Triptolide induces Sertoli cell apoptosis in mice via ROS/JNK-dependent activation of the mitochondrial pathway and inhibition of Nrf2-mediated antioxidant response

Abstract

Triptolide (TP), an oxygenated diterpene, has a variety of beneficial pharmacodynamic activities but its clinical applications are restricted due to severe testicular injury. This study aimed to delineate the molecular mechanisms of TP-induced testicular injury in vitro and in vivo. TP (5-50000 nmol/L) dose-dependently decreased the viability of TM4 Sertoli cells with an IC₅₀ value of 669.5-269.45 nmol/L at 24 h. TP (125, 250, and 500 nmol/L) dose-dependently increased the accumulation of ROS, the phosphorylation of JNK, mitochondrial dysfunction and activation of the intrinsic apoptosis pathway in TM4 cells. These processes were attenuated by co-treatment with the antioxidant N-acetyl cysteine (NAC, 1 mmol/L). Furthermore, TP treatment inhibited the translocation of Nrf2 from cytoplasm into the nucleus as well as the expression of downstream genes NAD(P)H quinone oxidoreductase1 (NQO1), catalase (CAT) and hemeoxygenase 1 (HO-1), thus abrogating Nrf2-mediated defense mechanisms against oxidative stress. Moreover, siRNA knockdown of Nrf2 significantly potentiated TP-induced apoptosis of TM4 cells. The above results from in vitro experiments were further validated in male mice after oral administration of TP (30, 60, and 120 mg·kg^-1·d^-1, for 14 d), as evidenced by the detected indexes, including dose-dependently decreased SDH activity, increased MDA concentration, altered testicle histomorphology, elevated caspase-3 activation, apoptosis induction, increased phosphorylation of JNK, and decreased gene expression of NQO1, CAT and HO-1 as well as nuclear protein expression of Nrf2 in testicular tissue. Our results demonstrate that TP activates apoptosis of Sertoli cells and injury of the testis via the ROS/JNK-mediated mitochondrial-dependent apoptosis pathway and down-regulates Nrf2 activation.

Figure 1

Effects of TP on cell viability and apoptosis in the TM4 Sertoli cells. (A) Chemical structure of TP. (B) Viability of cells with various concentrations of TP (5–10000 nmol/L) for 24 and 48 h was determined by MTT assay, respectively. (C) Apoptotic morphological changes were evaluated by fluorescent microscopy using Hoechst 33258 staining. Arrows indicate chromatin condensation and DNA fragmentation. (D) Apoptosis in TM4 cells was detected after 24 and 48 h of treatment with 125, 250 and 500 nmol/L TP by Annexin V-FITC/PI binding and measured by flow cytometry analysis. The chart illustrates apoptosis proportion from three separate experiments. (E) The mRNA expression of FSHR was detected by Real time PCR in TM4 cells. Data represent the mean±SD of three independent experiments. ^*P<0.05, ^**P<0.01 vs untreated control group.

Figure 2

Role of ROS generation in TP-induced apoptosis in TM4 cells. (A) Intracellular ROS levels were examined using DCF fluorescence by fluorescence microscope. Cells were pretreated with different concentrations of TP (125, 250, and 500 nmol/L), 500 nmol/L TP+1 mmol/L-acetylcysteine (NAC) and H₂O₂ (300 μmol/L) for 3 h, and incubated with DCFH-DA as the probe oxidized to DCF intracellular and emitting fluorescence. (B) The DCF fluorescence intensity was measured by a fluorescence spectrophotometer. (C) Effect of NAC on TP-induced cell death. Cell viability was estimated by MTT assay (data represent the mean±SD of six experiments in each group). (D) The MDA levels in culture medium of TP (125, 250, and 500 nmol/L) treatment for 12 h; (E) The intracellular SDH activations in TP (125, 250, 500 nmol/L) treatment for 12 h. Data represent the mean±SD of three independent experiments. ^*P<0.05, ^**P<0.01 vs untreated control cells. ^##P<0.01 vs TP (500 nmol/L) cells.

Figure 3

TP induces apoptosis through a mitochondria-mediated pathway. (A) Mitochondrial membrane potential is assessed by JC-1 staining. TM4 cells were cultured for 24 and 48 h in control medium or medium containing different concentrations of TP (125, 250, and 500 nmol/L) and relative Δψm ratios of red/green fluorescence were analyzed by flow cytometry; Quantification of Δψm expressed as a ratio of JC-1 aggregate to JC-1 monomer (red: green) fluorescence intensity. Each bar is the mean±SD. derived from three independent experiments (^*P<0.05, ^**P<0.01 vs untreated control cells). (B) Effect of TP in dose-dependent manner on the PARP, cleaved caspase-3, Cyt c, Bax and Bcl-2 protein expression in TM4 cells by Western blot experiment. β-Actin was used as a loading control to confirm equal loading; (C) The densitometric analyses of expression of Bax and Bcl-2 from three independent experiments. ^*P<0.05, ^**P<0.01 vs untreated control cells.

