| Purpose:Experiment 1:To establish the acne model of rabbit ear,observe the regulation of acne mixture on Th17/Treg cell number/proportion,and explore the regulation mechanism of acne mixture in maintaining body homeostasis;Experiment 2:To observe the regulatory effect of acne mixture on Hippo signaling pathway,explore the possible pathogenesis of acne,and seek new targets for drug treatment.Materials and methods:1.Grouping:24 male large-eared white rabbits from Japan were randomly divided into a blank group,a model group,a positive drug group,and an acne combination group after 1 week of adaptive feeding.2.Modeling:Except for the blank group,the rabbit ear acne model was established according to the Kligman modeling method in the other intervention groups once a day for 14 days,and then a biopsy was taken from the rabbit ear to determine whether the modeling was successful.3.Gavage:The acne combination group was given 20 ml of concentrated drug solution by gavage;the positive drug group was given20 ml of diluted clearing and darkening capsules by gavage;the blank group and the model group were given equal doses of saline once a day for 30d.4.Material was taken within 24 h after the last administration:blood was taken quickly from the abdominal aorta after intraperitoneal injection anesthesia,followed by static overdose anesthesia for execution,and the medial side of each rabbit ear was cut at 1x1 cm size tissue was cut near the ear canal and fixed at room temperature.The ear root lymph nodes were extracted,3～4/each for flow-through,and the rest were placed in-80℃refrigerator for freezing and storage.5.Detection:The changes of ear lesions and pathological changes under biopsy were observed by naked eye in each group,and the results were graded according to the grading criteria to evaluate the efficacy of acnes mixture;ELISA was used to detect the content of IL-10,IL-17,IFN-Y,TGF-β,IL-6 and TNF-αin serum;flow cytometry was used to detect the content and proportion of Th17 and Treg cells in CD4~+T cells;Western-blot was used to detect the effect of acne combination on The expression levels of MST1,YAP and TAZ were detected by Western-blot;the expression of STAT3m RNA,FOXP3m RNA,MST1m RNA,YAPm RNA and TAZm RNA were detected by QPCR.Results:1.General status observation:Compared with blank group,red papules,acne and nodules were observed in model group,accompanied by a small amount of bleeding and enlarged follicular orifice.Compared with the model group,the number of papules and the overflow of horn plugs were reduced in the drug intervention group.The morphology of the positive drug group was similar to that of the acne mixture group,but the phenomenon of follicular enlargement and peeling was still obvious.2.HE staining:the degree of keratinized follicle opening was significantly increased in the model group compared with the blank group;the area of inflammatory cell infiltration,the number of nodules,and the thickness of the ear epidermis were significantly reduced in the drug intervention group compared with the model group;combination group compared with the positive group,the acne combination group make the thickened granular layer and spinous layer become thinner,the inflammatory substance infiltration was improved.3.Effect of acne combination on the number/ratio of T-lymphocytes and their differentiated cytokines in acne affected bodies(1)ELISA results:compared with the blank group,the content of IL-17、IL-6、TNF-α、IFN-γwas increased(P<0.01 or P<0.05),while the content of IL-10 and TGF-βwas significantly decreased(P<0.01)in the remaining groups;compared with the model group,the content of IL-17、IL-6、TNF-α、IFN-γin the drug intervention group were reduced(P<0.01)and IL-10and TGF-βcontents were increased(P<0.01);compared with the positive drug group,the expression of IL-17、IL-6、TNF-α、IFN-γin the acne combination group were reduced(P<0.01),and IL-10 contents were increased(P<0.01),but TGF-βcontents of both of two groups were not statistically significant(P=0.084>0.05).(2)Flow cytometry results:the positive rate of Th17 cells to CD4~+T cells was increased(P<0.01)and Treg to CD4~+T cells was decreased(P<0.01)in the other groups compared to the blank group;the positive rate of Th17 cells to CD4~+T cells was decreased(P<0.01)and Treg to CD4~+T cells was increased(P<0.01)in the drug intervention group compared to the model group;compared with the positive drug group,the positive rate of Treg to CD4~+T cells was increased(P<0.01),but the positive of Th17 cells to CD4~+T cells of both of two groups were not statistically significant(P=0.076>0.05);compared with the blank group,the ratio of Th17/Treg in the other groups were increased(P<0.01 or P<0.05);compared with the blank group,the ratio of Th17/Treg in the drug intervention group were decreased(P<0.01);the ratio of Th17/Treg between the acne combination group and the positive drug group was not statistically significant(P=0.491>0.05).(3)QPCR results:compared with the blank group,there was a significant increase in the expression level of STAT3m RNA(P<0.01)and a significant decrease in the expression level of FOXP3m RNA(P<0.01)in the model group;after the intervention of acne combination as well as clearing and darkening capsules,compared with the model group,the expression level of STAT3m RNA decreased(P<0.01)and the expression level of FOXP3m RNA increased(P<0.01);compared with the positive drug group,the expression level of STAT3m RNA was decreased(P<0.01)and the expression level of FOXP3m RNA increased(P<0.01).4.Effect of acne combination on the main effectors of Hippo pathway in acne receptors(1)Western-blot assay results:compared with the blank group,the protein expression of MST1 was decreased(P<0.01)and the expression of YAP and TAZ was increased(P<0.01)in the model group;the protein expression of YAP and TAZ was down-regulated(P<0.01)and the expression of MST1 was up-regulated in the model group after the administration of acne combination and clearing acne capsule(P<0.01);compared with the positive drug group,the the protein expression levels of MST1 was up-regulated,the protein expression of YAP was down-regulated(P<0.01)in the acne combination group,the protein expression of TAZ in the acne combination group and the positive drug group was not statistically different(P=0.18>0.05).(2)QPCR results:compared with the blank group,the expression of MST1m RNA in the model group decreased significantly(P<0.01),while the expression levels of YAPm RNA and TAZm RNA increased significantly(P<0.01);compared with the model group,the expression of MST1m RNA in the drug intervention group was up-regulated(P<0.01),and the expression levels of YAPm RNA and TAZm RNA expression levels were down-regulated(P<0.01);compared with the positive drug group,the expression level of MST1m RNA was up-regulated(P<0.01)and the expression level of YAPm RNA and TAZm RNA were down-regulated(P<0.01).Conclusion:1.Acne Combination reduces the area of follicular expansion and infiltration of inflammatory factors,improves hyperkeratosis of follicular sebaceous ducts,reduces the thickness of the affected epidermal layer,and decreases the inflammatory response.2.Acne mixture can regulate Th17/Treg cell balance and restore immune homeostasis.3.Acne mixture can regulate Hippo pathway and prevent inflammatory infiltration.4.Acne mixture can interfere with Th17/Treg cell balance to control inflammation by interfering with Hippo pathway. |
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