X射线辐射远端效应诱发小鼠卵巢储备功能下降的实验研究

Diminished ovarian reserve induced by X-ray radiation-induced abscopal effects in mice:an experimental study

目的探讨X射线辐射远端效应(X-ray radiation-induced abscopal effects,X-RIAEs)对小鼠卵巢储备的影响及可能的作用机制。方法16只动情周期规律的6~8周龄雌性C57BL/6J小鼠分为对照组和照射组,8只/组,照射组小鼠麻醉后每日给予胸部局部区域8 Gy X射线照射,连续照射3 d,对照组小鼠仅给予麻醉处理。照射结束21 d后,检测两组小鼠动情周期、血清激素及促炎性因子水平、卵巢组织形态学变化;利用转录组测序技术(ribonucleic acid sequencing,RNA-seq)检测小鼠卵巢组织RNA转录组表达情况,筛选差异表达基因(differentially expressed genes,DEGs)后行基因本体论-生物学过程(gene ontology-biological processes,GO_BP)分析,通过应用实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)验证测序结果,免疫组织化学染色(immunohistochemistry,IHC)检测精卵发生特异性碱性螺旋环螺旋蛋白1(spermatogenesis-and oogenesis-specific basic helix-loop-helix-containing protein 1,SOHLH1)和中性粒细胞弹性蛋白酶(neutrophil elastase,NE)在卵巢组织内表达和定位情况。结果照射组小鼠动情周期紊乱,主要停滞于间期,照射组小鼠始基卵泡数量[10.50(1.25,12.75)]及生长卵泡数量[4.50(2.50,9.00)]均显著少于对照组小鼠[60.00(30.00,90.25),P<0.001;18.50(18.00,20.75),P<0.001],差异均具有统计学意义,而闭锁卵泡数量[56.00(45.25,98.75)]明显多于对照组[12.50(5.25,20.25)],差异具有统计学意义(P<0.001);照射组小鼠血清雌二醇水平[(70.28±5.27)pmol/L]、抗苗勒管激素水平[(104.00±6.98)μg/L]均明显低于对照组小鼠[(97.58±7.25)pmol/L,P=0.016;(129.70±8.39)μg/L,P=0.046],而照射组小鼠卵泡刺激素水平与对照组小鼠相比,差异无统计学意义(P=0.996);与对照组小鼠血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平[(31.61±12.89)μg/L]、白细胞介素(interleukin,IL)-1β水平[(52.75±2.06)μg/L]相比,照射组小鼠血清TNF-α水平[(488.30±36.20)μg/L]和IL-1β水平[(62.37±2.50)μg/L]均明显升高(P<0.001,P=0.018),照射组小鼠血清IL-6水平较对照组小鼠也呈上升趋势,但差异无统计学意义(P=0.301)。GO_BP分析结果显示,X-RIAEs诱导小鼠卵巢组织表达下调的DEGs主要参与卵泡发育过程,表达上调的DEGs主要参与卵巢组织炎症反应过程,RT-qPCR结果与测序结果一致。IHC结果显示,照射组小鼠卵巢组织SOHLH1阳性表达面积[(23.18±4.00)%]显著低于对照组[(65.90±6.28)%,P=0.005],而NE阳性表达面积[(30.73±4.00)%]显著高于对照组[(14.47±2.22)%,P=0.024]。结论X-RIAEs可诱发卵巢组织炎性反应,并抑制小鼠卵巢卵泡生长及发育过程,进而导致卵巢储备功能下降。

Objective To investigate the effect and possible mechanism of the X-ray radiation-induced abscopal effects(X-RIAEs)on the ovarian reserve of mice.Methods Totally sixteen female C57BL/6J mice aged 6-8 weeks with regular estrous cycle were randomly divided into the sham group(n=8)and irradiation group(n=8).After anesthesia,the mice in the irradiation group were irradiated with 8 Gy X-ray on the local area of the chest every day for 3 d,while the mice in the sham group were not irradiated.After irradiation 21 d,the estrous cycle,serum hormones,serum pro-inflammatory factors,and ovarian morphological changes were detected.Ribonucleic acid sequencing(RNA-seq)was used to detect the expression of transcriptional levels in mouse ovarian tissues.The differentially expressed genes(DEGs)were screened and analyzed by gene ontology-biological process(GO_BP).Real-time quantitative polymerase chain reaction(RT-qPCR)verified the sequencing results.The expression and localization of spermatogenesis-and oogenesis-specific basic helix-loop-helix-containing protein 1(SOHLH1)and neutrophil elastase(NE)in ovarian tissues were detected by immunohistochemistry(IHC).Results Compared with mice in the sham group,the irradiation group had a disordered estrous cycle,reduced primordial follicles[10.50(1.25,12.75)vs.60.00(30.00,90.25),P<0.001]and growing follicles[(4.50(2.50,9.00)vs.18.50(18.00,20.75),P<0.001],significantly increased atretic follicles[56.00(45.25,98.75)vs.12.50(5.25,20.25),P<0.001].The levels of serum estradiol[(70.28±5.27)pmol/L]and anti-Müllerian hormone[(104.00±6.98)μg/L]in the irradiation group were significantly lower than those in the sham group[(97.58±7.25)pmol/L,P=0.016;(129.70±8.39)μg/L,P=0.046],but the follicle-stimulating hormone(FSH)level in the irradiation group was not significantly different from that in the sham group(P>0.05).Compared with the sham group,the serum levels of TNF-α[(488.30±36.20)μg/L vs.(31.61±12.89)μg/L,P<0.001]and IL-1β[(62.37±2.50)μg/L vs.(52.75±2.06)μg/L,P=0.018]in the irradiation group were significantly increased,and the serum level of interleukin(IL)-6 in the irradiation group was also increased compared with the sham group,but the difference was not statistically significant(P>0.05).The results of GO_BP analysis showed that the down-regulated DGEs were mainly involved in the process of follicular development,and the up-regulated DGEs were involved in the inflammation process.The results of RT-qPCR were consistent with those of sequencing.The immunohistochemistry results showed that the positive expression area of SOHLH1 in the irradiation group[(23.18±4.00)%]was significantly lower than that of the sham group[(65.90±6.28)%,P=0.005],while the positive expression area of NE[(30.73±4.00)%]was significantly higher than that of the sham group[(14.47±2.22)%,P=0.024].Conclusion X-RIAEs can induce an inflammatory reaction in ovarian tissue and inhibit the growth and development of ovarian follicles in mice,which leads to a decrease in ovarian reserve.