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OACSTPCD

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,160 μ g/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 down-regulated,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.