乳脂肪球表皮生长因子8（MFG-E8）对脂多糖诱导小胶质细胞炎症反应的抑制作用及其机制

目的　探讨乳脂肪球表皮生长因子8（MFG-E8）对脂多糖（LPS）诱导小胶质细胞（BV-2细胞）炎症反应的抑制作用，并探讨可能作用机制。方法　CCK-8法检测0~200 mg·L^-1 MFG-E8对细胞活力影响。建立LPS诱导BV-2细胞视网膜变性疾病体外模型，分为对照组、LPS组、LPS + MFG-E8组、LPS + LY294002组和LPS + PDTC组。倒置显微镜观察BV-2细胞的形态改变，ELISA检测肿瘤坏死因子-α（TNF-α）的表达变化，Western blot检测TNF-α和白细胞介素-6（IL-6）的蛋白表达变化，及PI3K-Akt和NF-κB相关通路蛋白表达变化。结果　0 mg·L^-1、10 mg·L^-1、50 mg·L^-1、100 mg·L^-1和200 mg·L^-1的MFG-E8对BV-2细胞的活性无明显影响，细胞活力差异无统计学意义（P>0.05）；MFG-E8对LPS诱导的BV-2细胞形态变化具有一定的逆转作用；ELISA检测结果显示，LPS组BV-2细胞中TNF-α的含量较对照组明显升高（P<0.001），LPS + MFG-E8组BV-2细胞中TNF-α的含量较LPS组显著下降（P<0.001）。Western blot检测结果显示，LPS组BV-2细胞中TNF-α和IL-6蛋白相对表达量均显著高于对照组（均为P<0.05），LPS + MFG-E8组BV-2细胞中TNF-α和IL-6蛋白相对表达量均较LPS组明显降低（均为P<0.05）。LPS组BV-2细胞中NF-κB p-p65蛋白相对表达量明显高于对照组，p-PI3K、p-Akt蛋白相对表达量均明显低于对照组（均为P<0.001）；LPS + MFG-E8组BV-2细胞中NF-κB p-p65蛋白相对表达量较LPS组明显降低，p-PI3K、p-Akt蛋白相对表达量均较LPS组明显升高（均为P<0.001）。结论　MFG-E8可能是通过抑制NF-κB信号通路的激活，从而减轻LPS诱导的小胶质细胞炎症反应。

Inhibiting effect and mechanism of MFG-E8 on lipopolysaccharide-induced microglial inflammatory response

Objective　To investigate the inhibitory effect and mechanism of milk fat globule epidermal growth factor 8 (MFG-E8) on lipopolysaccharide (LPS)-induced microglia (BV-2 cell) inflammatory response.Methods　The effect of 0-200 mg·L^-1 MFG-E8 on cell viability was detected by CCK-8 kits. An in-vitro model of LPS-induced BV-2 cell retinal degeneration was established, dividing into five groups: control group, LPS group, LPS + MFG-E8 group, LPS + LY294002 group and LPS + PDTC group. The morphological change of BV-2 cells was observed by inverted microscope. The expression change of tumor necrosis factor α (TNF-α) was detected by ELISA. The changes in protein expression levels of TNF-α, interleukin-6 (IL-6), PI3K-AKT and NF-κB related pathways associated protein were detected by Western blot. Results　Different concentrations of MFG-E8 (0 mg·L^-1, 10 mg·L^-1, 50 mg·L^-1, 100 mg·L^-1 and 200 mg·L^-1) had no significant effect on the viability of BV-2 cells (P>0.05). MFG-E8 could reverse the morphological changes of BV-2 cells induced by LPS. ELISA results revealed that the concentration of TNF-α in BV-2 cells in the LPS group was significantly higher than that in control group (P<0.001). Compared with the LPS group, the concentration of TNF-α in BV-2 cells was significantly decreased in LPS + MFG-E8 group (P<0.001). Western blot showed that the relative protein expression levels of TNF-α and IL-6 in the LPS group were significantly higher than those in control group (both P<0.05). Compared with the LPS group, the relative protein expression levels of TNF-α and IL-6 were significantly decreased in LPS+ MFG-E8 group (both P<0.05). The relative protein expression level of NF-κB p-p65 in BV-2 cells in LPS group was significantly higher than that in the control group, while the relative protein expression levels of p-P13K and p-Akt were obviously lower than those in the control group (all P<0.001). The relative protein expression level of p-p65 in BV-2 cells in LPS + MFG-E8 group was lower than that in the LPS group, while the relative protein expression levels of p-P13K and p-Akt were obviously higher than those in the LPS group (all P<0.001). Conclusion　MFG-E8 may alleviate LPS-induced microglial inflammatory response by inhibiting the activation of NF-κB signaling pathway.