[Decline of secretory function of TM4 Sertoli cells stimulated by D-galactose in mice and its mechanism].

Objective To establish a model of decline in secretion of senescent TM4 cells in vitro induced by D-galactose (D-gal). Methods Different concentrations of D-Gal (25, 50, 100, 150, 200 and 250 mmol/L) were used to induce TM4 cell senescence. The viability of TM4 cells was detected by MTT assay. The cell cycle was analyzed by flow cytometry. The cell morphology was observed by light microscopy and the percentage of senescent cells was observed by senescence-associated β-galactosidase (SA-β-Gal) staining. The mRNA expression levels of P21, P16, glial-derived neurotrophic factor (GDNF) and stem cell factor (SCF) were detected by reverse transcription PCR. The protein expression levels of GDNF, SCF, nuclear factor erythroid 2 like 2 (NRF2), NAD(P)H dehydrogenase [quinone] 1 (NQO-1) and heme oxygenase-1 (HO-1) were detected by Western blot analysis. Results Compared with normal control group, D-Gal stimulation significantly decreased the cell viability in a concentration-dependent manner. The arrest of D-Gal-treated cells in G1 phase of cell cycle significantly increased, while it significantly decreased in S phase, and D-Gal induced cell cycle arrest at G0/G1 phase in TM4 cells. The percentages of SA-β-Gal positive cells increased significantly. The expression levels of P21 and P16 mRNAs were significantly up-regulated. The mRNA and protein levels of GDNF and SCF-1 were significantly down-regulated. Furthermore, the expression levels of oxidative stress-related protein NRF2, HO-1 and NQO-1 were significantly reduced. Conclusion The model of declined secretion function of senescent TM4 cells induced by D-Gal we established is stable and reliable. Its mechanism may be related to the down-regulation of NRF2 signaling pathway.