Bupivacaine reduces GlyT1 expression by potentiating the p-AMPKα/BDNF signalling pathway in spinal astrocytes of rats

Bupivacaine, a local anaesthetic, is widely applied in the epidural or subarachnoid space to clinically manage acute and chronic pain. However, the underlying mechanisms are complex and unclear. Glycine transporter 1 (GlyT1) in the spinal cord plays a critical role in various pathologic pain conditions. Therefore, we sought to determine whether bupivacaine exerts its analgesic effect by regulating GlyT1 expression and to determine the underlying mechanisms of regulation. Primary astrocytes prepared from the spinal cord of rats were treated with bupivacaine. The protein levels of GlyT1, brain-derived neurotrophic factor (BDNF) and phosphorylated adenosine 5′-monophosphate (AMP)-activated protein kinase α (p-AMPKα) were measured by western blotting or immunofluorescence. In addition, 7,8-dihydroxyflavone (7,8-DHF, BDNF receptor agonist) and AMPK shRNA were applied to verify the relationship between the regulation of GlyT1 by bupivacaine and the p-AMPKα/BDNF signalling pathway. After treatment with bupivacaine, GlyT1 expression was diminished in a concentration-dependent manner, while the expression of BDNF and p-AMPK was increased. Moreover, 7,8-DHF decreased GlyT1 expression, and AMPK knockdown suppressed the upregulation of BDNF expression by bupivacaine. Finally, we concluded that bupivacaine reduced GlyT1 expression in spinal astrocytes by activating the p-AMPKα/BDNF signalling pathway. These results provide a new mechanism for the analgesic effect of intrathecal bupivacaine in the treatment of acute and chronic pain.

www.nature.com/scientificreports/ treatment of acute and chronic pain [13][14][15][16] . In particular, inhibition of GlyT1 not only promotes glycine concentration in the glycine synaptic cleft but also reduces the expression of N-methyl-D-aspartate receptors (NMDARs) in the glutamate synapse to ameliorate neuropathic pain 17 . GlyT1 expression is modulated by various factors, one of which is that BDNF promotes GlyT1 endocytosis and degradation and inhibits GlyT1-mediated glycine uptake 18 . BDNF is one of the major neurotrophic factors in the development, maturation, and maintenance of the CNS. Moreover, it modulates the morphology of astrocytes by interacting with tropomyosin-receptor-kinase B (TrkB) 19,20 . Furthermore, BDNF expression is controlled by p-AMPKα in the CNS 21,22 . AMPK is an important intracellular energy sensor involved in energy metabolism. The specific phosphorylation of AMPK can activate transcriptional regulators to affect protein expression. Furthermore, local anaesthetics have been demonstrated to activate AMPK [23][24][25] .
Therefore, we speculated that bupivacaine might decrease GlyT1 expression by activating the p-AMPKα/ BDNF signalling pathway. To verify this hypothesis, we used primary astrocytes from the rat spinal cord to study the effect of bupivacaine on GlyT1, and the expression levels of GlyT1, BDNF, and p-AMPKα were detected in astrocytes after treatment with bupivacaine. In addition, we used 7,8-DHF or AMPK shRNA to modulate related protein expression. Finally, the results supported our hypothesis that bupivacaine decreased GlyT1 expression in astrocytes, which was associated with the activation of the p-AMPKα/BDNF signalling pathway. Thus, modulation of GlyT1 expression might represent a novel mechanism for the antinociceptive action of intrathecal bupivacaine.

Results
Bupivacaine regulated GlyT1 and BDNF expression in primary astrocytes. The regulation of GlyT1 and BDNF expression by bupivacaine was determined in primary astrocytes derived from the spinal cord of rats. Data from western blot analysis indicated a prominent decrease in GlyT1 expression at different concentrations of bupivacaine for 2 h (*p < 0.05, ***p < 0.001, Fig. 1A). In addition, the most significant decrease in GlyT1 expression was in the 3 mM bupivacaine group. The expression of BDNF was increased significantly after treatment with bupivacaine, especially in the 3 mM bupivacaine group (*p < 0.05, **p < 0.01, ***p < 0.001, Figs. 1B and 3B).

