| Purpose: In this study,the rabbit ear acne model was used as the research object.By detecting the changes of surface molecular expression of Langerhans cells(LCs)in ear root lymph nodes of rabbit ear acne model during the migration from epidermis to drainage lymph nodes And the expression state of subgroup specific gene transcription factors before activation by using Acne mixture decocion to intervine.in order to explore the inhibitory effect of Acne Mixture on LCs antigen presentation function and the regulation mechanism of Th1/Th2 and Th17/Treg balance,so as to provide scientific experimental basis for the clinical application of Chinese medicine.Material and method:Forty-eight SPF level New Zealand male white rabbits were randomly divided into blank group,model group,Acne mixture low dose group,Acne mixture medium dose group,Acne mixture high dose group and positive drug group.Except for the blank group,the other 5 groups were to make the rabbit ear acne model.Kligman method was used to prepare the rabbit ear acne model.The inner ear of the rabbit was located at the proximal end of the ear canal about 2.0cm x 2.0cm,and apply about 0.5ml coal tar with a medical cotton swab once a day for 2 weeks.After successful modeling,the low,medium and high dose groups of Chinese medicine were gavaged with 3ml of Acne mixture concentrated in different proportions,the positive drug group was gavaged with 3ml of diluted Qingre Anchuang capsule,and the blank group was gavaged with the same dose of normal saline,twice a day.On the 30 th day of the experiment,the blood of the abdominal aorta was collected after abdominal anesthesia,and then the air was injected into the ear vein for embolization.A 1cm perforator was used to collect skin tissue samples and 4-6 lymph nodes from the ear root from the opening near the ear canal of the right ear of big-ear rabbits and keep in the freezer of-70℃ to be measured.1.He staining was used to evaluate the success of the ear modeling of the rabbit acne model,andthe therapeutic effect of acne mixture on the ear of rabbit acne model from the perspective of histomathology.2.Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression levels of cytokines GM-CSF,TNF-α,IFN-γ,IL-12,IL-17,IL-22,IL-10,IL-3,IL-5,and TGF-β in the peripheral blood of rabbit acne models.3.Flow Cyto Metry(FCM)was used to detect the expression of CD207,MHC-II,CD86,OX40 L on the surface of LCS and OX40 of CD4~+T cells in the ear lymph nodes of rabbit acne model.4.Western-blot method was used to detect the protein expression levels of T-bet,Gata3,RORγ t,and Foxp3 in rabbit ear lymph nodes.5.Immunohistochemistry method was used to detect the protein expression levels of T-bet,Gata3,RORγ t,and Foxp3 in rabbit ear lymph nodes.Results: 1.Observation of hair follicle sebaceous glands under the pathological microstructure of rabbit ear tissue: Compared with model group,low,medium and high dose group,positive drug group ear tissue thickness of epidermis layer,pustules,nodules,cysts,and hair follicle expansion were significantly decreased.Keratin in a hair follicle unit is expelled from the skin surface through the opening of the hair follicle.The degree of keratinization of the cuticle and inner cuticle became thinner and softer,indicating that terminal differentiation and desquamation returned to normal.The thickened granular layer and spinous cell layer were significantly thinned,and the inflammatory infiltration in the superficial dermis was reduced.Compared with the positive drug group,there was no significant difference in the pathological improvement of ear tissue in the low,medium and high dose groups(P>0.05).2.ELISA method test result:(1)IL-3 level: Compared with blank group,IL-3 level in other groups was significantly increased(P < 0.01).Compared with model group,the level of IL-3 in intervention groups was significantly decreased(P < 0.01).There was no significant difference in the level of IL-3 between Acne mixture high-dose groups,medium-dose groups and positive groups(P > 0.05).(2)TNF-α level: Compared with blank group,TNF-α level in other groups was significantly increased(P < 0.01).Compared with model groups,the level of TNF-α in intervention groups was significantly decreased(P < 0.01).There was no significant difference in TNF-α levels between Acne mixture high-dose groups,Acne mixture medium-dose groups and positive groups(P > 0.05).(3)GM-CSF level: Compared with blank group,GM-CSF level in other groups was significantly increased(P < 0.01).Compared with model group,the level of GM-CSF in intervention groups was significantly decreased(P < 0.01).There was no significant difference in GM-CSF levels between Acne mixture high-dose,medium-dose groups and positive groups(P > 0.05).