Injurious Effect of Cisplatin on the Function of Hypothalamus-pituitary-adrenal/gonadal Axis in Mice and the Intervention Effect of Dehydroepiandrosterone

doi:10.12300/j.issn.1674-5817.2022.182

Abstract

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

CLC Number:

R-332

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.

Figures/Tables 7

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

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.

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.

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.

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.

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.

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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