Discussion
This study reported the actions of bupivacaine on GlyT1 protein expression and its further mechanism of regulation. In the CNS, GlyT1 was predominantly expressed in astrocytes, so primary astrocytes derived from the rat spinal cord were studied. Finally, we found that bupivacaine reduced GlyT1 expression in spinal astrocytes, which was associated with the potentiation of the p-AMPKα/BDNF signalling pathway. The analgesic effects of intrathecal bupivacaine have been described but cannot be explained solely by its blockade of voltage-gated Na + channels. In addition to blocking Na + channels, bupivacaine can also modulate many other membrane proteins, such as NMDA receptors 26,27 , nicotinic acetylcholine and 5-HT3A receptors 28 . www.nature.com/scientificreports/ In addition, GlyT1-mediated glycine uptake modulated by local anaesthetic has been investigated 7,29 . However, it has not yet been established whether the antinociceptive mechanism of intrathecal bupivacaine is related to GlyT1 regulation. Therefore, the aim of the study was to investigate the effect of bupivacaine on GlyT1 in vitro to elucidate the possible mechanism of the analgesic effect of intrathecal bupivacaine. The restoration of inhibitory glycinergic neurotransmission in the spinal cord plays a crucial role in the treatment of pathologic pain 30,31 . Glycinergic neurotransmission is controlled by GlyT1 and GlyT2. GlyT1 affects glycinergic neurotransmission by removing the glycine concentration at the glycinergic synaptic cleft, while GlyT2 mediates the reuptake of glycine into glycinergic nerve terminals. Inhibitions of GlyT1 and GlyT2 both have significant therapeutic effects on various pathologic pain conditions 32 . In particular, inhibition of GlyT1 can attenuate neuropathic pain by priming NMDA receptors for internalization 17,33,34 . In addition, it has been established that GlyT1 inhibitors can relieve pain sensitivity in different neuropathic pain models. This phenomenon is related to the enhancement of glycinergic neurotransmission and the inhibition of glutamatergic neurotransmission. A study in rats demonstrated that sciatic nerve constriction injury-induced neuropathic pain was attenuated by intrathecal GlyT1 inhibitors 35 . In addition, Armbruster et al. 36 found that GlyT1 inhibition by intraperitoneal or oral bitopertin (GlyT1 inhibitor) could effectively alleviate hyperalgesia in rats with neuropathic pain induced by peripheral nerve injury. Furthermore, in sciatic nerve ligation injury-induced neuropathic pain models, mechanical allodynia was ameliorated by intrathecal sarcosine (GlyT1 inhibitor) and GlyT1 knockdown of the spinal cord of mice 37 . The authors of these studies concluded that inhibition of GlyT1 in the spinal cord represents a new therapy for neuropathic pain. In this study, we found that bupivacaine dose-dependently reduced GlyT1 expression in spinal astrocytes at concentrations reported in previous studies 4,5 . According to www.nature.com/scientificreports/ our results, a possible mechanism of the bupivacaine-mediated antinociceptive effect is the inhibition of GlyT1 expression. Nevertheless, whether bupivacaine could regulate GlyT2 has to be elucidated in further investigations. It has been reported that many factors affect GlyT1 expression in astrocytes. Aroeira et al. reported that BDNF promoted GlyT1 internalization and degradation and suppressed GlyT1-mediated glycine uptake in astrocytes by acting on TrkB receptors 18 . Several investigations have been conducted to demonstrate that the  www.nature.com/scientificreports/ activation of protein kinase C (PKC) induced endocytosis and degradation of GlyT1 by promoting ubiquitination of GlyT1 38 . Additionally, another study proposed that calmodulin was involved in the regulation of GlyT1 surface expression and GlyT1-mediated glycine uptake 39 . In our study, we found that bupivacaine increased the expression of BDNF and decreased GlyT1 expression in astrocytes. Moreover, GlyT1 expression in astrocytes was reduced by 7,8-DHF. Therefore, we concluded that bupivacaine reduced GlyT1 expression by upregulating BDNF expression in astrocytes. However, whether bupivacaine reduces GlyT1 expression by modulating PKC or calmodulin remains to be studied. BDNF is one of the major neurotrophic factors produced by astrocytes to maintain the development and survival of neurons in the CNS. A previous study demonstrated that BDNF protected astrocytes from death through the TrkB signalling pathway and induced astrocytes to release neuroprotective factors 40 . In addition, BDNF plays an important role in astrocyte morphogenesis via astrocytic TrkB receptors 20,41,42 . Furthermore, many investigations have indicated that the expression of BDNF in brain astrocytes is promoted by the activation of AMPK 43,44 . AMPK is expressed ubiquitously and is a key regulator of metabolic pathways such as fatty acid and cholesterol synthesis 45 . AMPK is also active in CNS, and the activation of AMPK plays a neuroprotective or neurodegenerative role 46,47 . AMPK activation is regulated by various factors, previous studies have shown that AMPK is phosphorylated and activated by two major upstream kinases LKB1 and CAMKKβ in response to stimuli that increase intracellular AMP/ADP or calcium levels 48 . It has been reported that local anaesthetics have activating effects on AMPK 7,23 . Consistent with their findings, our study established that bupivacaine activated AMPK in spinal astrocytes, and bupivacaine increased BDNF expression by AMPK activation. But the mechanism of AMPK activation by bupivacaine remains to be further studied.
In conclusion, bupivacaine decreased GlyT1 expression in spinal astrocytes by potentiating the p-AMPKα/ BDNF signalling pathway. GlyT1 inhibition of bupivacaine on spinal astrocytes provides a new molecular mechanism for the analgesic effect of intrathecal bupivacaine. However, whether the mechanism of the antinociceptive effects of intrathecal bupivacaine is consistent with our in vitro study needs to be further clarified.

