顺铂对小鼠下丘脑-垂体-肾上腺/性腺轴功能的损伤作用及脱氢表雄酮的干预效应

doi:10.12300/j.issn.1674-5817.2022.182

摘要/Abstract

摘要：

目的研究顺铂（又名顺式-二氯二氨合铂，cis-dichlorodiamineplatinum，DDP）抑制小鼠类固醇激素合成的作用途径，并观察脱氢表雄酮（dehydroepiandrosterone，DHEA）的干预效应。方法将60只成年ICR小鼠随机分为3组：对照组、DDP建模组、DHEA治疗组，每组10只雄性和10只雌性小鼠。DDP建模组小鼠按2.5 mg·kg^-1·d^-1剂量腹腔注射DDP溶液，每3 d注射1次，共7次；造模同日，对照组小鼠腹腔注射等量的生理盐水；而DEHA治疗组小鼠在予以DDP处理的同时，按8.3 mg·kg^-1·d^-1剂量给予DHEA灌胃，连续给药21 d。通过旷场、抓力和转棒试验观察小鼠疲劳指标的变化，采用常规组织切片的HE染色法观察肾上腺、睾丸与卵巢组织形态的变化，运用高效液相色谱串联质谱方法检测小鼠血清类固醇激素的含量，运用实时荧光定量PCR和蛋白质印迹技术检测下丘脑、垂体、肾上腺、睾丸和卵巢组织中相关基因的mRNA和蛋白表达水平。结果与对照组比较，DDP建模组雄性与雌性小鼠的体重以及旷场运动能力均显著下降（P＜0.05），同时雌性小鼠的抓力和转棒时间也均显著下降（P＜0.05）；而DHEA治疗组小鼠的上述疲劳指标均显著改善（P＜0.05）。DDP建模组雄性小鼠睾丸组织中各级生精细胞排列紊乱且睾丸间质水肿，雌性小鼠卵巢组织中大量原始卵泡被激活，闭锁卵泡增多，卵泡颗粒细胞减少；而DHEA治疗组小鼠睾丸和卵巢受损伤表型均得到显著改善。与对照组比较，DDP建模组雄性与雌性小鼠血清睾酮、二氢睾酮含量均显著降低（P＜0.01），雄性小鼠血清孕烯醇酮含量下降而皮质酮含量显著升高（P＜0.05），雌性小鼠血清皮质酮含量显著降低（P＜0.05）；与DDP建模组比较，DHEA治疗组雄性小鼠的孕烯醇酮含量和雌性小鼠的孕酮含量均显著增高（P＜0.05），而雌性小鼠的孕烯醇酮含量和雄性小鼠的孕酮含量均显著降低（P＜0.05）。与对照组比较，DDP建模组雄性与雌性小鼠的肾上腺Cyp21a1、Cyp11a1基因和下丘脑Gnrh基因表达均显著下调（P＜0.05）；雌性小鼠肾上腺Hsd3b2基因，卵巢Star、Cyp11a1、Lhr基因，下丘脑Crh、垂体Pomc和Lhb基因表达均显著下调（P＜0.05）；雄性小鼠睾丸Star基因及StAR蛋白以及垂体Fshb和Lhb基因表达均显著下调（P＜0.05）。与DDP建模组比较，补充DHEA后，雄性小鼠肾上腺Cyp17a1基因和睾丸Cyp17a1、Lhr、Fshr基因表达均显著下调（P＜0.05）；雌性小鼠肾上腺Cyp11a1基因表达显著下调（P＜0.05），而肾上腺Hsd3b2基因和卵巢Star、Cyp11a1、Hsd3b2、Lhr基因，以及垂体Lhb基因表达均上调（P＜0.05）。结论 DDP间断给药可以抑制下丘脑-垂体-肾上腺皮质与性腺轴功能，并且雌性受抑制更显著；补充DHEA有助于调节体内类固醇激素水平的自身稳态。

