Paeoniflorin attenuated bupivacaine‐induced neurotoxicity in SH‐SY5Y cells via suppression of the p38 MAPK pathway
Long Chen | Qiushi Li | Hao Wang | Quan Chen | Yuanyuan Wu | You Shang
1 Department of Anesthesiology, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, , China
2 Department of Neurology, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning, China
1 | INTRODUCTION
Local anesthetics are widely used clinically because they can provide analgesia and antiarrhythmic action, but also improve bowel function after surgery or trauma and protect central nervous system.1-3 For instance, bupivacaine, an amide‐type local anesthetic, is commonly used for pain management, epidural or spinal anesthesia, and nerve blockade in clinical patients.4-6 However, accumu- lating evidence suggests that local anesthetics can induce toxicity to various tissues including nerve and cause postoperative neurological complications such as cauda equina syndrome, sensory disturbance, and motor paralysis, which strikingly limits their efficacy.7-9 For example, bupivacaine exposure resulted in the reduction of cell viability and the increase of lactate dehydrogenase release, apoptotic rate and cleaved caspase‐3 level in SH‐SY5Y cells.10 Bupivacaine triggered notable neuronal injuries in the spinal cord such as neuronal degeneration, edema, and vacuolation of myelin sheaths in rats.11
It has been proposed that p38 mitogen‐activated protein kinase (p38 MAPK), protein kinase B (Akt), caspase pathways and apoptosis signaling play vital roles in local anesthetics‐induced neurotoxicity.8,12,13 For instance, bupivacaine‐induced neurotoxicity has been demonstrated to be closely associated with the activation of p38 MAPK and apoptosis signaling.14-18
Recently, increasing studies indicated that bupivacaine‐ induced neurotoxicity could be weakened by some neuro- protective reagents such as dexamethasone,19 lithium,20 and α‐lipoic acid.21 Paeoniflorin (Figure 1A), a major active ingredient of paeonia, has been widely reported as a potential neuroprotective agent in neural injury models.22-24 However, it remains unknown whether paeoniflorin can alleviate bupivacaine‐induced neurotoxicity. Hence, the current study aimed to determine the roles and molecular mechanisms of paeoniflorin in bupivacaine‐induced neurotoxicity.
2 | MATERIALS AND METHODS
2.1 | Cell culture and treatment
Human neuroblastoma cell line SH‐SY5Y was purchased from Cell Bank of Chinese Academy of Science (Shanghai, China) and maintained in DMEM/F12 medium (Invitro- gen, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin (Hyclone, Logan, UT) and 100 μg/mL streptomycin (Hyclone) in 5% CO2 incubator at 37°C. SH‐SY5Y cells were pre‐treated with 10 μM SB203580 (purity ≥ 98%, Sigma‐Aldrich Co Ltd, Louis, MO) for 30 minutes or/and stimulated with bupivacaine (purity > 99%, Sigma‐Aldrich Co Ltd) or paeonifiorin (purity > 99%, National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) at the indicated concentration for 24 hours. Small interfering RNA targeting p38 (si‐p38) and the negative control (si‐con) were synthesized by Sangon Biotech (Shanghai, China). The siRNA sequences targeting p38 were as follow: forward, 5′‐AUG AAU GAU GGA CUG AAA UGG UCU G‐3′; reverse, 5′‐CAG ACC AUU UCA GUC CAU CAU UCA U‐3′. siRNA transfection was carried out using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol.
2.2 | 3‐(4,5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide assay
SH‐SY5Y cells (5 × 104 cells per well) were seeded into 96‐well plates and incubated overnight and then treated with bupivacaine (0.5, 1.0, and 1.5 mM) or paeonifiorin (10, 20, 40, 80, and 160 μM) for 24 hours. Subsequently, 10 μL 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) solution (5 mg/mL, Beyotime, Shanghai, China) was inoculated into each well of 96‐well plates and cultured for 4 hours at 37°C. Next, 150 μL dimethyl sulfoxide (DMSO, Sigma‐Aldrich) was added into each well. Optical densities were measured at 570 nm using a microplate reader (Bio‐ Tek, Winooski, VT).