Materials and methods
Primary cultures of astrocytes. Newborn Sprague-Dawley (SD) rats were purchased from the Laboratory Animal Center of Ningxia Medical University. All studies were approved by the Ethics Committee of Ningxia Medical University and performed according to the American Animal Protection Legislation. The study was carried out in compliance with the ARRIVE guidelines. 60 newborn rats were used in the experiment, and the age of the newborn rats was 1-2 days. Spinal cord primary astrocytes from 36 rats were used to study the effects of bupivacaine on GlyT1, BDNF and P-AMPK protein expression, and spinal cord primary astrocytes from 12 rats were applied to investigate the effect of 7,8-DHF on GlyT1 expression, spinal cord primary astrocytes from the remaining 12 mice were used to study the effect of bupivacaine on BDNF regulation after AMPK knockdown. The neonatal rats were anaesthetized by inhaling nitrous oxide. After disinfection with 75% ethyl alcohol, primary astrocytes of the spinal cord were isolated from newborn rats and cultured according to the work of Zhou et al. 49 . Briefly, the spinal cords were dissected and digested with 0.25% trypsin (Gibco) for 6 min at 37 °C, the reaction was terminated by adding medium containing foetal bovine serum, and the cell suspension was centrifuged at 1200 rpm for 5 min. Finally, the cell pellet was cultured in a mixed medium of DMEM/ F12 (1:1) with 10% FBS and 1% penicillin/streptomycin, and the cells were cultured in 75 cm 2 polylysine-coated flasks in the presence of 5% CO 2 . Nonastrocytes were detached from the flasks by shaking and were removed by changing the medium. Third-or fourth-passage cells were rendered quiescent through incubation in medium containing 0.5% FBS for 4 days prior to the experiments. Astrocytes were confirmed by their typical morphology and positive staining for the specific marker GFAP, and the purity of astrocytes was over 90% (see Supplementary Fig S5).

