Contributed equally
Ketamine is a widely used intravenous anesthetic; however, basic and clinical studies have demonstrated that prolonged exposure can cause irreversible injury to the immature human brain. Yes-associated protein (YAP) is the main effector of the Hippo signaling pathway, which serves an important role in regulating tissue homeostasis and organ size during development. However, whether YAP mediates ketamine-induced apoptosis is not completely understood. Based on the functions of YAP during apoptosis resistance and cell self-renewal regulation, the present study hypothesized that YAP serves a role during ketamine-induced apoptosis. An
Ketamine is an intravenous anesthetic with analgesic effects that is commonly used for pediatric surgery and superficial operations (
The Hippo signaling pathway is a protein kinase cascade composed of a series of protein kinases and various transcription factors, which is highly evolutionarily conserved from lower animals to mammals (
Based on the effects of YAP on cell proliferation and apoptosis, the present study hypothesized that YAP may serve a role in regulating ketamine-induced apoptosis. A human neuroblastoma cell line (SH-SY5Y), which has been widely used to study neurological disease and therapeutic effects, was used to investigate the impact of ketamine-induced toxicity (
The SHSY-5Y cell line (American Type Culture Collection) was cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 300 mg/l L-glutamine, 4.5 mg/l D-glucose, 10 mg/l sodium pyruvate, 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin solution. Cells were seeded (2×105 cells/ml) and maintained at 37°C in a 5% CO2 humidified incubator. The culture medium was changed every 2 days. For subsequent experiments, cells were treated with various concentrations of ketamine (Fujian Gutian Pharmaceutical Industry Co., Ltd.) and incubated at 37°C for 12 and 24 h.
The effects of different concentrations of ketamine (0, 400, 800, 1,200 and 1,600 µM) on cell proliferation-neurotoxicity (viability) were assessed by performing the Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.). It has been reported that high doses of ketamine are required to induce SH-SY5Y cell apoptosis and the pre-experiment results indicated that 1,600 µM induced cell death but not excessive cell death (
Lentiviral infection was used to increase the expression level of YAP in SH-SY5Y cells. LV5 (cat. no. A2831-3; Shanghai GenePharma Co., Ltd.) was used for YAP overexpression lentiviral particle production and LV5-negative control (NC) was used as the control for the YAP overexpression plasmid. LV5-based lentiviral vectors (1 µg/µl) were transfected into 293T cells at 80–90% confluence, 3.5×105/ml. After purification and titration, the viral supernatant was harvested at 48–72 h post-transfection, passed through a 0.45-mm filter and diluted 2:3 with fresh medium containing 8 mg/ml polybrene. At 80% confluence, the SH-SY5Y target cells were infected with the viral supernatant. At 48 h post-transfection, cells were selected using puromycin (1 mg/ml). SH-SY5Y cells were seeded (0.5×105 cells/well) into 24-well plates and incubated at 37°C for 24 h in a 5% CO2 humidified incubator. Subsequently, the cell culture medium was aspirated and the virus was diluted in DMEM containing 10% FBS and 5 µg/ml polybrene. Blank and negative control (treated with the LV5NC plasmid) groups were established. After incubation for 12–24 h at 37°C, the viral suspension was removed, and DMEM was added before incubation at 37°C with 5% CO2 for 24–48 h.
An siRNA was used to reduce the expression level of YAP in SH-SY5Y cells. The siRNAs (Shanghai GenePharma Co., Ltd.) used in the present study were as follows: YAP1-homo-1858 siRNA forward, CUGCCACCAAGCUAGAUAATT and reverse, UUAUCUAGCUUGGUGGCAGTT; and NC-siRNA forward, UUCUCCGAACGUGUCACGUTT and reverse, ACGUGACACGUUCGGAGAATT . The siRNA targeting YAP was fluorescently labeled and si-fluorescein amidite (FAM), which has an excitation wavelength of 480 nm and a emission wavelength of 520 nm, was used as the negative control. Cells were transfected with 20 µM YAP-siRNA or NC-siRNA using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C, according to the manufacturer's protocol. Briefly, 0.5–2×105 cells and 500 µl DMEM without penicillin/streptomycin was added to each well and incubated for 24 h. Lipofectamine® 2000 was diluted in 50 µl Opti-MEM I Reduced Serum medium (Gibco; Thermo Fisher Scientific, Inc.), mixed gently and incubated at room temperature for 5 min. Subsequently, 2 µl si-FAM diluted in 50 µl Opti-MEM I Reduced Serum medium was added to the Lipofectamine® 2000 dilution and the cells incubated for 20 min at room temperature; YAP-siRNA prepared in the same way. The time interval between transfection and subsequent experimentation was 48 h.
