This article is mentioned in:

Introduction

Propofol is always utilized as a sedative and anesthetic agent before medical procedures, which can modulate different GABA receptors in the central nervous system (1). Various studies have demonstrated that propofol can induce developmental neurotoxicity in mice (2,3) and induces the apoptosis of neuronal stem cells in the hippocampus of neonatal mice (4). Thus, the reproductive system in the fetal period can also be highly possible affected by anesthetic agents. It has been demonstrated that Leydig cells are derived from the neural system (5) and the essential role of Leydig cells is to produce testosterone (6). For normal reproductive functions in males, Leydig stem/progenitor cells are essential for testis development in the fetal period (7). It has been demonstrated that different factors influence the development of Leydig stem/progenitor cell in the fetal period, resulting in infertility (7). However, it remains unknown as to whether propofol induces the death of Leydig stem/progenitor cells, which would suppress male reproductive function and development.

Cell number regulation is essential in tissue homeostasis controlled by cell death, proliferation and differentiation (8). Cell death can be categorized into 3 categories based on morphological appearance, which includes necrosis, apoptosis and autophagy (9). Cell apoptosis is uniformly featured with nuclear condensation and fragmentation, cell shrinking, membrane blebbing and separation of the cellular components into apoptotic bodies (10). Apoptosis is regulated by 2 pathways, the extrinsic and intrinsic pathways, respectively (11). The extrinsic pathway is known as the death receptor pathway, stimulated by the activation of death receptors with pro-apoptotic ligands, such as Fas ligand (FasL), tumor necrosis factor (TNF)-α and TNF-related apoptosis-inducing ligand (TRAIL). As ligands bind, the intracellular death domains of the receptors associate with Fas-associated death domain (FADD), resulting in the death-induced signaling complex (DISC) recruitment, and hence leading to caspase-8 activation and further triggering caspase-3 or -7 activation (12). The intrinsic pathway is associated with the mitochondrial pathway, which is triggered by a number of stimuli (chemotherapeutic agents, radiation and growth factor withdrawal) leading to the release of cytochrome c from the mitochondria into the cytosol (13). Cytochrome c binds with the adapter molecule, apoptotic protease activating factor 1 (Apaf-1), and recruits procaspase-9 to form the apoptosome to further activate caspase-9 and caspase-3, orchestrating apoptosis (14). Both the intrinsic and extrinsic pathways can lead to the cleavage of poly(ADP-ribose) polymerase (PARP), which impedes the effects of DNA repair (11).

The mitogen-activated protein-serine/threonine kinase (MAPK) pathway plays a key role in regulating cell proliferation, growth, differentiation, invasion, migration and apoptosis (15). MAPKs include extracellular-regulated kinase (ERK)1/2, Janus kinase (JNK) and p38, which can be stimulated by phosphorylation, commencement with MAPK kinase kinase kinase (MAP3K) activation to control cell fate (16). The Akt signaling pathway is considered as a pro-survival pathway, which can inhibit apoptotic cascades and activate pro-survival signals (17). The Akt pathway can inhibit a number of pro-apoptotic Bcl-2 family members (Bad, Bax and Bim) and positively regulate anti-apoptotic pathways [nuclear factor (NF)-κB transcription factor], promoting the transcription of a number of anti-apoptotic genes, such as Bcl-2 and Bcl-xL (18). It has been illustrated that the reduction of Akt expression can induce the apoptosis of various cells (19).

TM3 cells are derived from 11-13-day-old testicular interstitial cells of neonatal BALB/c mouse, which can be stimulated by luteinizing hormone (LH) to secret testosterone (20-22), and TM3 cells exhibit progenitor cell characteristics (7,20). Consequently, TM3 cells are appropriate for use in the investigation of the apoptotic effects of propofol with mechanistic analyses. In the present study, propofol induced TM3 progenitor cell membrane blebbing with detachment phenomena, a decrease in cell number, activated the caspase cascade, activated MAPK pathways and inhibited Akt pathway, which promoted cell apoptosis. The findings of the present study demonstrated that propofol regulates TM3 mouse Leydig stem/progenitor cell apoptosis, which deepens the knowledge of the mechanisms through which anesthetic agent exposure can affect testicular normal development.

