Lipid rafts but not putative receptors mediate anandamide inhibited hepatic stellate cell proliferation

Qiao Yang, Yao-wen Zhang, Hong-yan Liu, Wang-xian Tang
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

An increasing body of evidence about anandamide (AEA) has indicated its role in the systemic circulation disturbance in cirrhosis. However, its precise physiological role in liver fibrosis remains to be elucidated. This study determined the mechanism of AEA on hepatic stellate cell (HSC). By using MTT, we investigate the effect of AEA and its CB1 and CB2 receptor antagonists (SR141716A and AM630) on the proliferation of HSC. Meanwhile, we adopted nuclear staining of Hoechst 33258, Western-blot and flow cytometry(FCM) to detect the mechanism of AEA on HSC. Because AEA could interact with lipid rafts (LRs) on cell membrane, the LRs on HSC were detected by confocal microscopy. We also studied if the membrane cholesterol depletory-methyl-β-cyclodextrin (MCD) exhibits an inhibitory effect on anandamide-induced HSC death. We present evidence that AEA trigger potently grow inhibition in HSC, in a concentration-dependent manner (5umol/L -20umol/L), showed by MTT. Nuclear staining, Western-bolt and Annexin V-PE/7AAD binding assay showed that AEA induce HSC necrosis but not apoptosis. Whereas, cannabinoid receptors antagonists failed to block anandamide-induced cell death. Moreover, LRs on HSC were shown by confocal microscopy, and MCD to counteract the anandamide-induced HSC death (P<0.05). In conclusion AEA could potently suppress the proliferation of HSC via the membrane cholesterol-LRs but not putative receptors, LRs may be functioning as anandamide receptors in HSC. (Supported by National Nature Science Foundation of China NO.30571627)