BCG诱导RAW264.7细胞脂肪酸氧化对细胞自噬和促炎因子表达的调控作用

旨在探究脂肪酸氧化（fatty acid oxidation，FAO）对BCG介导的RAW264.7细胞自噬和促炎因子表达的调控作用。用BODIPY染色和游离脂肪酸定量试剂盒检测BCG感染后RAW264.7细胞中脂滴聚集情况以及脂肪酸含量；Western blot检测BCG感染对肉毒碱棕榈酰基转移酶1A （CPT-1A）表达的影响；Etomoxir （100 μmol·L^-1）预处理细胞2 h后，BCG感染细胞6 h，检测RAW264.7细胞中BCG存留量，并用Western blot方法检测自噬相关蛋白（Beclin1、LC3-II）和溶酶体蛋白（Rab7）的表达情况；用免疫荧光方法和mRFP-GFP-LC3荧光双标腺病毒分别检测自噬小体聚集和自噬流；荧光定量PCR和ELISA分别检测促炎因子IL-1β、IL-6和TNF-α mRNA表达情况以及在细胞培养上清中的含量。结果显示，BCG感染促进RAW264.7细胞中脂滴聚集和CPT-1A的表达，而游离脂肪酸含量降低；Etomoxir预处理抑制了细胞中BCG存活，并上调了Beclin1、LC3-II和Rab7表达，且细胞中出现大量自噬小体聚集，自噬流增强，却抑制了促炎因子IL-1β、IL-6和TNF-α mRNA表达与分泌。综上表明，抑制FAO可促进BCG感染诱导的RAW264.7细胞自噬，并抑制BCG感染引起的炎症反应。

The Effect of BCG-induced RAW264.7 Cells Fatty Acid Oxidation on Autophagy and Pro-inflammatory Cytokines Expression

To investigate the effect of fatty acid oxidation (FAO) on autophagy and the expression of pro-inflammatory cytokines in BCG-infected RAW264.7cells. The accumulation of lipid droplets and the content of fatty acids inside RAW264.7 cells after BCG infection were detected by BODIPY staining and free fatty acid quantitative kit. The expression of carnitine palmityl transferase 1A (CPT-1A) in BCG-infected cells was detected by Western blot. After pretreatment with Etomoxir (100 μmol·L^-1) for 2 h, BCG was infected with RAW264.7 cells for 6 h, intracellular survival BCG was assessed by CFU assay, and the expression of Beclin1, LC3-II and lysosomal protein (RAB7) were detected by Western blot. The aggregation of autophagosomes and autophagy flux were detected by immunofluorescence and mRFP-GFP-LC3 adenovirus. Pro-inflammatory cytokines IL-1β, IL-6 and TNF-α mRNA and their concentration in cells culture supernatant were detected by fluorescence quantitative PCR and ELISA, respectively. The results showed that BCG infection promoted the accumulation of lipid droplets and the expression of CPT-1A in RAW264.7 cells, while free fatty acid content decreased. Etomoxir treatment inhibited intracellular BCG survival and elevated the expressions of Beclin1, LC3-II and RAB7. Besides, a large number of autophagosomes were aggregated and autophagy flux was enhanced, however, the mRNA expressions and secretion of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α were all inhibited. It is suggested that FAO inhibition promotes RAW264.7 cells autophagy and inhibits the inflammatory response induced by BCG infection.