关键词: 顺铂, 脱氢表雄酮, 睾丸, 卵巢, 类固醇激素, 小鼠

Abstract:

Objective To study the pathway of cis-dichlorodiamineplatinum (DDP) inhibiting the synthesis of steroid hormones in mice, and to observe the intervention effect of dehydroepiandrosterone (DHEA). Methods Sixty adult ICR mice were randomly divided into three groups: control group, DDP modeling group, and DHEA group, with 10 male and 10 female mice in each group. The DDP modeling group mice were intraperitoneally injected with DDP solution at a dose of 2.5 mg·kg^-1·d^-1, once every 3 days, a total of 7 times. On the same day of modeling, the control group mice were injected with an equal amount of physiological saline intraperitoneally. The DEHA treatment group mice were treated with DDP and given a dose of 8.3 mg·kg^-1·d ^-1 of DHEA by gavage for 21 consecutive days. The changes of fatigue indexes of mice were observed by open field, grip and rod rotation tests. The morphology changes of adrenal gland, testicular and ovarian tissue were observed by pathological section and HE staining. The levels of serum steroid hormones were detected by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The mRNA and protein expression levels of the related genes of the hypothalamus, hypophysis, adrenal, testis and ovary were tested by real-time fluorescent quantitative PCR (RT-qPCR) and Western blotting. Results Compared with control group, both male and female mice in DDP modeling group were significantly losing weight (P＜0.05), their abilities in horizontal movement and vertical movement decreased (all P＜0.05), and the stay time and grip also significantly decreased (all P＜0.05) in female mice. Indexes of fatigue were improved after DHEA supplement (all P＜0.05). In the DDP modeling group, the arrangement of spermatogenic cells at all levels in the testicular tissue was disordered and the testicular interstitial edema was observed, and a large number of primordial follicles in the ovarian tissue were activated, the number of atresia follicles increased, and the number of granulosa cells in the follicles decreased; while in the DHEA group, the damaged phenotype of testicles and ovaries was significantly improved. Compared with control group, the levels of serum testosterone and dihydrotestosterone in both male and female DDP modeling mice significantly decreased (P＜0.01), the pregnenolone was down-regulated but corticosterone was up-regulated significantly (P＜0.05) in male mice, the corticosterone was down-regulated significantly (P＜0.05) in female mice. Compared with the DDP group, after DHEA supplement, the pregnenolone in male mice and the progesterone in female mice increased significantly (P＜0.05), but the pregnenolone in female mice and the progesterone in male mice decreased significantly (P＜0.05). Compared with control group, the expression levels of Cyp21a1 and Cyp11a1 genes in the adrenal gland and Gnrh gene in the hypothalamus of male and female mice in the DDP modeling group significantly decreased (all P＜0.05); the expression levels of Hsd3b2 gene in the adrenal gland, Star, Cyp11a1, and Lhr genes in the ovaries, Crh, Pomc, and Lhb genes in the hypothalamus, pituitary, and pituitary of female mice significantly decreased (all P＜0.05); the expression levels of Star gene and StAR protein in the testicles of male mice, as well as Fshb and Lhb genes in the pituitary gland, were significantly down-regulated (all P＜0.05). After DHEA supplement, compared with the DDP modeling group, the mRNA expression levels of Cyp17a1 in the adrenal gland of male mice and Cyp17a1, Lhr and Fshr genes in testis were down-regulated significantly (P＜0.05); the expression level of Cyp11a1 gene in the adrenal gland of female mice was also decreased (P＜0.05); while the expression levels of Hsd3b2 gene in the adrenal gland, Star, Cyp11a1, Hsd3b2 and Lhr gene in the ovary, and Lhb gene in the pituitary gland were all up-regulated ( P＜0.05). Conclusion The function of hypothalamus-pituitary-adrenal/gonadal axis was inhibited by DDP intermittent injection, especially in female. Supplementation of DHEA can help regulate the homeostasis of steroid hormone levels.

