Study On Effect Of Astragaloside ? On Type2 Allegic Dermatitis

Allergic Inflammation is one of the major diseases in human race,primarily concerned with common clinical diseases,such as allergic rhinitis,asthma,dermatitis.The mobidity of Allergic Inflammation all around the world has been reported to be about 30%-40%,and is growing at the rate of more than 1%per year.In addition,the incidence rate of children increased most.The mobidity of allergic inflammation in our country is also rising year by year with the development of the level of modernization,industrialization and westernized lifestyle.Therefore,the influencing factors,the treatment of allergic inflammation and the mechanism became the focus of current research.ObjectivesAtopic dermatitis model is a representative model of allergic diseases.Our previous study found that extract of Yupingfengsan can inhibit allergic contact dermatitis(ACD),asthma and other Th2 allergic diseases effectively.Astragalus is the main herb in Yupingfengsan which plays a very clear role in immune regulation.However,if the important monomer components Astragaloside IV in Astragalus has effect on Th2 ACD,what is the function mechanism of AS-IV and whether AS-IV is an active ingredient in Yupingfengsan or not?All of these haven't been confirmed yet by experiments.Type2 allergic contact dermatitis induced by fluoresciniso thiocyate(FITC)in mice was utilized in this study to explore the function,effect stage and the mechanism of Astragaioside IV(AS-IV)on allergic inflammation.In addition,we use the model of TSLP produced by Epithelial cells co-cultured with poly(I:C)and TNF-a to further validate the AS-IV function in which way can we to provide data for the research on the substantial base of the anti-allergy effects caused by Yupingfengsan,the basis for clinical application and a new direction for the treatment of allergic diseases.Methods:1.The way to establish the model of Type 2 ACD.The BALB/c mice were topically sensitized on the shaved abdomens with 1.5%FITC solution at day 1 and day 2,and elicited on the right ear with 0.5%FITC at day 6.The control group was exposed to the same volume of solvent.Auricle swelling(ear thickness,ear weight),ear homogenates and histopathological examination were measured 24 hours after the elicitation.2.The way to establish the model of TSLP generation in initial stage of induction of Type 2 ACD.The BALB/c mice were topically smeared on the left and right ear with 0.5%FITC from day 1 to day 2,the control group was smeared with the same volume of solvent.Take a part of ear tissue to be tested on day 3.3.The method of administration of AS-IV.(1)The method of administration of AS-IV in whole processMice in AS-IV group were intragastric administered with Astragaioside ?(6.25,12.5,25,50 mg/kg)and the dexamethasone group were intraperitoneally injection of dexame-thasone(0.67 mg/kg)from day 1 to day 7,the control and model group were administered with normal saline,Auricle swelling was measured 1 hour after the administration on day 7.(2)The method of administration of AS-IV in the inductive phaseMice in AS-IV group were intragastric administered with Astragaioside ?(6.25,12.5,25,50 mg/kg)and the dexamethasone group were intraperitoneally injection of dexame-thasone(0.67 mg/kg)from day 1 to day 5,the control and model group were administered with normal saline,obtaining tissue on day 7.(3)The method of administration of AS-IV in the reaction phaseMice in AS-IV group were intragastric administered with Astragaioside ?(6.25,12.5,25,50 mg/kg)and the dexamethasone group was intraperitoneally injection of dexamethasone(0.67 mg/kg)1h before the day 6 elicitation,and 4h,8h and 23h after the elicitation.The control and model group were administered with normal saline.Auricle swelling was measured 24 h after the elicitation.(4)The method of administration of AS-IV in initial stage of induction phase.The BALB/c Mice in the AS-IV group were intragastric administered two days in advance with Astragaioside IV(6.25,12.5,25,50 mg/kg)and the dexamethasone group were intraperitoneally injection of dexamethasone(0.67 mg/kg)to day 3,the control and model group were administered with normal saline,obtaining tissue 1h after the administration on day3.4.The culture and identification of Human Keratinocyte(HKC).The foreskins were provided by Nanjing Jianguo Men's Hospital.The foreskins were aseptic processing and cells isolated.Detect the expression of cytokeratin 5 using immunocytochemistry staining method for cell identification when cell fusion reached 80%.5.The way to establish the model of TSLP generation in HKC stimulated by Poly(I:C).Cells were stimulated with poly(I:C)100 ?g/ml for 24 hours.Collect supernatant for ELISA.6.The way to establish the model of TSLP generation in HaCaT stimulated with Poly(I:C)and