Rearch On The Mechnism Of Huangqi-Fangfeng And Mahuang-Gancao Regarding To The Effect On Allergic Diseases

Medicine Pairs come from theory of traditional Chinese medicine which are the basic units representing the treatment principles of Chinese medicine.Their effectiveness has spoken for themselves as famous combinations used for thousands of years.Huangqi-Fangfeng and Mahuang-Gancao are the most basic ones using in the treatment of allergic diseases.As allergic diseases are becoming one of the most problems all over the world,the effectiveness of Chinese traditional medicine used in the clinic has aroused wide attention.This article aims to study and discover new targets in allergic diseases by focusing on these two medicine pairs.Based on the previous studies,main researchers carried out in this paper were as written as below:1.Study on the effect and different action channels of Huangqi-Fangfeng and Mahuang-Gancao medicine pairs on a Type 2 ACD murine modelBased on an murine model of Type 2 ACD model,we explored the effectiveness of two medicine pairs firstly.Then,we administrated in different phases separately aiming to find different action channels.2.Study on the chromatogram of Huangqi-Fangfeng and Mahuang-Gancao medicine pairsWe established HPLC chromatogram of two medicine pairs and discovered main ingredients in each pair by comparing with standards which provided foundations for further discovery focusing on the specific ingredients in these medicine pairs.3.Analysis of the effects and basic mechanisms of Huangqi-Fangfeng and Mahuang-Gancao medicine pairs and their main ingredients alleviating allergic diseases in vitroBased on the findings above,we focus on the effect of these two medicine pairs on three major immunocytes including epithelial cells,macrophages and lymphocytes.The major parts are as follows:(I)We discovered whether the medicine pairs could reduce the production of TSLP based on a ACD mouse model stilumated by FITC and a cell model stimulated by poly(I:C)with TNF-a.(II)We discovered the effect of the medicine pairs on the proliferation of macrophages as well as the influence on the inflammation and the balance between M1/M2 stimulated by LPS.(?)We also studied the effect of the medicine pairs on the proliferation of lymphocytes as well as the balance between Thl/Th2 stimulated with ConA.Results:1.Huangqi-Fangfeng and Mahuang-Gancao medicine pairs both could alleviate inflammation in Type 2 ACD modelWe found that both two medicine pairs used during whole process could reduce the ear swelling and pathologic structures and also reduce the expression of IL-4 and IL-13 in the supermatant of ear homogenate considerably.Huangqi-Fangfeng shows stronger effect than Mahuang-Gancao.2.Administrating in the inductive phrase or reaction phase both could alleviate inflammation in Type 2 ACD modelAfter treating type 2 ACD model mice with medicine pairs in different phrases,we found the inflammation could be reduced in varying degrees and Huangqi-Fangfeng administrated in reaction phrases shown the best effect.3.Determination of major ingredients in medicine pairs using HPLCWe detected 22 characteristic peaks at 254 nm in Huangqi-Fangfeng including claycosin-7-O-?-D-glucopyranoside,ononin,claycosin and formononetin,which are major ingredients in Huangqi and cimifugin and prin-O-glucosylcimifugin which are major ones in Fangfeng.We also detected 22 characteristic peaks at 210 nm and 24 characteristic peaks at 254 nm in Mahuang-Gancao,showing that ephedrine,pseudoephedrine,glycyrrhizic acid and liquiritin could be the most important parts in it.4.Huangqi-Fangfeng and Mahuang-Gancao medicine pairs both could reduce the expression of TSLP secreted by epithelial cells1)The results shown that these two medicine pairs could both reduce the expression of TSLP produced during initial phase.2)Medicine pairs are able to reduce the expression of TSLP in 16HBE stimulating by poly(I:C)with TNF-a.It is also shown that Huangqi-Fangfeng has stronger effect in this procedure.5.Mahuang-Gancao shows stronger effect on macrophagesBoth RAW264.7 cells and mouse peritoneal macrophages were used to test the proliferation effect of medicine pairs.Only Mahuang-Gancao could promote cell proliferation on mouse peritoneal macrophages considerably.Both two medicine pairs can reduce the expression of NO in LPS-induced cells model both in RAW264.7 and mouse peritoneal macrophages.On the other side,Mahuang-Gancao can reduce the production of TNF-a in both two LPS-induced cell models.Huangqi-Fangfeng didn't show the same effect.Medicine pairs could suppress the mRNA expression of marker genes of M1 macrophages including IL-6 and iNOS while no effect was seen on the marker genes of M2 macrophages including IL-10,ARG1 and MRC1.Besides,Mahuang-Gancao shows stronger effect and could also suppress the expression of IL-1?.6.T lymphocyte but not B lymphocyte could be one of the targets of Huangqi-Fangfeng and Mahuang-Gancao medicine pairsWe found that two medicine pairs could promote proliferation of naive lymphocyte or ConA-induced lymphocyte using MTT.Mahuang-Gancao shows better effect.On the other hand,medicine pairs didn't affect the proliferation in LPS-activated lymphocytes.These results predicted that T lymphocyte,but not B lymphocyte could be one of the targets which medicine pairs act on.Medicine pairs could probably rectify Thl/Th2 balance in the body of allergic diseases by down-regulating the expression of type 2 cytokines such as IL-4,IL-10 and GATA3.Conclusions:(1)Two medicine pairs could both alleviate type 2 allergic diseases and Huangqi-Fangfeng shown better therapeutic effect than Mahuang-Gancao.They might reduce inflammation by reducing the production of type 2 cytokines which could exaggerate inflammation or suppressing the expression of TSLP which is known as a key initial factor in allergic diseases.(2)Two medicine pairs could both suppress the mRNA expression of marker genes of M1 macrophages including IL-6 and iNOS while no effect was seen on the marker genes of M2 macrophages including IL-10,ARG1 and MRC1.They could also alleviate inflammation in LPS-induced macrophages by down-regulating the production of NO.While Mahuang-Gancao could reduce the expression of TNF-a and the gene expression of IL-1? at the same time which could be supposed that it could be another important way in suppressing inflammation by affecting macrophages.(3)Two medicine pairs both have mitogen-like effect on lymphocyte and could affect T lymphocyte but not B lymphocyte.Regulating the balance between Thl and Th2 is another possible way to intervene allergic diseases.