冷应激反应加重大鼠多囊卵巢综合征生殖功能障碍

Cold stress aggravates reproductive dysfunction in rats with polycystic ovary syndrome

目的研究冷应激刺激对多囊卵巢综合征(PCOS)大鼠生殖功能的影响及其作用机制。方法将大鼠分为对照组(Contral)、模型组、冷应激组、抑制剂组(给予HSP90抑制剂50 mg/kg)及联合组(冷应激联合HSP90抑制剂),每组10只。ELISA检测血清性激素及氧化应激水平、HE染色观察卵巢形态、TUNEL检测卵巢细胞凋亡、免疫印迹检测HSP90、NF-κB p65、IkB、TAK-1和RIP蛋白表达。结果与对照组FSH[(16.37±2.14)mU/mL]、LH[(9.32±1.46)nmol/L]、T[(1.23±0.17)nmol/L]比较,模型组FSH[(7.21±1.54)mU/mL]与联合组FSH[(7.25±1.51)mU/mL]降低(P<0.05),模型组LH[(20.34±2.81)nmol/L]、T[(1.58±0.26)nmol/L]与联合组LH[(20.28±2.85)nmol/L]、T[(1.56±0.27)nmol/L]升高(P<0.05);与模型组比较,抑制剂组FSH[(6.28±1.24)mU/mL]降低、LH[(22.08±2.73)nmol/L]、T[(1.64±0.25)nmol/L]升高(P<0.05),与抑制剂组比较,冷应激组FSH[(5.34±0.67)mU/m L]降低、LH[(23.17±2.95)nmol/L]与T[(1.79±0.31)nmol/L]升高(P<0.05)。与对照组比较,模型组与联合组SOD、GSH-Px水平降低,MDA水平升高(P<0.05),与模型组比较,抑制剂组SOD、GSH-Px水平升高,MDA水平降低(P<0.05),与抑制剂组比较,冷应激组SOD、GSH-Px水平降低,MDA水平升高(P<0.05)。对照组大鼠卵巢组织结构正常;模型组与联合组卵巢皮质黄体及卵泡减少,卵泡囊性扩张并有闭锁产生;冷应激组与抑制剂组大鼠的卵巢主要表现为大量的小窦卵泡、囊性卵泡和增大的卵泡膜细胞层,且以冷应激组更为严重。对照组、模型组、抑制剂组、冷应激组与联合组的细胞凋亡率分别为(5.62±0.58)%、(41.38±3.92)%、(52.67±4.22)%、(63.48±5.71)%、(40.62±4.28)%,对照组卵巢组织中细胞凋亡率低于模型组、联合组(F=168.200,P<0.001),抑制剂组卵巢组织中细胞凋亡率较模型组高(t=6.213,P<0.001),冷应激组细胞凋亡率较抑制剂组高(t=4.815,P<0.001)。与对照组比较,模型组大鼠卵巢组织中HSP90、NF-κB p65、IkB、TAK-1、RIP升高(P<0.05),与模型组比较,冷应激组与对照组大鼠卵巢组织中HSP90、NF-κB p65、IkB、TAK-1、RIP升高(P<0.05)。大鼠卵巢组织中HSP90与PI3K在冷应激刺激后的表达呈正相关关系(R=0.581,P<0.001)。结论冷应激加重PCOS大鼠生殖功能障碍。

Objective To investigate the effects of cold stress stimulation on reproductive function of rats with polycystic ovary syndrome(PCOS)and the underlying mechanism.Methods Rats were randomly divided into control group,model group,cold stress group,inhibitor group(HSP90 inhibitor 50mg/kg)and combination group(cold stress combined with HSP90 inhibitor),with 10 in each group.Serum sex hormone and oxidative stress levels were detected by enzyme-linked immunosorbent assay(ELISA).Ovarian morphology was observed by hematoxylin and eosin(H&E)staining.Apoptosis of ovarian cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL).Protein expressions of heat shock protein 90(HSP90),nuclear factor-kappaB(NF-kappaB)P65,inhibitor kB(IkB),transforming growth factor-beta-activated kinase 1(TAK1)and RNA immunoprecipitation(RIP)were detected by Western blot.Results Follicle-stimulating hormone(FSH,[7.21±1.54]mU/mL vs[7.25±1.51]mU/mL vs[16.37±2.14]mU/mL,P<0.05)in the model group and combination group was significantly lower than that of the control group.Luteinizing hormone(LH,[20.34±2.81]nmoL/L vs[20.28±2.85]nmoL/L vs[9.32±1.46]nmoL/L,P<0.05)and testosterone(T,[1.58±0.26]nmoL/L vs[1.56±0.27]nmoL/L vs[1.23±0.17]nmoL/L,P<0.05)in the model group and combination group were significantly higher than those of the control group.Compared with those of the model group,the inhibitor group showed significantly reduced FSH([7.21±1.54]mU/mL vs[6.28±1.24]mU/mL,P<0.05),and significantly increased LH([20.34±2.81]nmoL/L vs[22.08±2.73]nmoL/L,P<0.05)and T([1.58±0.26]nmoL/L vs[1.64±0.25]nmoL/L,P<0.05).Compared with those of the inhibitor group,the cold stress group showed significantly reduced FSH([6.28±1.24]mU/mL vs[5.34±0.67]mU/mL,P<0.05),and significantly increased LH([22.08±2.73]nmoL/L vs[23.17±2.95]nmoL/L,P<0.05)and T([1.64±0.25]nmoL/L vs[1.79±0.31]nmoL/L,P<0.05).Compared with those of the control group,the model group and the combination group showed significantly reduced superoxide dismutase(SOD)and glutathione peroxidase(GSH-Px),and significantly increased malondialdehyde(MDA)(P<0.05).Compared with those of the model group,the inhibitor group showed significantly increased SOD and GSH-Px,and significantly decreased MDA(P<0.05).Compared with those of the inhibitor group,the cold stress group showed significantly reduced SOD and GSH-Px,and significantly increased MDA(P<0.05).Rat ovarian tissue in the control group was normal.Rat ovarian tissue in the model group and combination group showed reduced cortical corpus luteum and follicles,cystic expansion of follicles,and atresia.Rat ovarian tissue in the cold stress group and inhibitor group mainly showed a large number of small antral follicles,cystic follicles and enlarged theca cell layers,which were much severer in the cold stress group.The cell apoptotic rate of the control group,model group,inhibitor group,cold stress group and combination group was(5.62±0.58)%,(41.38±3.92)%,(52.67±4.22)%,(63.48±5.71)%and(40.62±4.28)%,respectively.The cell apoptotic rate in the ovarian tissue of the control group was significantly lower than that of the model group and combination group(F=168.200,P<0.001).The cell apoptotic rate in the ovarian tissue of the inhibitor group was significantly higher than that of the model group(t=6.213,P<0.001).The cell apoptotic rate in the cold stress group was significantly higher than that of the inhibitor group(t=4.815,P<0.001).Compared with those of the control group,the expression levels of HSP90,NF-κB P65,IkB,TAK-1 and RIP in rat ovarian tissue of the model group were significantly upregulated(P<0.05).Compared with those of the model group,the expression levels of HSP90,NF-κB P65,IkB,TAK-1 and RIP in rat ovarian tissue of the cold stress group and control group were significantly upregulated(P<0.05).There was a positive correlation between the expressions of HSP90 and PI3K in rat ovarian tissue after cold stress stimulation(R=0.581,P<0.001).Conclusion Cold stress aggravates reproductive dysfunction in PCOS rats.