天津医药

天津医药 ›› 2021, Vol. 49 ›› Issue (2): 119-125.doi: 10.11958/20201991

• 细胞与分子生物学 • 上一篇    下一篇

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 #br#

饶超峰，薛笑楠，朱明英，罗富里   

  1. 1鹰潭市人民医院内分泌科（邮编335000）；2江西中医药大学附属医院肾病科

  • 收稿日期:2020-07-14 修回日期:2020-12-06 出版日期:2021-02-15 发布日期:2021-02-02
  • 通讯作者: 饶超峰 E-mail:lijun080708@163.com
  • 基金资助:
    江西省自然科学基金资助项目;鹰潭市科技计划项目

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

RAO Chao-feng, XUE Xiao-nan, ZHU Ming-ying, LUO Fu-li #br#   

  1. 1 Department of Endocrinology, Yingtan People’s Hospital, Yingtan 335000, China; 2 Department of Nephrology, Affiliated
    Hospital of Jiangxi University of Traditional Chinese Medicine
  • Received:2020-07-14 Revised:2020-12-06 Published:2021-02-15 Online:2021-02-02

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摘要：

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 28
mg/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。

关键词: 巨噬细胞, 外泌体, 足细胞, 自噬, 高糖

Abstract: ObjectiveTo 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- Ⅰ to LC3- Ⅱ in 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). ConclusionHG-treated macrophage derived exosomes can decrease podocyte activity and
increase podocyte damage, and its mechanism is related to the reduction of podocyte autophagy.

Key words: macrophages, exosomes, podocytes, autophagy, high glucose

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