2.3 | Apoptosis detection
Apoptosis was evaluated using the Annexin V‐FITC Apoptosis Detection Kit (Beyotime). Briefly, SH‐SY5Y cells treated as above were collected and washed with PBS three times, resuspended in 200 µL Annexin V‐FITC binding solution, and stained for 15 minutes with 10 µL Annexin V‐FITC solution and 5 µL propidium iodide (PI) solution in the dark at room temperature. Finally, the apoptotic rate of SH‐SY5Y cells was measured by a FACS‐ Calibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA).
2.4 | Caspase‐3 activity detection
Caspase‐3 activity was assessed using a caspase‐3 activity assay kit (Beyotime). Briefly, SH‐SY5Y cells treated as above were harvested, lysed and then centrifugated to obtain cell supernatant. Next, caspase‐3 substrate Ac‐ DEVD‐pNA at a final concentration of 0.2 mM was added into cell supernatant and then incubated for 1 hour. Finally, caspase‐3 activity was measured at 405 nm on a microplate reader (Bio‐Tek).
2.5 | Western blot assay
The treated SH‐SY5Y cells were harvested and lysed using RIPA buffer (Millipore, Billerica, MA) containing protease and phosphatase inhibitors (Thermo Fisher Scientific).
Then, protein concentration was determined using a BCA Protein Assay Kit (Pierce, Lockford, IL). Protein samples were separated by 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and then transferred to nitrocellulose (NC) membranes (Millipore). Afterwards, NC membranes were blocked with 5% skim milk and then immunoblotted overnight at 4°C with primary antibodies against Bcl‐2 (Cells Signaling Technology, Danvers, MA), Bax (Cells Signaling Technology), p38 MAPK (Cells Signaling Technology), phosphorylated‐p38 MAPK (p‐p38 MAPK, Cells Signaling Technology) and β‐actin (Cells Signaling Technology). After rinsing, the membranes were incubated for 1 hour at room temperature with goat anti‐ rabbit secondary antibody conjugated with horseradish peroxidase (Abcam, Cambridge, UK). Finally, protein signals were visualized using a Pierce™ ECL Western blot analysis substrate (Pierce) and quantified using Image J software (NIH, Bethesda, MD).
2.6 | Statistical analysis
Results are displayed as the mean ± standard deviation from three independent experiments. Statistical analysis was carried out using GraphPad software (GraphPad, San Diego, CA). Student t‐test or one‐way variance analysis was used for statistical analysis. P< 0.05 was considered as statistically significant.
3 | RESULTS
3.1 | Effect of bupivacaine on cell viability, apoptosis, and p38 MAPK pathway in SH‐SY5Y cells
We analyzed the cytotoxicity of bupivacaine in SH‐SY5Y cells. MTT assay showed that bupivacaine treatment at different concentrations (0.5, 1.0, and 1.5 mM) resulted in a reduction of SH‐SY5Y cell viability in a dose‐dependent manner (Figure 1B). Apoptotic rate of SH‐SY5Y cells was continuously elevated with the increase of bupivacaine concentration (Figure 1C). Furthermore, Western blot assay revealed that bupivacaine induced a concentration‐depen- dent increase of p‐p38 MAPK/p38 MAPK ratio in SH‐SY5Y cells (Figure 1D), suggesting that bupivacaine activated the p38 MAPK signaling in SH‐SY5Y cells. These results validated that bupivacaine induced neurotoxicity of SH‐SY5Y cells and activated the p38 MAPK signaling.
3.2 | Paeoniflorin attenuated bupivacaine‐induced viability inhibition in SH‐SY5Y cells
Next, we investigated the effect of paeoniflorin on SH‐SY5Y cell viability by MTT assay. The results disclosed that paeoniflorin at various concentrations (10, 20, 40, 80, and 160 μM) showed no significant effect on SH‐SY5Y cell viability (Figure 2A), suggesting that paeoniflorin had no cytotoxicity on SH‐SY5Y cells when its concentration reached to 160 μM. We further explored whether paeoniflorin could alleviate bupiva- caine‐induced neurotoxicity in SH‐SY5Y cells. As presented in Figure 2B, paeoniflorin weakened the inhibitory effect of bupivacaine on SH‐SY5Y cell viability in a dose‐dependent manner. Additionally, we found that paeoniflorin with the concentrations exceeding 40 μM had no further cytoprotective effect on SH‐SY5Y cells. Given this result, 40 μM paeoniflorin was used in the following experiments.
