P2X7 is involved in the anti-inflammation effects of levobupivacaine

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P2X7 is involved in the anti-inflammation effects of levobupivacaine Ya-Hsien Huang, MD,a,b Jiin-Cherng Yen, PhD,b Jie-Jen Lee, MD, PhD,c Jyh-Fei Liao, PhD,b Wen-Jinn Liaw, MD, PhD,d and Chun-Jen Huang, MD, PhDe,f,* a

Department of Anesthesiology, Mackay Memorial Hospital, Taipei, Taiwan Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan c Department of Surgery, Mackay Memorial Hospital, Taipei, Taiwan d Department of Anesthesiology, Tung’s Taichung MetroHarbor Hospital, Taichung, Taiwan e Department of Anesthesiology, Taipei Tzu Chi Hospital, Taipei, Taiwan f School of Medicine, Tzu Chi University, Hualien, Taiwan b

article info

abstract

Article history:

Background: We sough to elucidate whether purinergic P2X7 receptor is actively involved in

Received 3 April 2014

the effects of levobupivacaine on inhibiting microglia activation.

Received in revised form

Materials and methods: Microglia were treated with lipopolysaccharide (LPS, 50 ng/mL), LPS

30 June 2014

plus levobupivacaine (50 mM), or LPS plus levobupivacaine plus the P2X7 receptor agonist

Accepted 10 July 2014

Bz-ATP (100 mM) and denoted as the LPS, LPS þ Levo, and LPS þ Levo þ Bz-ATP group,

Available online 16 July 2014

respectively. Microglia activation was measured by assaying inflammatory molecules expression. Microglia activation was also measured by assaying neuronal cell viability

Keywords:

using coculture of microglia and neurons, as activated microglia may cause neuron injury.

Levobupivacaine

We also measured the levels of P2X7 receptor activation in microglia using ethidium up-

Microglia

take assay.

Purinergic P2X7 receptor

Results: Our data confirmed the effects of levobupivacaine on inhibiting inflammatory

Cell viability

molecules upregulation in activated microglia, as the concentrations of interleukin (IL)-1b, tumor necrosis factor a, IL-6, and macrophage inflammatory protein 2, of the LPS þ Levo group were significantly lower than those of the LPS group (all P < 0.05). Moreover, Bz-ATP significantly abrogated the inhibitory effects of levobupivacaine, as concentrations of IL-1b, tumor necrosis factor a, IL-6, and macrophage inflammatory protein 2 of the LPS þ Levo þ Bz-ATP group were significantly higher than those of the LPS þ Levo group (all P < 0.05). In contrast, neuronal cell viability of the LPS þ Levo group was significantly higher than those of the LPS and LPS þ Levo þ Bz-ATP groups (P ¼ 0.012 and 0.002). Moreover, levels of P2X7 receptor activation of the LPS and LPS þ Levo þ Bz-ATP groups were significantly higher than that of the LPS þ Levo group (P ¼ 0.003 and 0.006). Conclusions: P2X7 receptor is involved in the effects of levobupivacaine on inhibiting microglial activation. ª 2015 Elsevier Inc. All rights reserved.

* Corresponding author. Department of Anesthesiology, Taipei Tzu Chi Hospital, 289, Jianguo Rd., Sindian District, New Taipei City 231, Taiwan. Tel.: þ886 2 66289779x2639; fax: þ886 2 66289009. E-mail address: [email protected] (C.-J. Huang). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.07.020

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1.

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Introduction

Levobupivacaine is an amino amide group local anesthetic agent that possesses similar potency to its racemic parent bupivacaine [1]. Previous data indicated less cardiac and central nervous system (CNS) toxicity of levobupivacaine compared with bupivacaine [1]. Clinically, levobupivacaine has emerged as a safer alternative for bupivacaine [1]. Previous data also highlighted the potent anti-inflammation effects of levobupivacaine. For example, levobupivacaine could inhibit vascular flare response induced by bradykinin and substance P in human skin [2]. Rectal administration of levobupivacaine could mitigate colon lesion in rats with colitis [3]. Our recent data further confirmed that levobupivacaine could inhibit microglia activation induced by endotoxin [4]. However, the mechanism(s) underlying the effects of levobupivacaine on inhibiting microglia activation remains to be elucidated. Microglia are the resident immune cells of the CNS [5]. Activation of microglia constitutes a crucial CNS defense mechanism against invading pathogens [5]. However, microglia can in turn cause injury to neurons when activated in CNS infectious diseases [6e8]. The mechanisms involve the inflammation process triggered by activated microglia [6e8]. Previous data demonstrated that modulating the inflammation process triggered by activated microglia could provide beneficial effects against CNS infectious diseases [8,9]. Purinergic P2X7 receptor, a family member of the ionotropic ATPgated purinergic receptors, plays a crucial role in regulating proliferation and activation of microglia [10,11]. Previous data also highlighted the active involvement of P2X7 receptor in mediating endotoxin-induced microglia activation [12,13]. Judging from these data, we speculated that levobupivacaine may very likely act through inhibiting P2X7 receptor to exhibit its effects on inhibiting endotoxin-induced microglia activation. To elucidate further, we thus conducted this cellular study. Our hypothesis was that augmenting P2X7 receptor activation could abrogate the effects of levobupivacaine on inhibiting endotoxin-induced microglia activation.