After aspirating the culture media, cells were gently rinsed with PBS and fixed with 4% paraformaldehyde at 25°C for 15 min. Cells were rinsed twice with warm PBS and permeabilized using PBS containing 0.2% Triton X-100 and 0.2% BSA [Yeasen Biotechnology (Shanghai) Co., Ltd.] on ice for 15 min. Cells were blocked with PBS containing 0.02% Triton X-100 and 5% BSA for >30 min at room temperature. Subsequently, cells were incubated at 4°C overnight with an anti-cleaved caspase-3 (cat. no. 9661; 1:400; Cell Signaling Technology, Inc.) primary antibody. Following primary incubation, cells were incubated for 1 or 2 h at room temperature with Alexa 488-conjugated anti-rabbit IgG (cat. no. A11034; 1:300; Invitrogen; Thermo Fisher Scientific, Inc.) and 594-conjugated anti-rabbit IgG (cat. no. A21207; 1:300; Invitrogen; Thermo Fisher Scientific, Inc.) secondary antibodies, then cleaved caspase-3 protein labeled with the green fluorophore of the secondary antibody. Finally, nuclei were stained using DAPI (cat. no. 62248; 1:1,000; Thermo Fisher Scientific, Inc.) at room temperature for 10 min and sealed with 90% glycerin. Stained cells were visualized using a LSM88 confocal microscope (Zeiss GmbH) at magnifications of ×200, 400 and 630, ZEN 2.3 lite (Zeiss GmbH) was used for image capture and analysis.
SH-SY5Y cell apoptosis following ketamine treatment was analyzed by flow cytometry. Cells were seeded (3×105 cells/ml) into 6-well plates and incubated at 37°C with 5% CO2 for 24 h. Cells were treated with ketamine for 12 or 24 h at 37°C, harvested and washed twice with PBS. Subsequently, 5–10×104 cells were re-suspended in 1X binding buffer. To each 5-ml culture tube, 100 µl cell suspension (1×105 cells), 5 µl APC Annexin V and 5 µl 7-aminoactinomycin D (7-AAD) were added (cat. no. 561012, BD Pharmingen). Cells were incubated for 15 min at room temperature in the dark with gentle agitation. Subsequently, 400 µl 1X binding buffer was added to each tube and samples were analyzed by flow cytometry within 1 h. The following controls were used to set up compensation and quadrants: Unstained cells, cells stained with APC Annexin V and cells stained with 7-AAD, early and late apoptosis was assessed. FlowJo v10 (FlowJo LLC) was used for analysis and the model and supplier of the instrument used for flow cytometry was a Sony SA3800-Spectral Cell Analyzer (Sony Biotechnology).
Western blotting was performed as previously described (
All experiments were repeated at least three times. Data are presented as the mean ± SEM. Statistical analyses were performed using GraphPad Prism software (version 5.0; GraphPad Software, Inc.). Comparisons between two groups were analyzed using paired Student's t-test. Comparisons among multiple groups were analyzed using one-way or two-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.
The CCK-8 assay was used to investigate the optimum dose and exposure time of ketamine-induced cytotoxicity in SH-SY5Y cells. Different concentrations of ketamine induced cell toxicity, and cell viability was suppressed following treatment with ketamine for 12 and 24 h compared with the 0 µM group, particularly at a concentration of 1,600 µM. However, extensive cell death was observed when cells were treated with 2,000 µM ketamine (data not shown). Ketamine attenuated SH-SY5Y cell viability in a dose-dependent manner under the 24 h conditions. Short exposure to ketamine (12 h) also induced neurotoxicity in SH-SY5Y cells, but was less compared with 24 h. Nevertheless, cell viability was markedly decreased in a dose-dependent manner when the exposure time was extended to 24 h (P<0.01;
To further investigate whether ketamine-induced inhibition of cell viability was associated with apoptosis induction, western blotting assays were performed to measure the expression levels of apoptotic markers. SH-SY5Y cells treated with higher concentrations of ketamine displayed increased expression levels of cleaved caspase-3 and Bax, and decreased expression levels of Bcl-2 compared with the 0 µM group (
To investigate the potential role of YAP in regulating the activity and viability of ketamine-treated SH-SY5Y cells, the present study induced YAP overexpression by lentiviral infection. The rate of apoptosis was decreased in the YAP overexpression group compared with the NC group (P<0.001;
To improve the current understanding of the role of YAP in ketamine-induced apoptosis, western blotting was performed to measure the expression of apoptotic markers, including cleaved caspase-3, Bcl-2 and Bax. Following YAP overexpression, SH-SY5Y cells were treated with 800 or 1,600 µM ketamine for 24 h. Following YAP overexpression, the expression levels of cleaved caspase-3 and Bax were decreased (P<0.01;
Flow cytometry was performed to assess ketamine-induced alterations to SH-SY5Y cell apoptosis. Compared with the NC group, ketamine-induced apoptosis was reduced following YAP overexpression. Furthermore, ketamine treatment increased SH-SY5Y cell apoptosis in a dose-dependent manner; however, YAP overexpression decreased the rate of ketamine-induced apoptosis at each concentration compared with the NC group (
To further investigate whether YAP knockdown altered ketamine-induced cell apoptosis, YAP expression was knocked down using an siRNA. Transfection efficiency of the siRNA was confirmed by performing western blotting (P<0.01;
The present study used different dose of ketamine to decrease SH-SY5Y cell viability and proliferation. A number of previous studies have demonstrated that ketamine can induce irreversible neurotoxicity in the developing central nervous system (
Ketamine-induced neurotoxicity is associated with a number of signaling pathways. A previous study reported that the NLR family pyrin domain containing 3 inflammasome and apoptosis-associated speck-like protein and caspase-1 proteins regulate the cleavage and release of proinflammatory interleukin-1β (
The present study indicated that YAP may serve a crucial role in mediating ketamine-induced SH-SY5Y cell apoptosis. As a component of the Hippo pathway, YAP is involved in organ development via its effects on cell proliferation, apoptosis and migration (
However, the present study had a number of limitations. First, the YAP-associated neuroprotection mechanism was not investigated using an
In conclusion, the present study suggested that ketamine induced neurotoxicity and cell apoptosis in a dose-dependent manner, YAP regulation may serve as an important event during ketamine-induced neurotoxicity
Not applicable.