Key words: Cisplatin, Dehydroepiandrosterone, Testis, Ovary, Steroid hormone, Mice

中图分类号:

R-332

潘志强, 农淄心, 谢海纳, 彭佩克. 顺铂对小鼠下丘脑-垂体-肾上腺/性腺轴功能的损伤作用及脱氢表雄酮的干预效应[J]. 实验动物与比较医学, 2023, 43(3): 229-242.

Zhiqiang PAN, Zixin NONG, Haina XIE, Peike PENG. Injurious Effect of Cisplatin on the Function of Hypothalamus-pituitary-adrenal/gonadal Axis in Mice and the Intervention Effect of Dehydroepiandrosterone[J]. Laboratory Animal and Comparative Medicine, 2023, 43(3): 229-242.

图/表 7

表1 实时荧光定量PCR引物序列

Table 1 Primer sequences in real-time fluorescent quantitative PCR

基因名称 Gene name	引物序列 Primer sequence	产物大小 Length/bp
β-actin	F：5'-TGTTACCAACTGGGACGACA-3'; R：5'-GGGGTGTTGAAGGTCTCAAA-3'	165
Star	F：5'-TTGGGCATACTCAACAACCA-3'; R：5'-GAAACACCTTGCCCACATCT-3'	103
Cyp11a1	F：5'-ACTTCCGGTACTTGGGCTTT-3'; R：5'-GCTTGAGAGGCTGGAAGTTG-3'	201
Cyp11b1	F：5'-GTATCGAGAGCTGGCAGAGG-3'; R：5'-GGGTTGATGTCGTGTCAGTG-3'	140
Cyp21a1	F：5'-CTCCGGCTATGACATCCCTA-3'; R：5'-ACAGCCAAAGGATGGTGTTC-3'	151
Cyp17a1	F：5'-TGGTCATATGCATGCCAACT-3'; R：5'-CCCTTCTTCACGAGCACTTC-3'	132
Hsd3b2	F：5'-TCCAGCTCAGTTGATGTTGC-3'; R：5'-TGCCTTCTCAGCCATCTTTT-3'	129
Cyp19a1	F：5'-CTTTCAGCCTTTTGGCTTTG-3'; R：5'-ATTTCCACAAGGTGCCTGTC-3'	192
Hsd17b1	F：5'-GTTATGAGCAAGCCCTGAGC-3'; R：5'-AAGCGGTTCGTGGAGAAGTA-3'	112
Ar	F：5'-AAGCAGGTAGCTCTGGGACA-3'; R：5'-GAGCCAGCGGAAAGTTGTAG-3'	117
Fshr	F：5'-ATCACACATGCCATGCAACT-3'; R：5'-GTACGAGGAGGGCCATAACA-3'	199
Lhr	F：5'-CAATGGGACGACGCTAATCT-3'; R：5'-CTGGAGGGCAGAGTTTTCAG-3'	204
Esr1	F：5'-TTACGAAGTGGGCATGATGA-3'; R：5'-CCTGAAGCACCCATTTCATT-3'	117
Esr2	F：5'-GAAGCTGGCTGACAAGGAAC-3'; R：5'–AACGAGGTCTGGAGCAAAGA-3'	187
Gper1	F：5'-GTCAACCTTGCAGCCTTCTC-3'; R：5'-AGCACTGCTGAACCTGACCT-3'	198
Pomc	F：5'-CATTAGGCTTGGAGCAGGTC-3'; R：5'-CTTCTCGGAGGTCATGAAGC-3'	128
Fshb	F：5'-TCAGCTTTCCCCAGAAGAGA-3'; R：5'-CCGAGCTGGGTCCTTATACA-3'	249
Lhb	F：5'-CTCAGCCAGTGTGCACCTAC-3'; R：5'-GACCCCCACAGTCAGAGCTA-3'	150
GnRH	F：5'-AGCACTGGTCCTATGGGTTG-3'; R：5'-GGGCCAGTGCATCTACATCT-3'	221
Crh	F：5'-TCTCACCTTCCACCTTCTGC-3'; R：5'-AAGCGCAACATTTCATTTCC-3'	118

