间充质干细胞来源的外泌体miR-3614-5p通过抑制铁死亡改善模型大鼠先兆子痫进展的机制研究

Mechanism Study of Mesenchymal Stem Cell Derived Exosome miR-3614-5p to Improve the Progression of Preeclampsia in Model Rats by Inhibiting Iron Death

目的研究间充质干细胞来源外泌体微小核糖核酸-3614-5p(miR-3614-5p)对模型大鼠先兆子痫(preeclampsia,PE)进展的调节作用及相关机制。方法将36只SD大鼠(24只雌性和12只雄性)以雌雄比例2∶1合笼饲养自然受孕。24只妊娠大鼠随机分为假手术组(sham组)、PE模型组(PE组)和外泌体miR-3614-5p组(PE+exo组),每组8只。PE组通过皮下注射100 mg/kg的NG-硝基-L-精氨酸甲酯建立大鼠PE模型;PE+exo组构建PE模型,同时在第14天腹腔注射160μg/ml的外泌体悬液(0.5 ml/只/天),连续6天,实验持续21天;sham组则给予等量生理盐水。在妊娠第0,7,14和21天测量血压和尿蛋白浓度。RT-qPCR检测miR-3614-5p水平及B细胞淋巴瘤病-2(Bcl-2)、Bcl相关X蛋白(Bax)的mRNA水平;ELISA检测Caspase-3活性、活性氧(ROS)水平及丙二醛(MDA)、谷胱甘肽(GSH)和亚铁离子(Fe2+)含量;Western blot检测谷胱甘肽过氧化物酶4(GPX4)和溶质载体家族7成员11(SLC7A11)蛋白水平。结果与sham组大鼠相比,PE组大鼠的胎盘组织(0.43±0.05 vs 1.01±0.07)和外周血(0.51±0.07vs 1.01±0.12)中miR-3614-5p表达显著下调,差异具有统计学意义(t=19.070,10.180,均P<0.01)。与上清液相比,源自MSCs的外泌体中miR-3614-5p显著富集。与sham组相比,PE组大鼠第21天的舒张压(175.43±6.02 mmHg vs113.26±5.11 mmHg)、收缩压(123.57±5.63 mmHg vs 82.63±5.26 mmHg)及尿蛋白含量(175.48±13.21 mg/ml vs67.65±5.76 mg/ml)显著升高(t=22.606,16.440,23.168,均P<0.01);与PE组相比,PE+exo组舒张压(124.57±5.33mmHg vs 175.43±6.02 mmHg)、收缩压(89.76±3.88 mmHg vs 123.57±5.63 mmHg)及尿蛋白含量(97.69±7.23 mg/ml vs 175.48±13.21 mg/ml)显著降低,差异具有统计学意义(t=18.493,13.577,16.713,均P<0.01)。与sham组相比,PE组大鼠胎盘组织中Caspase-3活性(238.56%±13.22%vs 100.12%±5.93%)、Bax水平(3.18±0.71 vs 1.01±0.11)、ROS水平(387.65%±25.98%vs 100.51%±5.89%)、MDA含量(33.21±3.17 nmol/mg vs 14.83±2.69 nmol/mg)和Fe2+浓度(38.77±6.53 nmol/ml vs 17.51±3.15 nmol/ml)显著升高,而Bcl-2水平(0.47±0.08 vs 1.01±0.12)、GSH含量(4.12±1.22 nmol/mg vs 9.76±0.93 nmol/mg)、GPX4蛋白(0.48±0.06 vs 1.01±0.24)和SLC7A11蛋白(0.51±0.11vs 1.01±0.11)水平则显著降低(t=6.459~32.863,均P<0.01);与PE组相比,PE+exo组胎盘组织中Caspase-3活性(117.35%±8.67%vs 238.56%±13.22%)、Bax水平(1.13±0.45 vs 3.18±0.71)、ROS水平(128.73%±14.37%vs387.65%±25.98%)、MDA含量(18.13±3.89 nmol/mg vs 33.21±3.17 nmol/mg)和Fe2+浓度(19.05±3.45 nmol/ml vs38.77±6.53 nmol/ml)显著降低,而Bcl-2水平(1.04±0.11 vs 0.47±0.08),GSH含量(7.86±1.07 nmol/mg vs 4.12±1.22nmol/mg),GPX4蛋白(0.98±0.14 vs 0.48±0.06)和SLC7A11蛋白(1.11±0.09 vs 0.51±0.11)水平则显著升高,差异具有统计学意义(t=6.093~29.633,均P<0.01)。结论miR-3614-5p在PE模型大鼠的胎盘组织和外周血中显著下调。MSCs来源的外泌体miR-3614-5p通过抑制铁死亡改善大鼠的PE进展。MSCs来源的外泌体miR-3614-5p可能是PE治疗的一个新的潜在的生物标志物。

