Role Of FTO In Fatty Acid Regulation Of Macrophage Polarization And Lipid Droplet Formation

With the enrichment of material life,the problem of obesity has become With the enrichment of material life,the problem of obesity becomes more and more prominent.Obesity can induce chronic inflammation,and then induce a series of diseases such as diabetes and atherosclerosis.It has been found that fat content and fat related genes(FTO)play an important role in obesity,which can inhibit metabolism,reduce energy consumption efficiency,and inhibit the expression of pro-inflammatory factors.Macrophages play an important role in the immune process of the body.They play an important role in promoting inflammation by activating the classic M1 polarization,and choose the alternative M2 polarization to play an anti-inflammatory role.Because the different metabolism of cells can affect the function of cells,lipid metabolism may affect the anti-inflammatory function of macrophages.In order to better explore the role of FTO in macrophages,this study explored the effects of FTO on macrophage polarization and the molecular mechanism related to the regulation of lipid accumulation.In order to explore the role of FTO in regulating macrophage polarization,we used RAW264.7 macrophages as experimental model,and used LPS or IL-4 to induce M1 and M2 polarization of macrophages,respectively.According to the needs of the experiment,we transfected FTO overexpression vector,FTO mutant plasmid and si RNA that can knock down FTO.Then RT-q PCR was used to detect the relative m RNA expression levels of IL-1 ?,IL-6,TNF-? and arg1,MRC and MGL,which are the markers of M1 polarization,and Western blot was used to detect and verify the protein expression levels.The results showed that in the effect of FTO on macrophage polarization,overexpression of FTO could inhibit M1 polarization and promote M2 polarization of macrophages,thus inhibiting the pro-inflammatory and anti-inflammatory effects.On the contrary,knockdown of FTO can promote M1 polarization and inhibit M2 polarization,which is consistent with the effect of overexpression of FTO on macrophage polarization.In addition,this study also verified the effect of FTO demethylase activity on the polarization of macrophages.After transfection of FTO mutant plasmid,the expression level of M1 and M2 polarization markers of macrophages was detected.It was found that the RNA demethylase activity of FTO is necessary for FTO to play an anti-inflammatory role.In order to explore the role of FTO in the regulation of fatty acid on macrophage polarization,this study used the medium containing fatty acid to treat macrophages.Under the stimulation of fatty acids,the expression of FTO in macrophages is up-regulated,and FTO can regulate the polarization of macrophages,so we also detected the expression level of macrophage markers under M1 or M2 polarization.The experimental results show that fatty acids may affect the polarization of macrophages by up regulating FTO,that is,inhibiting M1 polarization of macrophages and then inhibiting its pro-inflammatory effect,It can promote M2 polarization of macrophage and enhance its anti-inflammatory effect.In order to further verify the experimental results,we knock down FTO to detect the expression of related genes again.The results are consistent with the above results,and its anti-inflammatory effect is weakened.Previous studies in our laboratory have shown that FTO can increase lipid droplet accumulation in hepatocytes,but the effect of FTO on lipid droplet accumulation in macrophages is not clear.Therefore,in order to explore the effect of FTO on the accumulation of lipid droplets in macrophages,we first carried out oil red O staining experiment.The results showed that the lipid droplets of macrophages increased when fatty acids were added to cell culture medium.Because lipid droplets can enter the cell through CD36 receptor on the surface of macrophage as a channel,we detected the expression level of CD36 and found that fatty acids can significantly up regulate the expression of CD36 on the surface of macrophage,so fatty acids may stimulate the up regulation of CD36 to promote the accumulation of lipid droplets in RAW264.7 cells.Knockdown of FTO reduced the accumulation of lipid droplets in macrophages induced by fatty acids,and down regulated the expression of CD36 m RNA.Therefore,we suggest that FTO may regulate the expression of CD36 to regulate the intake of fatty acids and the formation of lipid droplets in macrophages.In conclusion,our results show that FTO can influence the polarization of macrophages and the accumulation of lipid droplets induced by fatty acids,so as to regulate the anti-inflammatory effect of macrophages.