Materials and methods

Chemicals and reagents

Propofol, penicillin-streptomycin, ethylene diamine tetraacetic acid (EDTA), propidium iodide (P), MTT, RNase A, 30% acrylamide/Bis-acrylamide solution, Waymouth MB 752/1 medium, sodium orthovanadate, monoclonal antibody against β-actin (#A5441) and Triton X-100 were purchased from Sigma-Aldrich; Merck KGaA. EGTA, Tween 20, dimethyl sulfoxide (DMSO) and DS were purchased from Merck KGaA. An Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was purchased from Strong Biotech Corporation. Dulbecco's modified Eagle medium/F12, fetal bovine serum and trypsin-EDTA were purchased from Gibco; Thermo Fisher Scientific Inc. Potassium chloride, sodium chloride, Tris base and glycine 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid were purchased from J.T. Baker (Avantor Performance Materials). Donkey anti-rabbit (NEF81200-1EA) and anti-mouse (NEF82200-1EA) IgG conjugated with horseradish peroxidase (HRP) were purchased from PerkinElmer, Inc. A Micro BCA protein assay kit was purchased from Thermo Fisher Scientific, Inc. An enhanced chemiluminescence detection kit was purchased from EMD Millipore. Polyclonal antibodies against cleaved caspase-8 (#9429; 1/1,000), cleaved caspase-9 (#9509; 1/1,000), cleaved PARP (#9542; 1/1,000), phospho-JNK (#9251; 1/4,000), JNK (#9252; 1/1,000), phospho-ERK1/2 (#9101; 1/4,000), ERK1/2 (#9102; 1/4,000), phospho-p38 (#9215; 1/1,000), p38 (#9212; 1/4,000), phospho-Akt (#9271; 1/4,000) and Akt (#9272; 1/1,000) and phospho-mechanistic target of rapamycin (mTOR) (#2971; 1/1,000) were purchased from Cell Signaling Technology, Inc. ISOTON™ II Diluent was purchased from Beckman Coulter, Inc. Monoclonal antibody against cleaved caspase-3 (#9661; 1/1,000) was purchased from Cell Signaling Technology, Inc.

Cells and cell culture

TM3 cells were obtained from ATCC and sustained in DMEM/F12 medium with 10% FBS at 37˚C in a humidified incubator containing 95% air and 5% CO2 for all experiments.

Morphological observation

TM3 cells were seed at 6×105/ml and treated with various concentrations of propofol (0, 300, 350 and 400 µM) for 3 h, respectively. Propofol was diluted with DMSO. The changes in cell morphology were then investigated with an Olympus CK40 light microscopy at ×100 magnification and the images were logged using an Olympus DP20 digital camera (Olympus Corporation).

MTT cell viability assay

MTT assay is a colorimetric assay for measuring cell viability (23). TM3 cells were cultured at 8×103 cells. After reaching 70-80% confluence, cells were treated with various concentrations of propofol (0, 10, 50, 100, 300, 400, 500 and 600 µM) for 1, 3, 6, 12 and 24 h, respectively. Subsequently, each well was supplemented with MTT (0.5 mg/ml) for different periods of time followed by incubation at 37°C for 4 h. The media was discarded and 50 µl DMSO were added to dissolve the crystals using a shaker to vibrate the plate at 37°C for 20 min in the dark. Cell viability was examined at λ=570 nm using a VersaMax ELISA reader (Molecular Devices, Inc.), as previously described (23).

