Kaempferol counteracts bupivacaine‐induced neurotoxicity in mouse dorsal root ganglia neurons by regulating TRAF6‐dependent NF‐κB signaling

Abstract

Kaempferol (KA), a widely recognized anti‐oxidation and anti‐inflammation agent, has been reported to have neuroprotective effects. This work aimed to investigate whether KA protects mouse dorsal root ganglia (DRG) neurons against bupivacaine (BU)‐stimulated neurotoxicity and explore the underlying mechanisms. In this study, BU treatment suppressed DRG neuron viability and promoted LDH leakage, which was partially abated by KA. Besides, BU‐triggered DRG neuron apoptosis, and changes in Bax and Bcl‐2 levels were attenuated by KA treatment. In addition, pretreatment with KA substantially reduced interleukin (IL)‐6, IL‐1β, and tumor necrosis factor (TNF)‐α levels in BU‐treated DRG neurons. In addition, KA administration abrogated BU‐induced decline in CAT, SOD, and GSH‐Px levels, as well as the increase in the malondialdehyde level. Interestingly, we found that KA significantly attenuated BU‐induced TNF receptor‐associated factor 6 (TRAF6) upregulation as well as NF‐κB activation. Furthermore, oe‐TRAF6‐mediated TRAF6 overexpression promoted NF‐κB activation and partly abolished KA‐induced protection against BU‐triggered neurotoxic effects on DRG neurons. Our results revealed that KA mitigated BU‐induced neurotoxic effects on DRG neurons by deactivating the TRAF6/NF‐κB signaling.

1. INTRODUCTION

As a long‐acting anesthetic, bupivacaine (BU) is generally applied in peripheral‐nerve‐block, local‐infiltration, and intraspinal anesthesia for surgery as well as analgesia of post‐surgical or chronic pains in clinical use. ¹ , ² BU administration exhibits good analgesic effects; however, long‐term systemic absorption of BU may also lead to disorders in the central nervous system (CNS) and the cardiovascular system. ³ Although rare BU administration can induce neurotoxicity or irreparable neurological deficits in humans even at clinically acceptable concentrations, ⁴ resulting in symptoms, including hypoventilation, paralysis, and urinary incontinence. ⁵ Previous reports also suggest that inflammation, oxidative stress, and associated neuroapoptosis are deeply implicated in the pathogenetic process of BU‐induced neurotoxic effects. ⁶ Nevertheless, specific mechanisms of BU‐induced neurotoxicity are poorly known.

Kaempferol (KA), a natural polyphenol, is a common dietary flavonoid found in most foods of plant origin, including grapefruit, cabbage, and tea. ⁷ In view of the satisfactory efficacy of KA in inhibiting inflammatory response and oxidative stress reaction, it also gains a lot of attention in the field of medication for human diseases. ⁸ , ⁹ , ¹⁰ In addition, KA, in particular, exerts neuroprotective effects against many neurological diseases, such as Alzheimer's disease, ¹¹ ischemic stroke, ¹² Huntington's disease, ¹³ and Parkinson's disease. ¹⁴ Nevertheless, the effects of KA on BU‐triggered neurotoxicity are still poorly understood.

Tumor necrosis factor (TNF) receptor‐associated factor 6 (TRAF6), the most divergent member of the TRAF family, ¹⁵ is an important regulator in CNS disorders. ¹⁶ It has been reported that TRAF6 contributes to traumatic brain injury, ¹⁷ ischemic stroke, ¹⁸ and neuropathic pain ¹⁹ by regulating cellular processes, including inflammation, oxidative stress reaction, and/or apoptosis, in neurons. For instance, TRAF6 promotes LPS‐induced neurotoxicity by promoting BV‐2 microglia migration and PC12 cell death via NF‐κB and AP‐1 signaling pathways. ²⁰ TRAF6 ubiquitination contributes to microglia‐induced dopaminergic neurotoxicity. ²¹ TRAF6 upregulation and subsequent NF‐κB activation facilitate Aβ‐induced cognitive deficits and neuroinflammation. ²² Furthermore, TRAF6‐induced NF‐κB activation is deeply involved in acrylamide‐induced neurotoxicity in rats. ²³ All these studies indicate a promoting role of TRAF6 in neurotoxicity. Hence, TRAF6‐dependent NF‐κB signaling might play a critical role in KA‐mediated neuroprotective effects.