图1 DDP和DHEA处理后小鼠体重和疲劳指标的变化注：CON为对照组，DDP为DDP建模组，DHEA为脱氢表雄酮治疗组。与正常组比较，?P<0.05；与DDP建模组比较，#P<0.05。

Figure 1 Changes of body weight and fatigue indexes in mice after DDP and DHEA treatmentNote：CON indicates control group， DDP indicates cis-dichlorodiamineplatinum （cisplatin） modeling group， DHEA indicates dehydroepian-drosterone （DHEA） treatment after cisplatin modeling group. Compared with the control group， ?P<0.05； Compared with the cisplatin modeling group， #P<0.05.

图2 小鼠肾上腺（×200）、睾丸（×200）、卵巢（×100）组织病理形态学观察（HE染色）注：CON为对照组，DDP为DDP建模组，DHEA为脱氢表雄酮治疗组。肾上腺组织（图A～F，比例尺大小为50 μm）中，■表示肾上腺皮质；×表示肾上腺髓质；★表示球状带；◆表示束状带；▼表示网状带。睾丸组织（图G～I，比例尺大小为50 μm）中，○表示曲细精管；←表示精子细胞；→表示精原细胞；↑表示初级精母细胞；↓表示次级精母细胞；↘表示支持细胞；↖表示睾丸间质细胞。卵巢组织（图J～L，比例尺大小为100 μm）中，○表示卵泡腔；□表示黄体；▲表示闭锁卵泡；●表示白体；←表示原始卵泡；→表示初级卵泡；↑表示次级卵泡；↘表示卵泡膜层；↖表示颗粒细胞。

Figure 2 Pathological morphology of adrenal gland （×200）， testis （×200） and ovary （×100） in mice （HE staining）Note：CON indicates control group， DDP indicates cisplatin modeling group， DHEA indicates dehydroepiandrosterone （DHEA） treatment after cisplatin modeling group. In Fig. A-F （scale bar size is 50 μm）， ■ indicates adrenal cortex， × indicates adrenal medulla， ★ indicates zona glomerulosa， ◆indicates zona fasciculata， ▼ indicates zona reticularis. In Fig. G-I （scale bar size is 50 μm）， ○ indicates seminiferous tubule， ← indicate spermatocyte， → indicates spermatogonia， ↑ indicates primary spermatocyte， ↓ indicates secondary spermatocyte， ↘indicates sertoli cells， ↖ indicates leydig cells. In Fig. J-L （scale bar size is 100 μm）， ○ indicates follicular cavity， □ indicates corpus luteum， ▲ indicates atresia follicles， ● indicates corpus albicans of ovary， ← indicates primordial follicle， → indicates primary follicle， ↑ indicates secondary follicle， ↘ indicates follicle， ↖ indicates granulosa cells.

图3 小鼠血清皮质激素含量及肾上腺、下丘脑和垂体组织中皮质激素合成相关基因表达水平注：A～B，高效液相色谱串联质谱联用仪检测小鼠血清中11-脱氧皮质酮（DOC）和皮质酮（Cort）含量的结果；C～D，实时荧光定量PCR检测小鼠肾上腺组织中Cyp21a1和Cyp11b1 mRNA水平；E～G，蛋白质印迹法检测肾上腺组织中CYP11B1与CYP21A2蛋白表达及其相对表达量分析；H～I，实时荧光定量PCR检测小鼠下丘脑组织中促肾上腺皮质激素释放激素（Crh）和垂体组织中促肾上腺皮质激素（Pomc）mRNA水平。CON为对照组，DDP为DDP建模组，DHEA为脱氢表雄酮治疗组。与对照组比较，*P<0.05，**P<0.01；与DDP建模组比较，#P<0.05；与同组雄性小鼠比较， &P<0.05。

