Study On The Mechanism Of Anti-urticaria By Electroacupuncture Regulation Of P38MAPK And PI3K Pathways Based On The Concept Of "spleen-nutrient And Defense

Purpose:The curative effects of acupuncture on urticaria were investigated by observing the improvement of skin symptoms such as the diameter of locus coeruleus and the leakage of Evans blue reduction at the sensitized part of the urticaria rat models which were induced by electro-acupuncture at Xuehai and Quchi Points.The regulation of acupuncture via IgE mediated mast cell degranulation on immune-inflammatory response was analyzed by observing the changes of electroacupuncture pretreatment at Xuehai and Quchi points on skin histomorphological,the IgE mediated mast cells degranulation and the expression of inflammatory mediators such as histamine IL-4 IL-13 and TNF-α in urticaria rat models.The mechanism of improving urticaria symptoms by acupuncture-mediated mast cells degranulation was studied and provided a new experimental basis for treating urticaria by acupuncture-induced acupuncture by observing the effects of electroacupuncture pretreatment at Xuehai and Quchi points on the expression of p38MAPK、PI3K/PDK1/AKT signaling pathway in urticaria rat models.Materials and methods:There are three rats which were randomly selected from 35 healthy male SD rats(SPF).Allogeneic rat anti ovalbumin serum was prepared by combined injection of chicken egg albumin and immune adjuvant(BCG vaccine).After dilution,the serum was stored at-20℃.The remaining 32 rats were divided into 4 groups(n=8 each)using a random number table method:blank group model group electroacupuncture group and western medicine group.The electroacupuncture group was treated with electroacupuncture at bilateral Xuehai and Quchi points.The parameters of electroacupuncture were set as current 1mA,density wave,frequency 2Hz,and the needle was left for 20 min every time.The western medicine group was given loratadine diluted solution(lmg/kg)by gavage.The blank group and the model group were given the same dose of normal saline(lmg/kg)by gavage.Each group was given corresponding intervention once a day for 7 consecutive days.On the 5th day intervention hairs of 3×3 cm area on the back of rats in each group were cut and 0.1 ml of anti ovalbumin serum diluent was injected intradermal at 6 isometric sites which were 1.5 cm from both sides of the spine to obtain antibodies.30 minutes before sampling the mixed solution of chicken egg albumin and Evans blue dye(lml/100g)were injected into the tail vein to establish the urticaria rat models.The general states of rats in each group were observed.The diameter of skin wind masses in the sensitized parts in the back were measured by rulers.Ultraviolet spectrophotometer was used to measure the amount of Evans blue dye exudation in skin tissues.Toluidine blue staining was used to detecte the morphology and number of mast cells degranulation in skin tissues.HE staining was used to observe the histopathological changes in skin tissues.Immunohistochemistry was used to applied to determine the protein expressions of IgE and histamine in subcutaneous tissues and p38MAPK PI3K PDK1 and AKT in skin tissues.Western blot was used to detect the expressions of p38MAPK PI3K PDK1 and AKT in skin tissues.ELISA was used to detect the expression levels of IL-4 IL-13 and TNF-α in serum.Results:Thesis 1:Study on the preventive and therapeutic effect of acupuncture on urticaria rat model1 General changes of ratsDuring the adaptive feeding period the rats in each group had good mental state sensitive response active behavior shiny fur good eating and drinking,and normal defecation.After the establishment of urticaria model except of the blank group after intradermal injection of serum and tail vein injection of Evans blue and chicken egg albumin normal saline solution the rats of the rest three group showed blue spots and small solid papules at the injection site scratched the skin lesions in varying degrees licked the soles of their feet and rubbed each other which was most obvious in the model group and slightly lighter in the acupuncture group and western medicine group.2 Changes in the diameter of locus coeruleus on the back of ratsThere was no obvious diameter of locus coeruleus in the back hair removal area of rats in the blank group.Compared with the blank group there was a significant diameter of sensitized locus coeruleus in the back hair removal area of rats in the model group(P<0.01).Compared with the model group the diameter of sensitized locus coeruleus in the back hair removal area of rats was decreased significantly in electroacupuncture group and western medicine group(P<0.01).Compared with the electroacupuncture group the diameter of sensitized locus coeruleus in the back hair removal area