In this work, we proposed to evaluate the neuroprotective potential and underlying mechanisms of KA against BU‐induced neurotoxicity. A cell model of BU‐induced neurotoxicity was established based on mouse dorsal root ganglia (DRG) neurons to assess the neuroprotective effects of KA in vitro. To further elucidate the specific mechanism of KA‐mediated protection against neurotoxic effects of BU, the alteration and involvement of TRAF6 and NF‐κB signaling were focused on.

2. MATERIALS AND METHODS

2.1. Isolation and culture of DRG neurons

All animal procedures were permitted by the Ethics Committee of Liyang People's Hospital. In brief, DRG was retrieved from neonatal C57BL/6 mice and then dissociated to obtain DRG neurons, as previously described. ²⁴ DRG neurons were seeded into 6‐well plate and cultured in DMEM/F12 containing 10% FBS (ThermoFisher Scientific, USA) in a humidified incubator (5% CO₂; 37°C). DRG neurons were incubated for 5 days before further research.

2.2. Cell transfection

TRAF6 was PCR amplified from cDNA prepared by reverse transcription (RT) of RNA extracted from Bend.3 cells and then cloned into the pCMS‐EGFP vector to produce TRAF6 overexpression plasmid (oe‐TRAF6). The empty vector was used as the control vector (Vector). These plasmids were transfected into DRG neurons with Lipofectamine 3000 (Invitrogen, CA). Thereafter, DRG neurons were cultured for 48 h before further analyses.

2.3. Drug treatment

BU and KA were bought from Sigma Aldrich (USA). First, stock solutions of KA and BU were prepared in DMSO. Then, stock solutions were diluted in the culture medium to required concentrations.

To establish a cell model of BU‐induced neurotoxicity, DRG neurons were subject to BU at increased concentrations (0.5, 1.0, 1.5, or 2.0 mM) as previously described. ⁵ For KA treatment, DRG neurons were cultivated with KA (2.5, 5, and 10 μM) ²⁵ 2 h before BU treatment (1.5 mM).

2.4. Cell viability assay

CCK‐8 assay was performed to assess DRG neuron viability. Briefly, DRG neurons (5 × 10³ cells/well) were seeded into 96‐well plates. After 24 h incubation at 37°C, CCK‐8 reagent was added (10 μL/well) and DRG neurons were incubated for another 2 h at 37°C. The absorbance at 450 nm was measured with a microplate reader.

2.5. Measurement of oxidative stress factors

After the abovementioned treatments, DRG cell supernatants were collected to measure catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH‐Px), and malondialdehyde (MDA) levels via specific commercial kits (Nanjing Jiancheng Bioengineering Institute, China) as per the manufacturer's instructions.

2.6. ELISA

To test the levels of pro‐inflammatory cytokines (IL‐6, IL‐1β, and TNF‐α), commercial ELISA kits (R&D Systems, USA) were used. In brief, treated DRG neurons were centrifuged to collect culture supernatant. Then, IL‐6, IL‐1β, and TNF‐α levels were measured as per the manufacturer's instructions.

2.7. TUNEL

One‐step TUNEL assay apoptosis kit was used to detect DRG neuron apoptosis. In brief, treated DRG neurons were fixed by 4% paraformaldehyde, permeabilizated by 0.3% Triton X‐100, cultured with TUNEL reaction reagent in darkness, and then counterstained with DAPI. Afterwards, the DRG neurons were visualized with the IX70 fluorescence microscopy (Olympus, Japan). The apoptotic rate of DRG neurons was presented as the ratio of TUNEL‐positive DRG neurons to total DRG neurons.

2.8. Western blotting

Through lysing DRG neurons with RIPA buffer (Beyotime, China), total protein was first extracted. Then, the total protein was separated into equal amounts via SDS‐PAGE and transferred to a PVDF membrane. Thereafter, the membrane was blocked with 5% skimmed milk and incubated with primary antibodies (anti‐Bax, anti‐Bcl‐2, anti‐TRAF6, anti‐p‐NF‐κB‐p65, anti‐NF‐κB‐p65, and anti‐β‐actin) and secondary antibody (Abcam). The protein bands were visualized with an ECL system (Pierce, USA).

