MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

Abstract

Non-thermal plasma treatment is an emerging innovative technique with a wide range of biological applications. This study was conducted to investigate the effect of a non-thermal dielectric barrier discharge plasma technique on immature chicken Sertoli cell (SC) viability and the regulatory role of microRNA (miR)-7450. Results showed that plasma treatment increased SC apoptosis in a time- and dose-dependent manner. Plasma-induced SC apoptosis possibly resulted from the excess production of reactive oxygen species via the suppression of antioxidant defense systems and decreased cellular energy metabolism through the inhibition of adenosine triphosphate (ATP) release and respiratory enzyme activity in the mitochondria. In addition, plasma treatment downregulated miR-7450 expression and activated adenosine monophosphate-activated protein kinase α (AMPKα), which further inhibited mammalian target of rapamycin (mTOR) phosphorylation in SCs. A single-stranded synthetic miR-7450 antagomir disrupted mitochondrial membrane potential and decreased ATP level and mTOR phosphorylation by targeting the activation of AMPKα, which resulted in significant increases in SC lethality. A double-stranded synthetic miR-7450 agomir produced opposite effects on these parameters and ameliorated plasma-mediated apoptotic effects on SCs. Our findings suggest that miR-7450 is involved in the regulation of plasma-induced SC apoptosis through the activation of AMPKα and the further inhibition of mTOR signaling pathway.

Figure 1

Chicken SC viability and apoptosis after non-thermal plasma treatment. (A) Schematic of non-thermal DBD plasma reactor. (B) Dependence of the discharge power on the applied voltage. (C) Relative viability in SCs exposed to 11.7 kV for different durations. (D) Relative viability in SCs exposed to different potentials for 120 s. (E) Flow cytometric analysis of cell apoptosis in SCs exposed to 11.7 kV for different durations. (F) Flow cytometric analysis of cell apoptosis in SCs exposed to different potentials for 120 s. (G) JC-1 staining of SCs exposed to 22.0 kV of plasma for 120 s. Scale bar: 50 μm. (H) Relative green/red fluorescence intensity for JC-1 staining. Data are represented as the mean ± SD (n = 3 per group). *p < 0.05; **p < 0.01, according to one-way ANOVA and LSD test.

Figure 2

ROS production and antioxidant activity of plasma-treated SCs. Chicken SCs were exposed to 22.0 kV of non-thermal plasma for 120 s. (A) Imaging of SCs stained with DCFDA/MitoSOX Red/DAPI. Intracellular ROS production was detected by DCFDA staining and mitochondrial superoxide was detected by MitoSOX Red staining. DCFDA: green fluorescence; MitoSOX Red: red fluorescence; DAPI: blue fluorescence. Scale bar: 50 μm. (B) Relative fluorescence intensity for DCFDA staining. (C) Relative fluorescence intensity for MitoSOX Red staining. (D) Total ROS levels in SCs. (E) MDA level in SCs. Activities of (F) SOD, (G) CAT, and (H) GPx in SCs. Relative mRNA levels of (I) NOX4, NRF2, and KEAP1, and (J) SOD, CAT, GPx, and PRDX4. Data are represented as the mean ± SD (n = 3 per group). *p < 0.05; **p < 0.01, according to one-way ANOVA and LSD test.

Figure 3

Chicken SC protein expression. (A) Representative western blot analysis of protein bands in SCs exposed to 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S5. Relative protein levels of (B) NRF2, KEAP1, PRDX4, (C) ATP5A, (D) p-AMPKα/AMPKα, and (E) p-mTOR/mTOR in SCs exposed to plasma. (F) Representative western blot analysis of protein bands in SCs trasfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S6. Relative protein levels of (G) ATP5A, (H) p-AMPKα/AMPKα, and (I) p-mTOR/mTOR in transfected SCs. One independent replicate on western blot analysis of protein bands in SCs is presented in Supplementary Figure S7. Data are represented as the mean ± SD (n = 3 per group). *p < 0.05; **p < 0.01; ^##p < 0.01, according to one-way ANOVA and LSD test.

Figure 4

Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of plasma-treated SCs. Chicken SCs were exposed to 22.0 kV of non-thermal plasma for 120 s. (A) Imaging of SCs stained with a Cell Navigator Mitochondrial Staining Kit (green fluorescence). Scale bar: 50 μm. (B) Relative fluorescence intensity for mitochondrial staining. (C) NADH level. Activities of (D) cytochrome c oxidase and (E) ATPase synthase in the mitochondria of SCs. (F) ATP level in SCs. (G) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). *p < 0.05; **p < 0.01, according to one-way ANOVA and LSD test.

Figure 5

Expression of miR-7450 and mRNA levels of AMPKα and mTOR in SCs. (A) miR-7450 relative level and (B) relative mRNA levels of AMPKα and mTOR in SCs exposed to 22.0 kV of plasma for 120 s. (C) miR-7450 relative level and (D) relative mRNA levels of AMPKα and mTOR in SCs transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. RT-PCR analysis of a non-target gene (POU1F1) and an unrelated target gene (PDE10A) of miR-7450 in transfected SCs is presented in Supplementary Figure S4. Data are represented as the mean ± SD (n = 3 per group). *p < 0.05; **p < 0.01; ^##p < 0.01, according to one-way ANOVA and LSD test.

Figure 6

SC viability and apoptosis after miRNA transfection. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. (A) Relative viability of SCs. (B) Flow cytometric analysis of SC cell apoptosis. (C) JC-1 staining of SCs. Scale bar: 50 μm. (D) Relative green/red fluorescence intensity for JC-1 staining. Data are represented as the mean ± SD (n = 3 per group). *p < 0.05; **p < 0.01; ^##p < 0.01, according to one-way ANOVA and LSD test.

Figure 7

Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of transfected SCs. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. (A) Mitochondrial staining in SCs. Scale bar: 50 μm. (B) Relative fluorescence intensity for mitochondrial staining. (C) NADH level. Activities of (D) cytochrome c oxidase and (E) ATPase synthase in the mitochondria of SCs. (F) ATP level in SCs. (G) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). *p < 0.05; **p < 0.01; ^#p < 0.05; ^##p < 0.01, according to one-way ANOVA and LSD test.