TNF-?.Cells were stimulated with poly(I:C)100 ?g/ml and TNF-a 20 ng/ml for 24 hours.Collect supernatant for ELISA.7.The method of seeding plate of cells and administration of AS-IV on cells for immune-fluorescence assay.Cells were seeded in 48-well plate containing pieces of climbing.The cells were divided into seven groups,as control,stimulation,AS-IV,and negative groups.Each group has three wells.Add AS-IV with corresbonding concentration in each well of AS-IV groups.Incubate in 37 ? for 6 hours.After the pre-incubation,each group of cells(In addition to the control group)were stimulated with stimulation for 24 hours.Collect supernatant for ELISA.The climbing cells were collected for Immunofluorescence assay.8.Index detection.Measure the thickness of mouse ear and the weight of mouse ear.Use HE staining for Histopathological examination.IL-4,IL-13,IL-5,IL-9 and TSLP levels of ear tissue homogenates were.detected by ELISA.TSLP mRNA,TSLPR mRNA,NF-?B mRNA,TLRs(TLR2?TLR3?TLR4?TLR8?TLR9)mRNA,Jaggedl mRNA,Notchl mRNA of ear tissue were detected by real-time PCR.Immunofluorescence was used to detect the TSLP expression in cells.Results:1.Administration with AS-IV could inhibit Th2 cell-mediated allergic contact dermatitis in mice in whole process significantly.Compared with the normal group,ear swelling in model group was stimulated by FITC treatment significantly.Meanwhile,IL-4,IL-13,IL-5 and IL-9 levels increased remarkably in FITC treated mice.Wherein,AS-IV 25 mg/kg and 50 mg/kg evidently inhibited the auricle inflammation of the FITC treated mice.Furthermore,IL-4,IL-13,IL-5 and IL-9 levels in ear tissue homogenates were markedly decreased by AS-IV treatment.2.The effect stage of Astragaloside IV inhibits Type2 ACD is major in the induction phase.Administration with AS-IV in induction phase found that compared with the normal group,ear swelling in model group was stimulated by FITC treatment significantly and ear tissue local edema,angiectasis and lymphocytic infiltration were observed simultaneously.Meanwhile,IL-4,IL-13 and IL-9 levels increased remarkably in FITC treated mice.Wherein,AS-IV 25 mg/kg and 50 mg/kg evidently inhibited the auricle inflammation of the FITC treated mice.Pathohistological results indicated that AS-IV alleviated local edema and angiectasis of mice models and reduced lymphocytic infiltration significantly.Furthermore,IL-4,IL-13 and IL-9 levels in ear tissue homogenates were markedly decreased by AS-IV treatment.Administration with AS-IV in reaction phase.In conclusion,AS-IV only individual doses inhibited the ear weightness,IL-4 or IL-9 expression in ear tissue homogenates.Compared with the induction phase administer-ation,the reaction phase administration of AS-IV is not effective on Th2 ACD.Therefore,the effect stage of AS-IV suppressing Th2 ACD is major in the induction phase.3.AS-IV inhibits the expression of TSLP in mice ear tissue at the initial stage of induction phase,in HKC and HaCaT stimulated in vitro.In the model of FITC-induced TSLP production at initial stage of induction phase,compared with the normal group,the expression of TSLP in model group increased remarkably and the TSLP expression reduced significantly after giving Astragaloside ?.In addition,PCR results showed that TSLP significantly expression in model group,AS-IV evidently inhibited the expression of TSLP.In the model of TSLP production in HKC and HaCaT stimulated with poly(I:C)and TNF-?,compared with the normal group,the TSLP expression in model group increased remarkably.Each group of AS-IV evidently inhibited the TSLP expression.The Immunofluorescence results also showed that AS-IV evidently inhibited the TSLP expression in HKC and HaCaT.4.AS-IV regulates the main signaling molecules expression in TLRs-NF-?B signaling pathway and Notch signaling pathway.In vivo experiments about the model of TSLP generation at initial stage of induction phase,PCR results showed that NF-?B/TLR3 mRNA significantly expression in model group,AS-IV evidently inhibited the expression of NF-?B mRNA and downregulated the TLR3 mRNA expression.In addition,AS-IV also reduces the expression of TLR2 mRNA and TLR8 mRNA.Observing the Notch signaling pathway found that AS-IV has no significant effect on the expression of Jagged1 mRNA and Notch1 mRNA.Conclusion:1.Astragaloside IV could inhibit the Th2 cell-mediated mice allergic contact dermatitis induced by FITC significantly,and the effect stage is major in the induction phase.2.Astragaloside IV suppress the TSLP production in epithelial cells at initial stage of sensitization may be the important effect mechanism of AS-?,and the effect mechanism could be related with regulating TSLP through TLRs-NF-?B pathway.3.Astragaloside IV is one of the active ingredients in prevention and treatment of allergic diseased of Yupingfengsan.