Drugs and treatments. Bupivacaine hydrochloride was provided by Zhaohui Pharmaceutical (Shanghai,
China). Treatment with 7,8-dihydroxyflavone (7,8-DHF), a specific TrkB receptor (one of the BDNF receptors) agonists, mimicked the physiological effects of BDNF. It was purchased from MedChemExpress. Bupivacaine was diluted to 1 mM, 2 mM, and 3 mM with DMEM/F12 medium containing 0.5% FBS and then treated with primary astrocytes for 2 h. The concentration of bupivacaine was determined according to the research of Huang et al. 24 . Moreover, 7,8-DHF was diluted to 5 ng/mL, 10 ng/mL, and 20 ng/mL with DMEM/F12 medium containing 0.5% FBS and administered to primary astrocytes for 2 h. The concentration of 7,8-DHF was determined by CCK-8 assays for cell viability (see Supplementary Fig S6). Then, various experimental tests were carried out. Primary astrocytes were diluted to a density of 2 × 10 4 cells/ml, seeded in new plates and incubated until 70-80% confluent cells were reached. The viral vector containing shRNA was diluted to the corresponding concentration according to MOI = 50 and incubated with primary astrocytes for 48 h. Then, follow-up experiments Western blotting analysis. Primary cultured astrocytes were lysed in lysis buffer (KeyGEN Biotech, Nanjing, China) containing protease and phosphatase inhibitors for 30 min. After centrifugation, the supernatant was collected for protein quantification by the BCA protein assay (KeyGEN Biotech, Nanjing, China). The protein extracts with sodium dodecyl sulphate (SDS) sample buffer were heated in a 100 °C water bath for 10 min. The samples (40 μg sample -1 ) were loaded on 10% SDS-PAGE for electrophoresis, and then, the gel that contained the proteins was cut according to the protein molecular size. Next, the same proteins come from two pieces of gel were transferred onto a PVDF membrane. The membrane was blocked using 5% bovine serum albumin (BSA) in Tris-buffered saline with Tween (TBS-T; 10 mmol litre -1 Tris-HCl, 150 mmol litre-1 NaCl, 0.1% Tween; pH 8.0) for 2 h. Next, the membrane was probed overnight at 4 °C with primary antibodies including rabbit anti-GlyT1 (1:500; Proteintech Group, Inc.), rabbit anti-BDNF (1:500; Proteintech Group, Inc.), rabbit anti-AMPKα (1:500; Proteintech Group, Inc.), and rabbit anti-p-AMPKα (Thr172) (1:500; Affinity Biosciences, Inc.), followed by HRP-conjugated goat anti-rabbit IgG (1:3000; Proteintech Group, Inc.) for 2 h. Blots were visualized with an enhanced chemiluminescence (ECL) system (Pierce; Thermo Fisher Scientific, Inc.), and the protein bands were quantified by Quantity One software version 4.6.2. Normalization was performed with GAPDH expression.
Immunofluorescence staining and microscopy. For immunocytochemistry assays, primary astrocytes were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature. All the cells were blocked with 5% goat serum in 0.01 mM phosphate-buffered saline (PBS, pH 7.4) with 0.3% Triton-X-100 for 2 g h at room temperature and then incubated overnight at 4 °C with primary antibodies, including mouse anti-GlyT1 (1:25; Proteintech Group, Inc.), mouse anti-BDNF (1:50; Proteintech Group, Inc.), and rabbit anti-p-AMPKα (Thr172) (1:50; Affinity Biosciences, Inc.). Then, the cells were incubated with TRITC-conjugated goat anti-rabbit IgG or FITC-conjugated goat anti-mouse IgG (1:100; Proteintech Group, Inc.) for 2 h at room temperature. Finally, the cells were sealed with an antifluorescence attenuating tablet containing DAPI. Omission of the primary antibody served as a negative control. Images were captured by confocal laser scanning microscopy (FluoView FV 1000; Olympus, Tokyo, Japan). Image-Pro Plus 6.0 software was applied to quantify the fluorescence intensity of images, and GraphPad Prism 7.0 software was used to generate graphs.
Statistical analysis. Data were analysed using SPSS 22.0 statistical software. All data are expressed as the mean ± SEM. All experiments were repeated at least three times. One-way ANOVA was used to compare the means of multiple samples. Statistical significance of the results was defined at p < 0.05.
Ethics approval and consent to participate. The protocol was approved by the Ethics Committee of Ningxia Medical University (date from 2021.6.1 to 2023.5.31, protocol number 2020-834). All experimental processes complied with internationally accredited guidelines and ethical regulations.

Data availability
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.