(4)IL-5 level: Compared with blank group,IL-5 level in other groups was decreased,the difference was statistically significant(P < 0.01).Compared with model group,the level of IL-5 in intervention groups was increased,the difference was statistically significant(P < 0.05).Compared with the Acne mixture high-dose group of,the level of IL-5 in the Acne mixture medium-and low-dose groups and the positive group decreased,the difference was statistically significant(P < 0.01).There was no significant difference in the level of IL-5 between Acne mixture medium-dose,low-dose groups and positive groups(P > 0.05).(5)IL-17 level: Compared with blank group,IL-7 level in other groups was increased,the difference was statistically significant(P < 0.01).Compared with the model group,the level of IL-7 in the intervention groups was decreased,the difference was statistically significant(P < 0.01).Compared with the Acne mixture high-dose group,the IL-7 level in the Acne mixture low-dose group and the positive group was increased,and the difference was statistically significant(P < 0.01).There was no significant difference in the level of IL-7 between Acne mixture high-dose,medium-dose groups and positive groups(P > 0.05).(6)IL-22 level: Compared with blank group,IL-22 level in other groups was increased,the difference was statistically significant(P < 0.01).Compared with the model group,the level of IL-22 in the intervention groups was decreased,the difference was statistically significant(P < 0.01).Compared with the Acne mixture high-dose group,the IL-22 level in the Acne mixture low-dose group and the positive group was increased,the difference was statistically significant(P < 0.01).There was no significant difference in IL-22 level between Acne mixture high-dose,medium-dose groups and positive groups(P > 0.05).(7)IL-12 level: Compared with blank group,IL-12 level in other groups was increased,the difference was statistically significant(P < 0.01).Compared with the model group,the level of IL-12 in the intervention groups was decreased,the difference was statistically significant(P < 0.01).Compared with Acne mixture high-dose group,the IL-12 level in Acne mixture low-dose group and positive group was increased,the difference was statistically significant(P < 0.05).There was no significant difference in the level of IL-12 between Acne mixture low-dose group and positive group(P > 0.05).(8)IFN-γ level: Compared with blank group,IFN-γ level in other groups was increased,the difference was statistically significant(P < 0.01).Compared with model group,IFN-γ levels in intervention groups were decreased,the difference was statistically significant(P < 0.01).Compared with Acne mixture high-dose group,the levels of IFN-γ in Acne mixture medium-dose,low-dose and positive groups were increased,and the difference was statistically significant(P < 0.05).Compared with the Acne mixture medium-dose group,IFN-γ level in the Acne mixture low-dose group and the positive group was increased,and the difference was statistically significant(P < 0.01).There was no significant difference in IFN-γ levels between Acne mixture low-dose group and positive group(P > 0.05).(9)TGF-β level: Compared with blank group,TGF-β level in other groups was increased,the difference was statistically significant(P < 0.01).Compared with model group,the level of TGF-β in intervention groups was increased,and the difference was statistically significant(P < 0.05).Compared with the Acne mixture high-dose group,the TGF-β level in the Acne mixture medium-dose and low-dose groups was decreased,and the difference was statistically significant(P < 0.01).Compared with the Acne mixture medium-dose group,TGF-β level in the Acne mixture low-dose group and the positive group was increased,and the difference was statistically significant(P < 0.01).There was no significant difference in TGF-β level between Acne mixture high-dose group and positive group(P > 0.05).(10)IL-10 level: Compared with blank group,IL-10 level in other groups decreased,the difference was statistically significant(P < 0.01);Compared with model group,the level of IL-10 in intervention groups was increased,with statistical significance(P < 0.01).Compared with the Acne mixture high-dose group,the IL-10 level in the Acne mixture medium-dose,low-dose groups and the positive group decreased,the difference was statistically significant(P < 0.01).Compared with Acne mixture medium-dose group,the level of IL-10 in positive group was increased,the difference was statistically significant(P < 0.01).Compared with Acne mixture low-dose group,the level of IL-10 in positive group was increased,the difference was statistically significant(P < 0.01).2.Flow cytometry results:(1)CD207 protein level: Compared with blank group,the ratio of CD207 positive cells in other groups was higher(P < 0.01).Compared with model group,the ratio of CD207 positive cells in intervention groups decreased(P < 0.01).Compared with the high dose group,CD207 positive cells in Acne mixture medium-dose group,Acne mixture low-dose group and positive group were significantly increased(P < 0.01).Compared with the Acne mixture medium-dose group,the ratio of CD207 positive cells in the Acne mixture low-dose group and the positive group was increased(P < 0.05).Compared with Acne mixture low-dose group,the ratio of CD207 positive cells in Acne mixture positive group was decreased(P < 0.05).