Figure 4

Activation of JNK phosphorylation during TP-induced apoptosis in cultured TM4 cells. (A) Cells were cultured in 6-well plates until confluent, and the medium was replaced with 1% serum medium in the presence or absence of TP (125, 250 and 500 nmol/L) for 24 and 48 h. The cells were lysed and MAPKs proteins were analyzed Western blot. (B) Densitometry scanning analysis of ratio of p-JNK/total JNK, p-p38/total p38 and p-ERK1/2/total ERK1/2. Data represent the mean±SD of three independent experiments. ^*P<0.05, ^**P<0.01 vs untreated control cells. (C) Effects of JNK inhibitor SP600125, P38 inhibitor SB203580 and ERK inhibitor PD98059 on TP-induced apoptosis in cultured TM4 cells for 24 h. Cell viability was determined by MTT assay and data represent the mean±SD of six experiments in each group. ^*P<0.05 vs single TP treated cells.

Figure 5

TP induces apoptosis in TM4 cells through ROS/JNK/mitochondria-dependent apoptotic signals. (A) Apoptosis of cells was detected by flow cytometric analyses of Annexin V-FITC/PI staining after treatment with vehicle, 500 nmol/L TP, 500 nmol/L TP+NAC and 500 nmol/L TP+ SP600125 for 24 h. (B) Effects of JNK inhibitor SP600125 and NAC on TP-induced collapsion of mitochondrial membrane potential (Δψm) as assessed with the JC-1 stain. SP600125 or NAC were added to cell culture media with TP (500 nmol/L) to combine treatment for 24 h and were determined by flow cytometric analyses. Quantification of Δψm expressed as a ratio of JC-1 aggregate to JC-1 monomer (red: green) fluorescence intensity. (C) TM4 cells were treated with 500 nmol/L TP+NAC or 500 nmol/L TP+ SP600125 for 24 h. The protein expressions were assessed by Western blot and the β-actin was used as the loading control. Each bar is the mean±SD. derived from three independent experiments. ^*P<0.05, ^**P<0.01 vs untreated control cells. ^**P<0.01 as compared with TP group.

Figure 6

TP inhibit the transcriptional activation of Nrf2. The mRNA expressions of HO-1, NQO1 and CAT were detected by Real time PCR in TM4 cells (A) and testicular tissues (B). Data represent the mean±SD of three independent experiments. ^*P<0.05, ^**P<0.01 vs untreated control group. (C) Effects of TP-inhibit Nrf2 nuclear translocation analyzed by immunofluorescence staining. (D) TM4 cells were treated with TP (125, 250 and 500 nmol/L) for 6 h. The translocation of Nrf2 from cytoplasm into nucleus was detected by Western blot as well as β-actin and LaminB were used as loading control for cytoplasm or nucleus, respectively. (E) Representative blots of nuclear and cytosolic Nrf2 in testicular tissues; The β-actin and Lamin B were used as the loading control for cytoplasm or nucleus, respectively. (F) Influence of Nrf2 knockdown on the effect of TP induced cell death measured by MTT. (G) Influence of Nrf2 knockdown on the effect of TP induced accumulation of ROS was measured by a fluorescence Spectrophotometer. (H) Influence of Nrf2 knockdown on the effect of TP induced apoptosis in TM4 cells by Annexin V-FITC/PI binding and measured by flow cytometry analysis. The histogram illustrates apoptosis proportion from three separates experiments.

Figure 7

TP induced testicular injury in mice. (A) The testis indexes of mice after the treatment of TP for 14 days; (B) The MDA levels in testicular tissue; (C) The SDH activations in testicular tissue; (D) The SOD activations in testicular tissue; (E) Activations of caspase-3 in testicular tissue were determined by classical colorimetric method by commercial kits; (F) The mRNA expression of FSHR was detected by real-time PCR in testicular tissues. Data represent the mean±SD of eight independent experiments. ^*P<0.05, ^**P<0.01 vs untreated control. (G) Histological analysis [stained with H&E 200×] of testicular tissue in mice induced by TP for 14 days. (H) In situ detection of apoptotic cells in testis sections after the treatment of TP for 14 days by optical microscope using the TUNEL assay (normal cells: bluish violet; apoptotic cell: brown). (I) Effect of TP on p-JNK expressions in male mice testis sections were detected by immunocytochemistry (positive cell: claybank).

Figure 8

Overview of pathways for TP induced apoptosis of SCs.