Accepted Manuscript Lidocaine Attenuates LPS-Induced Inflammatory Responses in Microglia Tong Yuan , M.D. Zhiwen Li , M.D. Xinbai Li , M.D. Gaoqi Yu , M.D. Na Wang , M.D. Xige Yang , M.D. PII:

S0022-4804(14)00463-6

DOI:

10.1016/j.jss.2014.05.023

Reference:

YJSRE 12728

To appear in:

Journal of Surgical Research

Received Date: 15 December 2013 Revised Date:

23 April 2014

Accepted Date: 8 May 2014

Please cite this article as: Yuan T, Li Z, Li X, Yu G, Wang N, Yang X, Lidocaine Attenuates LPSInduced Inflammatory Responses in Microglia, Journal of Surgical Research (2014), doi: 10.1016/ j.jss.2014.05.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Revised Apr 23, 2014 Lidocaine Attenuates LPS-Induced Inflammatory Responses in

RI PT

Microglia Tong Yuan, M.D., Zhiwen Li, M.D., Xinbai Li, M.D., Gaoqi Yu, M.D., Na Wang, M.D., and Xige Yang*, M.D.

Running title: Lidocaine inhibits inflammatory response.

M AN U

Category: Trauma

SC

Department of Anesthesiology, First Hospital of Jilin University, Changchun, China

Author contributions

Xige Yang: conception, design, analysis and interpretation of data; writing the manuscript. Tong Yuan: conception, design, analysis and interpretation of data; data collection; critical revision of the manuscript; statistical expertise; obtaining funding; supervision.

TE D

Zhiwen Li: data collection; critical revision of the article. Gaoqi Yu: data collection; critical revision of the article.

EP

Xinbai Li: conception and design; critical revision of the article. Na Wang: conception and design; critical revision of the article.

AC C

*Corresponding Author: Xige Yang

Department of Anesthesiology, First Hospital of Jilin University No.71 Xinmin Street, Changchun, Jilin 130021, China Tel.: +86 431 84327413; Fax: +86 431 84327413 Email: [email protected]

1

ACCEPTED MANUSCRIPT

Abstract Background. Lidocaine has been used as a local anesthetic with anti-inflammatory properties, but its effects on neuroinflammation have not been well defined. In the present

RI PT

study, we investigated the prophylactic effects of lidocaine on lipopolysaccharide (LPS)-activated microglia and explored the underlying mechanisms.

Materials and methods. Microglial cells were incubated with or without 1 µg/mL LPS in

SC

the presence or absence of lidocaine, a p38 MAPK inhibitor (SB203580), an NF-κB inhibitor (PDTC), or small interfering RNA(si RNA). The protein and expression levels of

M AN U

inflammatory mediators, such as monocyte chemotactic protein-1 (MCP-1),nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) were measured using enzyme-linked immunosorbent assays and real-time polymerase chain reaction. The effect of lidocaine on NF-κB and p38 MAPK activation

TE D

was evaluated using enzyme-linked immunosorbent assays, Western Blot Analysis, and Electrophoretic Mobility Shift Assay.

Results. Lidocaine (≥ 2 µg/mL) significantly inhibited the release and expression of NO,

EP

MCP-1, PGE2, IL-1β, and TNF-α in LPS-activated microglia. Treatment with lidocaine also significantly inhibited the phosphorylation of p38 MAPK and the nuclear

AC C

translocation of NF-κB p50/p65, increased the protein levels of IκB-α. Furthermore, our study shows that the LPS-induced release of inflammatory mediators was suppressed by SB203580, PDTC and si RNA. Conclusions. Prophylactic treatment with lidocaine inhibits LPS-induced release of inflammatory mediators from microglia, and these effects may be mediated by blockade of p38 MAPK and NF-κB signaling pathways.

2

ACCEPTED MANUSCRIPT

Key Words: lidocaine, inflammation, microglia, lipopolysaccharides, NF-kappa B, p38

AC C

EP

TE D

M AN U

SC

RI PT

MAPK.

3

ACCEPTED MANUSCRIPT

Introduction Microglial cells are activated during neuropathological conditions (infection,

RI PT

inflammation, or injury) to restore CNS homeostasis [1], and participate in host defense and inflammation in the brain [2]. Once activated, microglia can promote neuronal injury through the release of inflammatory mediators, including nitric oxide (NO), monocyte chemotactic protein-1 (MCP-1), prostaglandin E2 (PGE2), interleukin-1β (IL-1β), and

SC

tumor necrosis factor-α (TNF-α) [3-5]. Studies have demonstrated that inhibiting the

M AN U

release of inflammatory mediators from microglia can attenuate the severity of Alzheimer’s disease, Parkinson’s disease, atherosclerosis, and multiple sclerosis [6, 7]. Thus, anti-inflammatory treatment via inhibition of microglial activation offers a potentially

effective

therapeutic

to

mitigate

the

progression

of

TE D

neuroinflammatory diseases.

approach

In vitro studies have shown that lidocaine, a common local anesthetic drug, has significant anti-inflammatory properties on various cell types, including monocytes,

EP

macrophages, and neutrophils [8-10]. Lidocaine has also been shown to modulate inflammatory cascades and provide protection from ischemic reperfusion injury [8, 11,

AC C

12] and septic peritonitis [13] in in vivo studies. Jeong et al. [14] reported that lidocaine significantly inhibited some of the inflammatory responses in microglia stimulated by LPS, although the detailed underlying mechanism has not yet been completely resolved. Recently, it was reported that the protective effect of lidocaine was associated with the inhibiton of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation and the downstream activation of nuclear factor-kappa B (NF-κB) signaling [15-18].

4

ACCEPTED MANUSCRIPT

Therefore, the anti-inflammatory effects of lidocaine treatment may be due to inhibition of these signaling pathways. In this study, we aimed to elucidate the mechanisms underlying these neuroprotective and anti-inflammatory actions of lidocaine by prophylactic treatment

RI PT

of LPS-activated primary microglial cultures.

Materials and methods

SC

Reagents

Lidocaine, SB203580 (p38 MAPK inhibitor), PDTC (NF-κB inhibitor), and LPS

M AN U

(Escherichia coli 0111:B4) were purchased from Sigma Aldrich (St. Louis, MO). Small interfering RNA (siRNA) for NF-κB p65, IκB-α, or p38MAPK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

TE D

Cell Culture and Treatment

Sprague-Dawley (SD) rats were obtained from the Experimental Animal Center of Central South University (Changsha, Hunan, China). Experiments were carried out

EP

according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and were approved by the Bioethics Committee of Central South University.