of rats was decreased in the western medicine group but there was no differences between the two groups(P>0.05).3 Changes of Evans blue dye exudation from rats back skinThere was no obvious Evans blue exudation on the back of rats in the blank group.Compared with the blank group the amount of Evans blue exudation from the back of rats was increased significantly in the model group(P<0.01).Compared with the model group the amount of Evans blue exudation from the back of rats was decreased significantly in electroacupuncture group and western medicine group(P<0.01).Compared with the electroacupuncture group the amount of Evans blue exudation from the back of rats was decreased in the western medicine group but there was no differences between the two groups(P>0.05).4 Histomorphological changes of rats dorsal skinHE staining was used to observe the back skin tissue of rats.The results showed that the structure of skin cells in the back hair removal area of rats in the blank group was clear and orderly and there was no inflammatory cell infiltration in the cell space of skin dermis.In the model group the skin cell space of the sensitized part of the back hair removal area was significantly swollen the cell structure level was significantly disordered and a large number of inflammatory cell infiltration was seen in the cell space of the skin dermis.Compared with the model group the edema of skin cell space the level of cell structure and the infiltration of inflammatory cells in tissue space in the sensitized area of back hair removal area of rats were reduced significantly in electroacupuncture group and western medicine group.Thesis 2:Effects of acupuncture on degranulation of mast cells and expression of IgE,histamine and serum inflammatory factors in subcutaneous tissue of urticaria rats1 Changes of IgE expression in subcutaneous dorsal tissue of rats in each groupImmunohistochemical method was used to detect the subcutaneous tissue of the back of rats.The results showed that the expression of IgE in the subcutaneous tissue of the back hair removal area of the blank group was less.Compared with the blank group the expression of IgE in the subcutaneous area of desensitized rats was increased significantly in the model group(P<0.01).Compared with the model group the expression of IgE in the subcutaneous tissue of the sensitized part of the back hair removal area of rats was decreased significantly in the electroacupuncture group and western medicine group(P<0.01).Compared with the electroacupuncture group there was no differences in the expression of IgE in the subcutaneous tissue of the sensitized part of the back hair removal area in the western medicine group(P>0.05).2 Changes of IL-4 and IL-13 expression in serum of rats in each groupThe results showed that the expression levels of serum IL-4 and IL-13 in the blank group were less.Compared with the blank group the expression levels of serum IL-4 and IL-13 was increased significantly in the model group(P<0.01).Compared with the model group the expression levels of serum IL-4 and IL-13 was decreased significantly in electroacupuncture group and western medicine group(P<0.05 or P<0.01).Compared with the electroacupuncture group there was no differences in the expression levels of serum IL-4 and IL-13 in the western medicine group(P>0.05).3 Changes of degranulation of mast cells in skin tissue of rats in each groupToluidine blue staining was used to observe the back skin tissue of rats.The results showed that the morphology of skin mast cells in the back hair removal area of rats in the blank group was stable and degranulation was rare.Compared with the blank group the degranulation rate of skin mast cells in the sensitized area of back hair removal area was increased significantly in the model group(P<0.01).Compared with the model group the degranulation rate of skin mast cells in the sensitized area of back hair removal area was decreased significantly in electroacupuncture group and western medicine group(P<0.01).Compared with the electroacupuncture group there was no differences in the degranulation rate of mast cells in the sensitized part of the back hair removal area in the western medicine group(P>0.05).4 Changes of histamine expression in subcutaneous dorsal tissue of rats in each groupImmunohistochemical method was used to detect the subcutaneous tissue of the back of rats.The results showed that the expression of histamine in the subcutaneous tissue of the back hair removal area of the blank group was less.Compared with the blank group the expression of histamine in the subcutaneous tissue of the sensitized part of the back hair removal area of rats was increased significantly in the model group(P<0.01).Compared with the model group the expression of histamine in the subcutaneous