巨噬细胞外泌体通过抑制自噬诱导高血糖对肾小球足细胞的损伤作用

Effects of exosomes from high glucose-treated macrophage on the injury of glomerular podocytes via inhibiting autophagy

目的观察高糖(HG)处理的Raw264.7巨噬细胞来源的外泌体(Exos)对足细胞的损伤作用,并探讨其作用机制是否与抑制足细胞自噬相关。方法将高糖处理Raw264.7细胞的外泌体(HG-Exos)和原代足细胞体外孵育24 h,检测Exos在原代足细胞的内化情况。原代足细胞用HG提取不同剂量的Exos(0、7、14、28、56、112 mg/L)处理,探讨HG-Exos对足细胞存活、自噬的影响。将原代足细胞分为对照组、HG-Exos组、氯喹组、HG-Exos+氯喹组、雷帕霉素组、HG-Exos+雷帕霉素组,探讨HG-Exos诱导的细胞毒性是否会受到自噬调节剂的影响。采用透射电镜观察自噬体生成情况;采用CCK-8法测定Exos对细胞活力的影响。采用Western blot法检测细胞中LC3B、Beclin 1、Nephrin蛋白的表达。结果与正常葡萄糖处理的Raw264.7细胞相比,HG处理的Raw264.7细胞产生的Exos数量显著增加(P<0.05)。在激光共聚焦显微镜下观察到PKH67标记的Exos位于足细胞的核周区中。CCK-8法检测显示,足细胞活力随HG-Exos刺激剂量的增加和时间的延长而降低。与0 mg/L HG-Exos组比较,7 mg/L、14 mg/L和28mg/L HG-Exos组足细胞中Nephrin、Beclin 1、LC3B蛋白表达水平均明显降低,且含有双层膜结构的自噬小体数量显著减少(P<0.05)。雷帕霉素组、HG-Exos+雷帕霉素组Nephrin、Beclin 1、LC3B蛋白表达水平及自噬体数量均明显高于HG-Exos组、氯喹组和HG-Exos+氯喹组(P<0.05)。结论高糖诱导的巨噬细胞Exos可降低足细胞活力以及增加足细胞损伤,其作用机制与降低足细胞自噬有关。

Objective To investigate the effect of high glucose(HG)-treated Raw264.7 macrophage derived exosomes(Exos)on the podocyte injury,and to explore whether its mechanism is related to the inhibition of autophagy.Methods The exosomes from high glucose-treated Raw264.7 cells(HG-Exos)and primary podocyte cells were incubated for 24 h,and the internalization of Exos in primary podocyte cells was detected.Primary podocytes were treated with different doses of Exos(0,7,14,28,56 and 112 mg/L)extracted by HG to investigate the effects of HG-Exos on podocyte survival and autophagy.The primary podocytes were divided into control group,HG-Exos group,chloroquine group,HG-Exos+chloroquine group,rapamycin group and HG-Exos+rapamycin group.The formation of autophagy was observed by transmission electron microscopy.The effects of HG-Exos on the proliferation of primary podocyte cells were evaluated by CCK-8 assay.The expressions of LC3B,Beclin 1 and Nephrin protein were detected by Western blot assay.Results Raw264.7 cells treated by the HG produced an increased number of Exos compared to Raw264.7 cells treated with normal glucose(P<0.05).Confocal laser microscopy showed that PKH67-labeled Exos were localized to the perinuclear region of podocytes.CCK-8 assay showed that the podocyte viability reduced dose-and time-dependently after exposure to HGExos.Western blot assay showed that HG-Exos decreased the accumulation of Nephrin,Beclin 1 and the conversion of LC3-Ⅰto LC3-Ⅱin podocytes.Compared to 0 mg/L HG-Exos group,the autophagic double-membrane compartments containing lamellar structures in podocytes were significantly increased in the 7,14 and 28 mg/L HG-Exos groups(P<0.05).The expressions of Nephrin,Beclin 1 and LC3B,and the number of autophagosomes were significantly higher in rapamycin group and HG-Exos group+rapamycin group than those of HG-Exos group,chloroquine group and HG-Exos+chloroquine group(P<0.05).Conclusion HG-treated macrophage derived exosomes can decrease podocyte activity and increase podocyte damage,and its mechanism is related to the reduction of podocyte autophagy.