AC C

Primary microglia were isolated from SD rats as previously described [19], and cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Grand Island, NE) supplemented with 10% fetal calf serum (Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco) and maintained in a 5% CO2 incubator at 37°C. After overnight incubation, the cells were washed 3 times and then transferred to 6-well polystyrene culture plates at 1 × 105 cells/mL (2 mL per well). Microglial cells were treated with lidocaine (0.2, 2, and

5

ACCEPTED MANUSCRIPT

20µg/mL) for 1 h and then stimulated with LPS (1 µg/mL) for 24 h, with a control group receiving 20µg/mL lidocaine only. In a second set of experiments, microglial cells were pre-incubated with 10 µM SB203580 or 100 µM PDTC for 1 h, followed by LPS treatment

RI PT

for 24 h. The dosages of lidocaine, SB203580, and PDTC were determined according to previous studies [20, 21].

SC

Cell Counting Kit-8 Assay (CCK-8)

Cell viability was determined using the Cell Counting Kit-8 Assay Kit (Beyotime,

M AN U

Jiangsu, China) [22]. Breifly, microglial cells were plated in 96-well plates at a density of 1 × 104 cells/well in 100 µL DMEM and pre-incubated with various concentrations of lidocaine for 1 h, followed by LPS (1 µg/mL) treatment for 24 h. Following treatment, 20 µL of CCK-8 reagent was added to each well and incubated for 2 h at 37°C. The

TE D

absorbance of the solution was measured using a microplate reader (Bio-Rad Laboratories,

NO Assay

EP

Hercules, CA) at a test wavelength of 450 nm and a reference wavelength of 630 nm.

AC C

NO concentrations in culture supernatants were determined using Griess reagent (Sigma Aldrich, St. Louis, MO). Briefly, microglial cells were pre-incubated with lidocaine (0.2, 2, and 20µg/mL) for 1 h, followed by LPS (1 µg/mL) treatment for 24 h. The supernatants were collected and mixed with an equal volume of Griess reagent. Samples were incubated at room temperature for 10 min, and absorbance was subsequently read at 540 nm using a microplate reader.

6

ACCEPTED MANUSCRIPT

MCP-1, PGE2 and Cytokine Assays Microglial cells were seeded in 6-well plates at a density of 4 × 105 cells/mL and pre-incubated with lidocaine (0.2, 2, and 20µg/mL) for 1 h, followed by LPS (1 µg/mL)

RI PT

treatment for 24 h. MCP-1,PGE2,TNF-α and IL-1β concentrations from 0.1 mL samples of culture supernatants were determined using an enzyme-linked immunosorbent assay (ELISA) (R & D Systems, Minneapolis, MN) following the manufacturer’s instructions

SC

and absorbance readings at 450 nm were determined on a microplate reader.

M AN U

Quantitative real-time polymerase chain reaction (qPCR) analysis

Total RNA was extracted from microglia using Trizol (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s instructions, and reverse-transcribed with MMLV reverse transcriptase (Promega, Madison, WI). Quantitative real-time polymerase

TE D

chain reaction (qPCR) was performed using a LightCycler 2.0 Real-Time PCR System (Roche Applied Science, Indianapolis, IN). cDNA was amplified using specific primers for inflammatory mediators gene expression and the results were normalized to β-actin

EP

gene expression. The relative mean fold-change of inflammatory mediators gene expression in the experimental group was calculated using the 2△△Ct method and compared

AC C

to the control group[23].

Protein Extraction

For making whole cell lysates, the cells were lysed in radioimmune precipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail (Roche). Nuclear and cytoplasmic fractionations were performed with NE-PER Cytoplasmic and Nuclear

7

ACCEPTED MANUSCRIPT

Protein Extraction Kit (Pierce Protein Research Products, Thermo Fisher Scientific, Rockford, IL) according to manufacturer’s protocol.

RI PT

NF-κB (p50/p65) Assay

Microglial cells were pre-treated with lidocaine (0.2, 2, and 20µg/mL) for 1 h and then stimulated with LPS (1 µg/mL) for 30 min. At the end of the stimulation period, cells

SC

were harvested and washed three times with cold phosphate-buffered saline (PBS) [22], followed by protein extraction using the NE-PER extraction reagent. The DNA binding

M AN U

activity of NF-κB (p50/p65) in the nuclear fraction was determined using an ELISA-based nonradioactive NF-κB p50/p65 transcription factor assay kit (Chemicon, Temecula, CA), according to the manufacturer’s instructions, and absorbances at 450 nm were measured.

TE D

IߢB-α and Phospho-IߢB-α Assay

Microglial cells were pre-treated with lidocaine (0.2, 2, and 20 µg/mL) for 1 h and then stimulated with LPS (1 µg/mL) for 30 min [24]. At the end of the stimulation period,

EP

cells were harvested and washed three times with cold PBS, followed by protein extraction

AC C

using the NE-PER extraction reagent. The protein extraction were subjected to IߢBߙ and phospho-IߢBߙ ELISA according to the manufacturer,s instructions (Cell signalling Technology, Inc) , and absorbances at 450 nm were measured.

MAPK Assays Microglial cells were pretreated with lidocaine (0.2, 2, and 20 µg/mL) for 1 h and then stimulated with 1 µg/mL LPS for 30 min [25-27]. At the end of the stimulation

8

ACCEPTED MANUSCRIPT

period, cells were washed with PBS and lysed using NE-PER extraction reagent (Pierce Protein Research Products, Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s protocol. Cell lysates were subjected to ELISA for phospho-p38 according

RI PT

to the manufacturer’s instructions (eBioscience, Beverly, MA) and absorbances at 450 nm were measured.

SC

siRNA Transfection Assay

Microglia cells (1×105 cells/ml) were plated in a 6-well plate, incubated for 48 h, and

or the nonsense control siRNA

M AN U

transfected with NF-κB p65-specific siRNA, IκB-α-specific siRNA, p38 MAPK-specific using the Lipofectamine 2000 reagent (Invitrogen,

Carlsbad, CA) according to the manufacturer’s instructions. The transfected cells were pre-incubated with lidocaine (20µg/mL) for 1 h, followed by LPS (1 µg/mL) treatment or

TE D

medium for 24 h.

