Endoplasmic reticulum stressed hepatocellular carcinoma promotes macrophage M2 polarization through exosome-toll-like receptor 4 pathway / 中国药理学通报

ABSTRACT

Aim To explore the effect of endoplasmic reticulum (ER) stress-related exosomes derived from hepatocellular carcinoma (HCC) cells on the M2 polarization of macrophages via Toll-like receptor 4 (TLR4) and the underlying mechanism. Methods Western blot experiments were conducted to evaluate the GRP78 levels in HCC tissues and HCC cells with or without pre-treatment of tunicamycin and the identification of the isolated exosomes. Immunohistochemical staining was also employed to detect the expression of GRP78 in HCC tissues. Immunofluorescence was used to confirm the effective absorbance of exosomes by macrophages. Flow cytometry ( FCM ) , Western blot and CBA kit were used to detect M2 polarization-asso ciated markers and the expression of TLR4 on macrophages. Affymetrix gene chip and bioinformatics analy-sis were used to screen the differential expression of mRNA in macrophages stimulated by ER stressed-relat- ed exosomes and TLR4 expression. Results In clinical samples, GRP78 expression in liver cancer tissues was higher than that in the adjacent tissues. In cell experiments, tunicamycin (2.5 jxmol • L"1 ) treatment for 24 hours in HCC cells could significantly increase the expression of ER stress marker protein GRP78 compared with control group. Western blot showed that HCC cell-derived exosomes positively expressed exosom- al marker proteins,such as CD63,TSG101 and HSP70, and negatively expressed Calnexin. Interestingly, HSP70-enriched exosomes released by ER-stressed HCC cells were observed. Laser confocal microscopy found that exosomes could be effectively taken up by RAW264.7 macrophages. Flow cytometry and Western blot further indicated that ER stress-related exosomes could significantly up-regulate the expression of CD206, transformed growth factor-beta (TGF-p) and arginase-1 (Arg-1) in macrophages. Meanwhile, these exosomes could promote the secretion of IL-6 and IL- 10,and up-regulate the expression of TLR4 in macrophages. Conclusions ER stressed-HCC cells could promote macrophage M2 polarization by releasing HSP70-enriched exosomes and activating TLR4 signaling.