2.

Materials and methods

2.1.

Cell culture and cell activation protocols

BV-2, an immortalized murine microglial cell line, can readily produce inflammatory molecules with endotoxin exposure [4]. This study thus used BV-2 cells to facilitate investigation. Using Dulbecco’s modified Eagle’s medium (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Life Technologies), BV-2 cells were grown and maintained in a humidified incubator (37 C) supplied with a gas mixture of 5% CO2/95% air. Then, confluent microglia (BV-2) were stimulated with gram (negative) endotoxin (lipopolysaccharide, LPS, 50 ng/mL, Escherichia coli Serotype 0127:B8; SigmaeAldrich, St. Louis, MO) to induce microglia activation. The dosage of LPS was determined according to our previous data that LPS at the dosage of 50 ng/mL

could readily induce microglia activation and upregulate inflammatory molecules expression in BV-2 cells [4].

2.2.

Experimental protocols

To elucidate the possible role of P2X7 receptor in mediating the anti-inflammatory effects of levobupivacaine, microglia were randomly allocated to receive LPS, LPS plus levobupivacaine (50 mM; Abbott Laboratories Ltd, Abbott Park, IL), or LPS plus levobupivacaine plus the potent P2X7 receptor agonist Bz-ATP (100 mM; SigmaeAldrich) and designated as the LPS, LPS þ Levo, or LPS þ Levo þ Bz-ATP group, respectively. Levobupivacaine and/or Bz-ATP were added immediately after LPS. The dosage of levobupivacaine was determined according to our previous data that levobupivacaine at the dosage of 50 mM could consistently inhibited upregulation of inflammatory molecules in endotoxin-activated microglia and, most of all, posted no significant effects on microglia cell viability in endotoxin-activated microglia [4]. The dosage of Bz-ATP was determined according to previous data that Bz-ATP at the dosage of 100 mM could significantly activate P2X7 receptor [14]. To serve as controls for the additives, another set of microglia were randomly allocated to receive phosphatebuffered saline (PBS; Life Technologies), PBS plus levobupivacaine (50 mM), or PBS plus levobupivacaine plus Bz-ATP (100 mM) and designated as the PBS, Levo, or Levo þ BzATP group, respectively. In addition, another set of microglia were randomly allocated to receive PBS, PBS plus Bz-ATP (100 mM), LPS (50 ng/mL), or LPS plus Bz-ATP (100 mM) (designated as the PBS, Bz-ATP, LPS, or LPS þ Bz-ATP group, respectively) to help confirm the effects of P2X7 receptor activation on modulating endotoxin-induced microglia activation.

2.3. Inflammatory molecules measurements using enzyme-linked immunosorbent assay We assayed the concentrations of inflammatory molecules to determine the levels of microglia activation, as microglia will readily produce inflammatory molecules, including interleukin (IL)-1b, tumor necrosis factor a (TNF-a), IL-6, and macrophage inflammatory protein 2 (MIP-2), on exposure to endotoxin [4]. For assaying inflammatory molecules, BV-2 cells were plated on six-well dishes (1e2  106 cells per well; Corning, Acton, MA). After reaction for 24 h, culture media from each group were harvested and then analyzed for the concentrations of IL-1b, TNF-a, IL-6, and MIP-2 using enzymelinked immunosorbent assay (enzyme-linked immunosorbent assay kits of IL-1b, TNF-a, IL-6, and MIP-2; R&D Systems, Minneapolis, MN).

