高糖环境下肾小管上皮细胞来源外泌体诱导巨噬细胞向肌成纤维细胞转化

Exosomes from high glucose-treated renal tubular epithelial cells induce macrophage-to-myofibroblast transformation

目的 探讨高糖环境下肾小管上皮细胞来源外泌体诱导巨噬细胞向肌成纤维细胞转化的作用与机制。方法 正常糖(5.5 mmol/L)及高糖(30.0 mmol/L)分别处理人肾小管上皮细胞(HK2)48 h,收集上清液提取并鉴定外泌体;观察人急性单核细胞白血病细胞株(THP-1)巨噬细胞是否吞噬PKH67标记的外泌体;通过检测诱导性氮氧化物合酶(iNOS)、α-平滑肌肌动蛋白(α-SMA)、甘露醇受体(CD206)的表达,以确定分化为M1和M2型巨噬细胞的最佳浓度及时间点;激光共聚焦检测CD206与α-SMA、IV型胶原(Col-IV)、纤维连接蛋白(FN)的荧光共表达。qRT-PCR与ELISA法测定转化生长因子受体-β1(TGF-β1)、白细胞介素(IL)-10、IL-6水平。Western blot测定TGF-β1、Smad3和p-Smad3蛋白表达。结果 上清液离心获取标本后检测到白细胞分化抗原群63(CD63)和肿瘤易感基因101蛋白(TSG101)阳性、内质网分子伴侣蛋白(Calnexin)阴性,确认为外泌体并且纯度较高;THP-1巨噬细胞能够吞噬各组外泌体;各组外泌体刺激的最佳浓度为40 mg/L、最佳时间为96 h。高糖外泌体刺激24 h巨噬细胞以M1型为主,96 h则以M2型为主。与正常糖组相比,高糖组M2型巨噬细胞CD206、α-SMA、Col-IV、FN的荧光表达均增强,TGF-β1、IL-10的表达和分泌水平均上调(均P<0.05),IL-6的表达和分泌则下调(P<0.05),TGF-β1、p-Smad3蛋白表达也显著升高(均P<0.05)。结论 高糖环境下HK2分泌的外泌体能够诱导M2型巨噬细胞向肌成纤维细胞转化,其机制可能与TGF-β1/Smad3信号通路的激活相关。

Objective To investigate the role and mechanism of macrophage-to-myofibroblast transition(MMT)induced by exosomes from high glucose-treated renal tubular epithelial cells.Methods Human renal tubular epithelial cells(HK2)were divided into glucose control group(5.5 mmol/L D-glucose)and high glucose group(30.0 mmol/L D-glucose)and cultured for 48 hours.The supernatant was collected by ultracentrifugation and identified by transmission electron microscopy and Western blot.PKH67 labelled exosomes were used to stimulate THP-1 macrophages.Laser scanning confocal microscopy was used to observe the phagocytosis process.The expression of inducible nitric oxide synthase(iNOS),α-SMA and CD206 was detected by flow cytometry and Western blot to determine the best concentration and time point.Laser scanning confocal microscopy was used to detect the fluorescence co-expression of rabbit polyclonal to mannose receptor(CD206),α-smooth muscle actin(α-SMA),collage type-Ⅳ(Col-IV)and fibronectin(FN).The expression of tumor growth factor-beta 1(TGF-β1),interleukin(IL)-6,IL-10 was separately detected by real-time quantitative PCR(qRT-PCR)and enzyme-linked immunosorbent assay(ELISA).The expression of TGF-β1,mothers against decapentaplegic homolog 3(Smad3),phosphorylated-smad 3(p-Smad3)in THP-1 macrophages was detected by Western blot.Results The expression of cluster of differentiation 63(CD63)and tumor susceptibility gene 101 protein(TSG101)was positive,while the Calnexin protein was negative in the supernatant,confirming that the specimen was exosomes with high purity.THP-1 macrophages could internalize each group of exosomes.40 mg/L exosomes for 96 hours were the best experimental condition.After being stimulated by high-glucose-exosomes,the percentage of M1 macrophages would climax in 24 hours,and the rate of M2 macrophages would climax in 96 hours.Immunofluorescence staining showed that exosomes released by HG-treated HK2 induced CD206,α-SMA,Col-IV and FN accumulation in cultured THP-1 macrophages.Compared with normal-glucose-exosomes,high-glucose-exosomes increased the expression of TGF-β1,IL-10 in M2 macrophages and decreased the expression of IL-6(all P<0.05).Moreover,TGF-β1 and p-Smad3 proteins expression also increased significantly(all P<0.05).Conclusion Exosomes secreted by renal tubular epithelial cells in high glucose environment can induce M2 macrophages transdifferentiate into myofibroblasts,and its mechanism may be related to activation of TGF-β1/Smad3 signal pathway.