Cell cycle analysis

TM3 cells were seeded at 6×105 with 2 ml culture medium, and treated with various concentrations of propofol (0, 100, 300 and 400 µM) for 3, 6, 12 and 24 h, respectively. Cells were collected through trypsin digestion and centrifugation (400 x g for 12 min at 4°C), and washed with isoton II and fixed with 70% ethanol for at least 2 h at -20°C. Following fixation, cells were washed with cold isoton II and collected by centrifugation (400 × g for 12 min at 4°C). Cell suspensions were then mixed with 100 µg/ml RNase and stained with 40 µg/ml propidium iodine (PI) solution for 30 min at 25°C. Stained cells were determined at λ=488 nm for PI detection by FACScan flow cytometer (BD Biosciences). SubG1 phase cells with lower DNA contents are considered as apoptotic cells (24). The percentages of sub-G1, S and G2/M phase cells were analyzed using FACStation v6.1x and Modfit LT v3.3 software (BD Biosciences).

Annexin V/PI double staining assay

After the TM3 cells were collected by trypsin, cell suspensions were centrifuged (400 × g for 12 min at 4°C). The pellets were resuspended with cold isoton II and centrifuged again (400 × g for 12 min at 4°C), and then mixed with 100 µl staining solution for 15 min at 25°C following the user's manual of Annexin V-FITC apoptosis detection kit from Srong Biotech Corporation. Stained cells were determined at λ=488 nm excitation using 515 nm band pass filter for FITC detection and >600 nm band pass filter for PI detection using a FACScan flow cytometer (BD Biosciences). The double-positive cells (late apoptotic), Annexin V single-positive cells (early apoptotic), PI single-positive cells (necrotic) and double-negative cells (viable) are illustrated in 4 quadrants (25,26). The percentage of cells in the 4 quadrants were analyzed using FACStation v6.1x software.

Protein extraction and western blot analysis

Following the treatments, cells in the medium were removed and washed with cold PBS. Attached cells were lysed by 20 µl lysis buffer (150 mM NaCl, 20 mM Tris pH7.5, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate and 2.5 mM sodium pyrophosphate) with proteinase inhibitor cocktail (cat. no. P5655; Sigma-Aldrich; Merck KGaA). The pellets were then resuspended with 10 µl lysis buffer and centrifuged at 12,000 × g for 12 min at 4°C. The supernatants were saved and stored at −80°C. The protein concentrations of cell lysates were analyzed by Lowry assay (27).

For western blot analysis, 30 µg protein were separated by 12% SDS-PAGE gel with running buffer (0.1% SDS, 192 mM glycine and 25 mM Tris; pH 8.3) at 25°C, and electrophoretically transferred to polyvinylidene difluoride membranes at 4°C. After blocking the membranes with 1% milk at 25°C for 3 h, the membranes were incubated with primary antibodies for 24 h at 4°C. The membranes were then washed 3 times and incubated with HRP-conjugated secondary antibodies (1/2,000) at 25°C for 1 h. Bands were detected using an enhanced chemiluminescence kit and the UVP EC3 BioImaging system (UVP, LLC). Quantification of the western blotting data was performed using ImageJ version 1.50 software (National Institutes of Health) (28).

Statistical analysis

Data are presented as the means ± standard error of the mean among 3 experiments. Significance of differences between the control and treatment groups were determined by one-way analysis of variance followed by all pairs Tukey's honestly significant difference (HSD) post-hoc test comparisons. Statistical analysis was accomplished using GraphPad Prism 6 software (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

Cell morphology is altered by propofol associated with the death of TM3 cells

TM3 cells were treated with various concentrations (0, 300, 350 and 400 µM) of propofol (Fig. 1) for 3 h, respectively. Morphological changes of cells were examined by light microscopy. The TM3 cells shrank in size following treatment with 300 µM propofol for 24 h (Fig. 1) and 350 µM propofol for 3 h (Fig. 1); in addition, treatment with 400 µM propofol for 3 h caused the detachment of most cells (Fig. 1). These results suggested that propofol can cause membrane blebbing in TM3 cells, which indicates that propofol can promote cell death possibly via apoptosis in TM3 mouse Leydig stem/progenitor cells.

Figure 1 Cell morphology is altered by propofol associated with the death of TM3 cells. Cells were treated with various concentrations of propofol (control, 300, 350 and 400 µM) for 3 h, respectively, and the morphological changes were examined by light microscopy (scale bar, 50 µm; white arrowhead, cells with membrane blebbing).