2.4. Analysis of nuclear factor kB activation using immunoblotting assay We also assayed nuclear factor kB (NF-kB) expression to determine the levels of microglia activation, as expression of inflammatory molecules is tightly regulated by the upstream transcription factor NF-kB [15]. To assay NF-kB activation, BV-

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2 cells were also plated on six-well dishes (1e2  106 cells per well). After reaction for 120 min, cells were harvested. The time point for cell harvesting was determined according to our previous data that the effects of levobupivacaine on inhibiting endotoxin-induced NF-kB activation were most prominent at 120 min after reaction [4]. NF-kB activation was analyzed using immunoblotting assay, as we have previously reported [16]. After cell harvesting, nuclear and cytosolic extracts of the BV-2 cells were prepared and then separated by gel electrophoresis. After separation, the proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). For nuclear extracts, the nitrocellulose membranes were incubated overnight at 4 C in primary antibody solution of phosphorylated NF-kB (p-NF-kB p65 [Ser536], 1:500 dilution; Cell Signaling Technology, Inc, Danvers, MA) or Histone H3 (internal standard, 1:500 dilution; Cell Signaling) to facilitate assaying NF-kB nuclear translocation. For cytosolic extracts, the membranes were incubated overnight (4 C) in primary antibody solution of phosphorylated inhibitor-kB (p-I-kBa [Ser32], 1:1000 dilution; Cell Signaling) or actin (internal standard; 1:5000 dilution; Millipore Corporation; Burlington, MA) to facilitate assaying IkB phosphorylation. Horseradish peroxidaseeconjugated anti-mouse IgG antibody (Amersham Pharmacia Biotec, Inc, Piscataway, NJ) was used as the secondary antibody. Bound antibody was detected by chemiluminescence (ECL plus kit; Amersham) and chemiluminescence film (Hyperfilm; Amersham). The protein band densities were quantified using densitometric technology (Scion Image for Windows; Scion Corp, Frederic, MD).

2.5.

Neuronal cell viability assay using MTT assay

Microglia activation will cause neuron injury [6e8]. This study thus used coculture of microglia (BV-2) and neurons (B35, an immortalized murine neuronal cell line) and a widely used MTT [3-(4,5-di methyl thiazol-2-yl)-2,5diphenyltetrazolium bromide] assay to facilitate neuronal cell viability measurement [17]. We also used a permeable transwell system (24-well plates, Transwell Permeable Supports; Corning), which allowed microglia-neuronal cells coculture without cellecell contact. Microglia (BV-2) were initially plated in the upper panel of each well of the 24-well plates (Transwell Permeable Supports) (approximate 1.5  104 cells per well) and treated as previously mentioned. After reaction for 24 h, the culture media were removed, and the upper panel of each well was relocated and mounted on a separate 24-well plate permeable transwell system with confluent neuronal cells (B35) cultured on the bottom of each well (approximate 3.75  104 cells per well). Then, aliquots of 500 mL of culture media were added in each well. After coculture for 24 h, the upper panel (BV-2) of each well was removed and aliquots of 50 mL of MTT solution (5 mg/ mL; SigmaeAldrich) were added to each well (B35). After incubation for 1 h (37 C), the culture media were removed and aliquots of 200 mL dimethylsulfoxide (SigmaeAldrich) were added to each well. After reaction for another 10 min on a shaker, the absorbance of each well at a wavelength of 570 nm was measured to assay neuronal cells viability.

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2.6. Assay of P2X7 receptor activation in microglia using ethidium bromide uptake assay Activation of P2X7 receptor will significantly increase plasma membrane permeability to organic solutes [18]. This study thus used ethidium bromide uptake assay to facilitate evaluation of P2X7 receptor activation in microglia [18]. In brief, BV-2 cells were plated in 96-well plates (Corning) and treated as previously mentioned. After reaction for 1 h, culture media were removed and aliquots of 100 mL dye uptake buffer (i.e., divalent-free NaCl bathing solution containing 150 mM Naþ, 5 mM Kþ, and 10 mM HEPES, adjusted to pH 7.4) were added to each well. Then, aliquots of 100 mM ethidium bromide dye (SigmaeAldrich) were added to each well. The plates were then placed in a microplate reader (Infinite 200 PRO series; Tecan Group Ltd, Switzerland) and excited with a wavelength of 310 nm and emission was collected at a wavelength of 580 nm. A series of preliminary studies performed in our laboratory revealed that changes in the levels of ethidium uptake in endotoxin-activated microglia stabilized at 20e30 min after reaction. We thus decided to collect data at time points of 0, 1, 5, 10, 20, and 30 min after reaction.