3.3 | Paeoniflorin weakened bupivacaine‐induced apoptosis in SH‐SY5Y cells
We further demonstrated that 40 μM paeoniflorin had no impact on cell apoptotic rate (Figure 3A), caspase‐3 activity (Figure 3B), Bcl‐2 and Bax level (Figure 3C) in SH‐SY5Y cells, indicating that paeoniflorin alone (40 μM) did not affect SH‐SY5Y cell apoptosis. Bupiva- caine (1 mM) markedly induced apoptosis of SH‐SY5Y cells, as evidenced by the increased apoptotic rate (Figure 3A), elevated caspase‐3 activity (Figure 3B), upregulated Bax level, and reduced Bcl‐2 level (Figure 3C) in SH‐SY5Y cells compared with untreated cells. Nevertheless, bupivacaine‐induced apoptosis of SH‐SY5Y cells was attenuated following the addition of paeoniflorin (Figure 3A‐C). Collectively, these findings suggested that paeoniflorin abated bupivacaine‐induced apoptosis in SH‐SY5Y cells.
3.4 | Paeoniflorin inhibited bupivacaine‐induced activation of p38 MAPK signaling in SH‐SY5Y cells
Western blot assay further showed that the ratio of p‐p38 MAPK/p38 MAPK had no significant difference in SH‐SY5Y cells treated with or without 40 μM paeoni- florin, indicating that paeoniflorin alone had no impact on p38 MAPK signaling (Figure 4). Paeoniflorin markedly undermined bupivacaine‐induced increase of p‐p38 MAPK/p38 MAPK ratio in SH‐SY5Y cells, indicating that paeoniflorin suppressed bupivacaine‐ induced activation of p38 MAPK signaling in SH‐SY5Y cells (Figure 4).
3.5 | Inhibition of p38 MAPK signaling reduced bupivacaine‐induced neurotoxicity in SH‐SY5Y cells
The p38 MAPK signaling inhibitor SB203580 was used to clarify whether the p38 MAPK signaling was involved in mediating the neurotoxicity of bupivacaine in SH‐SY5Y cells. Treatment with SB203580 decreased the ratio of p‐p38 MAPK/p38 MAPK (Figure 5A). MTT assay implicated that 10 μM SB203580 had no signifi- cant influence on cell viability of SH‐SY5Y cells relative to untreated cells, suggesting that SB203580 had no cytotoxicity on SH‐SY5Y cells (Figure 5B). Furthermore, inhibition of p38 MAPK signaling by SB203580 relieved bupivacaine‐mediated viability inhibition in SH‐SY5Y cells (Figure 5B). Pre‐treatment of SB203580 led to a reduction of apoptotic rate (Figure 5C), caspase‐3 activity (Figure 5D), and Bax level (Figure 5E), and an elevation of Bcl‐2 level (Figure 5E) in bupivacaine‐ treated SH‐SY5Y cells, suggesting that the inhibition of p38 MAPK signaling alleviated bupivacaine‐induced apoptosis in SH‐SY5Y cells. To further confirm the p38 MAPK signaling was involved in mediating the neuro-toxicity of bupivacaine, small interfering RNA targeting p38 (si‐p38) was used in this study. As shown in Figure 6A, the expression level of p38 was decreased 48 hours after transfection. Inhibition of p38 MAPK signaling by si‐p38 led to an increase in cell viability (Figure 6B), a reduction in apoptotic rate (Figure 6C), caspase‐3 activity (Figure 6D), and Bax level (Figure 6E), and an elevation in Bcl‐2 level (Figure 6E) in bupivacaine‐ treated SH‐SY5Y cells. These data suggested that inhibition of p38 MAPK signaling mitigated bupivacaine‐induced neurotoxicity in SH‐SY5Y cells.
