The Regulated Roles Of Acyl Coenzyme A Cholesterol Acyltransferases 1 In Macrophage Autophagy Induced By BCG Infection

Mycobacterium tuberculosis(Mtb)is a pathogen causing tuberculosis in humans and animals.Macrophages,as Mtb's main target cells and immune cells,play an important role in anti-Mtb infection.Studies have shown that macrophages can effectively eliminate Mtb through autophagy,however,Mtb can also evade the scavenging effect of macrophages autophagy through corresponding mechanisms.The acyl coenzyme A cholesterol acyltransferases(ACAT)protein family plays an important role in cholesterol absorption and metabolic balance in vivo.ACAT1 is a key enzyme in the esterification of free cholesterol and is widely found in animal tissues and cells.Studies have shown that high expression of ACAT1,affects intracellular cholesterol metabolism and promotes macrophage foaming,and can regulates macrophage autophagy.In order to explore the regulation mechanism of ACAT1 on autophagy of macrophages induced by Mtb,ACAT1 inhibitor Avasimibe and mTOR inhibitor Rapamycin were used to treat RAW264.7 and bovine alveolar macrophages(BAMs),and overexpressing/interfering with ACAT1 in RAW264.7 cells were adopted on the basis of BCG-infected macrophages in this study.The levels of ACAT1 protein expression,autophagy-related proteins expression and intracellular cholesterol levels were detected.The experimental results are as follows:1.BAMs and RAW264.7 cells were infected by BCG for 2h,6h,12h and 24h,with the expression of ACAT1 protein,cholesterol ester content and intracellular lipid droplets in macrophages decreased first and then increased over time and all reached the lowest at 12h post-infection in RAW264.7 cells and at 6h post-infection in BAMs,while free cholesterol changed conversely.With the prolongation of BCG infection time,the expression levels of autophagy-related proteins,Beclinl and LC3?/I increased first and then decreased over time,and all reached the highest at 12h post-infection in RAW264.7 cells and at 6h post-infection in BAMs.The results suggest that BCG infection with macrophages,ACAT1 may affect autophagy level by changing intracellular cholesterol level.2.The RAW264.7 stably transfected cell lines with ACAT1 overexpression/interference was successfully obtained by lentiviral transfection method and were verified by RT-PCR and western blot,which provided experimental materials for further study on the regulation of ACAT1 in BCG-induced macrophage autophagy.3.BAMs and RAW264.7 cells were treated with Avasimibe and infected by BCG,the expression of autophagy-related proteins,Beclinl,Atg5,Atg7 and LC3II/I were significantly increased at 12h post-infection in RAW264.7 cells and BAMs.The results indicated that inhibition of ACAT1 enhances BCG-induced autophagy in macrophages.4.Overexpressed ACAT1 RAW264.7 cells were infected by BCG,the expression of autophagy-related proteins,Beclinl,Atg5,Atg7 and LC3?/? and free cholesterol content were significantly decreased,while cholesterol ester content was significantly increased at 12h post-infection in overexpressed AC ATI RAW264.7 cells;after interfered ACAT1 RAW264.7 cells were infected with BCG,the expression of autophagy-related proteins,Beclinl,Atg5,Atg7 and LC3?/?and free cholesterol content was significantly increased,while cholesterol ester content was significantly decreased at 12h post-infection in interfered ACAT1 RAW264.7 cells.The results showed that ACAT1 enhanced autophagy by reducing intracellular cholesterol ester content after BCG infection of macrophages.5.BAMs and RAW264.7 cells were treated with Rapamycin and infected by BCG,the expression levels of ACAT1 protein was significantly down-regolated at 12h post-infection in RAW264.7 cells and BAMs.The results showed that after BCG infection of macrophages,PI3K/mTOR signaling pathway regulates the autophagy level of macrophages induced by BCG by affecting the expression of ACAT1.6.ACAT1 was interferened or inhibited,and BCG infection of macrophages,the expression levels of PI3K/mTOR signaling pathway-related proteins PI3K,mTOR^P-mTOR and P-Akt protein were significantly down-regulated at 12h post-infection;while ACAT1 was overexpressed,and BCG infection of overexpressed ACAT1 RAW264.7 cells,the expression of PI3K/mTOR signaling pathway-associated proteins PI3K,mTOR and P-mTOR were significantly up-regulated at 12h post-infection.These results indicated that ACAT1 regulates autophagy mediated by PI3K/mTOR signaling pathway after BCG infection of macrophages.In summary,the research results show,after BCG infection of macrophages,ACAT1 participates in the regulation of autophagy in macrophages by affecting intracellular cholesterol levels and synergistically interacting with PI3K/Akt/mTOR signaling pathway.