Viability of TM3 cells is reduced by propofol in a time- and dose-dependent manner

The viability of TM3 cells affected by propofol was investigated by MTT assay. TM3 cells were treated with propofol (0 to 600 µM) for 1, 3, 6, 12 and 24 h, respectively. The viability of the TM3 cells decreased significantly following treatment with propofol from 100 to 600 µM for 6 to 24 h (P<0.05) (Fig. 2). In fact, TM3 cell viability decreased to 49±8.1% with 300 µM propofol for 12 h treatment (Fig. 2).

Figure 2 Viability of TM3 cells was reduced by propofol in a time- and dose-dependent manner. Cells were treated with 0 (Control), 0.5% DMSO, 10, 50, 100, 300, 400, 500 and 600 µM propofol for 1, 3, 6, 12 and 24 h, respectively. MTT viability assay was used to determine cell viability. Data are illustrated as a percentage of cell growth relative to the control groups. Each data point represents the mean ± SEM of 3 separate experiments. *P<0.05, **P<0.01 and ***P<0.001 indicate statistically significant differences compared to the control group. DMSO, dimethyl sulfoxide.

Propofol regulates the TM3 cell cell cycle

TM3 cells were treated with various concentrations of propofol (0, 100, 300 and 400 µM) for 3, 6, 12 and 24 h, and the DNA contents in each treatment were investigated by flow cytometry. The data illustrated that the percentage of TM3 cells in the subG1 phase significantly increased by treatment with 400 µM propofol for 3 to 24 h (P<0.05) (Fig. 3A and B). Moreover, the percentage of TM3 cells in the G2/M phase markedly increased following treatment with 400 µM propofol for 3 and 12 h (P<0.05) (Fig. 3A and C).

Figure 3 Propofol regulates the TM3 cell cycle. Cells were treated with 0 (Control), 100, 300 and 400 µM propofol for 3, 6, 12 and 24 h, respectively. Cell were then fixed and stained with propidium iodide (PI) for cell cycle analysis, and (A and B) subG1 and (A and C) G2/M phase cells were then analyzed and illustrated, respectively. SubG1 phase cells contain less DNA content compared to normal cells, indicating apoptosis. Each data point represents the mean ± SEM of 3 separate experiments. *P<0.05 and ***P<0.001 indicate statistically significant differences compared to the control group.

Propofol induces the apoptosis of TM3 cells

The results revealed that propofol induced cell death with DNA fragmentation plus membrane blebbing and an increase in the number of TM3 cells in the subG1 phase. To further examine whether propofol induces the apoptosis of TM3 cells, Annexin V/PI double staining assay was used. It is acknowledged that percentages of double-positive cells (late apoptotic), Annexin V single-positive cells (early apoptotic), PI single-positive cells (necrotic) and double-negative cells (viable) are illustrated in 4 quadrants with the double staining illustrating different cell apoptotic phenomena (29). The cell distributions of viable, early apoptotic, necrotic and late apoptotic TM3 cells are illustrated in Fig. 4A, and the percentages of 4 different statuses among the cells treated with 0, 300, 350 and 400 µM propofol, respectively, are demonstrated in Fig. 4B. In addition, the number of Annexin V-positive TM3 cells (early and late apoptosis) was significantly induced by treatment with propofol at 350 and 400 µM for 24 h (P<0.05) (Fig. 4C). These data indicate that propofol induces the apoptosis of TM3 cells.

Figure 4 Propofol induces the apoptosis of TM3 cells. Cells were treated with 0 (Control), 300, 350 and 400 µM propofol for 24 h, respectively. (A) The apoptotic phenomenon of propofol-induced cells was examined by Annexin V/PI double staining assay. (B) The percentages of Annexin V and PI double positive cells represent late apoptotic cells; Annexin V single-positive cells represent early apoptotic cells; PI single-positive cells represent necrotic cells; and double-negative cells represent viable cells are shown in each group. (C) The differences of Annexin V-positive cells (early and late apoptotic status) was then analyzed between the treatment groups. Each data point represents the mean ± SEM of 3 separate experiments. *P<0.05 and ***P<0.001 indicate statistically significant differences compared to the control group.

