CeO₂NPs relieve radiofrequency radiation, improve testosterone synthesis, and clock gene expression in Leydig cells by enhancing antioxidation

Figures & data

Figure 1 Characterization of test material CeO₂NPs. (A) Scanning electron micrographs of CeO₂; (B) transmission electron micrographs of CeO₂; (C) size distribution histogram of CeO₂NPs; (D) the results of XRD test.

Abbreviations: CeO₂NPs, cerium oxide nanoparticles; XRD, X-ray powder diffractometry.

Table 1 Experimental design groups

Groups	Solvent	CeO2NPs	RF exposure
Blank	–	–	–
Solvent Control	+	–	–
CeO2NPs	+	+	–
RF 1 hr	+	–	+
RF 2 hrs	+	–	+
RF 4 hrs	+	–	+
CeO2NPs + RF 1 hr	+	+	+
CeO2NPs + RF 2 hrs	+	+	+
CeO2NPs + RF 4 hrs	+	+	+

Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation.

Figure 2 Distribution of SAR of the culture medium inside a standard petri dish (35-mm) exposed to 1,800 MHz radiofrequency fields (power densities, 200.27 μW/cm²).

Abbreviation: SAR, specific absorption rate.

Table 2 Primer sequence for real-time PCR

Genes	Primer sequence
Star	Sense: 5ʹ-GGCATACTCAACAACCAGAAGGC −3’
Antisense: 5ʹ-CTCCATGCGGTCCACAAGTTCTTC −3’
Cyp11a1	Sense: 5ʹ-CCGTGGATAACAGCAGCAGGAAC −3’
Antisense:5ʹ-CCAGCACAGATGGTCGCAGATAC −3’
Hsd-3β	Sense: 5ʹ-TCCACACTGCTGCTGTCATTGATG −3’
Antisense: 5ʹ-AGAAGATGAAGGCTGGCACACTTG −3’
Bmal 1	Sense: 5ʹ-CTCGGTCACATCCTACGACAAAC −3’
Antisense: 5ʹ-CACAGAAGCAAACTACAAGCCAAC −3’
Clock	Sense: 5ʹ-TGGTGACTGCCTATCCTACCTTCG −3’
Antisense: 5ʹ-TGCTGCTGCTGCTGCTGTTG −3’
Rorα	Sense: 5ʹ-CCACCTACTCCTGTCCTCGTCAG −3’
Antisense: 5ʹ-CTTCTGCACCTCGGCGTACAAG −3’
β-actin	Sense: 5′-TGGAATCCTGTGGCATCCATGAAAC-3’
Antisense: 5ʹ-TAAAACGCAGCTCAGTAACAGTCCG-3’

Figure 3 Stain of HSD-3β on primary leydig cells (A, 20×; B, 40×).

Abbreviation: HSD-3β, 3β-Hydroxysteroid dehydrogenase.

Figure 4 Effects of different dose CeO₂NPs on the proliferation rate of primary Leydig cells.

Notes: Compared with the 0 dose level (Solvent), the difference was significant as, *P<0.05, **P<0.01.

Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation.

Figure 5 Testosterone concentration in Leydig cells medium treated by CeO₂NPs, RF and CeO₂NPs + RF.

Notes: Compared with the Solvent Control group, the difference was significant as, *P<0.05, **P<0.01; Compared with the RF group at same exposure time, the difference was significant as, # P<0.05.

Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation.

Figure 6 CAT activity in Leydig cells treated by CeO₂NPs, RF, and CeO₂NPs + RF.

Notes: Compared with the Solvent Control group, the difference was significant as, *P<0.05, **P<0.01; Compared with the RF group at same exposure time, the difference was significant as, # P<0.05, ## P<0.01.

Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation; CAT, catalase.

Figure 7 MDA content in Leydig cells treated by CeO₂NPs, RF and CeO₂NPs + RF.

Notes: Compared with the Solvent Control group, the difference was significant as, **P<0.01; compared with the RF group at same exposure time, the difference was significant as, ## P<0.01.

Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation; MDA, malondialdehyde.

Figure 8 T-AOC levels in Leydig cells treated by CeO₂NPs, RF, and CeO₂NPs + RF.

Notes: Compared with the Solvent Control group, the difference was significant as, *P<0.05, **P<0.01; Compared with the RF group at same exposure time, the difference was significant as, ## P<0.01.

Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation; T-AOC, total antioxidation capacity.

Figure 9 The mRNA expression of testosterone synthase genes (Star, Cyp11a1, Hsd-3β) in Leydig cells treated by CeO₂NPs, RF, and CeO₂NPs + RF.

Notes: Compared with the Solvent Control group, the difference was significant as, *P<0.05, **P<0.01; Compared with the RF group at same exposure time, the difference was significant as, # P<0.05, ## P<0.01.

Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation.

Figure 10 The clock genes (Clock, Bmal1, Rorα) mRNA expression from Real-time PCR in Leydig cells treated by CeO₂NPs, RF, and CeO₂NPs + RF.

Notes: Compared with the Solvent control group, the difference was significant as, **P<0.01; Compared with the RF group at same exposure time, the difference was significant as, ## P<0.01.

Abbreviations: CeO₂NPs, cerium oxide nanoparticles; RF, radiofrequency radiation.