Immunofluorescence Confocal Microscopy Assay

EP

For the detection of intracellular location of NF-κB p65, microglia cells were cultured on sterile glass cover slips in 24 well plates and treated with lidocaine and LPS as described

AC C

above. At 60min after the LPS treatment, cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS. After rinsing, cells were blocked with 3% BSA in PBS for 1 h and incubated with rabbit anti-NF-κB p65 antibodies. (1:200, Santa Cruz Biotechnology, Santa Cruz) overnight at 4°C. After washing, cells were incubated with FITC-conjugated goat anti-rabbit IgG (1:400, Pierce, Rockford, IL) for 1 h and counterstained with 4, 6-diamidino-2-phenylindole (DAPI, Roche, Shanghai, China)

9

ACCEPTED MANUSCRIPT

for the identification of nuclei. After washing with PBS, the cover slips were mounted with antifade mounting medium (Beyotime, China) on slides, and the cells were observed with a

RI PT

confocal microscope Olympus Fluoview FV500.

Western Blot Analysis

Equal amounts of cytoplasmic, nuclear, or whole cell extracts were electrophoresed

SC

on sodium dodecyl sulfate-polyacrylamide gels, and then transferred onto a polyvinylidene difluoride membrane (Millipore). The transformed membrane was blocked for 1 h and

M AN U

incubated with indicated primary antibodies (Santa Cruz Biotechnology) at 4°C overnight. The primary antibodies used were as follows: rabbit anti-β-actin (1:1000), p65 (1:1000), Lamin B (1:1000), p38 (1:1000) and mouse anti-phosphorylated p38 antibody (1:1000). The membrane was washed three times with Tris-buffered saline containing 0.05% Tween

TE D

20 (TBST) for 10 min and incubated with anti-rabbit or anti-mouse IgG-horseradish peroxidase (1:5000, Pierce) at room temperature for 1 h. The Supersignal West Pico chemiluminescent substrate system (Pierce) was used to detect immunoreactive bands. The

EP

intensity of protein bands after western blotting were quantitated by using Quantity One

AC C

Version 4.6.3 Image software (Bio-Rad) and normalized against proper loading controls.

Electrophoretic Mobility Shift Assay (EMSA) Nuclear extracts were prepared as described above. Oligonucleotides corresponding to the NF-κB (5’-AGTTGAGGGGACTTTCCCAGGC-3’) binding site consensus sequences were synthesized and end-labeled with biotin by Invitrogen. EMSAs were performed using the LightShift chemiluminescent EMSA kit (Pierce). Briefly, 20 fmol of

10

ACCEPTED MANUSCRIPT

biotin-labeled, double strand probe was incubated for 20 min at room temperature in 20 µl of EMSA binding buffer containing 2.5% glycerol, 5 mM MgCl2, 50 ng/µl poly (dI-dC), 0.05% Nonidet P-40, and 6 µg of nuclear proteins. For competition EMSA, 200-fold (4

RI PT

pmol) excess unlabeled, double strand probe was added to the binding reaction. The DNA-nuclear protein complexes were resolved by electrophoresis in 6% nondenaturing polyacrylamide gel in 0.5 × Tris-borate-EDTA (TBE) buffer at 100 V. Gels were then

SC

electroblottedonto Hybond nylon membranes (GE Healthcare) at 380 mA for 50 min. The membranes were then cross-linked for 15 min with the membrane face down on a

M AN U

transilluminator at 312 nm, and the biotinylated protein-DNA bands were detected with HRP-conjugated streptavidin using the chemiluminescent nucleic acid detection system (Pierce).

TE D

Statistical Analysis

All data are presented as the mean ± standard deviation (SD) of the results obtained from three independent experiments. Differences between each group were assessed by

EP

One-way analysis of variance followed by Student-Newman-Keuls post hoc tests. P < 0.05 was considered statistically significant. All data were analyzed using SPSS version 13.0

AC C

statistical software (SPSS, Chicago, IL).

Results

Lidocaine is not toxic to primary rat microglia. The cytotoxic effects of lidocaine were evaluated with the Cell Counting Kit-8 (CCK-8) assay by measuring the viability of primary rat microglia after 24 h incubation

11

ACCEPTED MANUSCRIPT

with lidocaine in the presence or absence of LPS (1 µg/mL). There were no significant differences in cell viability between controls and cells treated with 0.2, 2, or 20 µg/mL lidocaine (Fig. 1). These data indicate that the doses of lidocaine used do not reduce cell

RI PT

viability after 24 hr.

Lidocaine inhibits LPS-induced release and expression of NO and MCP-1.

SC

As shown in Fig. 2, LPS induced a marked increase in the release of NO and MCP-1 (P < 0.05), which was significantly attenuated with lidocaine (2 and 20 µg/mL)

M AN U

treatment (P < 0.05, Fig. 2A, B). The release of NO and MCP-1 was lower in lidocaine (20 µg/mL) treatment group than in lidocaine (2 µg/mL) treatment group (P < 0.05, Fig. 2A, B). Furthermore, quantitative real-time PCR analysis on primary rat microglia showed that LPS markedly induced iNOS and MCP-1 mRNA expression after 24 hr (P < 0.05).

TE D

Treatment with lidocaine (2 and 20 µg/mL) inhibited this LPS-induced iNOS and MCP-1 mRNA expression in a dose-dependent manner (P < 0.05, Fig. 2C, D). The expression of iNOS and MCP-1 mRNA was lower in lidocaine (20 µg/mL) treatment group than in

EP

lidocaine (2 µg/mL) treatment group (P < 0.05, Fig. 2C, D). Importantly, lidocaine itself

AC C

did not alter the release and expression of NO and MCP-1 from microglial cells.