2.9. Statistical analyses

With the GraphPad Prism 6.0 software, all statistical analyses in this work were performed with differences assessed via Student's t test (2 groups) or one‐way ANOVA (>2 groups). Each experiment was repeated three times with results expressed as mean ± standard deviation (SD). Any difference with p < 0.05 was deemed significant in statistics.

3. RESULTS

3.1. KA diminishes BU‐induced inhibition of DRG neuron viability

To establish a cell model of BU‐induced neurotoxicity, DRG neurons were exposed to BU at increased concentrations. Then, CCK‐8 was conducted to measure DRG neuron viability. As exhibited in Figure 1A, BU dose‐dependently decreased DRG neuron viability. 1.5 mM was selected as the BU concentration for the following experiments. To determine whether KA pretreatment protects DRG neurons against BU‐caused neurotoxicity, DRG neurons were pretreated with KA at increased concentrations for 2 h and subject to 1.5 mM BU for 24 h. Intriguingly, BU‐stimulated decline in DRG neuron viability was notably rescued by KA pretreatment in a concentration‐dependent way (Figure 1B). Similarly, lactate dehydrogenase (LDH) assay manifested that BU exposure resulted in a remarkable increase in the LDH release in DRG neurons, which was abated by KA treatment in a dose‐dependent manner (Figure 1C). These results implied that KA might exert neuroprotective effects on BU‐challenged DRG neurons.

FIGURE 1.

KA diminishes BU‐induced inhibition of DRG neuron viability. (A) DRG neurons accepted BU (0.5, 1.0, 1.5, or 2.0 mM) treatment. DRG neuron viability was detected via CCK‐8. (B and C) DRG neurons were divided into Control, BU, BU+KA (2.5 μM), BU+KA (5 μM), and BU+KA (10 μM) groups. Briefly, DRG neurons were first treated with KA (2.5, 5, and 10 μM) and exposed to BU (1.5 mM). DRG neuron viability and LDH leakage were detected via CCK‐8 and LDH assays. Data are presented as mean ± SD (n = 3). *p < 0.05 and **p < 0.01.

3.2. KA suppresses BU‐stimulated DRG neuron apoptosis

To look into the influence of KA on BU‐caused cell apoptosis, the TUNEL assay was utilized to assess DRG neuron apoptosis. As determined by the TUNEL assay, BU exposure led to a high apoptotic rate in DRG neurons, whereas KA pretreatment considerably reduced BU‐induced apoptosis in DRG neurons to a concentration‐dependent extent (Figure 2A). The balance of Bax (pro‐apoptotic protein) and Bcl‐2 (anti‐apoptotic protein) was crucial for the regulation of cell viability and apoptosis. ²⁶ Then, Bax and Bcl‐2 protein levels were detected by western blotting. Consistent with TUNEL assay results, BU treatment alone markedly caused an increase in the Bax level and a dramatic decrease in the Bcl‐2 level; however, KA concentration‐dependently pretreatment reversed the trend of Bax and Bcl‐2 levels in BU‐challenged DRG neurons (Figure 2B). Altogether, KA could inhibit BU‐stimulated DRG neuron apoptosis.

FIGURE 2.

KA suppresses BU‐stimulated DRG neuron apoptosis. (A) DRG neuron apoptosis in each group was assessed via the TUNEL assay. (B) Bax and Bcl‐2 protein levels were detected by Western blotting. Data are presented as mean ± SD (n = 3). *p < 0.05 and **p < 0.01.

3.3. KA alleviates BU‐stimulated inflammatory responses in DRG neurons

Pro‐inflammatory cytokine (IL‐6, IL‐1β, and TNF‐α) levels were determined through ELISA assay. It was shown that IL‐6, IL‐1β, and TNF‐α levels were obviously increased in DRG neurons in response to BU exposure as compared with untreated control; nevertheless, KA treatment dose‐dependently reversed this change for the above‐estimated inflammatory factors (Figure 3A–C). These data implied the protective effect of KA against BU‐induced neurotoxicity in DRG neurons via inhibiting the secretion of pro‐inflammatory cytokines.

FIGURE 3.

KA alleviates BU‐stimulated inflammatory responses in DRG neurons. (A–C) IL‐6, IL‐1β, and TNF‐α levels in cell supernatant from each group were detected via the ELISA assay. Data are presented as mean ± SD (n = 3). *p < 0.05 and **p < 0.01.