Propofol activates the caspase cascade associated with the apoptosis of TM3 cells

The extrinsic and intrinsic caspase cascade is an imperative inducer of apoptotic pathway (30). As demonstrated above, propofol induced the apoptosis of TM3 cells. Thus, the present study then determined whether propofol induces apoptosis by activating caspase extrinsic and intrinsic pathways to induce the cleavage of caspase-9, caspase-8, caspase-3 plus PARP. The results revealed that treatment with 350 and 400 µM propofol for 3 h significantly induced the expression of cleaved caspase-8 (Fig. 5A and B). Treatment with 350 and 400 µM propofol for 3 and 6 h also significantly induced the expression of cleaved caspase-9 (Fig. 5A and C), and treatment with 350 µM propofol for 3 h and 400 µM propofol for 3, 6 and 12 h significantly induced the expression of cleaved caspase-3 (Fig. 5A and D). It was also found that treatment with 350 µM propofol for 3 h significantly induced the expression of cleaved PARP (P<0.05) (Fig. 5A and E). These data illustrate that caspase pathways were activated by propofol to stimulate apoptosis in TM3 cells.

Figure 5 Propofol activates the caspase cascade associated with the apoptosis of TM3 cells. Cells were treated with various concentrations of propofol (control, 300, 350 and 400 µM) for 3, 6, 12 and 24 h, respectively. (A) Cleaved caspase-8 (43/18 kDa), cleaved caspase-9 (39/37 kDa), cleaved caspase-3 (17/19 kDa) and cleaved PARP (85-90 kDa) were examined by western blot analyses. The integrated optical densities (IOD) of (B) cleaved caspase-8, (C) cleaved caspase-9, (D) cleaved caspase-3 and (E) cleaved PARP proteins were normalized to β-actin (43 kDa) in each lane, respectively. Each data point represents the mean ± SEM of 3 separate experiments. *P<0.05, **P<0.01 and ***P<0.001 indicate statistically significant differences compared to the control group. C, control.

It should be noted that treatment with 400 µM propofol for 24 h significantly reduced the expression of cleaved PARP (P<0.05) (Fig. 5A and E), suggesting that treatment of the TM3 cells for a long period of time with a high concentration of propofol suppressed the apoptotic phenomenon. In addition, the results revealed that treatment with 350 µM propofol for 3 h activated the caspase cascade in TM3 cells (Fig. 5), but not in the MA-10 cells, as previously demonstrated (26), suggesting that the TM3 cells are more sensitive to propofol compared to MA-10 cells.

Propofol activates MAPK pathways associated with the apoptosis of TM3 cells

A previous study demonstrated that the MAPK pathways control cell proliferation, growth and apoptosis (15). In the present study, to examine whether propofol regulates MAPK pathways to induce TM3 cell apoptosis, MAPK proteins were examined by western blot analysis. The results demonstrated that treatment with 300 µM propofol for 12 h and 350 µM propofol for 3, 6 and 12 h significantly induced the expression of phospho-JNK (P<0.05) (Fig. 6A and B). In addition, treatment with 300 µM propofol for 3 h and 350 µM propofol for 3 and 6 h significantly induced the expression of phospho-ERK (P<0.05) (Fig. 6A and C). It was also found that treatment with 350 µM propofol for 3, 6 and 12 h, and 400 µM propofol for 3 h significantly induced the expression of phospho-p38 (P<0.05) (Fig. 6A and D). These data clearly illustrate that propofol activates MAPK pathways to stimulate the apoptosis of TM3 cells.