Figure 3 The content of serum corticosteroid and the expression levels of corticosteroid synthesis-related genes in adrenal， hypothalamic， and pituitary tissues of miceNote：A-B， The contents of 11-deoxycorticosterone （DOC） and corticosterone （Cort） in mouse serum were determined by high-performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）； C-D， The expression levels of Cyp21a1and Cyp11b1 mRNA in adrenal tissue were determined by real-time fluorescent quantitative PCR （RT-qPCR）； E-G， The expression levels of CYP11B1 and CYP21A2 protein in adrenal tissue were determined by Western blotting； H-I， The expression levels of corticotropin releasing hormone （Crh） mRNA in hypothalamus and pro-opiomelanocortin-alph （Pomc） mRNA in pituitary were determined by RT-qPCR. CON indicates control group， DDP indicates cisplatin modeling group， DHEA indicates dehydroepiandrosterone （DHEA） treatment after cisplatin modeling group. Compared with control group， *P<0.05， **P<0.01； Compared with the DDP group， #P<0.05； Compared with male mice in the same group， &P<0.05.

图4 小鼠血清孕烯醇酮含量及肾上腺、睾丸与卵巢组织中孕烯醇酮合成相关基因表达水平注：A，高效液相色谱串联质谱联用仪检测小鼠血清孕烯醇酮（Preg）含量的结果；B～C，实时荧光定量PCR检测小鼠肾上腺组织中类固醇激素急性调节蛋白（Star）和P450scc裂解酶编码基因Cyp11a1的mRNA转录水平；D～E，实时荧光定量PCR检测小鼠睾丸与卵巢组织中Star和Cyp11a1 mRNA水平；F～L，蛋白质印迹法检测小鼠肾上腺、睾丸与卵巢组织中18ku转位蛋白（TSPO）、StAR与CYP11A1蛋白及其相对表达量分析。CON为对照组，DDP为DDP建模组，DHEA为脱氢表雄酮治疗组。与对照组比较，*P<0.05，**P<0.01；与DDP建模组比较，#P<0.05，##P<0.01。

Figure 4 The content of serum pregnenolone and the expression levels of pregnenolone-synthesis-related genes in adrenal， testis and ovary tissues of miceNote：A， The contents of pregnenolone （Preg） in mouse serum was determined by high-performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）； B-C， The expression levels of steroidogenic acute regulatory protein （Star） and Cyp11a1 mRNA in adrenal tissue were determined by real-time fluorescent quantitative PCR （RT-qPCR）； D-E， The expression levels of Star and Cyp11a1 mRNA in testis and ovary were determined by RT-qPCR； F-L， The expression levels of translocatorprotein （TSPO）， StAR and CYP11A1 proteins in adrenal， testis and ovary tissues were determined by Western blotting. CON indicates control group， DDP indicates cisplatin modeling group， DHEA indicates dehydroepiandrosterone （DHEA） treatment after cisplatin modeling group. Compared with control group， *P<0.05， **P<0.01； Compared with the DDP group， #P<0.05， ##P<0.01.

图5 小鼠血清孕酮、17-羟孕烯醇酮和17-羟孕酮含量以及肾上腺、睾丸与卵巢组织中合成相关基因表达注：A～C，高效液相色谱串联质谱联用仪检测小鼠血清孕酮（Prog）、17-羟孕烯醇酮（17-OH Preg）和17-羟孕酮（17-OH Prog）含量的结果；D～E，实时荧光定量PCR检测小鼠睾丸和卵巢组织中17α-羟化酶编码基因Cyp17a1和3β-羟基类固醇脱氢酶2（Hsd3b2）mRNA水平；F～G，实时荧光定量PCR检测小鼠肾上腺组织中Cyp17a1和Hsd3b2 mRNA水平。CON为对照组，DDP为DDP建模组，DHEA为脱氢表雄酮治疗组。与对照组比较，*P<0.05；与DDP建模组比较，#P<0.05；与同组雄性小鼠比较， &P<0.05。

