Study On Phlegm Syndrome Mechanism Of Coronary Heart Disease From The Metabolism Mechanism Of Glycosaminoglycan GAG

Research background and purposeThe glycosaminoglycan GAG and proteoglycan PG in extracellular matrix ECM can explain the characteristics of "phlegm" about "sticky" in many aspects,such as "retention" pathogenic factors.It is a good breakthrough point for studying the pathogenesis of"phlegm" syndrome.In particular,chondroitin sulfate CS/sulfate dermatan DS PG,as a widely existing biological macromolecule in ECM,plays a role in coronary heart disease.It has important pathogenicity.Many literatures have studied the levels of GAG and PG in the aortic wall under pathological and physiological conditions.This paper also explores the differences and possible mechanism between phlegm syndrome and non phlegm syndrome in patients with coronary heart disease through GAG.MethodsThe patients were from the Cardiology Department,University Town Branch of Guangdong Province Traditional Chinese medical Hospital.The content of serum TGF-?1 with phlegm syndrome was determined by ELISA assay,,the expression of CD44v6 was determined by Q-PCR,and the expression of CD44 was confirmed by Western blot.The sera GAG of the patients with phlegm syndrome and non phlegm syndrome of coronary heart disease and the GAG of the mice vascular smooth muscle cells cultured by sera were extracted.The content of GAG was determined by phenol sulfate method,carbazole sulfate method and cellulose acetate membrane electrophoresis method.The GAG was extracted after the intervention of traditional Chinese medicine monomer peiminine,hesperidin and PlatycodinD on MOVAS modeled by oxLDL.The content of GAG was determined by carbazole sulfate method.The time rule of GAG secretion was determined by Alcian blue staining and the type of GAG secreted at 24 and 48 hours was determined by lectins assay.The type of PG was determined by Western Blot.The GAG was extracted after the intervention of SB525334(for ALK),wortmannin(for PI3K),mk2206(for Akt)on MOVAS cultured by sera with phlegm syndrome and non phlegm syndrome of coronary heart disease.The enzyme of CSPG----ADAMTS4 and enzymatic fragment of versican by ADAMTS1/4 were confirmed by Western blot.The proliferation rate of MOVAS was determined by bFGF-GAG-FGFR ternary complex method.The expression of NF-?B and collagen hydrolase FAP in the downstream of PI3K/Akt was confirmed by Western blot.ResultsCompared with non phlegm syndrome,there was no significant difference in serum GAG of phlegm syndrome.After the serum of phlegm syndrome patients with similar blood glucose level acts on the smooth muscle cells,the GAG is higher than that of non phlegm syndrome patients,and the content of GAG extracted from MOVAS modeled by oxLDL is higher.The content of GAG extracted from modeling MOVAS intervened by three kinds of Chinese medicine monomers all reduces to close to the normal control group.The phenomenon that GAG increases in MOVAS cultured by serum of phlegm syndrome has been proved to exist directly by various methods and indirectly by intervention of Chinese medicine.Further analysis on the time rule of GAG secretion showed that this phenomenon mainly occurred in MOVAS cultured by serum with phlegm syndrome after 48 hours,the increased GAG was mainly CS,the corresponding CSPG was versican and biglycan;compared with non phlegm syndrome,the serum TGF-?1 with phlegm syndrome was increased;the trend of MOVAS intervened by TGF-?1 alone and MOVAS cultured by phlegm syndrome serum was the same;Inhibited Smad pathway,the effect of increasing GAG extracted from MOVAS cultured by phlegm syndrome was still higher than that by non phlegm syndrome group,but Inhibited PI3K pathway,the effect of increasing GAG extracted from MOVAS cultured by phlegm syndrome was eliminated.The increasing expression of ADAMTS4 and enzymatic fragment in MOVAS cultured by serum of phlegm syndrome indicates that TGF-?1 makes the GAG in MOVAS cultured by serum of phlegm syndrome higher than that of non phlegm syndrome by down regulating the degradation of CSPG.The increasing of ternary complex,NF-x B p65 and FAP,CD44 and CD44v6 in phlegm syndrome group showed that phlegm syndrome serum could induce the proliferation of MOVAS,ECM remodeling and inflammatory reaction.ConclusionsThe increase of TGF-?1 in the serum of patients with phlegm syndrome of coronary heart disease may cause the increase of GAG-CSPG,versican and biglycan by activating Smad pathway of vascular smooth muscle cells,and further increase of phlegm syndrome compared with non phlegm syndrome by inhibiting the degradation of these PG by down regulating ADAMTS4 through PI3K/Akt pathway.Expectorant herbs can reverse the increase of GAG in oxLDL model.PI3K/Akt is an important pathway for cell proliferation(especially tumor development).Different downstream signals can cause 1.Proliferation of vascular smooth muscle cells 2.Elevation of matrix hydrolase such as FAP 3.Inflammatory response recruited by NF-kB.Among them,the increase of CD44(especially CD44v6)in the blood of patients with phlegm syndrome also proves the high inflammatory state of patients with phlegm syndrome.