骨髓间充质干细胞来源的外泌体对老龄小鼠术后认知功能的影响及SIRT_(1)/NF-κB信号通路在其中的作用

Effect of exosomes derived from bone mesenchymal stem cells on postoperative cognitive function and SIRT_(1)/NF-κB signaling pathway in aged mice

目的评价骨髓间充质干细胞来源的外泌体(BMSCs-EXO)对老龄小鼠术后认知功能的影响及沉默信息调节因子1(SIRT1)/NF-κB信号通路在其中的作用。方法采用差速离心法提取BMSCs-EXO,并进行鉴定。健康雄性C57BL/6老龄小鼠20只,18月龄,体质量35~40 g,采用随机数字表法分为4组(n=5),假手术组(Sham组):仅备皮消毒不进行剖腹探查术;手术组(O组):行剖腹探查术;BMSCs-EXO组:术前1 h尾静脉注射BMSCs-EXO 50μg;EX527(SIRT1抑制剂)组:术前1~3 d每天腹腔注射EX5275 mg/kg,术前1 h尾静脉注射BMSCs-EXO 50μg。于术后1 d开始行Morris水迷宫实验。术后5 d水迷宫实验结束后处死小鼠,取海马组织,观察小鼠海马CA1区组织病理学变化,采用qRT-PCR法检测TNF-α、IL-6和IL-1β的mRNA表达,采用Western blot法检测SIRT1和NF-κB p65的表达。结果与Sham组比较,O组逃避潜伏期延长,穿越原平台位置次数和原平台象限游泳时间减少,海马TNF-αmRNA、IL-6 mRNA和IL-1βmRNA表达上调,SIRT1表达下调,NF-κB p65表达上调(P<0.05),海马CA1区发生病理学改变。与O组比较,BMSCs-EXO组逃避潜伏期缩短,穿越原平台位置次数和原平台象限游泳时间增加,海马TNF-αmRNA、IL-6 mRNA和IL-1βmRNA表达下调,SIRT1表达上调,NF-κB p65表达下调(P<0.05),海马CA1区病理改变减轻。与BMSCs-EXO组比较,EX527组逃避潜伏期延长,穿越原平台位置次数和原平台象限游泳时间减少,海马TNF-αmRNA、IL-6 mRNA和IL-1βmRNA表达上调,SIRT1表达下调,NF-κB p65表达上调(P<0.05),海马CA1区病理改变程度加重。结论BMSCs-EXO可改善老龄小鼠术后认知功能,机制可能与激活SIRT1/NF-κB信号通路有关。

Objective To evaluate the effect of exosomes derived from bone mesenchymal stem cells(BMSCs-EXO)on the postoperative cognitive function and silent infomation regulator 1(SIRT1)/nuclear factor kappa B(NF-κB)signaling pathway in aged mice.Methods BMSCs-EXO were isolated by differential centrifugation method and then identified.Twenty healthy male C57BL/6 aged mice,aged 18 months,weighing 35-40 g,were divided into 4 groups(n=5 each)using a random number table method:sham operation group(Sham group),operation group(O group),BMSCs-EXO group and EX527(SIRT1 inhibitor)group.The abdomen regions were shaved for sterilization without exploratory laparotomy in Sham group.Exploratory laparotomy was performed in O group.BMSCs-EXO 50μg was injected through the tail vein at 1 h before surgery in BMSCs-EXO group.EX5275 mg/kg was intraperitoneally injected daily at 1-3 days before surgery,and BMSCs-EXO 50μg was injected through the tail vein at 1 h before surgery in EX527 group.Morris water maze test was used to evaluate the learning and memory ability for 5 consecutive days staring from the 1st day after surgery.Mice were sacrificed at 1 h after the end of Morris water maze test on day 5 after surgery,and the hippocampal tissues were collected for observation of the pathological changes of hippocampal CA1 region and for determination of the expression of tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6)and IL-1βmRNA(quantitative real-time polymerase chain reaction)and SIRT1 and NF-κB p65(by Western blot).Results Compared with Sham group,the escape latency was significantly prolonged,the times of original platform crossing were decreased,the swimming time spent in the original platform quadrant was shortened,the expression of tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6)and IL-1βmRNA was up-regulated,the SIRT1 expression was down-regulated,the NF-κB p65 expression was up-regulated(P<0.05),and the pathological changes of hippocampal tissues in CA1 region were found in O group.Compared with O group,the escape latency was significantly shortened,the times of original platform crossing were increased,the swimming time spent in the original platform quadrant was prolonged,the expression of TNF-α,IL-6 and IL-1βmRNA was down-regulated,the expression of SIRT1 was up-regulated,the expression of NF-κB p65 was down-regulated(P<0.05),and the pathological changes of hippocampal tissues in CA1 region were significantly attenuated in BMSCs-EXO group(P<0.05).Compared with BMSCs-EXO group,the escape latency was significantly prolonged,the times of original platform crossing were decreased,the swimming time spent in the original platform quadrant was shortened,the expression of TNF-α,IL-6 and IL-1βmRNA was up-regulated,the SIRT1 expression was down-regulated,the NF-κB p65 expression was up-regulated(P<0.05),and the pathological changes of hippocampal tissues in CA1 region were accentuated in EX527 group.Conclusions BMSCs-EXO can improve the postoperative cognitive function in aged mice,and the mechanism may be associated with the activation of SIRT1/NF-κB signaling pathway.