2.7.

Statistical analysis

Data of inflammatory molecules, NF-kB, and cell viability were analyzed using one-way analysis of variance (ANOVA) with Tukey post hoc test to determine the between-group difference. Data of ethidium uptake were analyzed with repeatedmeasures ANOVA with Bonferroni corrections to examine the within-group (time) effect, between-group effect, and the group by time interaction effect. Data were presented as mean  standard deviation. The significance level was set at 0.05. A commercial software package (SPSS 11.5 for Windows, SPSS Science, Chicago, IL) was used for data analysis.

3.

Results

3.1. P2X7 receptor activation augmented inflammatory molecules upregulation in activated microglia PBS or Bz-ATP alone posted no significant effects on inflammatory molecules expression, as the concentrations of IL-1b, TNF-a, IL-6, and MIP-2 of the PBS and Bz-ATP group were low (data not shown). As expected, LPS significantly upregulated inflammatory molecules expression in microglia, as the concentrations of the IL-1b, TNF-a, IL-6, and MIP-2 of the LPS group were significantly higher than those of the PBS group (all P < 0.001). Moreover, these effects of endotoxin could be significantly augmented by Bz-ATP, as the concentrations of IL-1b, IL-6, and MIP-2 of the LPS þ Bz-ATP group were significantly higher than those of the LPS group (all P < 0.001, Fig. 1A, C, and D). In addition, ethidium uptake of the LPS þ Bz-ATP group measured at 5, 10, 20, and 30 min after reaction was significantly higher than that of the LPS group (all P < 0.001, Fig. 1E), indicating that Bz-ATP could also augment P2X7 receptor activation induced by endotoxin.

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Fig. 1 e Relative concentrations of inflammatory molecules, including (A) IL-1b, (B) TNF-a, (C) IL-6, and (D) MIP-2. Concentrations of inflammatory molecules were measured using enzyme-linked immunosorbent assay. (E) Activity of P2X7 receptor measured at 0, 1, 5, 10, 20, and 30 min after reaction using ethidium uptake assay. LPS: the lipopolysaccharide (50 ng/mL) group. LPS D Bz-ATP: the LPS plus the potent P2X7 receptor agonist Bz-ATP (100 mM) group. Data were mean ± standard deviations. For inflammatory molecules, the LPS group was used as the control. *P < 0.05 the LPS D BzATP group versus the LPS group.

3.2. P2X7 receptor activation abrogated the effects of levobupivacaine on inhibiting inflammatory molecules upregulation in activated microglia Levobupivacaine or levobupivacaine plus Bz-ATP posted no significant effects on inflammatory molecule expression, as the concentrations of IL-1b, TNF-a, IL-6, and MIP-2 of the Levo

and Levo þ Bz-ATP groups were low (data not shown). In contrast, levobupivacaine significantly inhibited the upregulation of inflammatory molecules induced by endotoxin, as the concentrations of the IL-1b, TNF-a, IL-6, and MIP-2 of the LPS þ Levo group were significantly lower than those of the LPS group (P ¼ 0.043, P < 0.001, P ¼ 0.044, and P ¼ 0.002, respectively; Fig. 2AeD). Moreover, these effects of

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Fig. 2 e Relative concentrations of inflammatory molecules. The concentrations of inflammatory molecules, including (A) IL1b, (B) TNF-a, (C) IL-6, and (D) MIP-2, in endotoxin-activated microglia were measured using enzyme-linked immunosorbent assay. LPS: the lipopolysaccharide (50 ng/mL) group. LPS D Levo: the LPS plus levobupivacaine (50 mM) group. LPS D Levo D Bz-ATP: the LPS plus levobupivacaine (50 mM) plus the potent P2X7 receptor agonist Bz-ATP (100 mM) group. Data were mean ± standard deviations. The LPS group was used as the control. *P < 0.05 versus the LPS group. #P < 0.05 the LPS D Levo D Bz-ATP group versus the LPS D Levo group.

levobupivacaine could be abrogated by Bz-ATP, as the concentrations of IL-1b, TNF-a, IL-6, and MIP-2 of the LPS þ Levo þ Bz-ATP group were significantly higher than those of the LPS þ Levo group (P < 0.001, P ¼ 0.010, P < 0.001,

and P < 0.001, respectively; Fig. 2AeD). In addition, the concentration of IL-6 (but not IL-1b, TNF-a, and MIP-2) of the LPS þ Levo þ Bz-ATP group was significantly higher than that of the LPS group (P ¼ 0.005; Fig. 2C).