4 | DISCUSSION
Combined treatment of bupivacaine and some neuroprotective drugs has been reported as an effective approach to enhance anesthesia efficiency and abate bupivacaine‐ induced neurotoxicity.11,25 Curcumin markedly reduced bupivacaine‐induced cell injury and apoptosis in SH‐SY5Y cells by activating Akt Signaling.26 The combination of dexmedetomidine plus bupivacaine prolonged the duration of sensory and motor blockade, and reduced neurotoxicity as compared with bupivacaine alone in Sprague‐Dawley rats received bilateral sciatic nerve blocks.27 In the current study, we validated that bupivacaine induced the reduction of cell viability in a dose‐dependent manner in SH‐SY5Y cells, which was in accordance with previous findings.10,25,28 Also, the pro‐ apoptosis effect of bupivacaine was further confirmed in SH‐SY5Y cells, as demonstrated by the increase of apoptotic rate, caspase‐3 activity, Bax expression, and the decrease of Bcl‐2 level in bupivacaine‐treated cells relative to untreated cells. The pro‐apoptosis effect of bupivacaine was also observed in rat neural cell line RT4‐ D6P2T and mouse neuroblastoma cell line Neuro2a.16,17
Previous studies also disclosed that bupivacaine could induce the activation of p38 MAPK in Neuro2a cells,17,18 SH‐SY5Y cells15 and adult rat DRG neurons.14 Consistently, our study showed that bupivacaine (0.5‐1.5 mM) induced a concentration‐dependent enhancement of p‐p38 MAPK/p38 MAPK ratio in SH‐SY5Y cells, suggest- ing that bupivacaine could induce the activation of p38 MAPK signaling.
Paeoniflorin, a water‐soluble monoterpene glycoside, has multiple pharmacological effects such as anti‐ inflammatory, anti‐depressant, and neuroprotective effects on nervous system.29-32 Previous studies pointed out that paeoniflorin could protect against neural injury or neurotoxicity induced by some stimulations such as Aβ25‐35,22 glutamate,33 H2O2,34 and cerebral ischemia.23
Paeoniflorin inhibited lipopolysaccharide (LPS)‐induced hippocampal cell death and reduced the generation of proinflammatory factors such as interleukin (IL)‐1β and nitric oxide (NO) in organotypic hippocampal slice cultures.29 Our study showed that paeoniflorin at the concentration range of 10 to 160 μM had no significant effect on SH‐SY5Y cell viability. Functional analyses manifested that paeoniflorin attenuated bupivacaine‐ mediating inhibition on SH‐SY5Y cell viability. It has been reported that neuroprotective effects of paeoniflorin are closely linked to the increase of anti‐apoptotic proteins and the reduction of pro‐apoptotic mole- cules.23,31,32 Our data also unveiled that paeoniflorin attenuated bupivacaine‐induced increase of apoptotic rate, caspase‐3 activity, and Bax level, and ameliorated bupivacaine‐induced reduction of Bcl‐2 level in SH‐SY5Y cells, indicating that paeoniflorin weakened bupivacaine‐ induced apoptosis by regulating apoptosis‐related pro- teins in SH‐SY5Y cells. In a word, these data indicated that paeoniflorin relieved bupivacaine‐induced neuro- toxicity in SH‐SY5Y cells.
Previous findings suggested that p38‐MAPK pathway might be involved in mediating neuroprotective effects of paeoniflorin.24,35 Paeoniflorin exerted the protective effect on ischemia‐induced brain injury in rats by inactivating p38 MAPK/nuclear factor‐κappa B (NF‐κB) signaling in rats.35 Hence, we further explored whether the neuroprotective effect of paeoniflorin on bupivacaine‐ induced SH‐SY5Y cell injury was mediated by the p38 MAPK pathway. Our results showed that paeoniflorin alone had no effect on the p38 MAPK signaling. However, paeoniflorin inhibited bupivacaine‐induced activation of p38 MAPK signaling. Our study further showed that p38 MAPK inhibitor SB202190 or small interfering RNA targeting p38 abrogated bupivacaine‐ induced viability inhibition and apoptosis in SH‐SY5Y cells, which was in line with previous studies.15,17 Thus, we inferred from these results that paeoniflorin abated bupivacaine‐induced neurotoxicity by inactivating the p38 MAPK signaling.
Collectively, these data provided the evidence that NSC 178886 attenuated bupivacaine‐induced neurotoxi- city in SH‐SY5Y cells via suppression of the p38 MAPK pathway. To our knowledge, our study is the first report to elucidate the neuroprotective effects of paeoniflorin on bupivacaine‐induced neural injury, providing a potential strategy to reduce the side effects of bupivacaine.