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To observe the effect of Nε-carboxymethyl-Lysine (CML) on apoptosis in foam cells.
The present study consisted of three in vitro investigations. ①RAW264.7 cells were generated by incubating with 50μg/mL oxLDL and various contents of CML for 48 h at 37°C, whereas control group were incubated without oxLDL for the same period, so as to test the hypothesis that CML could induce macrophages transformation into foam cells and the apoptosis of macrophages and select the best dose of it. ②RAW264.7 cells were randomly assigned into four groups: control group, oxLDL group, CML group and oxLDL plus CML group. After that, relative indexes of endoplasmic reticulum stress, such as GRP78, P-PERK, P-eIF2ɑ, ATF-4, CHOP and caspase-3 were detected. ③The transmission pathway mediated the specific mechanism of CML-induced apoptosis via endoplasmic reticulum stress was investigated in RAW264.7 cells under high-lipid and pro-apoptotic condition. Oil red O staining, plasma lipid and glucose measurement, intracellular cholesterol content assay, annexin V-FITC/PI double-staining,flow cytometry, immunohistochemical staining and Western blot were then performed.
Oil red O staining and cholesterol oxidase method demonstrated CML could dramatically increase the free cholesterol, cholesterol ester and total cholesterol content in foam cells. Western blot indicated CML could significantly promote the expression of RAGE, CD36, GRP78, P-PERK, P-eIF2ɑ, ATF-4, CHOP and caspase-3, but down-regulate the expression of ABCA1. Further investigation suggested that 10 μmol/L CML up-regulated the content of the above endoplasmic reticulum stress-related indexes, which were dramatically higher than that of 50 μg/mL oxLDL. Aannexin V-FITC/PI double-staining, flow cytometry analysis as well as immunocytochemistry staining showed CML could efficiently up-regulate the total rate of apoptotic cells and the expression ofactivated caspase-3 in macrophage-derived foam cells. Further investigation blocked caspase-12, JHP, CHOP or RAGE by special antibodies or inhibited them by antagonists by incubating with CML and oxLDL. Then the semi-quantitative analysis indicated that CHOP pathway could mainly transmit the CML-induced endoplasmic reticulum stress in foam cells and caspase-12 may play a minor role in apoptosis signal.
CML-induced apoptosis in RAW264.7 macrophages under high-lipid conditions may mainly transmit through CHOP pathway. Further study may target on the inhibition of CML/RAGE pathway so as to search for effective therapy in preventing atherosclerosis in diabetics.