Lidocaine inhibits LPS-induced release and expression of inflammatory mediators. To examine the effect of lidocaine treatment on the release of inflammatory

mediators (e.g., PGE2, TNF-αand IL-1β), primary rat microglia were incubated with lidocaine (0.2, 2, and 20 µg/mL) in the presence or absence of LPS (1 µg/mL). As shown in Fig. 3, LPS induced a marked increase in the release of inflammatory mediators (P < 0.05),

12

ACCEPTED MANUSCRIPT

which was significantly attenuated with lidocaine (2 and 20 µg/mL) treatment (P < 0.05, Fig. 3A, B, C). The release of inflammatory mediators was lower in lidocaine (20 µg/mL) treatment group than in lidocaine (2 µg/mL) treatment group (P < 0.05, Fig. 3A, B,

RI PT

C).Furthermore, quantitative real-time PCR analysis on primary rat microglia showed that LPS markedly induced inflammatory mediators mRNA expression after 24 hr (P < 0.05). Treatment with lidocaine (2 and 20 µg/mL) inhibited this LPS-induced inflammatory

SC

mediators mRNA expression in a dose-dependent manner (P < 0.05, Fig. 3D, E, F). The expression of inflammatory mediators mRNA was lower in lidocaine (20 µg/mL)

M AN U

treatment group than in lidocaine (2 µg/mL) treatment group (P < 0.05, Fig. 3D, E, F). Importantly, lidocaine itself did not alter the release and expression of these mediators from microglial cells.

TE D

Lidocaine inhibits LPS-induced NF-κB activation.

To further delineate the mechanism by which lidocaine inhibits inflammatory responses in LPS-activated primary microglia, we next examined the activation of NF-κB

EP

signaling. ELISA assays showed that LPS treatment increased NF-κB p50/p65 DNA binding activity in primary microglial cells. Furthermore, treament with lidocaine

AC C

significantly reduced the level of NF-κB activation in LPS-activated primary microglia in a dose-dependent manner (P < 0.05, Fig. 4A). The activation of NF-κB signaling was lower in lidocaine (20 µg/mL) treatment group than in lidocaine (2 µg/mL) treatment group (P < 0.05, Fig. 4A). Fluorescence microscopy assay showed that LPS treatment caused obvious translocation of NF-κB p65 from the cytoplasm into the nucleus 60 min after activation, whereas the lidocaine treatment (20 µg/mL) reduced this (Figure 4B).

13

ACCEPTED MANUSCRIPT

EMSA showed that NF-κB binding activities were induced by LPS treatment, whereas pretreatment with lidocaine (20 µg/mL) markedly reduced the LPS-induced DNA-binding activity of NF-κB (Figure 4C). To further verify the p65 nuclear

RI PT

translocation data, we analyzed the microglial cells by western blotting and found that pretreatment of cells with 20 µg/mL lidocaine prevented p65 nuclear localization induced

SC

by LPS (Figure 4D).

M AN U

Lidocaine increases the IκB-α level in LPS-stimulated microglia.

Inhibition of NF-κB signaling may occur through a variety of mechanisms, one of which may involve the enhanced expression of IκB-α, which forms an inactive cytoplasmic complex with the p65-p50 heterodimeric complex. We determined the protein

TE D

levels of IκB-α by ELISA analysis. Interestingly, treament with lidocaine significantly increased the level of IκB-α in LPS-activated primary microglia (P < 0.05, Fig. 5A). The level of IκB-α was higher in lidocaine (20 µg/mL) treatment group than in lidocaine (2

EP

µg/mL) treatment group (P < 0.05, Fig. 5A). Because activation of NF-κB requires IκB-α phosphorylation and degradation, we assessed whether lidocaine affected the protein level

AC C

of phosphorylated IκB-α. Our data show that lidocaine significantly inhibited the phosphorylation levels of IκB-α in LPS-activated primary microglia (P < 0.05, Fig. 5B). The phosphorylation levels of IκB-α was lower in lidocaine (20 µg/mL) treatment group than in lidocaine (2 µg/mL) treatment group (P < 0.05, Fig. 5B).These results indicate that lidocaine inhibited LPS-induced NF-κB activation by preventing IκB-α phosphorylation and degradation in primary microglia.

14

ACCEPTED MANUSCRIPT

Lidocaine inhibits LPS-induced p38 MAPK activation. We assayed the effect of lidocaine on the activation of the p38 MAPK signaling

RI PT

pathway by examining the phosphorylation levels of p38 MAPK. ELISA analysis of cells pretreated with lidocaine (0.2, 2, and 20 µg/mL) for 24 h and then stimulated with 1 µg/mL LPS for 30 min showed that LPS increased the phosphorylation of p38 MAPK, and that

SC

pre-treatment with lidocaine significantly attenuated this effect (P < 0.05,Fig. 6A). The activation of p38 MAPK signaling was lower in lidocaine (20 µg/mL) treatment group than

M AN U

in lidocaine (2 µg/mL) treatment group (P < 0.05, Fig. 6A).To further verify the phosphorylation of p38 MAPK data, we analyzed the microglial cells by western blotting and found that pretreatment of cells with 20 µg/mL lidocaine prevented phosphorylation of

TE D

p38 MAPK induced by LPS (Figure 6B).

NF-kB inhibitor suppresses LPS-induced release of inflammatory mediators. To clarify the role of NF-κB activation in the release of inflammatory mediators (e.g.,

EP

NO, PGE2, TNF-α, IL-1β and MCP-1), microglial cells were treated with LPS in the presence of 100 µM PDTC (an NF-κB inhibitor) and lidocaine (20 µg/mL). We found that

AC C

LPS-induced release of NO, PGE2, TNF-α, IL-1β and MCP-1 was significantly inhibited by both PDTC pretreatment and lidocaine pretreatment alone (*P < 0.05, Fig. 7A-E), furthermore, when both lidocaine pretreatment and PDTC pretreatment were performed to the LPS-induced microglial cells, the inflammatory mediators releasing was most strongly inhibited (#P < 0.05, Fig. 7A-E). These results implicate NF-κB activation in the release of inflammatory mediators from LPS-activated microglia, and suggest that the inhibition of

15

ACCEPTED MANUSCRIPT

signaling pathways leading to this NF-κB activation is a mechanism for the anti-inflammatory effects of lidocaine treatment and the effects of lidocaine pretreatment

RI PT

may be not completely removed by blocking NF-κB signaling pathway.

P38 MAPK inhibitor suppresses LPS-induced release of inflammatory mediators.

