ACT001 was kindly provided by Tianjin Shangde Pharmaceutical Margin Technology Co., Ltd. Microglial BV-2 cells were obtained from China Center for Type Culture Collection. Ultrapure lipopolysaccharide (LPS), HEK Blue TLR4 293 cells and HEK-Blue Selection were obtained from Invivogen. Phospha-Light™ SEAP Reporter Gene Assay System was purchased from Applied Biosystems. Dual-Glo Luciferase Assay System was purchased from Promega. Crystal violet, 2, 3-diaminonaphthalene, protease inhibitor cocktails and anti-b-actin antibody were purchased from Sigma-Aldrich. Dulbecco's Modified Eagle Medium (DMEM), TRIzol, RIPA buffer were purchased from Thermo Fisher Scientific. Fetal bovine serum was purchased from PAN-Seratech. RNeasy Mini Kit, RT² Easy First Strand cDNA Synthesis Kit, PCR primers and SYBR Green PCR Master Mix were obtained from Qiagen. Primary MD2 antibody, anti-Iba1 antibody and anti-GFAP antibody were purchased from Abcam. Primary antibodies targeting MyD88, p38 MAPK, NF-κB p65, ERK (1/2), IKK-β, SAPK/JNK, phospho-NF-κB p65, phospho-ERK (1/2), phospho-SAPK/JNK, phospho-IKK-α/β, and phospho-p38 MAPK antibodies were obtained from Cell Signaling Technology.
Fluorescence titrations of MD2 with ACT001
MD2 expression and purification were performed as described previously [17, 18]. Fluorescence titrations of MD2 with ACT001 were performed on a Cary Eclipse spectrofluorometer. All measurements were carried out at room temperature using a 2×10 mm quartz cell with MD2. The fluorescence titration was carried out at a wavelength of 280 nm to excite the Tyr and Trp residues in MD2. Emission at 310-450 nm was measured. 0.5 mM MD2 was titrated with different concentrations of ACT001 and fluorescence intensity at 337 nm was plotted against ACT001 concentration.
Saturation transfer difference (STD) NMR measurement
MD2 was prepared in a phosphate buffer in D2O (75 mM potassium phosphate, 150 mM sodium chloride, pH 7.5) and ACT001 was dissolved in DMSO-d6 (50 mM) as stock solution. Saturation transfer difference NMR spectroscopy experiments were performed to investigate ligand-protein interactions. NMR spectra were acquired at 25 oC in a Bruker Avance III-600 MHz (proton frequency) spectrometer with a conventional inverse 5 mm probe head with z-gradients using standard Bruker pulse programs. Samples containing 400 μM ACT001 in the absence or presence of MD2 (4 μM) in D2O buffer were used for NMR spectra data acquisition.
In Silico Simulations
System preparation and docking
The structure of ACT001 was drawn through GaussView 6  and optimized by Gaussian 09  software using the B3LYP density functional method and 6-31G (d, p) basis set. The X-ray structure of MD2 was extracted from TLR4/MD2 complex (PDB ID: 2Z64) and was used for molecular docking and molecular dynamics simulations. Missing hydrogen atoms of MD2 were added by Maestro under pH 7.0 . Molecular docking was conducted through Autodock Vina 1.1.2 in a box of 50 × 60 × 50 Å3, which covers MD2 protein completely . The most favorable binding site was searched and located by the Iterated Local Search Globule Optimizer [23, 24]. MD2 was considered rigid and ACT001 was regarded as semi-flexible during molecular docking. Ten docking poses were generated by Autodock Vina 1.1.2 and ranked according to their affinity with MD2. Of all the docking poses, the pose with the best affinity to MD2 was chosen for further simulations.
Molecular dynamics simulation
MD2 alone (apo-MD2) and the best docking pose of MD2 interacting with ACT001 were further studied through molecular dynamics simulations by the NAMD2.12 package  with AMBER ff03 force field [26, 27]. R.E.D was used to optimize and fit the atomic charges of ACT001 based on the quantum mechanics calculations . The general AMBER force field (GAFF) was used to treat other atomic parameters . A TIP3P model of a water box was used to solvate all solutes with a distance of 10 Å between the protein and the edge of the box. Na+ and Cl- ions were added to neutralize the system with a concentration of 0.15 M. Energy minimization was performed for 5000 steps first and the system was heated to 310 K in 310 ps with 1 ns equilibration. The system was further run in the isothermal-isobaric (NPT) ensemble at a temperature of 310 K for 400 ns. SHAKE algorithm was used to restrain all bonds involving hydrogen . Calculations of long-range electrostatic interactions were performed by the Particle-mesh Ewald (PME) summation method . Langevin dynamics was used to keep the temperature of the system at 310 K with the collision frequency of 5 ps–1 and the pressure was set at 1 atm with Nosé–Hoover Langevin piston method .