(2)MHC-II protein level: Compared with blank group,the ratio of MHC-II positive cells in other groups was higher(P < 0.01);Compared with model group,the ratio of MHC-II positive cells in intervention groups decreased(P < 0.01);Compared with the Acne mixture high-dose group,the MHC-II positive cells ratio in the Acne mixture medium-dose,Acne mixture low-dose and positive groups of acne mixture was significantly increased(P < 0.01);Compared with Acne mixture medium-dose group,MHC-II positive cell ratio of Acne mixture low-dose group and positive group was increased(P < 0.05).Compared with Acne mixture low-dose group,MHC-II positive cell ratio of positive group was decreased(P < 0.05).(3)CD86 protein level: Compared with blank group,the ratio of CD86-positive cells in other groups was higher(P < 0.01);Compared with model group,the ratio of CD86 positive cells in intervention groups decreased(P < 0.01).Compared with the Acne mixture high-dose group,CD86 positive cells in the Acne mixture medium-dose group,Acne mixture low-dose group and the positive group were significantly increased(P < 0.01).Compared with the Acne mixture medium dose group,the ratio of CD86 positive cells in the Acne mixture low dose and positive group was increased(P < 0.05).Compared with Acne mixture low-dose group,the ratio of CD86 positive cells in positive group decreased(P < 0.05).(4)OX40 protein level: Compared with blank group,the ratio of OX40 positive cells in other groups was significantly higher(P < 0.01).Compared with model group,the proportion of OX40 positive cells in intervention groups was significantly decreased(P < 0.01).Compared with the Acne mixture high-dose group,the proportion of OX40 positive cells in Acne mixture medium-dose,Acne mixture low-dose and positive group was significantly increased(P < 0.01).Compared with the Acne mixture medium-dose group,the proportion of OX40 positive cells in the Acne mixture low-dose group and the positive group was significantly increased(P < 0.05).Compared with Acne mixture low-dose group,the proportion of OX40 positive cells in positive group was significantly increased(P < 0.05).(5)OX40L protein level: Compared with blank group,the ratio of OX40 L positive cells in other groups was significantly higher(P < 0.01).Compared with model group,the proportion of OX40 L positive cells in intervention groups was significantly decreased(P < 0.01).Compared with the Acne mixture high-dose group,the proportion of OX40 L positive cells in Acne mixture medium-dose,Acne mixture low-dose and positive group was significantly increased(P < 0.01).Compared with Acne mixture medium-dose group,the proportion of OX40 L positive cells in Acne mixture low dose-group and positive group was significantly increased(P < 0.05).Compared with acne mixture low-dose group,the proportion of OX40 L positive cells in positive group was significantly increased(P < 0.05).4.Western blot test results:(1)T-bet protein level: Compared with blank group,the relative expression of T-bet in model group was up-regulated(P < 0.01).Compared with the model group,the relative expression of T-bet in the intervention group was down-regulated(P < 0.05).There was no significant difference in the relative expression of T-bet in the Acne mixture low-,medium-,high-dose groups and the positive groups(P >0.05).(2)Gata3 protein level: Compared with blank group,the relative expression of Gata3 in model group was down-regulated(P < 0.01).Compared with the model group,the relative expression of Gata3 in the intervention group was up-regulated(P < 0.01).Compared with the Acne mixture high-dose group,the relative expression of Gata3 protein in the Acne mixture low-dose group was down-regulated(P < 0.01).Compared with the Acne mixture medium-dose group,the relative expression of Gata3 in the Acne mixture low-dose group was down-regulated(P < 0.01).There was no significant difference in the relative expression of Gata3 between the Acne mixture high-dose,Acne mixture medium-dose groups and the positive groups(P > 0.05).(3)RORγt protein level: Compared with blank group,the relative expression of RORγt in model group was up-regulated(P < 0.01).Compared with model group,the expression of RORγt in Acne mixture high-dose,Acne mixture low-dose groups and positive groups was down-regulated(P < 0.05).Compared with the Acne mixture high-dose group,the relative expression of RORγt in Acne mixture medium-dose,Acne mixture low-dose groups and positive groups was down-regulated(P < 0.05).There was no significant difference in the relative expression of RORγt in the Acne mixture medium-dose,Acne mixture low-dose and positive groups(P > 0.05).