| ObjectiveTo study the effects of lipid rafts on the process of inhibition in proliferation of Hepatic Stellate Cell through cannabinoid-2 receptors caused by AEA in rat.Methods1. Cell viability was investigated using 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay after different concentration of AEA and Methyl-β-cyclodextrin (MCD) treated.2. Cnr2-ShRNA was designed and transfected to hepatic stellate cells. Then, Cell viability was investigated by using MTT assay after different concentration of AEA and MCD treated.3. The levels of p-P38MAPK and p-JNK proteins in hepatic stellate cells were determined after different concentration of AEA and MCD treated by Western blot.4. Subcellular localization of lipid rafts and CB2 receptors was investigated by immunofluorescence. 5. The amount of CB2 receptors in lipid rafts was detected by Western blot after isolating lipid rafts from hepatic stellate cells by sucrose density gradient centrifugation.Results1. Cnr2-ShRNA was designed successfully. MTT assay showed either Cnr2-ShRNA cells or MCD treatment cells had noticeably higher ratio of cell proliferation compared with control cells. Interestedly, the ratio of cell proliferation was not obvious difference between Cnr2-ShRNA transfected cells and control cells with MCD treatment.2. The levels of p-P38MAPK and p-JNK proteins depended on the concentration of AEA and they can be antagonistic by MCD partially.3. LRs and CB2 receptors were co-localizated in memebrane of in Hepatic Stellate Cells, but CB2 receptors was little in LRs before anandmide's stimulation.Conclusions1. Lipid rafts are required for the process of inhibition in the proliferation of HSC caused by AEA through CB2 receptors. This process was associated with the p-P38MAPK and p-JNK signaling pathway.2. It is possible to be an effective treatment for liver fibrosis by controlling of lipid rafts, CB2 receptor and related signal in future targetment.
|
PDF Full Text Request