| Objective:Based on the fact that atopic dermatitis is an inflammatory pruritus disease and mast cells participate in the occurrence of pruritus,a variety of animal and cell models were used to evaluate the anti-inflammatory and antipruritic effects of Danggui Kushen pill,improve the efficacy of chronic pruritus of damp-heat atopic dermatitis and regulate the mechanism of mast cell degranulation,so as to provide scientific basis for clinical medication and guide clinical medication.Methods:1.The acute inflammation model of Kunming mice induced by xylene was used to calculate the degree of ear swelling and the inhibition rate of ear swelling,and to preliminarily evaluate the anti-inflammatory effect of Danggui Kushen pill;The acute itching models of mice induced by dextran-40 and 4-AP were used to observe the scratching times of mice and detect the levels of LTB4 and histamine in the serum of 4-AP mice,and to preliminarily evaluate the antipruritic effect of Danggui Kushen pill;C48/80 induced mouse skin pruritus model was used to observe the scratch of mice,the morphological changes of mast cells and the expression of TPS were observed by TB staining and IHC staining respectively,and the expression level of Histamine in skin homogenate was detected by ELISA,to evaluate the intervention effect of Danggui Kushen pill on mouse pruritus induced by mast cell degranulation.2.The model of chronic pruritus of atopic dermatitis of damp-heat syndrome was established by using damp-heat syndrome superimposed with MC903.The scratch of mice was observed,the weight of ear piece was measured,the degree of ear swelling was calculated,the organ index was calculated,the pathological changes of ear and the infiltration of mast cells were observed by HE staining and TB staining respectively,and the TSLP,IL-4,IL-13,IL-31,IL25,IL-33 in the ear lesions were detected by IHC staining,The expression of IgE,TSLP,IL4,IL-13 in mouse serum and TSLP,IL-4,IL-13,IL-31,IL-25,IL-33,Histamine,LTB4,PGD2,IFN-y in ear lesions were detected by ELISA.To evaluate the anti-chronic pruritus effect of Danggui Kushen pill.3.The model of mast cell degranulation was established by stimulating RBL-2H3 cells with C48/80.Colorimetry was used to detect the content of β-Hex in the supernatant and ELISA was used to determine the content of Histamine and TPS in the supernatant,and the intervention effect of Danggui Kushen Pill containing serum on RBL-2H3 degranulation was evaluated.4.RBL-2H3 cells were induced by IgE to establish a degranulation model.Colorimetric method is used to detect the supernatant β-Hex release;ELISA kit detects the expression of IL-4,IL-13,Histamine,TPS,MIP-lα,TNF-α,TSLP,IL-31 in the supernatant;The expression of IL-4,IL-13,Histamine,TPS,MIP-1α,TNF-α,TSLP,IL-31 mRNA in cells of each group was detected by qPCR;Western blotting method was used to detect the expression of JAK-STAT pathway related proteins(p-JAK1/JAK1,p-JAK2/JAK2,pSTAT3/STAT3,p-STAT5/STAT5,p-STAT6/STAT6)in cells of each group.Results:1.Effect of Danggui Kushen Pill on acute inflammation and pruritus model mice(1)Inhibit the degree of ear swelling in mice with acute inflammation;(2)Reduce the scratching times of acute pruritus mice caused by Dextran-40;(3)Improve the scratching times of 4-AP-induced acute pruritus mice and reduce the expression of Histamine and LTB4 in serum;(4)Reduce the scratching times of C48/80 skin pruritus model mice,reduce the number of mast cells in the skin lesions,and inhibit the expression of Histamine and TPS.2.Effect of Danggui Kushen Pill on Damp-heat AD pruritus mice(1)Improve the yellow hair and loose stool of mice;(2)Inhibition of body weight and body temperature decline in mice;(3)Inhibit the swelling degree of mouse ear and the increase of right ear mass;(4)Reduce the times of scratching and improve the itching of mice;(5)Improve the pathology of AD-like lesions and inhibit the infiltration of mast cells;(6)Inhibit the expression level of IgE,TSLP,IL-4,IL-13 in serum and TSLP,IL-4,IL13,IL-31,IL-25,Histamine,LTB4,PGD2,IFN-y in ear piece.3.Effect of serum containing Danggui Kushen pill on C48/80 stimulated RBL-2H3 degranulation(1)The drug concentrations that had no significant effect on RBL-2H3 cell viability were Keto-10%,DGKSW-20%,DGKSW-15%and DGKSW-10%;(2)Inhibition of C48/80 stimulation of RBL-2H3 degranulation β-Release of β-Hex,Histamine,TPS and LTB4.4.Effect of serum containing Danggui Kushen pill on IgE-induced RBL-2H3 cell degranulation(1)Inhibition of RBL-2H3 cell degranulation release β-Hex;(2)Reduce IL-4,IL-13,Histamine,TPS,MIP-1α,TNF-α,TSLP IL-31 in the supernatant;(3)Inhibit the expression of IL-4,IL-13,Histamine,TPS,MIP-1α,TNF-α,TSLP,IL31 mRNA in activated cells;(4)Suppress the expression of p-JAK1/JAK1,p-JAK2/JAK2,p-STAT5/STAT5,pSTAT6/STAT6 proteins in the JAK-STAT pathway.Conclusion:Danggui Kushen pill can inhibit the release of type 2 cytokines with pro-inflammatory and itchy properties,inhibit the infiltration of mast cells and release of itching media from degranulation,and improve the itching of AD mice.It is preliminarily believed that Danggui Kushen pill may improve AD pruritus by regulating IgE-mediated RBL-2H3 degranulation by JAK-STAT. |
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