Flurochloridone-induced apoptosis via IRE1α-JNK signaling pathway in mice testicular cells and TM4 cells

Background Flurochloridone (FLC) can induce apoptosis in Sertoli cells, but the specific mechanism remains unknown.

Objective To investigate the testicular cell apoptosis in mice as well as apoptosis and activation of endoplasmic reticulum stress in TM4 cell line induced by FLC through in vivo and in vitro study designs respectively, and study the role of inosital-requiring enzyme 1α (IRE1α)-c-Jun N-terminal kinase (JNK) signaling pathway in the process of FLC-induced apoptosis in TM4 cells through intervention study design.

Methods Testicular tissues were collected from male C57BL/6 mice which were treated with 3, 15, 75, and 375 mg·（kg·d）⁻¹ FLC by oral perfusion for 28 d. Apoptosis was observed by TUNEL staining, and the levels of apoptosis-related proteins were detected by Western blotting, including B-cell lymphoma-2 (Bcl-2), Bcl-2 interacting mediator of cell death (Bim), and Bcl-2 associated X protein (Bax). In the in vitro study, TM4 cells were treated with different concentrations of FLC (40, 80, and 160 μmol·L⁻¹) for 6 h, then apoptosis rate was detected by flow cytometry, and the levels of apoptosis-related proteins (Bcl-2, Bim, and Bax) and endoplasmic reticulum stress-related proteins glucose regulated protein 78 (GRP78), phosphorylated-protein kinase R like endoplasmic reticulum kinase (p-PERK), activating transcription factor 6 (ATF6), phosphorylated-inosital-requiring enzyme 1α (p-IRE1α), and phosphorylated-JNK (p-JNK) were measured by Western blotting. In the intervention study, TM4 cells were pretreated with IRE1α phosphorylation inhibitor 4μ8C and JNK phosphorylation inhibitor SP600125 for 6 h, then treated with 160 μmol·L⁻¹ FLC for 6 h. The levels of apoptosis-related proteins and endoplasmic reticulum stress-related proteins were measured by Western blotting, and cell viability was detected by cell counting kit-8.

Results After the male C57BL/6 mice orally exposed to FLC for 28 d, apoptosis occurred in the seminiferous tubule. The protein expression level of Bcl-2, apoptosis inhibitor, was decreased in the 75 and 375 mg·(kg·d)⁻¹ groups (P<0.05), and the protein expression levels of Bim and Bax, apoptosis promoters, were increased in the 75 and 375 mg·(kg·d)⁻¹ groups respectively (P<0.05). The percentages of apoptotic cells in the 0, 40, 80, and 160 μmol·L⁻¹ FLC groups were 2.7%±0.2%, 4.8%±1.3%, 9.4%±0.3%, and 13.2%±0.2%, respectively, increased significantly compared with the control group (P<0.05). The protein expression level of Bcl-2 also was decreased in the 160 μmol·L⁻¹ FLC group (P<0.05), while the levels of Bim and Bax were increased in both of the 80 and 160 μmol·L⁻¹ groups (P<0.05). The expression levels of endoplasmic reticulum stress-related proteins (GRP78, p-PERK, ATF6, p-IRE1α, and p-JNK) were increased (P<0.05) or showed a rising trend in TM4 cells. Pre-treatment with 4μ8C (25 and 50 μmol·L⁻¹) and SP600125 (10 and 20 μmol·L⁻¹) significantly down-regulated the protein expression levels of GRP78, p-IRE1α, p-JNK, and Bax induced by FLC (P<0.05) or in a downward trend. Both of the inhibitors alleviated the decreased cell viability induced by FLC (P<0.05) or in alleviating fashion.

Conclusion FLC could induce apoptosis in mice testis and TM4 cell apoptosis through activating endoplasmic reticulum stress and IRE1α-JNK signaling pathway.