Objective To investigate the regulatory effects of exosome microRNA-3614-5p(miR-3614-5p)derived from mesenchymal stem cells on the progression of preeclampsia(PE)in model rats and its related mechanisms.Methods Thirty-six SD rats(24 females and 12 males)were housed in cages at a female-to-male ratio of 2:1 for natural conception.Twenty-four pregnant rats were randomly divided into sham group(sham group),PE model group(PE group)and exosome miR-3614-5p group(PE+exo group),with 8 rats in each group.The PE model was established by subcutaneous injection of 100 mg/kg NG-nitro-L-arginine methyl ester in PE group.PE model was constructed in PE+exo group.Meanwhile,160μg/ml exosome suspension(0.5 ml/individual/day)was intraperitoneally injected on the 14th day for 6 consecutive days,and the experiment lasted for 21 days.Sham group was given an equal amount of normal saline.Blood pressure and urinary protein concentration were measured on days 0,7,14 and 21 of pregnancy.The levels of miR-3614-5p,B lymphoblastoma-2(Bcl-2)and Bcl-associated X protein(Bax)mRNA were detected by RT-qPCR.The activity of Caspase-3,the levels of reactive oxygen species(ROS)and the content of malondialdehyde(MDA),glutathione(GSH)and ferrous ion(Fe2+)were detected by ELISA.Western blot was used to analyze the protein levels of the iron death-related protein glutathione peroxidase 4(GPx4)and solute carrier family 7 member 11(SLC7A11).Results Compared with the sham group,the expression of miR-3614-5p in the placental tissues(0.43±0.05 vs 1.01±0.07)and peripheral blood(0.51±0.07 vs 1.01±0.12)of rats in the PE group was downregulated,with significant differences(t=19.070,10.180,all P<0.01).Compared with supernatant liquid phase,miR-3614-5p in exosomes derived from MSCs was enriched.Compared with sham group,the diastolic blood pressure(175.43±6.02 mmHg vs 113.26±5.11 mmHg),systolic blood pressure(123.57±5.63 mmHg vs 82.63±5.26 mmHg)and urinary protein content(175.48±13.21 mg/ml vs 67.65±5.76 mg/ml)of rats in PE group were increased on the 21st day with statistical significante between groups(t=22.606,16.440,23.168,all P<0.01).Compared with PE group,diastolic blood pressure(124.57±5.33 mmHg vs 175.43±6.02 mmHg),systolic blood pressure(89.76±3.88 mmHg vs 123.57±5.63 mmHg)and urinary protein content(97.69±7.23 mg/ml vs 175.48±13.21 mg/ml)in PE+exo group were decreased,and the differences between groups were significant(t=18.493,13.557,16.713,all P<0.01).Compared with sham group,Caspase-3 activity(238.56%±13.22%vs 100.12%±5.93%),Bax level(3.18±0.71 vs 1.01±0.11),ROS level(387.65%±25.98%vs 100.51%±5.89%),MDA content(33.21±3.17 nmol/mg vs 14.83±2.69 nmol/mg)and Fe2+concentration(38.77±6.53 nmol/ml vs 17.51±3.15 nmol/ml)in placenta tissue of PE group were increased,while Bcl-2 level(0.47±0.08 vs 1.01±0.12),GSH content(4.12±1.22 nmol/mg vs 9.76±0.93 nmol/mg),GPX4 protein(0.48±0.06 vs 1.01±0.24)and SLC7A11 protein(0.51±0.11 vs 1.01±0.11)levels were decreased(t=6.459~32.863,all P<0.01);Caspase-3 activity(117.35%±8.67%vs 238.56%±13.22%),Bax level(1.13±0.45 vs 3.18±0.71),ROS level(128.73%±14.37%vs 387.65%±25.98%),MDA content(18.13±3.89 nmol/mg vs 33.21±3.17 nmol/mg)and Fe2+concentration(19.05±3.45 nmol/ml vs 38.77±6.53 nmol/ml)in placental tissues of PE+exo group were decreased,while Bcl-2 level(1.04±0.11 vs 0.47±0.08),GSH content(7.86±1.07 nmol/mg vs 4.12±1.22 nmol/mg),GPX4 protein(0.98±0.14 vs 0.48±0.06)and SLC7A11 protein(1.11±0.09 vs 0.51±0.11)levels were increased compared with PE group,with significant differences between groups(t=6.093~29.633,all P<0.01).Conclusion In the placental tissues and peripheral blood of PE rats,miR-3614-5p was down-regulated.Exosomes overexpressing miR-3614-5p derived from MSCs suppressed PE progression in rats by inhibiting ferroptosis.These results suggested that exosomes miR-3614-5p derived from MSCs may be a novel potential biomarker for PE treatment.