To clarify the role of p38 MAPK signaling in the release of inflammatory mediators

SC

(e.g., NO, PGE2, TNF-α, IL-1β and MCP-1), microglial cells were treated with LPS in the presence of 10 µM SB203580 (a p38 MAPK inhibitor) and lidocaine (20 µg/mL). We

M AN U

found that LPS-induced release of NO, PGE2, TNF-α, IL-1β and MCP-1 was significantly inhibited by both SB203580 pretreatment and lidocaine pretreatment alone (*P < 0.05, Fig. 8A-E), furthermore, when both lidocaine pretreatment and SB203580 pretreatment were performed to the LPS-induced microglial cells, the inflammatory mediators releasing was

TE D

most strongly inhibited (#P < 0.05, Fig. 8A-E). These results implicate p38 MAPK as a likely effector of lidocaine’s inhibition of LPS-induced inflammatory signaling and the effects of lidocaine pretreatment may be not completely removed by blocking p38 MAPK

EP

signaling pathway.

AC C

NF-kB p65 siRNA suppresses LPS-induced expression of inflammatory mediators. We further confirmed the results by knocking down NF-κB p65 in microglia cells using specific siRNA of NF-κB p65. We found that silencing NF-κB p65 with siRNA inhibited the expression levels of iNOS, PGE2, TNF-α, IL-1β and MCP-1 mRNA in LPS-stimulated primary microglia (*P < 0.05, Fig. 9A-E) and the lidocaine treatment strongly increased the inhibition effect of siRNA (#P < 0.05, Fig. 9A-E).

16

ACCEPTED MANUSCRIPT

IκB-α siRNA increases LPS-induced expression of inflammatory mediators. We further confirmed the results by knocking down IκB-α in microglia cells using

RI PT

specific siRNA of IκB-α. We found that silencing IκB-α with siRNA increased the expression levels of iNOS, PGE2, TNF-α, IL-1β and MCP-1 mRNA in LPS-stimulated primary microglia (*P < 0.05, Fig. 10A-E) and the lidocaine treatment inhibited the

SC

increased mRNA expression of iNOS, PGE2, TNF-α, IL-1β and MCP-1 caused by LPS and

M AN U

siRNA (#P < 0.05, Fig. 10A-E).

P38 MAPK siRNA suppresses LPS-induced expression of inflammatory mediators. We further confirmed the results by knocking down p38 MAPK in microglia cells using specific siRNA of p38 MAPK. We found that silencing p38 MAPK with siRNA

TE D

inhibited the expression levels of iNOS, PGE2, TNF-α, IL-1β and MCP-1 mRNA in LPS-stimulated primary microglia (*P < 0.05, Fig. 11A-E) and the lidocaine treatment

Discussion

EP

strongly increased the inhibition effect of siRNA (#P < 0.05, Fig. 11A-E).

AC C

The present results show that 2 and 20 µg/mL lidocaine significantly inhibited the

release of inflammatory mediators such as NO, MCP-1,PGE2, IL-1β and TNF-α in LPS-activated primary microglia in vitro. Cell viability was not affected, indicating that treatment with lidocaine was anti-inflammatory rather than cytotoxic. The data also suggest that the prophylactic inhibitory effects of lidocaine occur via inhibition of p38 MAPK and NF-κB pathways. It has been proposed that candidate drugs that downregulate

17

ACCEPTED MANUSCRIPT

these inflammatory mediators would alleviate the progression of neurodegenerative diseases caused by activated microglia [28-30]. Therefore, the inhibition of inflammatory

RI PT

mediators shown in this study suggests that lidocaine could be beneficial in the treatment of such neurodegenerative diseases.

NO, MCP-1, PGE2 and pro-inflammatory cytokines, such as TNF-α and IL-1β, have been implicated as important mediators in the process of inflammation [25]. There

SC

are several lines of evidence showing that these factors are released following microglial

M AN U

activation induced by CNS injury or infection [31-33]. Previous studies have also shown that pro-inflammatory cytokines upregulate the transcription of iNOS, a key enzyme for the synthesis of NO, in activated microglial cells. Therefore mechanisms regulating the expression of iNOS genes may help to effectively reduce NO release [31-34]. In this

TE D

study, we demonstrate that lidocaine pretreatment significantly inhibits NO release in LPS-activated primary microglia, as well as attenuates the expression iNOS mRNA. NF-κB is known to play a critical role in the regulation of inflammatory processes and to

EP

coordinate the expression of pro-inflammatory enzymes and cytokines, such as iNOS, MCP-1, PGE2, IL-1β and TNF-α [35-37]. Furthermore, blockade of transcriptional

AC C

activity by NF-κB in microglia suppresses their expression [38-40], and therefore many putative anti-inflammatory therapies seek to block NF-κB activity. Previous studies have indicated that lidocaine exerts anti-inflammatory effects by inhibition of the NF-κB pathway [15, 18, 41]. For instance, lidocaine inhibited LPS-induced release of high-mobility group box 1 protein from macrophages [18], and attenuated LPS-induced acute lung injury [15] through the inhibition of NF-κB activation. Therefore, we explored

18

ACCEPTED MANUSCRIPT

the inhibitory effect of lidocaine on NF-κB activity in microglia using p65 nuclear translocation assays. Our results support these previous findings, demonstrating that lidocaine inhibits NF-κB activation in LPS-activated microglia, reducing the release of

RI PT

inflammatory mediators. In our study, the fluorescence microscopy assay, electrophoretic mobility shift assay and western blotting results all demonstrated that lidocaine inhibited NF-κB p65 nuclear localization induced by LPS, so we believe lidocaine can affect

SC

canonical NF-κB signaling pathway. When both lidocaine pretreatment and PDTC

pretreatment were performed to the LPS-induced microglial cells, the inflammatory

M AN U

mediators releasing was most strongly inhibited (#P < 0.05, Fig. 7A-E), which indicated the effects of lidocaine pretreatment may be not completely removed by blocking NF-κB signaling pathway, lidocaine may also involve some other pathways that regulated by releasing inflammatory mediators.

