The Mechanism Of Acidic Environment Affecting The Synthesis And Release Of Cellular Ganglioside GM3

Cardiovascular disease and tumor are the two main types of diseases that seriously threaten human beings.Atherosclerosis is the root cause of some cardiovascular diseases(such as coronary heart disease,myocardial infarction and stroke,which are the most well-known ones),and tumors are also extremely harmful to people.Gangliosides are a class of sialic acid-containing glycolipids,and GM3 is a type of ganglioside.A large body of evidence shows that the content of ganglioside GM3 is significantly increased in atherosclerotic plaques and certain tumor tissues.Atherosclerosis and tumors have another feature in common: both have an acidic extracellular microenvironment.Moreover,a large number of studies have reported that the acidic microenvironment plays an important role in the formation and development of atherosclerosis and tumors,affecting the activity and function of the relevant cells living in it.We speculate that the acidic microenvironment may lead to the high expression and release of cellular ganglioside GM3.In order to verify the above speculation,we set different acidic conditions(p H7.0,6.5,6.0,5.5,5.0,4.5;p H7.4 was used as a neutral control),and four kinds of cells(including mouse macrophage Raw264.7,Human hepatoma cells SMMC-7721,mouse colon cancer cells CT26,and human umbilical vein endothelial cells(HUVEC)were probed for GM3 expression and release.first,we tested the tolerance of cells to different acidic conditions at different times(6 hours,8 hours,12 hours),and found that the viability of various types of cells was significantly increased after six hours of treatment under acidic conditions of p H 4.5.Influence.In order to explore the changes of intracellular ganglioside GM3 under acidic conditions,we used anti-GM3 antibody to perform immunofluorescence staining on GM3 molecules on the cell membrane surface,and then observed and quantitatively analyzed by fluorescence confocal microscopy and flow cytometry.The environment can significantly increase the expression of GM3 on the surface of different cell lines,and the fluorescence of cells reaches the strongest at p H 6.5-5.5.To determine the effect of acidic environment on intracellular GM3 content and GM3 released to the outside of cells,gangliosides were extracted from cells and supernatants,and GM3 concentrations were measured using high performance liquid chromatography.The results showed that the intracellular GM3 content was significantly higher than that of the neutral group(p H 7.4)under acidic conditions,and more GM3 was released to the outside of the cells.In order to explore the underlying mechanism of the effect of acidic environment on the expression and release of cellular GM3,we first measured the m RNA expression of GM3 synthase(GM3S or ST3GAL5)using real-time quantitative PCR,and found that acidic environment can lead to the increase of GM3 S expression.The gene silencing of intracellular GM3 S was further performed by RNA interference technology,and the GM3 synthase agonist valproic acid(VPA)was used as a positive control.The above results indicated that the elevation of intracellular GM3 under acidic conditions was achieved by promoting the expression of GM3 synthase.A large body of literature reports that acidity and hypoxia are interdependent in atherosclerotic plaques and tumor microenvironments.Hypoxia-inducible factor HIF-1α(hypoxia-inducible factor 1,HIF-1α)is expressed under hypoxic conditions.We found that acidic conditions increased the m RNA expression of HIF-1α in mouse macrophages(RAW264.7).To further explore the relationship between acid-induced expression and release of GM3 and hypoxia-inducible factor HIF-1α,we used the overexpression vector and inhibitor of HIF-1α and the RNA interference vector and agonist of GM3 synthase by real-time quantitative PCR technology.and Western Blot Western blotting technology for further verification.We found that the m RNA and protein of GM3 synthase and HIF-1α m RNA and protein had a synergistic relationship with each other.The above results suggest that the effect of acidic environment on the expression and release of cellular GM3 is likely to be closely related to the hypoxiainducible factor-related signaling pathway.In conclusion,the results of this paper show that the acidic environment can promote the expression and release of ganglioside GM3 by regulating the expression of GM3 synthase,and it is closely related to the signaling pathway related to hypoxiainducible factor.The results of the study preliminarily verified our hypothesis and also implied a potential close relationship between the acidic microenvironment and the high GM3 microenvironment in atherosclerotic plaques or some tumor tissues.This study is the first to elucidate the potential formation mechanism of high GM3 microenvironment in atherosclerosis or tumor tissue and its relationship with the acidic microenvironment,which has important scientific significance for in-depth understanding of the pathological mechanism of these diseases.