The Study On Mechanism Of Yupingfeng San's Anti-inflammatory Effect In Contact Dermatitis Mice Based On Network Pharmacology

Contact dermatitis is one kind of delayed-type hypersensitivity which is T cell-medidated immune response.Effect T cells binding with the specific antigen can cause mononuclear cell infiltration and tissue damage and these inflammatory response usually needs 24-72h to develop.This kind of inflammatory response has nothing to do with antibodies.They are just caused by cytokines and cytotoxic medium which are secreted by effect T cells and macrophage.Yupingfeng San is a famous Chinese traditional medicine which is composed of Astragali Radix(Astragalus membranaceus,Huangqi)?Atractylodis Macrocephalae Rhizoma(Rhizoma Atractylodis Macrocephalae,Baizhu)and Saposhnikoviae Radix(Saposhnikovia divaricate,Fangfeng).This formula has already been used for more than a thousand years,and it's used for clinical treatment of allergic rhinitis,upper respiratory tract infections and some other allergic diseases.Firstly,we constructed CD and DTH-related protein-protein interaction network and YPFS active ingredients-protein network.Then,we merged the two networks and constructed CD and DTH-related YPFS bioactive ingredients-protential targets interaction network.We got 23 proteins from the network which may be key node proteins.They were HSP90AA1,ESR1,CDK2,ELANE,F2,PPP1CC,LCK,PCK1,NR3C1,PGR,CCNA2,SRC,BTK,ABL1,CASP1,DPP4,JAK2,KIT,PRKCQ,GRB2,PYGL and HPGDS.Results revealed that 13 conpounds may help to improve CD through Hsp90 among the 20 compounds we use to do reverse molecular docking.Heat shock protein 90(Hsp90)is a highly conserved molecular chaperone protein widely found in cells.We merged the 23 proteins with Hsp90's client proteins,and got 6 proteins:SRC,CDK2,LCK,PRKCQ,CCNA2 and NR3C1.So Hsp90 may improve CD as a target as well as a chaperone protein to influent the stability of other targets.We here study YPFS's anti-inflammatory mechanism using its active ingredients on DNFB-induced model of contact dermatitis.We ues ELISA,qPCR and flow cytometry to detect the change of cytokine content on both cell level and molecular level.We also get the pathological section of ear tissue to observe each ingredient's improvement on inflammation.The protein level of Hsp90,Akt and the correlative protein in the NF-?B signal pathway are measured by western blot.Our result showed that the active ingredients have anti-inflammatory effects on the model mouse.Astragaloside ? and Atractylenolide I showed better improvement than Prim-O-glucosylcimifugin and 5-O-methylvisamminal.Results of serum ELISA revealed that Astragaloside ? and Atractylenolide I can significantly reduce IFN-y level.Results of spleen qPCR revealed that Astragaloside IV and Atractylenolide I can significantly reduce IL-2,IFN-y and TNF-a mRNA level.Western blot analysis in the mouse spleen tissues displayed that Astragaloside?? and Atractylenolide I can significa ntly reduce the protein level of Hsp90 and Akt,and then inhibit the activation of NF-?B.The protein level of Hsp90 of macrophages rises after stimulated by lipopolysaccharide(LPS).We use LPS to stimulate Raw264.7 for 24h after incubation with four active ingredients of YPFS for 1.5h.Then we detect the level of TNF-a and NO in cell culture medium.The protein level of Hsp90,Akt and the correlative protein in the NF-?B signal pathway was measured by western blot.Results show that active ingredients of YPFS can reduce TNF-a and NO released from macrophages after stimulating by LPS.Astragaloside ? and Atractylenolide I show obvious improvement of inflammation.The WB results also show that Astragaloside ? and Atractylenolide I can significantly reduce the protein level of Hsp90 and Akt,and then inhibit the activation of NF-?B.Above all,YPFS may improve inflammation in CD via Hsp90.And YPFS may impact the stability of Akt via Hsp90 and suppress the activation of NF-?B in the downstream.