Figure 6 Propofol activates MAPK pathways associated with the apoptosis of TM3 cells. Cells were treated with various concentrations of propofol (control, 300, 350 and 400 µM) for 3, 6, 12 and 24 h, respectively. (A) Phospho-JNK (p-JNK; 54/46 kDa), JNK (54/46 kDa), phospho-ERK (p-ERK; 44/42 kDa), ERK (44/42 kDa), phospho-p38 (p-P38; 43 kDa) and p38 (43 kDa) were examined by western blot analysis. The integrated optical densities (IOD) of (B) p-JNK, (C) p-ERK and (D) p-p38 proteins were normalized to β-actin (43 kDa) in each lane, respectively. Each data point represents the mean ± SEM of 3 separate experiments. *P<0.05, **P<0.01 and ***P<0.001 indicate statistically significant differences compared to the control group. C, control.

Propofol suppresses the Akt pathway associated with the apoptosis of TM3 cells

A previous study demonstrated that the Akt pathway inhibit apoptotic signal cascades and activate pro-survival signal cascades for cell survival (17). Thus, in the present study, to further examine whether propofol stimulates TM3 cell apoptosis through the inhibition of the Akt pathway, phospho-Akt, Akt, phospho-mTOR and mTOR expression patterns were examined by western blot analysis. The data demonstrated that treatment with 350 µM propofol for 3 h, and 400 µM propofol for 3 and 6 h significantly reduced phospho-Akt expression (P<0.05) (Fig. 7A and B). In addition, treatment with 400 µM propofol for 24 h reduced phospho-mTOR expression (P<0.05) (Fig. 7A and C). These observations suggested that propofol induced apoptosis by inhibiting the Akt pathway in TM3 cells.

Figure 7 Propofol suppresses the Akt pathway associated with the apoptosis of TM3 cells. TM3 cells were treated with various concentrations of propofol (control, 300, 350 and 400 µM) for 3, 6, 12 and 24 h, respectively. (A) Phospho-Akt (p-Akt; 60 kDa), Akt (60 kDa), phospho-mTOR (p-mTOR; 289 kDa) and mTOR (289 kDa) were examined by western blot analysis. The integrated optical densities (IOD) of (B) p-Akt and (C) p-mTOR proteins were normalized to β-actin (43 kDa) in each lane, respectively. Each data point represents the mean ± SEM of 3 separate experiments. *P<0.05, **P<0.01 and ***P<0.001 indicate statistically significant differences compared to the control group. C, control.

Discussion

A previous study indicated that propofol induced neurotoxicity (31). Moreover, studies have further demonstrated that propofol cause developmental neurotoxicity in mice (2,3) and induces the apoptosis of neuronal stem cells in the hippocampus in neonatal mice (4). It is highly possible that propofol disrupts the development of Leydig stem/progenitor cells during the fetal and neonatal periods. Thus, in the present study, TM3 mouse Leydig stem/progenitor cells were used to examine the effects of propofol with a brief mechanistic investigation; it was found that propofol induced TM3 cell apoptosis.

Apoptosis is associated with intense transformations in the cellular architecture, and the activation of caspase cascades weakens the cell cytoskeleton, causing morphological modifications, such as membrane blebbing and cell shrinkage (32). The findings of the present study demonstrated that propofol induced membrane blebbing and cell shrinkage, illustrating that propofol affects the cytoskeleton and induces morphological changes to stimulate cell death associated with the apoptosis of TM3 cells. The authors have previously demonstrated that membrane blebbing occurs in MA-10 cells treated with a higher concentration (400 µM) of propofol (26). However, in the present study, a lower concentration (350 µM) of propofol was needed in the TM3 cells, indicating that the TM3 cells were more sensitive to propofol than the MA-10 cells. In a cell viability assay, it was observed TM3 cell viability decreased to 49% following treatment with 300 µM propofol for 12 h. However, as previously demonstrated, MA-10 cell viability decreased to 67% following treatment with 300 µM propofol for 12 h (26). These phenomena indicate that the TM3 cells are more sensitive to propofol than the MA-10 cells, which is similar to the morphological results.

The substantial increase in subG1 phase cell numbers with propofol treatment illustrated that propofol regulates the distribution of the cell cycle to induce DNA fragmentation and the apoptosis of TM3 cells. Of note, propofol induced G2/M phase arrest in the TM3 cells, but not in the MA-10 cells (26). Previous studies have demonstrated that the increase in subG1 and G2/M phase arrest results in cell death through apoptosis (33,34). Henceforth, the results of the present study suggest that propofol-induced apoptosis may be associated with cell cycle regulation.

