Establishment And Application Of Mediated Anti-Inflammatory Drug Screening Model Based On ICAM-1 Signaling Pathways

Inflammation is a complex biological response of the body to harmful stimuli(such as pathogens,damaged cells or irritants).Although inflammation is the body's defense mechanism,excessive stagnation can also cause damage to the body.Glucocorticoid anti-inflammatory drugs and non-steroidal anti-inflammatory drugs are commonly used as an anti-inflammatory drugs,both have provedpotent anti-inflammatory effects in clinical practice.However,long-term administration of high-doses may cause many adverse ffects on the body.Therefore,it is necessary to establish a screening platform for anti-inflammatory drugs,and to find novel anti-inflammatory drugs with low toxicity and high efficiency from Chinese herbal medicines and natural products.In this study,an engineered yeast was constructed,and an anti-inflammatory drug screening platform was established based on the intercellular adhesion molecule-1(ICAM-1)signaling pathway using the human microvascular endothelial cell(HMEC-1).A library of Chinese herbal medicine components was established,and the anti-inflammatory compounds were selected and isolated by activity tracking.The main results are as follows:1.Based on yeast surface two-hybridization,an engineered yeast with a two-channel protein expression system was established: lymphocyte function-associated antigen 1(LFA-1)?L I domain was expressed on the yeast surface for binding to ICAM-1 and intracellular expression of green fluorescent protein GFP was used for fluorescence indication.It was found that lipopolysaccharide(LPS)induced inflammatory reaction in HMEC-1 cells,and ICAM-1 was significantly up-regulated.Under the microscope,it was observed that HMEC-1 cells were specifically bound to engineered yeast which was detected by microplate reader.The expression abundance of ICAM-1 in HMEC-1 cells was evaluated according to the fluorescence intensity.Anti-inflammatory compounds inhibited the expression of ICAM-1 in HMEC-1 cells induced by LPS,decreased the binding of engineered yeast to HMEC-1 cells,and decreased the fluorescence intensity.Therefore,the constructed yeast was used as a bridge and the expression of ICAM-1 was target anti-inflammatory drug screening model.2.Anti-inflammatory activity screening of 1340 compounds by anti-inflammatory drug screening model,identified two compounds with anti-inflammatory activity: Methyllycaconitine citrate(MLA)and Cytochalasin D(CytoD),an inducing inflammatory compound: Phorbol 12,13-dibutyrate(PDBU).The activity of these three compounds was further investigated to validate their activity by detecting the expression level of inflammatory factor gene and protein,and NF-?Bp65 protein nuclear localization.Results exhibited that CytoD showed anti-inflammatory activity in the mouse sepsis model while MLA didn't show anti-inflammatory activity.on the other hand,PDBU did not induce systemic inflammation in mice and onlyable to induce local inflammation,the ear swelling model.Furthermore,our established library of Chinese herbal medicines led to identified two components E10 and E11 with the best anti-inflammatory activity.However,upon investigating of anti-inflammatory activity of these two compounds in-vivo,compound E11 showed moderate anti-inflammatory activity in the mouse sepsis model while E10 did not show any anti-inflammatory activity.Check out the chinese herbal medicines library table,E10 and E11 are the initial separation components of Solanum lyratum Thunb.3.The activity tracking separation study of Solanum lyratum Thunb.was conducted by an anti-inflammatory drug screening model.The anti-inflammatory drug screening platform was selected for providing anti-inflammatory activity guidance,and all the parts isolated at each stage were quantitatively evaluated for anti-inflammatory activity.Based on the activity,one compound diosgenin3-O-?-Lrhamnopyranosyl-(1?2)-?-D-glucopyranosiduronic acid was isolated and identified with anti-inflammatory activity in Solanum lyratum Thunb.