Fig. 3 e Representative gel photography and relative densitometric data of phosphorylated NF-kB p65 (p-NF-kB p65) protein concentrations in the nuclear extracts and phosphorylated inhibitor-kBa (p-I-kBa) protein concentrations in the cytosolic extracts of endotoxin-activated microglia using immunoblotting assay. The p-NF-kB p65 protein concentrations were normalized by Histone H3. The p-I-kBa protein concentrations were normalized by actin. LPS: the lipopolysaccharide (50 ng/ mL) group. LPS D Levo: the LPS plus levobupivacaine (50 mM) group. LPS D Levo D Bz-ATP: the LPS plus levobupivacaine (50 mM) plus the potent P2X7 receptor agonist Bz-ATP (100 mM) group. Data were mean ± standard deviation. The LPS group was used as the control. *P < 0.05 versus the LPS group. #P < 0.05 the LPS D Levo D Bz-ATP group versus the LPS D Levo group.

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Levo and Levo þ Bz-ATP groups (data not shown). In contrast, endotoxin-activated microglia caused significant injury to neuronal cells, as neuronal cells viability of the LPS group was significantly lower than that of the PBS group (P < 0.001, Fig. 4). These effects of endotoxin was mitigated by levobupivacaine, as neuronal cells viability of the LPS þ Levo group was significantly higher than that of the LPS group (P ¼ 0.012, Fig. 4). In addition, the protective effects of levobupivacaine were abrogated by Bz-ATP, as neuronal cells viability of the LPS þ Levo þ Bz-ATP group was significantly lower than that of the LPS þ Levo group (P ¼ 0.002, Fig. 4). Moreover, neuronal cells viabilities of the LPS þ Levo þ Bz-ATP and LPS groups were comparable (Fig. 4).

3.5. Levobupivacaine significantly inhibited P2X7 receptor activation in activated microglia Fig. 4 e Relative neuronal cells viability. Neuronal cells (B35) were cocultured with microglia using a permeable transwell system. Neuronal cells viability was measured using MTT (3-(4,5-di methyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. PBS: the phosphate-buffered saline group. LPS: the lipopolysaccharide (50 ng/mL) group. LPS D Levo: the LPS plus levobupivacaine (50 mM) group. LPS D Levo D Bz-ATP: the LPS plus levobupivacaine (50 mM) plus the potent P2X7 receptor agonist Bz-ATP (100 mM) group. Data were mean ± standard deviations. The PBS group was used as the control. *P < 0.05 versus the PBS group. #P < 0.05 versus the LPS group. yP < 0.05 the LPS D Levo D Bz-ATP group versus the LPS D Levo group.

3.3. P2X7 receptor activation abrogated the effects of levobupivacaine on inhibiting NF-kB activation in activated microglia PBS, Levobupivacaine, or levobupivacaine plus Bz-ATP posted no significant effects on NF-kB activation, as the concentrations of p-NF-kB p65 in nuclear extracts and p-IkBa in cytosolic extracts of the PBS, Levo, and Levo þ Bz-ATP groups were low (data not shown). Endotoxin significantly induced NF-kB activation, as the concentrations of p-NF-kB p65 and p-IkBa of the LPS group were significantly higher than those of the PBS group (both P < 0.001). These effects of endotoxin were significantly mitigated by levobupivacaine, as the concentrations of p-NF-kB p65 and p-IkBa of the LPS þ Levo group were significantly lower than those of the LPS groups (P < 0.001 and P ¼ 0.011, Fig. 3). Bz-ATP could abrogate these effects of levobupivacaine, as the concentrations of p-NF-kB p65 and p-IkBa of the LPS þ Levo þ Bz-ATP group were significantly higher than those of the LPS þ Levo group (P < 0.001 and P ¼ 0.038, Fig. 3). Moreover, the concentrations of p-NF-kB p65 and p-IkBa of the LPS þ Levo þ Bz-ATP and LPS group were comparable (Fig. 3).

