| Objective: To investigate the improvement effect of acupuncture on urticaria rat model by observing the area of locus coeruleus,histomorphology,serum IgE and histamine levels of skin tissue on the back of rats,and to detect the changes of PLC?/ DAG/PKC levels in skin tissue,so as to study the mechanism of acupuncture improving urticaria rat model.It can enrich the modern mechanism of acupuncture intervention in urticaria,and provide experimental basis for clinical acupuncture treatment of urticaria.Materials and methods: Thirty five healthy male SD rats(SPF grade): three rats were randomly selected to prepare anti ovalbumin serum,and the remaining 32 rats were randomly divided into blank group,model group,acupuncture group and Western medicine group,with8 rats in each group.1.Preparation of anti ovalbumin serum: the anti ovalbumin serum was prepared by the method of ovalbumin(chicken)combined with BCG injection;the anti ovalbumin serum was diluted 1 ? 10 and frozen at-20 ? for standby;2.Intervention methods: the rats in each group were treated with acupuncture or western medicine for 7 days,once a day.(1)Methods and parameters of acupuncture intervention: according to the "experimental acupuncture" rat acupoint positioning,bilateral Quchi and Xuehai were selected for acupuncture treatment,the rats were fixed on a special fixator when awake,0.20 mm × 25 mm filiform needle was used,the needle was vertically inserted for 3-5 mm,and the electro acupuncture instrument was connected,the density wave was selected,and the frequency was2 Hz,current intensity 1m A,with slight tremor of the limb connected to the electroacupuncture instrument as the degree,keep the needle for 20 min/time.(2)Western medicine intervention method: Taking loratadine as the control treatment,according to the standard of adult dosage of 10 mg/d,according to the equivalent dose ratio of human and animal surface area conversion,the dosage of Loratadine Tablets was diluted and then gavaged.3.Establishment of model: the rat model of urticaria was established by passive cutaneous anaphylaxis(PCA).Injection of anti ovalbumin serum diluent: 48 hours before sampling,the rats in each group were lightly anesthetized with chloral hydrate,and their hair was sheared within 1.5 cm from the spine on both sides of the dorsal midline,and the injection points of each group were marked with marker pen;the diluted anti ovalbumin serum was injected symmetrically into the skin of the model group,acupuncture group and Western medicine group at the marked points on both sides of the spine.The blank group was only given the same amount of 0.9% sodium chloride solution.48 hours later,the rats in each group were injected with 0.9% sodium chloride solution(1ml/100g)containing 1 mg/ml ovalbumin and0.5% Evans blue via tail vein for antigen challenge to prepare urticaria rat model.4.Sampling and detection: 30 minutes after antigen challenge,the rats in each group were killed,and the skin,subcutaneous loose connective tissue and serum of the sensitized part on the back were taken.The diameter of locus coeruleus and Evans blue exudation were measured.The histopathological changes of skin were observed by HE staining.The expression levels of PLC ?,DAG and PKC protein in mast cells of subcutaneous loose connective tissue were detected by immunohistochemistry.5.Statistical analysis: the experimental results were expressed by(mean ± standard deviation),and were statistically analyzed by SPSS 26.0 software.The comparison between the groups was performed by one-way ANOVA,and P < 0.05 showed statistical significance.Results:1.Changes in the diameter of blue spot on the back of rats in each group: no blue spot appeared on the back of rats in the blank group;compared with the blank group,the blue spot on the back of rats in the model group increased significantly(P < 0.01);compared with the model group,the blue spot on the back of rats in the acupuncture group and Western medicine group decreased significantly(P < 0.01);compared with the acupuncture group,the blue spot on the back of rats in the western medicine group decreased slightly,but the difference was not statistically significant The difference was statistically significant(P > 0.05).2.Changes of Evans blue exudation on the back of rats in each group: Evans blue exudation on the back of rats in blank group was very little;compared with blank group,Evans blue exudation on the back of rats in model group increased significantly(P < 0.01);compared with model group,Evans blue exudation on the back of rats in acupuncture group and Western medicine group decreased significantly(P < 0.01);compared with acupuncture group,Evans blue exudation on the back of rats in western medicine group decreased significantly(P < 0.01)The amount of blue exudation decreased slightly,but the difference was not statistically significant(P > 0.05).3.HE staining was used to detect the histomorphological changes of the back skin of rats in each group: in the blank group,the cells in the epidermal layer of the back skin tissue of rats were arranged regularly,the hierarchical structure was very clear,and there was no obvious inflammatory cell infiltration in the dermis;compared with the blank group,the keratinization of the epidermal layer of the back skin tissue of rats in the model group was obvious,the intercellular space was significantly edema,the hierarchical structure was abnormal disorder,and there was no obvious inflammatory cell infiltration in the dermis A large number of inflammatory cells infiltration was observed;compared with the model group,the cuticle of the skin tissue on the back of the rats in the acupuncture group and the western medicine group became thinner,and the tissue space and inflammatory cell infiltration were significantly reduced.4.The expression changes of IgE and histamine in the back skin tissue of rats in each group were detected by ELISA: the expression levels of IgE and histamine in the skin tissue of rats in the blank group were less;compared with the blank group,the expression levels of IgE and histamine in the skin tissue of rats in the model group were significantly increased(P < 0.01);Compared with the model group,the expression levels of IgE and histamine in the skin tissue of rats in the acupuncture group and Western medicine group were significantly increased(P < 0.01);Compared with the acupuncture group,the expression levels of IgE and histamine in skin tissue of Western medicine group decreased slightly,but the difference was not statistically significant(P > 0.05).5.The protein expression levels of PLC ?,DAG and PKC in subcutaneous connective tissue mast cells of rats in each group were detected by immunohistochemistry: the protein expression levels of PLC ?,DAG and PKC in subcutaneous connective tissue mast cells of rats in the blank group were less;compared with the blank group,the protein expression levels of PLC ?,DAG and PKC in subcutaneous connective tissue mast cells of rats in the model group were significantly higher(P < 0.01);compared with the model group,the protein expression levels of PLC?,DAG and PKC in acupuncture group and Western medicine group were significantly lower(P < 0.01);Compared with the acupuncture group,the expression levels of PLC?,DAG and PKC in the western medicine group were slightly decreased,but the difference was not statistically significant(P > 0.05).Conclusion:Acupuncture at Quchi and Xuehai can regulate the PLC?/DAG/PKC signaling pathway of mast cells,reduce the expression levels of IgE and histamine in skin tissue to improve degranulation of mast cells,reduce the infiltration of inflammatory cells in skin tissue to reduce the skin inflammatory reaction of urticaria animal model,and then improve the area of blue spot and Evans blue exudation in skin tissue of allergic area on the back of rat model,so as to achieve the goal of prevention and treatment Objective to treat urticaria. |
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