In an Annexin V/PI double staining assay, the data also revealed the stimulation of apoptosis by propofol in a dose-dependent manner in TM3 cells, signifying that propofol actually induced TM3 cell apoptosis. However, propofol induced necrosis in TM3 cells in a dose-dependent manner, but not in the MA-10 cells (26). It has been shown that an overdose of propofol causes endothelial cytotoxicity by apoptosis plus necrosis (35). Thus, the findings of the present study are in accordance with those of other studies. It would be of interest to further investigate the mechanisms related to the stimulation of necrosis by propofol in TM3 cells.

It has been demonstrated that apoptosis principally commences by extrinsic and intrinsic signals, followed by the activation of the caspase cascade (11). The findings of the present study demonstrated that propofol activated caspase extrinsic and intrinsic signals to promote the apoptosis of TM3 cells. However, the same apoptotic effect with the activation of caspase pathways occurred in TM3 cells at a lower concentration (350 µM) as in the MA-10 cells with a higher concentration (400 µM) (26). Therefore, it was again observed that the TM3 cells were more sensitive to propofol than the MA-10 cells.

Studies have demonstrated that the MAPK pathways (ERK1/2, JNK and p38) regulated the apoptotic pathway to induce the apoptosis of various cell types (15,36-38). In fact, JNK can cause a shift to autophagy from apoptosis to regulate the survival of choriocarcinoma cells (37) and JNK can prevent the apoptosis of acute myeloid leukemia cells (39). It has also been demonstrated that ERK is able to promote apoptosis by facilitating cell cycle arrest in response to DNA damage in different cell types (38) and p38 can suppress caspase-3 activity in neural cells to induce apoptosis (36,40). Studies have indicated that propofol can inhibit the MAPK pathways in different cell types to inhibit cell migration and inflammation (41,42). The findings of the present study demonstrated that propofol significantly increased the phosphorylation of ERK, JNK and p38 in TM3 cells, suggesting that propofol can stimulate the MAPK pathways to induce the apoptosis of TM3 cells. It should be noted that propofol did reduce the expression of total MAPKs, as propofol induced p-JNK, p-ERK and p-p38 expression levels. These phenomena indicate that porpofol may cause the instability of MAPKs, which would result in MAPK degradation in TM3 cells. Further studies are warranted to reveal the underlying mechanisms.

Akt signaling can prevent the apoptotic signal and stimulate pro-survival signals to maintain cell survival (17). It has been demonstrated that PI3K/Akt pathway activation is highly associated with the formation of certain types of cancer (43). Actually, the function of propofol in the Akt pathway remains undetermined. Studies have found that propofol suppresses Akt signaling in macrophages to stimulate apoptosis (44), as propofol can activate the Akt pathway to protect rat cardio-myocytes from doxorubicin-induced toxicity (45). The present study found that propofol reduced the phosphorylation of Akt in TM3 cells. Thus, it is suggested propofol attenuates the activity of Akt and mTOR to stimulate the apoptosis of TM3 cells.

In conclusion, the present study demonstrates that propofol induces cell apoptosis through the activation of caspases and MAPKs pathways and the inhibition of the Akt pathway in TM3 Leydig stem/progenitor cells. This indicates that exposure to anesthetic agents can affect testicular normal Leydig cell development.

Funding

The present study was supported by grants from the Ministry of Science and Technology MOST 105-2320-B-006- 028-MY3 (to BMH) and the Chi Mei-NCKU hospital grant CMNCKU10811 (to FCK and BMH), Taiwan, R.O.C.

Availability of data and materials

The data used and analyzed in this study are always accessible from authors on reasonable request.

Authors' contributions

FCK, YCC and SCW contributed to conducting all the experiments with statistical analysis. ECS and BMH contributed to all the experimental designs plus rationale setup, data analysis, manuscript writing, and ensuring the accuracy integrity of the whole study. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

References