TE D

LPS has been shown to activate MAPKs in microglia [42]. The mitogen-activated protein kinases (MAPKs), which include the extracellular signal-regulated kinases (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 MAPK, are intracellular enzymes which allow

EP

cells to respond to extracellular stimuli. These kinases, p38 MAPK in particular, have also been shown to be important upstream modulators for MCP-1, PGE2, iNOS and

AC C

inflammatory cytokine (TNF-α and IL-1β) production in many cell types [43, 44]. Therefore, experiments were performed to determine whether lidocaine could suppress activation of p38 MAPK to induce anti-inflammatory effects in LPS-activated primary microglia. Although further studies are needed to validate the role of p38 MAPK in the regulation of various inflammatory mediators in microglia, our data implicate the p38 MAPK signaling pathway, as direct inhibition with SB203580 suppressed the release of

19

ACCEPTED MANUSCRIPT

NO,MCP-1, PGE2, IL-1β, and TNF-α. Furthermore, we demonstrate that lidocaine is a potent inhibitor of p38 MAPK, and it suppressed the release of inflammatory cytokines from LPS-activated microglia, which is similar with the effects of SB203580. In addition,

RI PT

the ELISA and western blotting assay both demonstrated that lidocaine inhibited p38

MAPK phosphorylation, indicated that lidocaine suppressed the upstream of p38 MAPK signaling pathway.

SC

In conclusion, the present study demonstrates that 2 and 20 µg/mL lidocaine exhibits anti-inflammatory activity by suppressing the release and expression of NO,

M AN U

MCP-1, PGE2, IL-1β and TNF-α in LPS-activated primary microglia in a dose-dependent manner, mediated in part through inhibition of p38 MAPK and NF-κB activation. Lemmen et al. ever reported that the plasma concentrations of lidocaine at 2 to 5 µg/mL are an accepted therapeutic range for antiepileptogenic effects in man [45]. Although the brain

TE D

lidocaine concentrations corresponding to this plasma therapeutic range are still unknown, our findings suggest that protection of microglial cells by lidocaine may occur at clinically relevant concentrations. In this study, we only investigated the prophylactic effects of

EP

lidocaine. Although the previous study has showed that lidocaine protects and inhibits cytokine production from activated microglial cells when applied after the LPS treatment

AC C

[14], a further study to determine the therapeutic role of lidocaine after LPS stimulation is needed. Taken together, these findings further describe the mechanism for the anti-inflammatory properties of lidocaine in LPS-activated microglial cells. Additionally, the absence of cytotoxic effects indicates that the addition of lidocaine treatment for LPS-mediated sepsis syndrome and neuroinflammatory disease would be beneficial.

20

ACCEPTED MANUSCRIPT

[6] [7] [8]

[9] [10]

[11]

[12]

[13]

[14]

21

RI PT

SC

[5]

M AN U

[4]

TE D

[3]

EP

[2]

AC C

[1]

References Streit WJ. Microglia as neuroprotective, immunocompetent cells of the CNS. Glia 2002;40:133. Kaur G, Han SJ, Yang I, Crane C. Microglia and central nervous system immunity. Neurosurg Clin N Am 2010;21:43. Graeber MB, Streit WJ. Microglia: biology and pathology. Acta Neuropathol 2010;119:89. Takeuchi H, Jin S, Wang J, et al. Tumor necrosis factor-alpha induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner. J Biol Chem 2006;281:21362. Stone DK, Reynolds AD, Mosley RL, Gendelman HE. Innate and adaptive immunity for the pathobiology of Parkinson's disease. Antioxid Redox Signal 2009;11:2151. Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev 2007;87:315. Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH. Mechanisms underlying inflammation in neurodegeneration. Cell 2010;140:918. Tasi WC, Petersen-Jones SM, Huang PY, Lin CT. The neuroprotective effects of lidocaine and methylprednisolone in a rat model of retinal ischemia-reperfusion injury. J Vet Med Sci 2012;74:307. Hollmann MW, Durieux ME. Local anesthetics and the inflammatory response: a new therapeutic indication? Anesthesiol 2000; 93:858. Caracas HCPM, Maciel JVB, de Souza MMG, et al. The use of lidocaine as an anti-inflammatory substance: a systematic review. J Dent 2009; 37:93. Kaczmarek DJ, Herzog C, Larmann J, et al. Lidocaine protects from myocardial damage due to ischemia and reperfusion in mice by its antiapoptotic effects. Anesthesiol 2009; 110:1041. Lan W, Harmon D, Wang J, Ghori K, Shorten G, Redmond P. The effect of lidocaine on in vitro neutrophil and endothelial adhesion molecule expression induced by plasma obtained during tourniquet-induced ischaemia and reperfusion. Eur J Anaesthesiol 2004;21:892. Gallos G, Jones DR, Nasr SH, et al. Local anesthetics reduce mortality and protect against renal and hepatic dysfunction in murine septic peritonitis. Anesthesiol 2004; 101:902. Jeong HJ, Lin D, Li L, Zuo Z. Delayed treatment with lidocaine reduces mouse microglial cell injury and cytokine production after stimulation with lipopolysaccharide and interferon gamma. Anesth Analg 2012;114:856.

ACCEPTED MANUSCRIPT

[19]

[20]

[21]

[22]

[23] [24]

[25]

[26]

22

RI PT

SC

M AN U

[18]

TE D

[17]

EP

[16]

Feng G, Liu S, Wang G-L, et al. Lidocaine attenuates lipopolysaccharide-induced acute lung injury through inhibiting NF-κB activation. Pharmacol 2007; 81:32. Lee PY, Tsai PS, Huang YH, et al. Inhibition of toll-like receptor-4, nuclear factor-κB and mitogen-activated protein kinase by lignocaine may involve voltage-sensitive sodium channels. Clin Exp Pharmacol Physiol 2008; 35:1052. Su D, Gu Y, Wang Z, Wang X. Lidocaine attenuates proinflammatory cytokine production induced by extracellular adenosine triphosphate in cultured rat microglia. Anesth Analg 2010;111:768. Wang HL, Zhang WH, Lei WF, Zhou CQ, Ye T. The inhibitory effect of lidocaine on the release of high mobility group box 1 in lipopolysaccharide-stimulated macrophages. Anesth Analg 2011;112:839. Akundi RS, Candelario-Jalil E, Hess S, et al. Signal transduction pathways regulating cyclooxygenase-2 in lipopolysaccharide activated primary rat microglia. Glia 2005;51:199. Heese K, Fiebich BL, Bauer J, Otten U. NF-kappaB modulates lipopolysaccharide-induced microglial nerve growth factor expression. Glia 1998;22:401. Zhan J, Zhang ZZ, Chen C, Chen K, Wang YL. Penehyclidine hydrochloride attenuates LPS-induced iNOS production by inhibiting p38 MAPK activation in endothelial cells. Mol Biol Rep 2012;39:1261. Gao F, Ding B, Zhou L, Gao X, Guo H, Xu H. Magnesium sulfate provides neuroprotection in lipopolysaccharide-activated primary microglia by inhibiting NF-kappaB pathway. J Surg Res 2013. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nature protocols 2008;3:1101. Zhu C, Xiong Z, Chen X, et al. Artemisinin attenuates lipopolysaccharide-stimulated proinflammatory responses by inhibiting NF-kappaB pathway in microglia cells. PloS one 2012;7:e35125. Park HY, Kim GY, Choi YH. Naringenin attenuates the release of pro-inflammatory mediators from lipopolysaccharide-stimulated BV2 microglia by inactivating nuclear factor-kappaB and inhibiting mitogen-activated protein kinases. Int J Mol Med 2012;30:204. Park HY, Kim ND, Kim GY, et al. Inhibitory effects of diallyl disulfide on the production of inflammatory mediators and cytokines in