3.4. KA ameliorates oxidative stress reaction of BU‐challenged DRG neurons

Next, the effect of KA on BU‐induced oxidative stress reaction of DRG neurons was investigated. It was found that BU treatment led to a substantial decline of CAT (Figure 4A) and SOD (Figure 4B) activities as well as GSH‐Px level (Figure 4C) and a distinct increase of MDA level (Figure 4D) in DRG neurons; however, KA pretreatment markedly reversed these changes caused by BU in a dosage‐dependent manner. The above results demonstrated that KA partially attenuated BU‐induced oxidative stress reaction in DRG neurons.

FIGURE 4.

KA ameliorates oxidative stress reaction of BU‐challenged DRG neurons. (A–D) CAT, SOD, GSH‐Px, and MDA levels in cell supernatant were detected via commercial kits. Data are presented as mean ± SD (n = 3). *p < 0.05 and **p < 0.01.

3.5. KA reverses BU‐induced TRAF6 upregulation and NF‐κB activation

Notably, KA was found to exert its anti‐inflammatory effects by decreasing TRAF6 expression in diabetic mice. ²⁷ This raises the possibility that KA mitigates BU‐induced neurotoxicity by regulating TRAF6. Moreover, it is widely recognized that inhibition of the TRAF6/NF‐κB pathway is an efficient way to protect against neurotoxicity. ²³ Thus, the levels of TRAF6 and NF‐κB‐related proteins (NF‐κB‐p65 and p‐NF‐κB‐p65) were determined by western blotting. As indicated in Figure 5A, BU stimulation led to an increase in the TRAF6 protein level; however, such an increase was reversed with the increase in the KA concentration. Likewise, western blotting results also showed that the p‐NF‐κB‐p65 level was significantly upregulated after the BU stimulation, while KA pretreatment concentration‐dependently eliminated the impact of BU treatment (Figure 5B). The above data indicated that the protective effects of KA against BU‐induced inflammation, oxidative stress and apoptosis in DRG neurons might be regulated via inactivation of the TRAF6/NF‐κB signaling pathway.

FIGURE 5.

KA reverses BU‐induced TRAF6 upregulation and NF‐κB activation. (A) TRAF6 protein level was measured. (B) NF‐κB‐p65 and p‐NF‐κB‐p65 protein levels were measured. Data are presented as mean ± SD (n = 3). *p < 0.05 and **p < 0.01.

3.6. TRAF6‐dependent NF‐κB activation impairs the neuroprotective effects of KA

To confirm the involvement of the TRAF6/NF‐κB pathway in KA‐mediated protection against BU‐induced cytotoxicity in DRG neurons, Control, BU, BU+KA, and BU+KA (10 μM)+oe‐TRAF6 groups were established. As illustrated by Western blotting results, oe‐TRAF6 transfected DRG neurons exhibited a dramatic increase in the TRAF6 protein level compared with the Vector group (Figure 6A). It was shown that KA pretreatment reversed BU‐stimulated p‐NF‐κB‐p65 upregulation, while TRAF6 overexpression partly abolished the inhibitory effects of KA on BU‐triggered NF‐κB‐p65 phosphorylation (Figure 6B), indicating that TRAF6 eliminated KA‐mediated NF‐κB activation in BU‐stimulated DRG neurons. Furthermore, TRAF6 upregulation significantly abated the impacts of KA on cell viability, apoptosis, inflammatory response, and oxidative stress reaction of BU‐stimulated DRG neurons (Figure 6C–M). Thus, KA prevented BU‐induced neurotoxicity by inhibiting the TRAF6‐activated NF‐κB signaling pathway.

FIGURE 6.

TRAF6‐dependent NF‐κB activation impairs the neuroprotective effects of KA. DRG neurons were divided into Control, BU, BU+KA (10 μM), and BU+KA (10 μM)+oe‐TRAF6 groups. For the last group, DRG neurons were transfected with oe‐TRAF6, pretreated with KA, and exposed to BU. (A) TRAF6 protein level was measured. (B) NF‐κB‐p65 and p‐NF‐κB‐p65 protein levels were measured. (C–M) DRG neuron viability (C); LDH release (D); cell apoptosis (E); Bax and Bcl‐2 protein levels (F); IL‐6 (G), IL‐1β (H), and TNF‐α (I) levels; as well as CAT, SOD, GSH‐Px, and MDA levels (J–M) were detected. Data are presented as mean ± SD (n = 3). *p < 0.05 and **p < 0.01.

