Scavenger Receptor A Pathway Inhibits Endoplasmic Reticulum Stress-induced Autophagy In Macrophage

Objectives:The aims of this study were to determine the effects of fucoidan, one of the ligands of scavenger receptor A, on ER stress-induced macrophage autophagy and the mechanisms underlying the process. This research would be useful to provide the theoretical evidence of SR-A in the macrophage apoptosis during the development of atherosclerosis.Methods:This study first used thapsigargin (Tg) to copy of endoplasmic reticulum stress model in the RAW264.7 cell line. We used Western blot to measure the expression of LC3 in the RAW264.7 cells incubated by Tg or by Tg plus fucoidan. To confirm the role of fucoidan on ER stresse-induced autophagy, we used the laser confocal microscope to observe the formation of autophagosomes in the different treatments. On the basis of the findings of fucoidan playing the role in the ER stress-induced autophagy in macrophages, we further explored the mechanisms during the processes. Western blot was used to detect the phophorylation activity of potential signaling molecules, such as AKT,mTOR,p70S6K. Rapmycin, the mTOR inhibitor, was used later to estimate the role of AKT/mTOR/p70S6K pathway playing in the fucoidan treatment of ER stress-induced autophagy in macrophages. The AnnexinV/PI staining and Western blot were used to detect the apoptosis of macrophages treated by Tg plus fucoidan when AKT/mTOR/p70S6K pathway was inhibited. In the end, we used the peritoneal macrophages of SR-A gene knock out and wild type mice to determine the role of SR-A on fucoidan-activated AKT/mTOR/p70S6K pathway.Results:We used the RAW264.7 cell line for exploration first. We measured the expression of LC3 in the RAW264.7 cells incubated by Tg or by Tg plus fucoidan using western blot methods. It came out that the expression of LC3-â…¡in ER stressed macrophages is inhibited by addition of fucoidan. We then confirmed the results using the laser confocal microscope to observe the formation of autophagosomes in the different treatments, and the result is consistent with the findings of western blot, ER stressed formation of autophagosomes was inhibited by addition of fucoidan. On the basis of the findings,we concluded that fucoidan could inhibit ER stressed autophagy.Then,we further explored the mechanisms during the processes.We tried to examine the activation of the mTOR pathway. We found the phosphorylation of AKT,mTOR and p70S6K could be obviously activated by treatment of Tg plus fucoidan,compared with treatment of Tg only at 4 hours after treatment.From the results, we deduced that fucoidan could activate the mTOR pathway.In order to detect the indicated effectiveness of inhibition of ER stress-induced autophagy by fucoidan,we use Rapmycin to block the AKT/mTOR/p70S6K pathway. The AnnexinV/PI staining and Western blot assays revealed that when the AKT/mTOR/p70S6K pathway was inhibited, fucoidan would lose the effects in inhibition of ER stress-induced autophagy in macrophages and reversal of the pro-apoptosis of macrophages induced by ER stress plus fucoidan.At the end of the experiment, we used the peritoneal macrophages from SR-A gene knock out mice and wild type mice to detect the role of SR-A in the fucoidan-activated AKT/mTOR/p70S6K pathway. It came out that the AKT/mTOR/p70S6K pathway could be activated by treatment of Tg plus fucoidan,compared with treatment of Tg in wild type macrophages, but there wasn't significant activation of AKT/mTOR/p70S6K pathway in the SR-A-/- macrophages.So,we could conclude that the activation of AKT/mTOR/p70S6K pathway by fucoidan was mediated by SRA.Conclusion:Fucoidan, as one of the ligands of scavenger receptor A, can inhibit the ER stress-induced autophagy through AKT/mTOR/p70S6K pathway, and promote the apoptosis of ER stressed macrophages.