双酚S和双酚F对雄性生殖系统损伤信号通路的生物信息学分析及其实验验证

doi:10.13481/j.1671-587X.20250220

Abstract

Abstract:

Objective To investigate the pathways involved in bisphenol S （BPS） and bisphenol F （BPF） induced male reproductive injury by bioinformatics methods and experimental verification. Methods Bioinformatics analysis was conducted to screen the genes related to male reproductive system diseases associated with BPF and BPS from the Comparative Toxicogenomics Database （CTD）. Functional enrichment using Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis were performed to predict potential signaling pathways and key genes. Cell counting kit-8（CCK-8） method was used to assess the cell viability in various groups treated with different concentrations of BPS and BPF （1×10^-3， 1×10^-2， 1×10^-1， 1×10⁰，1×10¹， and 1×10² μmol·L^-1）. TM3 cells were divided into control group （0.1% DMSO）， different doses of BPS groups， and different doses of BPF groups. The cells were treated with 20， 40， and 80 μmol·L^-1 of BPS and BPF for 72 h， respectively. Real-time fluorescence quantitative PCR （RT-qPCR） and Western blotting method were used to detect the expression levels of key genes mRNA and proteins in various groups. Results The bioinformatics analysis results revealed that 507 and 447 male systemic disease genes related to BPS and BPF were screened by CTD， respectively. The GO enrichment analysis results showed that the selected genes were primarily enriched in biological processes （BP） such as reproductive system development and reproductive structure development. The KEGG pathway analysis results indicated that these genes were significantly enriched in signaling pathways such as phosphatidylinositol 3-kinase/protein kinase B （PI3K/AKT）， hypoxia-inducible factor-1（HIF-1）， and cellular senescence（P<0.001）. The CCK-8 method results showed that compared with control group， the cell viabilities in 1×10² μmol·L^-1 BPF and BPS groups were significantly decreased （P<0.05）， while the viabilities of TM3 cells in other groups had no significant changes （P>0.05）. After BPS treatment， compared with control group， the expression levels of PI3K， AKT， hypoxia-inducible factor-1α （HIF-1α）， and CREB-binding protein （CBP） mRNA in low， medium， and high doses of BPS groups were decreased （P<0.05）， the expression levels of PI3K protein were decreased （P<0.05）， the expression levels of B-cell lymphoma-2-associated X protein （Bax） protein were increased （P<0.05）， and the expression levels of serine protease inhibitor clade B， member 10 （SERPINB10） mRNA were increased （P<0.01）； the expression levels of Bax and intraflagellar transport 80 homolog （IFT80） mRNA in the cells in medium and high doses of BPS groups were increased （P<0.05）； the expression levels of B-cell lymphoma-2 （Bcl-2） mRNA and protein in low and high doses of BPS groups were decreased （P<0.05）； the expression levels of additional sex combs like 2 （ASXL2） mRNA in low and medium doses of BPS groups were decreased （P<0.01）. After BPF treatment， compared with control group， the expression levels of Bcl-2， HIF-1α， and structural maintenance of chromosomes protein 1B （SMC1B） mRNA in low， medium， and high doses of BPF groups were decreased （P<0.05）， and the expression levels of IFT80 mRNA （P<0.01） and Bax protein （P<0.01） were increased； the expression levels of PI3K， AKT， and ring finger protein 130 （RNF130） mRNA in low and high doses of BPF groups were decreased （P<0.05）； the expression level of CBP mRNA in medium dose of BPF group was decreased （P<0.05）， while the expression level of RNF130 mRNA was increased （P<0.05）； the expression levels of PI3K and Bcl-2 proteins in high dose of BPF group were decreased （P<0.05）. Conclusion BPF and BPS may cause cell cytotoxicity and impair male reproductive health through PI3K/AKT and HIF-1 signaling pathways. RNF130 and SMC1B may be important targets for their induction of male reproductive toxicity.

Key words: Bisphenol S, Bisphenol F, Comparative toxicogenomics database, Male reproduction damage, Leydig cells

CLC Number:

R114

Yu SHI,Jingzhi LI,Hongqiang CHEN,Shimeng ZHOU,Na WANG,Jia CAO,Li YIN,Wenbin LIU. Bioinformatics analysis and experimental validation of BPS and BPF on signaling pathways of male reproductive system damage[J].Journal of Jilin University(Medicine Edition), 2025, 51(2): 460-470.

Figures/Tables 10

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References 28

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Transcript	Primer	Sequence (5′-3′)
PI3K	m-PI3K-F	CCATTACAAAGAAAGCCGGACTC
	m-PI3K-R	GGGGGCAGTGCTGGTG
AKT	m-AKT-F	GCCGCCTGATCAAGTTCTCC
	m-AKT-R	TTCAGATGATCCATGCGGGG
Bcl-2	m-Bcl-2-F	GACTGAGTACCTGAACCGGC
	m-Bcl-2-R	AGTTCCACAAAGGCATCCCAG
HIF-1α	m-HIF-1α-F	CGCCTCTGGACTTGTCTCTT
	m-HIF-1α-R	CGACGTTCAGAACTCATCCTATTTT
CBP	m-CBP-F	GGAAAGCCTGCCAAGTTGC
	m-CBP-R	ACAGTCATGTCGTGTGCAGT
Bax	m-Bax-F	CTGGATCCAAGACCAGGGTG
	m-Bax-R	GTGAGGACTCCAGCCACAAA
RNF130	m-RNF130-F	CCTTGCCGGTGACAGTTCC
	m-RNF130-R	CCTTCTTCAAAACCATTCTACCTTG
SERPINB10	m-SERPINB10-F	GTCCGACTTCCAGACCCTTG
	m-SERPINB10-R	TTGTGGTTCTGCCCCAAAGT
ASXL2	m-ASXL2-F	CGACTTCCCCTTTCTCCCAC
	m-ASXL2-R	GTTCTGACTGACTGGGAGGC
SMC1B	m-SMC1B-F	AGCTACAACTGTGTGGCTCC
	m-SMC1B-R	AAGAATGGAGCAGGCCGAAA
IFT80	m-IFT80-F	TCCTGGAACTTGAGTGAATACAA
	m-IFT80-R	ACTTGACGATCTGGTGGTCG
β-actin	m-β-actin-F	ACTATTGGCAACGAGCGGTT
	m-β-actin-R	GCCACAGGATTCCATACCCAA