(4)Foxp3 protein level: Compared with blank group,the relative of Foxp3 in model group was down-regulated(P < 0.01).Compared with the model group,Foxp3 relative expression in the intervention group was up-regulated(P < 0.01).Compared with the Acne mixture high-dose group,the relative expression of Foxp3 in the Acne mixture low-dose group was decreased(P < 0.05).Compared with the Acne mixture medium-dose group,the relative expression of Foxp3 in the Acne mixture low-dose group and the positive group was down-regulated(P < 0.05).There was no significant difference in the relative expression of Foxp3 between the Acne mixture low-dose group and the positive group(P > 0.05).5.Immunohistochemical test results:(1)T-bet protein level: Compared with blank group,the average optical density(IOD)of T-bet in model group was significantly increased(P < 0.01).Compared with the model group,the IOD of T-bet was significantly reduced in the intervention group(P < 0.05).Compared with the Acne mixture high-dose group,the IOD of T-bet in Acne mixture medium-dose,Acne mixture low-dose groups and positive groups were significantly increased(P < 0.05).There was no significant difference in the IOD of T-bet in Acne mixture medium-dose,Acne mixture low-dose groups and positive groups(P>0.05).(2)Gata3 protein level: Compared with blank group,the IOD of Gata3 in model group was significantly reduced(P < 0.05).Compared with the model group,the IOD of Gata3 in the intervention group was significantly increased(P < 0.01).Compared with the Acne mixture high-dose group,the IOD of Gata3 in Acne mixture medium-and low-dose groups was significantly increased(P < 0.05).There was no significant difference in the IOD of Gata3 between the Acne mixture high-dose and the positive group(P > 0.05).(3)RORγt protein level: Compared with blank group,the IOD of RORγt in model group was significantly increased(P < 0.01).Compared with the model group,the IOD of RORγt was significantly reduced in the intervention group(P < 0.05).Compared with the Acne mixture high-dose group,the IOD of RORγt in Acne mixture low-dose group were significantly increased(P < 0.05).There was no significant difference in the IOD of RORγt in the Acne mixture high-dose,Acne mixture medium-dose group and the positive groups(P > 0.05).(4)Foxp3 protein level: Compared with blank group,the IOD of Foxp3 in model group was significantly reduced(P < 0.01).Compared with the model group,the IOD of Foxp3 in the intervention group was significantly increased(P < 0.05).Compared with the Acne mixture high-dose group,the IOD of Foxp3 protein in the Acne mixture low-dose group and the positive group was significantly increased(P < 0.01).There was no significant difference in the IOD of Foxp3 in the Acne mixture high-and medium-dose groups(P > 0.05).Conclusion: 1.Acne mixture can reduce the thickness of the epidermal layer of rabbit ears,pustules,nodules,cysts and hair follicle opening dilation.The degree of keratinization of the epidermis and inner wall corneum became thin and soft,indicating that the terminal differentiation returned to normal.The results indicated that Acne mixture can inhibit the accumulation of keratin in hair follicle funnel,reduce the proliferation and infiltration of inflammatory cells in the superficial dermis,and improve the appearance of acne skin lesions.2.Phenotypes of acne-like lesions are associated with high expression of inflammatory factors.Acne mixture can down-regulate the expression levels of inflammatory mediators GM-CSF,IL-3 and TNF-α in serum of rabbits,and inhibit the secretion of pro-inflammatory factors,so as to realize the anti-inflammatory effect on the rabbit ear acne model.3.Acnes mixture can down-regulate the expression of CD207,MHC-II and CD86 on the surface of LCs cells and the expression of OX40 on CD4~+T cells in lymph nodes.It can interferes with the migration and antigen presentation of LCs from the lesion to the drainage lymph nodes,suggesting that Acne Mixture can inhibit the immune function of LCs,and thus inhibit the inflammatory response of acne.4.Acne mixture can correct Th1/Th2 and Th17/Treg migration and regulate the differentiation of CD4~+T cell subsets.The mechanism of such behavior may be related to the decreased expression of Th1/Th17 specific cytokines IFN-γ,IL-12,IL-17 and IL-22,and the up-regulation of Th2/Treg specific cytokines IL-5,IL-10 and TGF-β.5.Acne mixture can improve the expression of acne inflammation by regulating the expression level of specific transcription factors of CD4~+T cell subsets,balancing the polarization shift between Th1/Th2 and Th17/Treg to achieve endogenous homeostasis.It was further proved that Acne Mixture can regulate the differentiation of T lymphocyte subsets. |
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