.?2?Thickness of cuticle:a.Comparison among groups:On the 14th day of treatment?F=10.618,P<0.001?,and on the 28th day of treatment?F=8.065,P=0.001?,the differences in thickness of cuticle among the four groups were all statistically significant;b.Comparison within groups:The average thickness of cuticle in the model control group,the 2%super molecular salicylic acid treatment group and the 30%super molecular salicylic acid treatment group on the 28th day was thinner than that of the 14th day?t=7.020,P=0.001;t=10.363,P<0.001;t=8.243,P<0.001?,and the differences were all statistically significant?P<0.05?.?3?Diameter of sebaceous gland:a.Comparison among groups:On the 14th day of treatment?F=25.562,P<0.001?,and on the 28th day of treatment?F=30.794,P<0.001?,the differences in diameter of sebaceous gland among the four groups were all statistically significant;b.Comparison within groups:the diameter of sebaceous gland in model control group,positive control group,2%super molecular salicylic acid treatment group and 30%super molecular salicylic acid treatment group on the 28th day of treatment was smaller than that on the 14th day of treatment,and the differences were all statistically significant?t=4.649,P=0.006;t=11.049,P<0.001;t=7.290,P=0.001;t=5.076,P=0.004?.?4?Hair follicle area:a.Comparison among groups:On the28th day of treatment,the differences hair follicle area among the four groups were all statistically significant?F=14.375,P<0.001?.b.Comparison within the group:the hair follicle area of the positive control group,2%super molecular salicylic acid treatment group and 30%super molecular salicylic acid treatment group on the 28th day was smaller than that on the 14th day,and the differences were all statistically significant?t=3.963,P=0.011;t=6.891,P=0.001;t=5.183,P=0.004?.?5?The degree of hair follicle keratosis:a.Comparison among groups:On the 28th day of treatment,the differences in the degree of hair follicle keratosis among the four groups were all statistically significant??2=14.750,P=0.002?;b.Comparison within groups:The degree of hair follicle keratosis in the 28th day of treatment in the positive control group was less than that on the14th day of treatment,and the differences were all statistically significant?Z=-2.236,P=0.025?.?6?Inflammatory infiltration degree of dermis:a.Comparis-on among groups:On the 28th day of treatment,the differences in inflammatory infiltration degree of dermis among the four groups were all statistically significant??2=13.860,P=0.003?;b.Comparison within the group:On the 28th day of treatment,the true dermatitis infiltration degree in the 30%super molecular salicylic acid treatment group was less than that on the 14th day of treatment,and the differences were all statistically significant?Z=-2.000,P=0.046?.?5?Immunohistochemical staining results:?1?The average expression level of IL-17 protein:a.Comparison among groups:There were no statistical differences in average expression level of IL-17 protein among the four groups on the 14th day of treatment?F=1.324,P=0.295?and 28th day of treatment?F=1.637,P=0.213?.b.Comparison within the group:the average expression level of IL-17 protein in the model control group and 2%super molecular salicylic acid treatment group on the 28th day was lower than that on the 14th day,and the differences were all statistically significant?t=2.730,P=0.04;t=4.727,P=0.005?.?2?The average expression level of IL-6 protein:a.Comparison among groups:The differences in average expression level of IL-6 protein among the four groups were all statistically significant on the 14th day?F=22.733,P<0.001?and 28th day?F=17.613,P<0.001?;b.Comparison within the group:the average expression level of IL-6 protein in the model control group,positive control group,2%super molecular salicylic acid treatment group and 30%super molecular salicylic acid treatment group on the 28th day was lower than that on the 14th day,and the differences were all statistically significant?t=4.223,P=0.008;t=7.442,P=0.001;t=3.866,P=0.012;t=6.744,P=0.001?.?3?The average expression level of TGF-?1 protein:a.Comparison among groups:The differenc-es in average expression level of TGF-?1 protein among the four groups were all statistically significant on the 28th day?F=4.776,P=0.011?.b.Comparison within the group:the average expression level of TGF-?1 protein in 28th day of treatment in positive control group was lower than that in 14th day of treatment,and the differences were all statistically significant?t=3.392,P=0.019?.Conclusion:1.The expression levels of Th17 cells,IL-17,IL-23,IL-6,TGF-?1 in peripheral blood of patients with moderate and severe acne were abnormally increased,while the proliferation of Treg cells was insufficient,and the proportion of Th17/Treg was unbalanced,which suggested that Th17/Treg cell axis and related inflammatory factors might play a corresponding role in the development of acne,and it might be a new target for acne treatment.2.The models of acne rabbit ears were successfully constructed by Kligman method,and the expression of IL-17,IL-6,TGF-?1 protein in the skin lesions of the models was increased in varying degrees.3.2%super molecular salicylic acid treatment group?once a day?and 30%super molecular salicylic acid treatment group?once a week?can reduce the thickness of epidermis,cuticle,sebaceous gland,hair follicle and inflammatory infiltration degree of dermis of the rabbit ear acne model in varying degrees,but the degree of hair follicle keratosis is not significantly reduced.4.The expression level of IL-6 and TGF-?1 in the 2%and30%supermolecular salicylic acid treatment groups could be reduced in different degrees,but the expression level of IL-17 was not significantly reduced.5.2%supermolecular salicylic acid treatment group?once a day?and30%supermolecular salicylic acid treatment group?once a week?have therapeutic effect on experimental rabbit ear acne,and the therapeutic effect is better on the 28th day than on the 14th day;in terms of anti-keratinization and anti-inflammatory ability,2%supermolecular salicylic acid treatment group?once a day?has certain tendentious advantage. |
PDF Full Text Request