3.4. P2X7 receptor activation abrogated the effects of levobupivacaine on protecting neuronal cells against injury caused by activated microglia Neuronal cells viability of the PBS group was high (Fig. 4), indicating that PBS-treated microglia posted no significant injury to neuronal cells. Similar pictures were observed in the

Repeated-measures ANOVA revealed that the trend of ethidium uptake was significantly different among the LPS, LPS þ Levo, and LPS þ Levo þ Bz-ATP groups (P < 0.001).The levels of ethidium uptake in microglia of the LPS group reached plateau at around 5e10 min after reaction (Fig. 5). Similar pattern was observed in the LPS þ Levo and LPS þ Levo þ Bz-AP groups (Fig. 5). However, the levels of ethidium uptake measured at 5, 10, 20, and 30 min after reaction of the LPS þ Levo group were significantly lower than those of the LPS groups (P ¼ 0.001, P < 0.001, P ¼ 0.003, and P ¼ 0.005, respectively, Fig. 5), indicating that levobupivacaine significantly suppressed the effects of LPS on activating P2X7 receptor. Moreover, the levels of ethidium uptake of the LPS þ Levo þ Bz-ATP group measured at 5, 10, 20, and 30 min after reaction were significantly higher than that of the LPS þ Levo group (P ¼ 0.008, P ¼ 0.003, P ¼ 0.006, and P ¼ 0.004, respectively, Fig. 5), indicating that these effects of levobupivacaine could be abrogated by Bz-ATP. In contrast, the levels of ethidium uptake of the LPS þ Levo þ Bz-ATP and LPS groups were comparable (Fig. 5).

4.

Discussion

Data from this study, in concert with those previous ones [4,9,12,13], confirmed that endotoxin can readily induce microglia activation and cause robust production of inflammatory molecules, including cytokine (e.g., IL-1b, TNF-a, and IL-6) and chemokines (e.g., MIP-2). Moreover, the robust production of inflammatory molecules can subsequently cause neuron injury [6e8]. This concept is also confirmed by this study, as our data revealed that the viability in neuronal cells cocultured with endotoxin-treated microglia was significantly lower than that in neuronal cells cocultured with PBS-treated microglia. Together, these data demonstrated that endotoxininduced microglia activation is a sensitive cell model and can be used to facilitate investigation in areas related to CNS infection. Data from this study further demonstrated that levobupivacaine could inhibit microglia activation, as our data revealed that levobupivacaine could mitigate the upregulation of inflammatory molecules in endotoxin-activated microglia. These data were consistent with our previous data [4].

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Fig. 5 e Activity of purinergic P2X7 receptor in endotoxinactivated microglia. Activity of P2X7 receptor was measured at 0, 1, 5, 10, 20, and 30 min after reaction using ethidium uptake assay. LPS: the lipopolysaccharide (50 ng/ mL) group. LPS D Levo: the LPS plus levobupivacaine (50 mM) group. LPS D Levo D Bz-ATP: the LPS plus levobupivacaine (50 mM) plus the potent P2X7 receptor agonist Bz-ATP (100 mM) group. Data were mean ± standard deviation. *P < 0.05 versus the LPS group. #P < 0.05 the LPS D Levo D Bz-ATP group versus the LPS D Levo group.

Moreover, data from this study demonstrated that levobupivacaine could alleviate neuron injury induced by activated microglia. These data, in concert with those previous ones [2e4], provide clear evidence to confirmed the potent antiinflammation effects of levobupivacaine. Previous data revealed that inhibition of microglia could protect neuronal tissues in inflamed and diseased brain [12]. In line with this notion, we speculate that incorporation of levobupivacaine as part of the therapies may be a beneficial therapeutic strategy against CNS infection. As levobupivacaine is regularly used in clinical settings, data from this study thus should have profound clinical relevance. As aforementioned, purinergic P2X7 receptor plays a crucial role in regulating the activation of microglia [10e13]. This concept is confirmed by the present study, as our data revealed that P2X7 receptor activation induced by Bz-ATP could augment the effects of endotoxin on activating microglia. Judging from these data, we thus hypothesized that P2X7 receptor may be involved in mediating the effects of levobupivacaine on inhibiting microglia activation. Data from this study revealed that endotoxin could induce P2X7 receptor activation in microglia and this P2X7 receptor activation could be inhibited by levobupivacaine. Furthermore, our data revealed that the potent P2X7 receptor agonist Bz-ATP could abrogate the effects of levobupivacaine and subsequently resume the activity of P2X7 receptor in endotoxin-activated microglia. Our data further demonstrated that Bz-ATP could also abrogate the effects of levobupivacaine on mitigating upregulation of inflammatory molecules in endotoxinactivated microglia. Resuming the activity of P2X7 receptor induced by Bz-ATP could also abrogate the protective effects