AC C

[15]

ACCEPTED MANUSCRIPT

[32] [33] [34] [35]

[36] [37] [38]

[39] [40] [41]

[42]

23

RI PT

SC

[31]

M AN U

[30]

TE D

[29]

EP

[28]

AC C

[27]

lipopolysaccharide-activated BV2 microglia. Toxicol Appl Pharmacol 2012;262:177. Huang YH, Yen JC, Lee JJ, Liao JF, Liaw WJ, Huang CJ. Suppressive effects of levobupivacaine on endotoxin-induced microglial activation. J Surg Res 2013;184:989. Garden GA, Moller T. Microglia biology in health and disease. J Neuroimmune Pharmacol 2006;1:127. Sugama S, Takenouchi T, Fujita M, et al. Differential microglial activation between acute stress and lipopolysaccharide treatment. J Neuroimmune 2009; 207:24. Schwartz M, Shechter R. Systemic inflammatory cells fight off neurodegenerative disease. Nat Rev Neurol 2010;6:405. Das S, Basu A. Inflammation: a new candidate in modulating adult neurogenesis. J Neurosci Res 2008;86:1199. Gebicke-Haerter PJ. Microglia in neurodegeneration: molecular aspects. Microsc Res Tech 2001;54:47. Perry VH, Nicoll JA, Holmes C. Microglia in neurodegenerative disease. Nat Rev Neurol 2010;6:193. Rivest S. Molecular insights on the cerebral innate immune system. Brain Behav Immun 2003;17:13. Wright JG, Christman JW. The role of nuclear factor kappa B in the pathogenesis of pulmonary diseases: implications for therapy. Am J Respir Med 2003;2:211. Atreya I, Atreya R, Neurath MF. NF-kappaB in inflammatory bowel disease. J Intern Med 2008;263:591. Baker RG, Hayden MS, Ghosh S. NF-kappaB, inflammation, and metabolic disease. Cell Metab 2011;13:11. Shin WS, Szuba A, Rockson SG. The role of chemokines in human cardiovascular pathology: enhanced biological insights. Atheroscler 2002; 160:91. Nam N-H. Naturally occurring NF-B inhibitors. Mini Rev Med Chem 2006; 6:945. Luqman S, Pezzuto JM. NFkappaB: a promising target for natural products in cancer chemoprevention. Phytother Res 2010;24:949. Lang A, Ben Horin S, Picard O, et al. Lidocaine inhibits epithelial chemokine secretion via inhibition of nuclear factor κB activation. Immunobiol 2010; 215:304. Pyo H, Jou I, Jung S, et al. Mitogen-activated protein kinases activated by lipopolysaccharide and beta-amyloid in cultured rat microglia. Neuroreport 1998; 9:871.

ACCEPTED MANUSCRIPT

[44]

AC C

EP

TE D

M AN U

SC

[45]

Kim YJ, Hwang SY, Oh ES, et al. IL-1β, an immediate early protein secreted by activated microglia, induces iNOS/NO in C6 astrocytoma cells through p38 MAPK and NF κB pathways. J Neurosci Res 2006; 84:1037. Munoz L, Ammit AJ. Targeting p38 MAPK pathway for the treatment of Alzheimer's disease. Neuropharmacology 2010;58:561. Lemmen LJ, Klassen M, Duiser B. Intravenous lidocaine in the treatment of convulsions. JAMA 1978;239:2025.

RI PT

[43]

24

ACCEPTED MANUSCRIPT

Figure Legends Figure 1. Lidocaine does not affect microglial cell viability. Microglial cells were

RI PT

treated with lidocaine (Lido: 0, 0.2, 2, and 20 µg/mL) for 24 h. Cell viability was assessed using a CCK-8 assay. Data are expressed as mean ± SD from three independent

SC

experiments.

Figure 2. Lidocaine suppresses the release and expression of NO and MCP-1 from

M AN U

LPS-activated microglial cells. The levels of (A) NO and (B) MCP-1 in microglial cell culture media after treatment with LPS and lidocaine (Lido: 0, 0.2, 2, and 20 µg/mL) were measured using ELISA kits.The expression of (C) iNOS mRNA and (D) MCP-1 mRNA was assessed by qPCR following treatment with LPS and lidocaine (Lido: 0, 0.2, 2, and 20 µg/mL). Data are expressed as mean ± SD from three independent experiments. *P < 0.05

TE D

compared with LPS treatment alone, #P < 0.05 compared with LPS and lidocaine (Lido: 2

EP

µg/mL) treatment.