4. DISCUSSION

Recently, convincing evidence has demonstrated that BU, as a local anesthetic, can induce severe neurotoxicity even at a normal clinical dose. ²⁸ However, the mechanisms by which BU triggers neurotoxicity have not been elucidated precisely. Previous studies have proven that BU administration can induce neurotoxicity in diverse types of neurons in the spinal cord, including DRG neurons, so DRG neurons have been widely used in the establishment of in vitro experimental models for BU‐induced neurotoxicity. ²⁹ Hence, we established a cellular model of BU‐induced neurotoxicity based on DRG neurons in this work.

Numerous studies have demonstrated the neuroprotective properties of KA. ³⁰ For instance, KA mitigates pyroptosis and alleviates neuroinflammation in secondary spinal cord injury. ³¹ Besides, KA ameliorates neuropathic pain by inhibiting the TLR4/NF‐ĸB signaling pathway as a neuroprotectant. ³² Also, KA protects against α‐synuclein‐induced neurotoxicity. ³³ In line with these findings, we demonstrated that KA pretreatment inhibited BU‐induced decrease in the DRG neuron viability and increase in the LDH leakage, decreased BU‐caused DRG neuron apoptosis, reduced the BU‐triggered production of pro‐inflammatory cytokines (IL‐6, IL‐1β, and TNF‐α), and abrogated the levels of oxidative stress indicators (CAT, SOD, GSH‐Px, and MDA) in BU‐stimulated DRG neurons. Hence, these results revealed that KA substantially prevented DRG neurons against BU‐induced neurotoxicity by enhancing cell viability and repressing apoptosis, inflammation, as well as oxidative stress.

As BU‐induced neurotoxicity is largely attributed to increased neural apoptosis, inflammation, and oxidative stress, ⁶ relevant signaling pathways could be potential therapeutic targets. Accumulating data suggest that TRAF6 plays important roles in CNS disorders. ¹⁴ Researchers have discovered that TRAF6 exhibits pro‐inflammatory, pro‐apoptotic, and pro‐oxidant effects by activating the NF‐κB signaling. ³⁴ Besides, it has been proven that TRAF6‐dependent NF‐κB pathway is involved in the alleviation of anesthetic‐induced neurotoxicity. ³⁵ In addition, KA mitigates acute lung injury by inhibiting TRAF6 polyubiquitination and impairing NF‐κB activation. ³⁶ Consistently, our data showed that BU treatment increased the TRAF6 level and promoted NF‐κB activation in DRG neurons, which was attenuated by KA treatment. Additionally, TRAF6 addition substantially abated the impact of KA‐induced suppression on NF‐κB‐p65 phosphorylation of BU‐triggered DRG neurons. Moreover, the suppressive effects of KA on BU‐caused reduction in viability and increase in LDH release, apoptosis, inflammation, and oxidative stress were attenuated in TRAF6‐overexpressing DRG neurons. Collectively, it was indicated that KA mitigated BU‐induced neurotoxic effects via regulating the TRAF6‐dependent NF‐κB signaling pathway.

5. CONCLUSION

To sum up, this study demonstrated that KA could protect DRG neurons from BU‐induced neurotoxic effects through enhancing cell viability and repressing apoptosis, inflammation, and oxidative stress by restraining TRAF6‐activated NF‐κB signaling. Consequently, our findings suggested that KA could be a promising neuroprotectant against BU‐induced neurotoxic effects. One major limitation of the study is that the BU concentrations applied for the experiments are inconsistent with the BU concentrations in clinical use. In future studies, further investigation will be performed with clinically acceptable BU concentrations to optimize the dosing regimens of KA for the prevention and treatment of BU‐induced neurotoxicity.

CONFLICT OF INTEREST STATEMENT

All authors declare no conflict of interest.

Chen L, Zhuang K. Kaempferol counteracts bupivacaine‐induced neurotoxicity in mouse dorsal root ganglia neurons by regulating TRAF6‐dependent NF‐κB signaling. Kaohsiung J Med Sci. 2023;39(7):710–717. 10.1002/kjm2.12682