多囊卵巢综合征大鼠氧化应激状态下血小板反应蛋白-1和三羧酸循环因子表达变化及意义

Changes and significances of thrombospondin-1 and tricarboxylic acid cycle factors under oxidative stress in polycystic ovary syndrome rats

目的 观察来曲唑诱导的多囊卵巢综合征(PCOS)模型大鼠氧化应激状态下血小板反应蛋白-1(TSP-1)、异柠檬酸脱氢酶1(IDH1)、ATP柠檬酸裂解酶(ACLY)表达变化,探讨N-乙酰半胱氨酸(NAC)对PCOS大鼠氧化应激的改善作用。方法 24只雌性SD大鼠随机分为PCOS组16只和对照组8只。对照组给予羧甲基纤维素钠溶液灌胃,PCOS组给予来曲唑+羧甲基纤维素钠溶液灌胃,均连续21 d。PCOS模型制备成功后,随机分为治疗组和模型组各8只。治疗组给予NAC溶液灌胃,模型组给予等剂量生理盐水灌胃,均连续28 d。第28天禁食,次日取腹主动脉血,采用ELISA法检测睾酮(T)、黄体生成素(LH)、卵泡刺激素(FSH)、雌二醇(E2)水平并计算LH/FSH,采用WST-1法检测超氧化物歧化酶(SOD)活力,采用TBA法测定丙二醛(MDA)含量;然后摘取大鼠双侧卵巢,观察卵巢外观及组织形态学改变,采用WST-1法检测SOD活力,采用TBA法检测MDA含量,采用Western blot法检测TSP-1、IDH1、ACLY蛋白相对表达量。结果 (1)模型组血清T[(118.49±67.98)ng/L]、LH[(18.76±11.32)u/mL]水平,LH/FSH(7.85±8.66)及MDA含量[(10.53±1.46)mmol/L]均高于对照组[(28.37±8.99)ng/L、(5.02±1.92)u/mL、0.69±0.55、(3.56±1.21)mmol/L]和治疗组[(60.03±19.64)ng/L、(8.82±1.83)u/mL、1.58±0.68、(6.50±1.02)mmol/L](P<0.05),血清FSH水平[(3.09±1.25)u/mL]及SOD活力[(81.50±10.20)u/mL]均低于对照组[(10.56±6.55)、(132.81±3.03)u/mL]和治疗组[(6.03±1.49)、(147.85±3.79)u/mL](P<0.05)。(2)模型组大鼠卵巢苍白,体积增大,表面可触及大小不均一的卵泡,镜下见卵巢未发育成熟的小卵泡以及部分闭锁卵泡、颗粒细胞层稀疏;治疗组卵巢体积缩小、颜色红润、表面平滑,镜下见呈囊状扩大的卵泡数量减少、颗粒细胞层增加。(3)模型组卵巢组织MDA含量[(20.24±3.10)nmol/mg port]高于对照组[(4.80±1.83)nmol/mg port]和治疗组[(12.20±1.89)nmol/mg port](P<0.05),SOD活力[(23.82±4.06)u/mg port]低于对照组[(39.35±3.03)u/mg port]和治疗组[(37.34±4.72)u/mg port](P<0.05)。(4)模型组卵巢组织TSP-1(0.38±0.09)、ACLY(0.56±0.16)、IDH1(0.45±0.09)蛋白相对表达量均低于对照组(0.97±0.15、1.32±0.32、1.03±0.19)和治疗组(0.68±0.07、1.06±0.21、0.72±0.11)(P<0.05)。结论 PCOS大鼠血清及卵巢组织氧化应激指标呈异常表达,NAC可减轻PCOS大鼠的氧化应激、改善PCOS表型;PCOS大鼠氧化应激下低TSP-1表达可能与三羧酸循环相关因子异常表达有关。

Objective To observe the changes of thrombospondin-1(TSP-1),isocitrate dehydrogenase 1(IDH1),and ATP-citrate lyase(ACLY)underoxidativestress in rat models withletrozole induced polycystic ovary syndrome(PCOS),and to explore the improvement role of N-acetylcysteine(NAC)on oxidative stress in PCOS rats.Methods Twenty-four female SD rats were randomly divided into PCOS group(n=16)and control group(n=8).Control group received gavage with sodium carboxymethyl cellulose solution,while PCOS group received gavage with letrozole+sodium carboxymethyl cellulose solution,for 21 consecutive days.After PCOS models were established,PCOS group was randomly divided into treatment group and model group,with 8 rats each.Treatment group received gavage with NAC solution,and model group was given an equivalent volume of normal saline,for 28 consecutive days.On the next day after fasting for 28 d,the abdominal aortic blood was collected to detect the levels of testosterone(T),luteinizing hormone(LH),follicle stimulating hormone(FSH),and estradiol(E,)by ELISA,and LH/FSH was calculated.The superoxide dismutase(SOD)activity was detected by WST-1 method,and malondialdehyde(MDA)content was detected by TBA method.The bilateral ovaries were extracted to observe the appearance of the ovaries.The SOD activity was detected by WST-1 method,the MDA content was detected by TBA method,and the relative expressions of TSP-1,IDHl and ACLY proteins were detected by Western blot.Results(1)The serum levels of T,LH,LH/FSH and MDAwere higher in model group[(118.49±67.98)ng/L,(18.76±11.32)u/mL,7.85±8.66,(10.53±1.46)mmol/L]than thosein control group[(28.37±8.99)ng/L,(5.02±1.92)u/mL,0.69±0.55,(3.56±1.21)mmol/L]andtreatmentgroup[(60.03±19.64)ng/L,(8.82±1.83)u/mL,1.58±0.68,(6.50±1.02)mmol/L](P<0.05),and the serum FSH level and SOD activity were lower in model group[(3.09±1.25),(81.50±10.20)u/mL] than those in control group[(10.56±6.55),(132.81±3.03)u/mL] and treatment group[(6.03±1.49),(147.85±3.79)u/mL](P<0.05).(2)Inmodel group,theovarieswerepale in color and large in size,with unevenly sized follicles palpable on the surface;the microscopy showed more immature small follicles and partially occluded follicles,with sparse granulosa cell layers.In treatment group,the ovaries were red in color and reduced in size with smooth surface;the microscopy showed a decrease in number of follicles with cystic enlargement and an increase in granulosa cell layers.(3)The MDA content in ovarian tissues was higher in model group[(20.24±3.10)nmol/mg port]than that in'control group[(4.80±1.83)nmol/mg port]and treatment group[(12.20±1.89)nmol/mg port](P<0.05),the SOD activity was lower in model group[(23.82±4.06)u/mg port]than that in control group[(39.35±3.03)u/mg port]and treatment group[(37.34±4.72)u/mg port](P<0.05).(4)The relative expressions of TSP-1,ACLY andIDH1 proteins were lower inmodel group(0.38±0.09,0.56±0.16,0.45±0.09)than those in control group(0.97±0.15,1.32±0.32,1.03±0.19)and treatment group(0.68±0.07,1.06±0.21,0.72±0.11)(P<0.05).Conclusions The oxidative stress indicators in serum and ovarian tissue of PCOS rats are abnormally expressed.NAC can reduce oxidative stress and improve PCOS morphology.The low level of TSP-1 under oxidative stress in PCOS rats may be correlated with the abnormal expression of tricarboxylic acid cycle factors.