Figure 5 The content of serum progesterone， 17-hydroxypregnenolone and 17-hydroxyprogesterone as well as the expression levels of their synthesis-related genes in adrenal， testis and ovary tissues of miceNote：A-C， The contents of progesterone （Prog）， 17-hydroxypregnenolone （17-OH Preg） and 17-hydroxyprogesterone （17-OH Prog） in mouse serum were determined by high-performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）； D-E， The expression levels of Cyp17a1 and 3-beta-hydroxysteroid dehydrogenase type 2 （Hsd3b2） mRNA in testis and ovary were determined by real-time fluorescent quantitative PCR （RT-qPCR）； F-G， The expression levels of Cyp17a1 and Hsd3b2 mRNA in adrenal tissue were determined by RT-qPCR. CON indicates the control group， DDP indicates cisplatin modeling group， DHEA indicates dehydroepian-drosterone （DHEA） treatment after cisplatin modeling group. Compared with the control group， *P<0.05； Compared with the DDP group， #P<0.05； Compared with male mice in the same group， &P<0.05.

图6 小鼠血清雄激素水平及下丘脑-垂体-性腺轴相关基因表达水平注：A～C，高效液相色谱串联质谱联用仪检测小鼠血清雄烯二酮（Andro）、睾酮（T）和二氢睾酮（DHT）含量的结果；D～F，实时荧光定量PCR检测小鼠下丘脑组织中促性腺激素释放激素（Gnrh）以及垂体组织中促卵泡刺激素β亚基（Fshb）、黄体生成素β亚基（Lhb）mRNA表达水平；G～I，实时荧光定量PCR检测小鼠睾丸和卵巢组织中促卵泡刺激素受体（Fshr）、黄体生成素受体（Lhr）、雄激素受体（Ar）、雌激素受体（Esr1、Esr2）和G蛋白偶联雌激素受体1（Gper1）mRNA表达水平；J，实时荧光定量PCR检测小鼠卵巢组织中芳香化酶编码基因Cyp19a1和17β-羟基类固醇脱氢酶1（Hsd17b1）mRNA表达水平。CON为对照组，DDP为DDP建模组，DHEA为脱氢表雄酮治疗组。与对照组比较，?P<0.05，??P<0.01；与DDP建模组比较，#P<0.05；与同组雄性小鼠比较， &&P<0.01。

Figure 6 The content of serum androgen and the expression levels of hypothalamus-pituitary-adrenal/gonadal axis-related genes in adrenal， testis and ovary tissues of miceNote：A-C， The contents of androstenedione （Andro）， testosterone （T） and dihydrotestosterone （DHT） in mouse serum were determined by high-performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS）； D-F， The expression levels of gonadotropin-releasing hormone （Gnrh） mRNA in hypothalamus tissue， folicular stimulating hormone beta subunit （Fshb） and luteinizing hormone subunit beta （Lhb） mRNA in pituitary were determined by real-time fluorescent quantitative PCR （RT-qPCR）； G-I， The expression levels of folicular stimulating hormone receptor （Fshr）， luteinizing hormone receptor （Lhr）， androgen receptor （Ar）， estrogen receptor （Esr）1/2 and G-protein coupled estrogen receptor 1 （Gper1） mRNA in testis and ovary were determined by RT-qPCR； L， The expression levels of Cyp19a1 and 17-beta-hydroxysteroid dehydrogenase type 1 （Hsd7b1） mRNA in mouse ovarian tissue by RT-qPCR. CON indicates control group， DDP indicates cisplatin modeling group， DHEA indicates dehydroepiandrosterone （DHEA） treatment after cisplatin modeling group. Compared with control group， ?P<0.05， ??P<0.01； Compared with the DDP group， #P<0.05； Compared with male mice in the same group， &&P<0.01.

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