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of levobupivacaine on alleviating neuron injury induced by activated microglia. Together, these data provide clear evidence to confirm our hypothesis. These data also highlight the active involvement of P2X7 receptor in the effects of levobupivacaine on inhibiting microglia activation induced by endotoxin. Judging from these data, we further speculate that levobupivacaine may very likely act through inhibiting P2X7 receptor activation to exert its protective effects. P2X7 receptor is a family member of the ionotropic ATP-gated purinergic receptors that its activation could significantly increase intracellular Ca2þ concentrations via facilitating extracellular Ca2þ influx and mobilizing endoplasmic reticulum Ca2þ pool, which in turn could eventually induce neurotoxicity and cell death [10,14,19]. Previous data further revealed that the increase in intracellular Ca2þ concentrations plays an essential role in mediating the detrimental effects of P2X7 receptor activation, as inhibiting extracellular Ca2þ influx could mitigate cell death induced by P2X7 receptor activation [19]. Previous data also revealed that the transient increase in intracellular Ca2þ could significantly activate the crucial transcription factor NF-kB and subsequently upregulate the expression of inflammatory molecules [20]. Our previous data have confirmed that levobupivacaine could inhibit NF-kB activation in endotoxin-activated microglia [4]. Data from this study further confirmed that the effects of levobupivacaine on inhibiting endotoxin-induced NF-kB activation could be abrogated by Bz-ATP. Together, these data support the concept that the effects of levobupivacaine on inhibiting NF-kB activation may be related to its effects on inhibiting P2X7 receptor activation and the subsequent increase in intracellular Ca2þ concentrations. This concept is also supported by previous data that P2X7 receptor positively regulates NF-kB activation [21]. This study does have certain limitations. First, our data revealed that levobupivacaine could exert significant antiinflammation effects at subclinical dosage (i.e., 50 mM), as clinical dosages of levobupivacaine for regional anesthesia are approximately 2e15 mM (or 0.0625%e0.5%) [22,23]. Of note, previous data indicated that local anesthetic agents may exert toxicity to Schwann cells in a dose-dependent manner [24]. Previous data further indicated that bupivacaine could cause Schwann cell injury even at subclinical dosages (i.e., 500e1000 mM or 0.015%e0.03%) [24]. The question of whether levobupivacaine can also cause Schwann cell injury at clinical or subclinical dosages remains unstudied. However, judging from our data that the dosage of levobupivacaine could exert significant anti-inflammation effects is far below its clinical dosages, we believe that the toxicity of levobupivacaine at such a slow dosage, if any, should be insignificant. Second, data from this study confirmed the involvement of P2X7 receptor in mediating the anti-inflammation effects of levobupivacaine. However, the possible roles of the other family members of the purinergic receptors, e.g., P2X4 receptor, in this regard remain unstudied. Third, levobupivacaine is a potent blocker of the voltage-gated Naþ channels and the Ltype Ca2þ channel [25,26]. Previous data indicated that inhibition of the voltage-gated Naþ channels and the L-type Ca2þ channel could exert certain anti-inflammation effects [27,28]. Judging from these data, we speculate that the voltage-gated Naþ channels and/or the L-type Ca2þ channel may also

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involve in mediating the anti-inflammation effects of levobupivacaine. More studies are needed before further conclusions can be drawn.

Acknowledgment The authors would like to express our appreciations to Professors Yen-Jen Sung and Huey-Jen Tsay from National YangMing University, Taipei, Taiwan, and Professor Yuh-Chiang Shen from National Research Institute of Chinese Medicine, Taiwan for their generosity in supplying BV-2 cells for this study. Authors’ contributions: Y.H.H. contributed to the conception and design, analysis and interpretation, data collection, and writing the article. J.C.Y., J.J.L., J.F.L., and W.J.L. contributed to the data collection, analysis and interpretation, and writing the article. C.J.H. contributed to the conception and design, analysis and interpretation, data collection, writing the article, critical revision of the article, and obtaining funding. This work was supported by a grant from Taipei Tzu Chi Hospital (TCRD-TPE-101-13) awarded to C.J.H.

Disclosure The authors reported no proprietary or commercial interest in any product mentioned or concept discussed in this article.

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