tissue of the sensitized part of the back hair removal area of rats was decreased significantly in the electroacupuncture group and western medicine group(P<0.01).Compared with the electroacupuncture group there was no differences in the expression of histamine in the subcutaneous tissue of the sensitized part of the back hair removal area in the western medicine group(P>0.05).5 Changes of TNF-α expression in serum of rats in each groupELISA was used to detect the serum of rats.The results showed that the expression level of serum TNF-α in the blank group was less.Compared with the blank group the expression level of serum TNF-α was increased significantly in the model group(P<0.01).Compared with the model group the expression level of serum TNF-α was decreased significantly in electroacupuncture group and western medicine group(P<0.01).Compared with the electroacupuncture group there was no differences in the expression level of serum TNF-α in the western medicine group(P>0.05).Thesis 3:Effect of acupuncture on degranulation mechanism of mast cells in urticaria rats1 Expression changes of p38MAPK、PI3K/PDK1/AKT signal pathway related proteins in back skin of rats in each group(immunohistochemical method)1.1 Changes of p38MAPK protein expression in back skin of rats in each groupThe expression of p38MAPK in skin mast cells in the back hair removal area of rats in the blank group was less.Compared with the blank group the expression of p38MAPK in the skin mast cells of the sensitized part of the back hair removal area of rats was significantly increased in the model group(P<0.01).Compared with the model group the expression of p38MAPK in skin mast cells in the sensitized area of back hair removal area of rats was decreased significantly in electroacupuncture group and western medicine group(P<0.05).Compared with the electroacupuncture group there was no differences in the expression of p38MAPK in the skin mast cells of the sensitized part of the back hair removal area in the western medicine group(P>0.05).1.2 Changes of PI3K protein expression in back skin of rats in each groupThe expression of PI3K in skin mast cells in the back hair removal area of rats in the blank group was less.Compared with the blank group the expression of PI3K in skin mast cells in the sensitized area of back hair removal area of rats was significantly increased in the model group(P<0.01).Compared with the model group the expression of PI3K in skin mast cells in the sensitized area of back hair removal area of rats was decreased significantly in electroacupuncture group and western medicine group(P<0.01).Compared with the electroacupuncture group there was no differences in the expression of PI3K in the skin mast cells of the sensitized part of the back hair removal area in the western medicine group(P>0.05).1.3 Changes of PDK1 protein expression in back skin of rats in each groupThe expression of PDK1 in skin mast cells in the back hair removal area of rats in the blank group was less.Compared with the blank group the expression of PDK1 in the skin mast cells of the sensitized part of the back hair removal area of rats was increased significantly in the model group(P<0.01).Compared with the model group the expression of PDK1 in skin mast cells at the sensitized part of back hair removal area of rats was decreased significantly in electroacupuncture group and western medicine group(P<0.05).Compared with the electroacupuncture group there was no differences in the expression of PDK1 in the skin mast cells of the sensitized part of the back hair removal area in the western medicine group(P>0.05).1.4 Changes of AKT protein expression in back skin of rats in each groupThe expression of AKT in skin mast cells in the back hair removal area of rats in the blank group was less.Compared with the blank group the expression of AKT in skin mast cells in the sensitized area of back hair removal area of rats was increased significantly in the model group(P<0.01).Compared with the model group the expression of AKT in skin mast cells at the sensitized part of back hair removal area of rats was decreased significantly in electroacupuncture group and western medicine group(P<0.05).Compared with the electroacupuncture group there was no differences in the expression of AKT in the skin mast cells of the sensitized part of the back hair removal area in the western medicine group(P>0.05).2 Expression changes of p38MAPK、PI3K/PDK1/AKT signal pathway related proteins in back skin of rats in each group(Western blot method)2.1 Changes of p38MAPK protein expression in back skin of rats in each groupThe expression of p38MAPK in skin mast cells in the back hair removal area of rats in the blank group was less.Compared with the blank group the expression of p38MAPK in the skin mast cells