L-瓜氨酸对子痫前期中的氧化应激和滋养层细胞侵袭的调控机制研究

The Regulatory Mechanism of L-Citrulline on Oxidative Stress and Trophoblast Invasion in Preeclampsia

目的:研究L-瓜氨酸(L-Cit)对子痫前期(PE)大鼠模型的治疗作用,及其对氧化应激和滋养层细胞侵袭的影响。方法:建立N-硝基-L-精氨酸甲酯(L-NAME)诱导的PE大鼠模型后,将大鼠分为Control组、PE组、PE+低、中和高剂量L-Cit组(依次为L-L-Cit、M-L-Cit和H-L-Cit组,剂量依次为100、200和500 mg/kg),n=6,连续给药7 d。在孕第21 d时,检测各组大鼠收缩压(SBP)、24 h尿蛋白、血清超氧化物歧化酶(SOD)和丙二醛(MDA)水平,并对胎盘组织进行苏木精伊红(HE)染色。将HTR-8/Svneo细胞分为Control组、缺氧/复氧(H/R)组、H/R+L-Cit组和H/R+L-Cit+sc-221593组,分别对细胞进行H/R处理,并使用L-Cit(200μg/m L)和特异性ERK1/2抑制剂sc-221593(10μmol/L)培养细胞48 h。通过Transwell测定细胞侵袭。通过Western blotting检测total-ERK1/2、p-ERK1/2、total-p38、p-p38、基质金属蛋白酶(MMP)-2和MMP-9的蛋白表达。结果:与PE组比较,L-L-Cit组、M-L-Cit组和H-L-Cit组的SBP和24 h尿蛋白水平均降低(P<0.05);胎盘组织形态明显改善;血清SOD升高,MDA降低(P<0.05);胎盘组织中ERK1/2和p38的磷酸化水平及MMP-2和MMP-9的蛋白表达水平均升高(P<0.05)。与H/R组比较,H/R+L-Cit组的侵袭细胞数量升高(P<0.05);ERK1/2和p38的磷酸化水平及MMP-2和MMP-9的蛋白表达水平均升高(P<0.05)。与H/R+L-Cit组比较,H/R+L-Cit+sc-221593组的上述变化均被逆转(P<0.05)。结论:L-Cit可能通过激活ERK/JNK通路减轻PE中的氧化应激并促进滋养层细胞侵袭,从而减轻PE症状。

Objective:To investigate the therapeutic effect of L-Citrulline(L-Cit)on preeclampsia(PE)rat model and its effect on oxidative stress and trophoblast cell invasion.Methods:After establishing the PE rat model induced by N-nitro-L-arginine methyl ester(L-NAME),the rats were divided into Control group,PE group,PE+low,middle and high dose L-Cit groups(L-L-Cit,M-L-Cit and H-L-Cit groups in sequence,with doses of 100,200 and 500 mg/kg in sequence),n=6,continuous administration for 7 days.On the 21st day of pregnancy,systolic blood pressure(SBP),24-hour urinary protein,serum superoxide dismutase(SOD)and malondialdehyde(MDA)were measured,and placental tissue was stained with hematoxylin-eosin(HE).The HTR-8/Svneo cells were divided into Control group,hypoxia/reoxygenation(H/R)group,H/R+L-Cit group and H/R+L-Cit+sc-221593 group.The cells were treated with H/R,respectively,and cultured with L-Cit(200μg/m L)and the specific ERK1/2 inhibitor sc-221593(10μmol/L)for 48 h.Cell invasion was determined by Transwell assay.The protein expressions of total-ERK1/2,p-ERK1/2,total-p38,p-p38,matrix metalloproteinase(MMP)-2 and MMP-9 in placental tissues and cells were detected by Western blotting.Results:Compared with PE group,the SBP and 24 h urinary protein levels of the rats in L-L-Cit group,M-L-Cit group and H-L-Cit group were decreased(P<0.05);the placental tissue morphology was significantly improved;serum SOD was increased,while MDA was decreased(P<0.05);the phosphorylation levels of ERK1/2 and p38 and the protein expression levels of MMP-2 and MMP-9 in placental tissue were increased(P<0.05).Compared with H/R group,the number of invasive cells in H/R+L-Cit group increased(P<0.05);the phosphorylation levels of ERK1/2 and p38 and the protein expression levels of MMP-2 and MMP-9 increased(P<0.05).Compared with the H/R+L-Cit group,the above changes in the H/R+L-Cit+sc-221593 group were reversed(P<0.05).Conclusions:L-Cit may alleviate the symptoms of PE by reducing oxidative stress in PE and promoting trophoblast invasion by activating the ERK/JNK pathway.