Figure 3. Lidocaine suppresses the release and expression of inflammatory mediators

AC C

from LPS-activated microglial cells. The levels of (A) PGE2, (B) TNF-α and (C) IL-1β in microglial cell culture media after treatment with LPS and lidocaine (Lido: 0, 0.2, 2, and 20 µg/mL) were measured using ELISA kits. The expression of (D) PGE2 mRNA, (E) TNF-αmRNA and (F) IL-1β mRNA was assessed by qPCR following treatment with LPS and lidocaine (Lido: 0, 0.2, 2, and 20 µg/mL). Data are expressed as mean ± SD from three independent experiments. *P < 0.05 compared with LPS treatment alone, #P < 0.05 compared with LPS and lidocaine (Lido: 2 µg/mL) treatment. 25

ACCEPTED MANUSCRIPT

Figure 4. Lidocaine suppresses LPS-induced activation of NF-κB p65 in microglial cells. Microglial cells were pre-incubated with lidocaine (Lido: 0.2, 2, and 20 µg/mL) for 1

RI PT

h and then stimulated with LPS (1 µg/mL) for 30 min. NF-κB activity was determined from extracted nuclear proteins using the NF-κB p50/p65 transcription factor assay kit (A). Lidocaine inhibiting the LPS-induced translocation of NF-κB p65 from the cytoplasm into

SC

the nucleus was observed by fluorescence microscopy assay (B). The DNA-binding activity of NF-κB was detected by EMSA (C). The NF-κB p65 nuclear translocation was

M AN U

further verified by western blotting (D). Data are expressed as mean ± SD from three independent experiments. *P < 0.05 compared with LPS treatment alone, #P < 0.05 compared with LPS and lidocaine (Lido: 2 µg/mL) treatment.

TE D

Figure 5. Lidocaine suppresses LPS-induced degradation and phosphorylation of IκB-α in microglial cells. Microglial cells were pre-incubated with lidocaine (Lido: 0.2, 2, and 20 µg/mL) for 1 h and then stimulated with LPS (1 µg/mL) for 30 min. The

EP

LPS-induced degradation and phosphorylation of IκB-α were analyzed by ELISA (A and B). Data are expressed as mean ± SD from three independent experiments. *P < 0.05

AC C

compared with LPS treatment alone, #P < 0.05 compared with LPS and lidocaine (Lido: 2 µg/mL) treatment.

Figure 6. Lidocaine suppresses LPS-induced activation of p38 MAPK in microglial cells. Microglial cells were pre-incubated with lidocaine (Lido: 0.2, 2, and 20 µg/mL) for 1 h and then stimulated with LPS (1 µg/mL) for 30 min. P38 MAPK activation was

26

ACCEPTED MANUSCRIPT

determined using a phospho-p38 MAPK ELISA and Western Blot(A and B). Data are expressed as mean ± SD from three independent experiments. *P < 0.05 compared with LPS treatment alone, #P < 0.05 compared with LPS and lidocaine (Lido: 2 µg/mL)

RI PT

treatment.

Figure 7. NF-κB inhibitor suppressed release of LPS-induced inflammatory

SC

mediators in microglial cells. Microglial cells were incubated with 100 µM PDTC for 1 h and/or then treated with lidocaine (Lido: 20 µg/mL) and LPS (1 µg/mL) for 24 h. (A) The

M AN U

NO concentration in the culture medium was measured by the Griess reagent. The levels of (B) PGE2, (C) TNF-α, (D) IL-1β and (E) MCP-1 in the culture media were measured using ELISA kits. Data are expressed as mean ± SD from three independent experiments. *P < 0.05 compared with LPS treatment alone, #P < 0.05 compared with LPS, lidocaine (Lido:

TE D

20 µg/mL), and PDTC (100 µM) treatment.

Figure 8. P38 MAPK inhibitor suppressed release of LPS-induced inflammatory

EP

mediators in microglial cells. Microglial cells were incubated with 10 µM SB203580 for 1 h and then treated with lidocaine (Lido: 20 µg/mL) and 1 µg/mL LPS for 24 h. (A) The

AC C

NO concentration in the culture medium was measured by the Griess reagent. The levels of (B) PGE2, (C) TNF-α, (D) IL-1β and (E) MCP-1 in the culture media were measured using ELISA kits. Data are expressed as mean ± SD from three independent experiments. *P < 0.05 compared with LPS treatment alone, #P < 0.05 compared with LPS, lidocaine (Lido: 20 µg/mL), and SB203580 (10 µM) treatment.

27

ACCEPTED MANUSCRIPT

Figure 9. NF-κB p65 siRNA suppressed LPS-induced iNOS, PGE2, TNF-α, IL-1β and MCP-1 mRNA expression in microglial cells. Microglial cells were transfected with

RI PT

siRNA for NF-κB p65 or control, and then incubated with lidocaine (Lido: 20 µg/mL) and LPS (1 µg/mL) for 24 h. The expression of (A) iNOS mRNA, (B) PGE2 mRNA, (C) TNF-α mRNA , (D) IL-1β mRNA and (E) MCP-1mRNA was assessed by qPCR following

SC

treatment with LPS. Data are expressed as mean ± SD from three independent experiments. *P < 0.05 compared with LPS treatment alone, #P < 0.05 compared with LPS, lidocaine

M AN U

(Lido: 20 µg/mL), and NF-κB p65 siRNA treatment.

Figure 10. IκB-α siRNA incresased LPS-induced iNOS, PGE2, TNF-α, IL-1β and MCP-1 mRNA expression in microglial cells. Microglial cells were transfected with

TE D

siRNA for IκB-α or control, and then incubated with lidocaine (Lido: 20 µg/mL) and LPS (1 µg/mL) for 24 h. The expression of (A) iNOS mRNA, (B) PGE2 mRNA, (C) TNF-α mRNA , (D) IL-1β mRNA and (E) MCP-1mRNA was assessed by qPCR following

EP

treatment with LPS. Data are expressed as mean ± SD from three independent experiments. *P < 0.05 compared with LPS treatment alone, #P < 0.05 compared with LPS, lidocaine

AC C

(Lido: 20 µg/mL), and IκB-α siRNA treatment.

Figure 11. P38 MAPK siRNA suppressed LPS-induced iNOS, PGE2, TNF-α, IL-1β and MCP-1 mRNA expression in microglial cells. Microglial cells were transfected with siRNA for p38 MAPK or control, and then incubated with lidocaine (Lido: 20 µg/mL) and LPS (1 µg/mL) for 24 h. The expression of (A) iNOS mRNA, (B) PGE2 mRNA, (C) TNF-α

28

ACCEPTED MANUSCRIPT

mRNA , (D) IL-1β mRNA and (E) MCP-1mRNA was assessed by qPCR following treatment with LPS. Data are expressed as mean ± SD from three independent experiments. *P < 0.05 compared with LPS treatment alone, #P < 0.05 compared with LPS, lidocaine

AC C

EP

TE D

M AN U

SC

RI PT

(Lido: 20 µg/mL), and p38 MAPK siRNA treatment.

29

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT