胎盘源性外泌体miR-122-5p改善妊娠糖尿病脐静脉内皮细胞功能损伤的机制研究

Mechanism of placental exosomal miR-122-5p in alleviating the functional injury of umbilical vein endothelial cells in gesta-tional diabetes mellitus

目的探讨胎盘源性外泌体(Exo)miR-122-5p改善妊娠糖尿病(GDM)小鼠脐静脉内皮细胞(MUVECs)功能损伤的机制。方法取妊娠小鼠腹腔注射0.25%链脲佐菌素溶液以诱导GDM模型(GDM组),对照组小鼠腹腔注射等体积0.9%氯化钠溶液,检测两组FPG和胰岛素水平验证模型。采用HE染色观察小鼠胎盘组织形态,qRT-PCR法检测miR-122-5p表达情况,Western blot法检测核苷酸结合结构域富含亮氨酸重复序列和含热蛋白结构域受体3(NLRP3)炎症小体相关蛋白[包括NLRP3、凋亡相关斑点样蛋白、半胱氨酸蛋白酶-1]表达。采用差速离心法获得胎盘源性Exo,并采用透射电子显微镜和纳米颗粒追踪分子对Exo表征进行鉴定;与MUVECs共培养后,采用免疫荧光法观察MUVECs对Exo的摄取。采用噻唑蓝法、流式细胞术、Transwell实验和血管形成实验检测MUVECs细胞活性、凋亡能力、迁移能力和成管能力。结果GDM组小鼠FPG水平明显高于对照组(P<0.001),胰岛素水平低于对照组(P<0.001),提示模型构建成功。与对照组相比,GDM组小鼠胎盘组织出现明显损伤,miR-122-5p表达水平降低,NLRP3炎症小体相关蛋白表达水平均明显升高,差异均有统计学意义(均P<0.05)。MUVECs能对胎盘源性Exo进行摄取,且GDM小鼠胎盘Exo上调了MUVECs中miR-122-5p表达水平(P<0.001)。GDM小鼠胎盘Exo共培养的MUVECs较对照小鼠胎盘Exo共培养的MUVECs细胞活性增强,凋亡能力减弱,迁移能力和成管能力均增强,且NLRP3炎症小体相关蛋白表达水平均明显下降,差异均有统计学意义(均P<0.05)。过表达miR-122-5p的MUVECs细胞活性增强,凋亡能力减弱,迁移能力和成管能力均增强,NLRP3炎症小体相关蛋白表达水平降低,差异均有统计学意义(均P<0.05);抑制miR-122-5p表达后,MUVECs细胞活性减弱,凋亡能力增强,迁移能力和成管能力均减弱,NLRP3炎症小体相关蛋白表达水平均明显升高,差异均有统计学意义(均P<0.05)。结论胎盘源性Exo的miR-122-5p对GDM MUVECs起到保护作用,且可能通过调控NLRP3炎症小体的活化来改善MUVECs的损伤。

Objective To explore the protective effect of miR 122-5p in placenta-derived exosome(Exo)on mouse umbilical vein endothelial cells(MUVECs)in gestational diabetes mellitus(GDM)model and its molecular mechanism.Methods GDM model was induced by 35 mg/kg peritoneal injection of 0.25%streptozotocin in pregnant mice,and the same amount of normal saline was injected intraperitoneally in control mice.FPG and insulin levels were detected in two groups to validate the models.The placental morphology of mice was observed by HE staining.qRT-PCR was used to detect the expression of miR 122-5p.Western blotting was used to detect the expressions of nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor 3(NLRP3)inflammasome-associated proteins[including NLRP3,apoptosis associated speck-like protein containing CARD,and Caspase 1].The placenta-derived Exo was obtained by differential centrifugation and characterized by transmission electron microscopy and nanoparticle tracking molecule.Immunofluorescence was used to observe Exo uptake by MUVECs after MUVECs co-cultivation.MUVECs cell activity,apoptosis level,migration ability and tube formation ability were detected by 3-(4,5-dimethyl 2-thiazolyl)-2,5-diphenyl tetrazolium bromide,flow cytometry,Transwell and angiogenesis assays,respectively.Results The FBG of mice in GDM group was significantly higher than that of the control group(P<0.001),while the peripheral blood insulin level was significantly lower(P<0.001),and the glucose metabolism was abnormal,indicating successful modeling.Compared with the control group,the placental tissue of mice in GDM group was significantly damaged,the expression of miR 122-5p was significantly decreased,and the expression of NLRP3 inflammasome-related protein was significantly increased(all P<0.05).MUVECs could take up placenta-derived Exo,and placental Exo of GDM mice up-regulated the expression level of miR 122-5p in MUVECs(P<0.001).Compared with MUVECs co-cultured with normal mouse placental Exo,MUVECs co-cultured with GDM mouse placental Exo showed higher cell activity,lower apoptosis level,stronger migration and tubular formation ability,and significantly decreased NLRP3 inflammasome-related protein expression(all P<0.05).In the experiment of interfering the expression of miR 122-5p in MUVECs,compared with the control group,the activity of the MUVECs overexpressing miR 122-5p was significantly increased,the apoptosis level was decreased,the migration and tubular formation ability was increased,and the expression of NLRP3 inflammasome-related protein was significantly decreased(all P<0.05).After inhibiting the expression of miR-122-5p,the activity of MUVECs was significantly decreased with increased apoptosis level,and decreased migration and tubular formation ability,and the expression of NLRP3 inflammasome-related protein was significantly increased(all P<0.05).Conclusion miR-122-5p in placenta-derived Exo has a protective effect on MUVECs in GDM mice,which may improve the damage of MUVECs by regulating the activation of NLRP3 inflammasome.