of the sensitized part of the back hair removal area of rats was increased significantly in the model group(P<0.01).Compared with the model group the expression of p38MAPK in skin mast cells in the sensitized area of back hair removal area of rats was decreased significantly in electroacupuncture group and western medicine group(P<0.05).Compared with the electroacupuncture group there was no differences in the expression of p38MAPK in the skin mast cells of the sensitized part of the back hair removal area in the western medicine group(P>0.05).2.2 Changes of PI3K protein expression in back skin of rats in each groupThe expression of PI3K in skin mast cells in the back hair removal area of rats in the blank group was less.Compared with the blank group the expression of PI3K in skin mast cells in the sensitized area of back hair removal area of rats was increased significantly in the model group(P<0.01).Compared with the model group the expression of PI3K in skin mast cells in the sensitized area of back hair removal area of rats was decreased significantly in electroacupuncture group and western medicine group(P<0.01).Compared with the electroacupuncture group the expression of PI3K in the skin mast cells of the sensitized part of the back hair removal areaof rats was decreased significantly in the western medicine group(P<0.05).2.3 Changes of PDK1 protein expression in back skin of rats in each groupThe expression of PDK1 in skin mast cells in the back hair removal area of rats in the blank group was less.Compared with the blank group the expression of PDK1 in the skin mast cells of the sensitized part of the back hair removal area of rats was increased significantly in the model group(P<0.01).Compared with the model group the expression of PDK1 in skin mast cells at the sensitized part of back hair removal area of rats was decreased significantly in electroacupuncture group and western medicine group(P<0.05).Compared with the electroacupuncture group the expression of PDK1 in the skin mast cells of the sensitized part of the back hair removal area of rats was decreased significantly in the western medicine group(P<0.05).2.4 Changes of AKT protein expression in back skin of rats in each groupThe expression of AKT in skin mast cells in the back hair removal area of rats in the blank group was less.Compared with the blank group the expression of AKT in skin mast cells in the sensitized area of back hair removal area of rats was significantly increased in the model group(P<0.01).Compared with the model group the expression of AKT in skin mast cells in the sensitized area of back hair removal area of rats was decreased significantly in electroacupuncture group and western medicine group(P<0.01).Compared with the electroacupuncture group there was no differences in the expression of AKT in the skin mast cells of the sensitized part of the back hair removal area in the western medicine group(P>0.05).Conclusions:1.Electroacupuncture pretreatment at Xuehai and Quchi points can significantly minish the diameter of locus coeruleus in the back sensitized parts in the urticaria rat models reduce the amount of Evans blue exudation improve the skin histomorphological changes and effectively improve the urticaria rat models.2.Electroacupuncture pretreatment at Xuehai and Quchi points can significantly reduce the degranulation of mast cells in the back sensitization sites down-regulate the expressions of IgE and histamine in subcutaneous tissues in the back hair-removal areas and reduce the expressions of IL-4 IL-13 and TNF-α in serum to regulate the ige-mediated mast cells degranulation and reduce inflammation reactions mediated by IL-4 IL-13 and TNF-α.3.Electroacupuncture pretreatment at Xuehai and Quchi Points can significantly reduce the expressions of p38MAPK PI3K PDK1 and AKT which indicates that electroacupuncture can regulate the IgE mediated degranulation of mast cells the release of histamine IL-4 IL-13 TNF-α and other inflammatory mediators by the p38MAPK、PI3K/PDK1/AKT signaling pathway which can inhibite the inflammatory responses and improving urticaria symptoms.4.Electroacupuncture pretreatment at Xuehai and Quchi points can demonstrate the scientific connection between the immune-inflammatory response based on mast cell degranulation and the location of urticaria in Spleen and Yingwei.Acupuncture at the precise and few acupoints regulated the IgE-mediated degranulation of mast cells represented by urticaria and improved the modern biological mechanism of acupuncture regulating key effector cells(mast cells)to play an anti-allergic role to some extent and verified the scientific hypothesis of this experiment.Meanwhile the guiding significance of the thought of "Spleen-Ying wei" in clinical disease diagnosis and treatment was enlarged.