脂肪分化相关蛋白2对BCG诱导小鼠传代巨噬细胞自噬的调控作用

doi:10.11843/j.issn.0366-6964.2023.05.034

摘要/Abstract

摘要： 本研究旨在探讨脂肪分化相关蛋白2(perilipin 2,PLIN2)在卡介苗(bacillus calmette-gurin,BCG)感染的小鼠巨噬细胞RAW264.7中对自噬的调控作用。利用小干扰RNA敲减小鼠巨噬细胞RAW264.7中PLIN2的表达,结合BCG感染,通过免疫印迹、流式细胞术和免疫荧光等技术,检测胞内PLIN2、自噬、内质网应激及脂肪酸代谢相关因子指标的表达变化。结果显示:BCG感染显著上调巨噬细胞RAW264.7中PLIN2的表达(P＜0.05),极显著上调自噬相关蛋白ATG5(P＜0.01)、ATG12(P＜0.001)及LC3(P＜0.001)的表达,并伴随着细胞内中性脂质的蓄积。敲减PLIN2能使BCG感染的巨噬细胞RAW264.7中自噬相关蛋白ATG5(P＜0.05)和ATG12(P＜0.05)显著下调以及LC3(P＜0.001)极显著下调,细胞自噬率极显著降低(P＜0.001),并极显著降低细胞内中性脂质的含量(P＜0.001)。同时,敲减PLIN2显著下调CHOP(P＜0.05),并极显著下调ATF4(P＜0.001)内质网应激相关蛋白表达,细胞内Ca²⁺浓度极显著降低(P＜0.001)。此外,利用内质网应激激动剂Tunicamycin持续激活内质网应激通路后,无论是否敲减PLIN2,BCG感染的巨噬细胞内自噬相关蛋白表达均无显著差异(P＞0.05)。BCG感染巨噬细胞上调了PLIN2的表达,而PLIN2通过上调细胞内脂肪酸含量,进而级联激活PERK-eIF2α-ATF4-CHOP内质网应激信号通路,诱导细胞自噬发生。

关键词: PLIN2, BCG, 小鼠巨噬细胞RAW264.7, 内质网应激, 细胞自噬

Abstract: The aim of this study was to investigate the role of perilipin 2(PLIN2) in the regulation of autophagy in BCG infected mouse macrophages RAW264.7. In this study, we used small interfering RNA technology to knock down the expression of PLIN2 in murine macrophages RAW264.7, combined with BCG infection, and detected intracellular PLIN2 protein expression changes and indicators of autophagy, endoplasmic reticulum stress and fatty acid metabolism-related factors by immunoblotting, flow cytometry and immunofluorescence. BCG infection significantly upregulated the expression of PLIN2 (P<0.05) and highly significantly upregulated the expression of autophagy-associated proteins ATG5 (P<0.01), ATG12 (P<0.001) and LC3 (P<0.001) in macrophages RAW264.7 and was accompanied by intracellular neutral lipid accumulation. Knockdown of PLIN2 significantly downregulated autophagy-related proteins ATG5 (P<0.05) and ATG12 (P<0.05) and LC3 (P<0.001) in BCG-infected macrophages RAW264.7, significantly reduced autophagy rate (P<0.001) and significantly decreased intracellular neutral lipid content (P<0.001). At the same time, knockdown of PLIN2 significantly downregulated CHOP (P<0.05) and highly significantly downregulated ATF4 (P<0.001) endoplasmic reticulum stress-related protein expression, and intracellular Ca²⁺ concentration was highly significantly reduced (P<0.001). Finally, we treated cells with the endoplasmic reticulum stress agonist Tunicamycin and found no significant difference in intracellular autophagy-related protein. BCG infection of mouse macrophages RAW264.7 promoted the expression of PLIN2 and increased the accumulation of neutral lipids, which in turn activated the PERK-eIF2α-ATF4-CHOP signaling pathway in the endoplasmic reticulum stress pathway and induced the onset of cellular autophagy.

Key words: PLIN2, BCG, macrophage RAW264.7, endoplasmic reticulum stress, cellular autophagy

中图分类号:

王崇年, 于嘉霖, 宫照乾, 吴晓玲, 邓光存. 脂肪分化相关蛋白2对BCG诱导小鼠传代巨噬细胞自噬的调控作用[J]. 畜牧兽医学报, 2023, 54(5): 2134-2146.

WANG Chongnian, YU Jialin, GONG Zhaoqian, WU Xiaoling, DENG Guangcun. Regulation of BCG-induced Autophagy in Macrophages RAW264.7 by PLIN2[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2134-2146.

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