The present study was supported by the Natural Science Foundation of Shanghai (grant no. 18ZR1443100), the Shanghai Jiao Tong University School of Medicine, Innovation Center of Translational Medicine Collaboration (grant no. TM201729) and the National Natural Science Foundation of China (grant no. 81401279).
The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.
YC and YL designed the study, performed the experiments, analyzed the data and wrote the manuscript. ZY contributed to the design of the study, provided financial support and revised the manuscript. LW made substantial contributions to the conception and design of the work and supervised the experiments. JW, YW and WX aquired and analyzed the data. Jl and ZK made substantial contributions to conception and design of the current study. ZK, JL and YL drafted the manuscript and revised it critically for important intellectual content, approved the final version of the manuscript and provided financial support. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Effects of ketamine on SH-SY5Y cell viability. The Cell Counting Kit-8 assay was used to assess SH-SY5Y cell viability following treatment with ketamine for (A) 12 and (B) 24 h. The 0 µM ketamine group served as the negative control, H2O2 was used to cause cell apoptosis, its effect on inducing cell death was not significant at the concentration of 400 µM (NS). *P<0.05, **P<0.01 and ***P<0.001, as indicated. NS, not significant.
Ketamine induces neurotoxicity in a dose-dependent manner by mediating apoptosis. (A) Protein expression levels of typical apoptotic markers, including cleaved caspase-3, Bcl-2 and Bax, were (A) determined by western blotting and (B) semi-quantified. (C) Cleaved caspase-3 expression levels (green) in SH-SY5Y cells following treatment with ketamine for 24 h were determined by immunofluorescence (scale bar, 50 µm) and (D) quantified. (E) SH-SY5Y cell apoptosis following treatment with ketamine was assessed by flow cytometry. *P<0.05, **P<0.01 and ***P<0.001, as indicated. 7-AAD, 7-aminoactinomycin D; AV, Annexin V; APC, allophycocyanin.
YAP overexpression reverses ketamine-induced reductions in SH-SY5Y cell viability. (A) Confocal microscopy indicated that different concentrations of ketamine (0, 400, 800, 1,200, 1,600 µM) induced apoptosis, as indicated by the breakdown of the cell nucleus. The arrow represents the broken down cell nucleus (scale bar, 20 µm). (B) Apoptotic cells were counted to calculate the rate of apoptosis following YAP overexpression. The Cell Counting Kit-8 assay was used to investigate the effects of YAP overexpression on SH-SY5Y cell viability following treatment with ketamine for (C) 12 or (D) 24 h. *P<0.05, **P<0.01 and ***P<0.001 vs. NC. YAP, yes-associated protein; NC, negative control.
YAP overexpression decreases apoptotic protein production in SH-SY5Y cells. YAP-knockdown SH-SY5Y cells were treated with ketamine for 24 h. YAP and cleaved caspase-3 protein expression levels were (A) determined by western blotting and (B) semi-quantified. Bax and Bcl-2 protein expression levels were (C) determined by western blotting and (D) semi-quantified. *P<0.05, **P<0.01 and ***P<0.001, as indicated. YAP, yes-associated protein; NC negative control.
YAP overexpression decreases ketamine-induced SH-SY5Y cell apoptosis. Following YAP overexpression, SH-SY5Y cells were treated with 0, 800 or 1,600 µM ketamine for 24 h. (A) SH-SY5Y cell apoptosis was assessed by flow cytometry. (B) Cleaved caspase-3 expression (red) was determined by confocal microscopy (scale bar, 50 µm) and (C) quantified. YAP, yes-associated protein; 7-AAD, 7-aminoactinomycin D; AV, Annexin V; APC, allophycocyanin; NC, negative control.
YAP knockdown aggravates ketamine-induced apoptosis. Following siRNA-mediated YAP knockdown, SH-SY5Y cells were treated with ketamine for 24 h. Protein expression levels were (A) determined by western blotting and semi-quantified for (B) YAP, (C) cleaved caspase-3, (D) Bax and (E) Bcl-2. *P<0.05 and **P<0.01, as indicated. YAP, yes-associated protein; siRNA, small interfering RNA; NC, negative control.