Group	Cell viability
Group	（t/h）24	48	72
Control	1.00±0.06	1.00±0.05	1.00±0.02
BPF [c_B/μmol·L^-1]
1×10^-3	0.99±0.05	0.97±0.08	0.98±0.02
1×10^-2	1.05±0.03	0.87±0.04	0.92±0.01
1×10^-1	0.92±0.06	0.88±0.03	0.90±0.01
1×10⁰	0.99±0.03	0.90±0.03	0.87±0.02
1×10¹	0.86±0.03	0.83±0.04	0.84±0.01
1×10²	0.88±0.07^*	0.33±0.01^**	0.18±0.02^**

Group	Cell viability
Group	(t/h) 24	48	72
Control	1.00±0.05	1.00±0.02	1.00±0.02
BPS [c_B/μmol·L^-1]
1×10^-3	0.94±0.05	0.96±0.05	0.97±0.02
1×10^-2	0.89±0.09	0.99±0.03	0.91±0.02
1×10^-1	0.81±0.04	0.94±0.05	0.91±0.03
1×10⁰	0.83±0.07	0.92±0.04	0.91±0.02
1×10¹	0.83±0.03	0.90±0.03	0.92±0.02
1×10²	0.84±0.05^**	0.71±0.02^**	0.70±0.04^**

Group	PI3K	AKT	Bcl-2	HIF-1α	CBP	Bax
Control	1.00±0.01	1.00±0.04	1.00±0.01	1.00±0.02	1.00±0.05	1.00±0.01
BPS
Low dose	0.77±0.09^*	0.47±0.02^**	0.90±0.02^*	0.26±0.03^**	0.71±0.02^*	0.69±0.02^*
Medium dose	0.47±0.01^*	0.35±0.04^**	0.95±0.02	0.46±0.02^**	0.75±0.03^**	1.41±0.04^**
High dose	0.87±0.04^*	0.34±0.03^**	0.80±0.01^**	0.46±0.02^**	0.68±0.03^**	1.16±0.04^**

Group	PI3K	AKT	Bcl-2	HIF-1α	CBP	Bax
Control	1.00±0.09	1.00±0.04	1.00±0.01	1.00±0.02	1.00±0.05	1.00±0.01
BPF
Low dose	0.76±0.01^**	0.81±0.03^*	0.51±0.01^*	0.64±0.04^**	0.94±0.01	1.08±0.03
Medium dose	0.91±0.02	0.89±0.07	0.49±0.01^**	0.55±0.03^**	0.88±0.01^*	1.02±0.04
High dose	0.81±0.06^*	0.84±0.01^*	0.60±0.00^**	0.75±0.05^**	0.92±0.01	1.11±0.04

Bioinformatics analysis and experimental validation of BPS and BPF on signaling pathways of male reproductive system damage

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Group	PI3K	Bcl-2	Bax
BPS
Control	1.00±0.01	1.00±0.03	1.00±0.13
Low dose	0.83±0.01^*	0.81±0.08^*	2.37±0.01^*
Medium dose	0.76±0.11^**	0.95±0.04	2.72±0.39^*
High dose	0.51±0.09^***	0.79±0.04^*	3.25±0.58^**
BPF
Control	1.00±0.02	1.00±0.02	1.00±0.02
Low dose	0.85±0.14	0.95±0.03	1.50±0.02^△△
Medium dose	0.94±0.13	0.92±0.03	1.45±0.01^△△
High dose	0.57±0.14^△	0.53±0.03^△△	1.83±0.01^△△

Group	RNF130	SERPINB10	ASXL2	SMC1B	IFT80
Control	1.00±0.25	1.00±0.06	1.00±0.03	1.00±0.02	1.00±0.02
BPS
Low dose	1.11±0.13	1.51±0.03^**	0.73±0.02^**	0.78±0.09	1.01±0.03
Medium dose	1.13±0.19	1.67±0.05^**	0.87±0.03^**	1.01±0.05	1.25±0.06^**
High dose	1.52±0.21	2.08±0.17^**	0.96±0.04	1.27±0.12	1.17±0.06^*

Group	RNF130	SERPINB10	ASXL2	SMC1B	IFT80
Control	1.00±0.17	1.00±0.01	1.00±0.04	1.00±0.12	1.00±0.02
BPF
Low dose	0.20±0.10^**	0.83±0.02^*	0.77±0.08^*	0.45±0.01^**	1.46±0.04^**
Medium dose	1.18±0.01^*	1.08±0.08	0.78±0.03^*	0.40±0.01^*	1.49±0.07^**
High dose	0.15±0.06^**	0.94±0.21	0.79±0.07	0.60±0.02^**	1.53±0.04^**

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