| The harm to human health caused by advanced glycation end products has received more and more attention,especially the chronic inflammatory response caused by the interaction of AGEs and RAGE.The chronic inflammatory response induced by AGEs leads to a high level of inflammatory response during the repair of human wounds,resulting in impaired wound healing.Caffeic acid and chlorogenic acid are catechol compounds which are widely existed in nature,and have good anti-inflammatory activity.In this thesis,based on the natural activity of caffeic acid and chlorogenic acid,the effects of caffeic acid and chlorogenic acid on the AGEs-induced HUVEC inflammatory response and the involved inhibition mechanisms were investigated via cellular and spectroscopic techniques to extend the application of caffeic acid and chlorogenic acid in the field of wound healing.The main results are shown as follow:(1)Target the HUVEC,the reasonable concentration range of AGEs-BSA,caffeic acid and chlorogenic acid to HUVEC was investigated and has been used to construct the HUVEC inflammatory reaction system.The inflammatory response system: the HUVEC was treated with 200 ?g/mL of AGEs-BSA to produce inflammation and the controlled group of HUVEC was treated with the same concentration of BSA.The effect of caffeic acid and chlorogenic acid on the inflammatory reaction of HUVEC cells induced by AGEs-BSA was treated with 10 ?M,20 ?M caffeic acid and chlorogenic acid.(2)Based on the signaling pathway activated by AGEs-RAGE interactions,the anti-inflammatory effects of caffeic acid and chlorogenic acid on the inflammatory factors of ICAM-1,VCAM-1,MCP-1,TNF-?,IL-1? were mainly investigated.At the RNA expression level,the effects of caffeic acid and chlorogenic acid on the expression of inflammatory genes in HUVEC were investigated and we found that caffeic acid and chlorogenic acid can inhibit ICAM-1,VCAM-1,and MCP-1 mRNA expression in HUVEC cells induced by AGEs-BSA and the suppressed activity of caffeic acid on the expression of ICAM-1 and MCP-1 mRNA is stronger than the one of chlorogenic acid.In addition,the effect of caffeic acid and chlorogenic acid on the expression of inflammatory cytokines and the positive feedback loop of AGEs-RAGE in HUVEC cells was investigated and we found that caffeic acid and chlorogenic acid can inhibit increased TNF-? and IL-1? expression of HUVEC induced by AGEs-BSA and the suppressed activity of caffeic acid on the expression of TNF-? and IL-1? is stronger than the one of chlorogenic acid.In addition,the caffeic acid and chlorogenic acid can block the positive feedback loop of AGEs-RAGE.In summary,the suppressed activity of caffeic acid on the inflammation induced by AGEs-BSA is stronger than the one of chlorogenic acid.Finally,the effects of caffeic acid and chlorogenic acid on inflammatory signal pathway of HUVEC cells were investigated and we found that the caffeic acid and chlorogenic acid can inhibit the involved kinases expression in the the inflammatory signaling pathway of ROS/p38MAPK/NF-?B and result in the inhibition of AGEs-BSA-induced HUVEC inflammatory response.(3)The interaction of caffeic acid in combination with AGEs-BSA or BSA was investigated by fluorescence spectroscopy and UV-Vis spectrophotometry.the quenching mechanism of the interaction caffeic acid in combination with AGEs-BSA or BSA was studied by Stern-Volmer equation and ultraviolet-visible spectrophotometry and we found that the interactive mechanism of caffeic acid in combination with AGEs-BSA or BSA is a static quenching mechanism.In addition,linear regression is performed according to the binding constant formula to fit the value of n and we found that caffeic acid has only one binding site with AGEs-BSA or BSA,the binding molar ratio is 1:1,and the binding ability of AGEs-BSA and caffeic acid is stronger than the binding ability of BSA and caffeic acid.In the other hand,the thermodynamic parameters of the interaction of caffeic acid in combination with AGEs-BSA or BSA were calculated and we found that the interaction model of caffeic acid in combination with AGEs-BSA or BSA was mainly hydrophobic interaction force.Finally,the effect of caffeic acid on the conformation of AGEs-BSA or BSA was studied by synchronous fluorescence spectroscopy and we found that the effect of caffeic acid on the conformation of AGEs-BSA or BSA was minimal.(3)Suppressed mechanisms of caffeic acid and chlorogenic acid on the inflammation induced by AGEs-BSA were investigated.Caffeic acid and chlorogenic acid can inhibit the inflammatory responses induced by AGEs-BSA via intracellular mechanisms(the signaling pathway:AGEs-RAGE/ROS/MAPK/ NF-?B).In addition,there may be extracellular mechanisms involved in this process,one is the hydrophobic interaction of caffeic acid and chlorogenic acid with AGEs-BSA,the other one is that the corresponding quinones derived the oxidation of caffeic acid and chlorogenic acid trap the CML and result in the changed charges of the AGEs-BSA,then the interactions of AGEs and RAGE are affected.These achievements extend the application of caffeic acid and chlorogenic acid in the field of anti-inflammatory and provide theoretical support for the inhibition of AGEs-BSA induced inflammatory response by caffeic acid and chlorogenic acid,which broden the application of caffeic acid and chlorogenic acid in the field of wound healing. |
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