The RMSD (root-mean-square deviation) and RMSF (root-mean-square fluctuation) analyses were performed through VMD  and Bio3D package , respectively. The interactions between MD2 and ACT001 were analyzed by LigPlot+  and PyMol  software. The ratio of hydrophobic SASA (solvent accessible surface areas) in buried SASA was calculated as specified before .
Nitric oxide (NO) assay
BV-2 cells were cultured in supplemented DMEM (10% FBS, 50 U/mL penicillin and 50 mg/mL streptomycin) and seeded at a density of 4×104 cells per well in 96-well plates. After overnight incubation, media was aspirated and changed to DMEM media without FBS. Cells were then treated with LPS (200 ng/mL) and the indicated concentrations of ACT001. The NO concentration in the culture supernatant was determined by the 2, 3-diaminonaphthalene-based fluorescent method as described .
Cell Viability Assay
Cellular viability was determined by the crystal violet staining method and CCK-8 Kit as described [18, 36].
Secreted embryonic alkaline phosphatase (SEAP) assay
SEAP assay was performed as described .
Dual-luciferase NF-κB reporter assay
Dual-luciferase NF-κB reporter assay was performed as described .
BV-2 cells were seeded at 4×105 cells/well in 100 mm culture dishes. After 24 h incubation, cells were stimulated by LPS (200 ng/mL) and indicated concentrations of ACT001 for 1 h. Cells were washed twice with ice-cold PBS and lysed in 1 mL Co-IP lysis buffer (25 mM Tris pH 8.0, 150 mM KCl, 5 mM EDTA, 0.5% NP-40) with a complete protease inhibitor cocktail, 1 mM DTT and 1 mM PMSF by incubating on ice for 30 min. Cell supernatant was collected via centrifugation at 12,000 g at 4 °C for 12 min and incubated with corresponding primary antibody at 4 °C overnight. Washed magnetic beads were then incubated with the samples at room temperature for 1 h. The magnetic beads were washed twice with PBS and boiled with 50 μL 2 × SDS sample buffer at 100 °C for 8 min for immunoblotting.
Immunoblotting was performed as described .
BV-2 cells were seeded at a density of 4×105 cells/well in 6-well plates. After overnight incubation, BV-2 cells were treated with LPS (200 ng/mL) and indicated concentrations of ACT001 for 6 h. Total RNA was isolated from BV-2 cells using TRIzol reagent and cDNA was generated with an oligo (dT) primer. Primer sequences are shown in Table 1. The ribosomal protein L27 gene RPL27 was used as the internal control. qPCR was performed on a TOptical Real-Time qPCR Thermal Cycler (Analytik Jena, Thuringia, Germany) using the SYBR Green method following cycling conditions: preheating at 95°C for 3 min and 40 cycles of amplification at 95 oC (10s); 60 oC (10s); 72 oC (15s). The data were analyzed by the 2−ΔΔCT method and were normalized to RPL27.
In vivo study
Animals and drug treatment
Pathogen-free adult male Sprague-Dawley rats (300-350 g) were used in all experiments (Liaoning Changsheng Biotechnology, China). Rats were housed in temperature-controlled (20 ± 2 °C) and light-controlled (12-h light-dark cycle; lights on at 7:00 am) rooms with standard rodent food and water available ad libitum and allowed to habituate to the holding facility for ≥ 1 week before experimentation.
Animals were randomly divided into three groups. Rats in sham group (n = 6) and CCI group (n = 9) were intravenously administrated with 0.9 % saline, while rats in the CCI + ACT001 group (n = 9) were intravenously administrated with 50 mg/ kg ACT001 (dissolved in 0.9 % saline), once a day from the 2nd day to 42nd day after surgery.
CCI induced neuropathic pain
Neuropathic pain was induced using chronic constriction injury (CCI) surgery as described previously . Briefly, rats were anesthetized and maintained with isoflurane. The left sciatic nerve was gently exposed. Four ligations were tied loosely around the sciatic nerve with sterile chromic gut sutures. The sham group animals were proceeded with the same surgery but without the ligation. All animals were monitored postoperatively until fully ambulatory before returning to their home cage and checked daily for any sign of infection. No such cases occurred in this study.
Animals received at least two days of habituation in the test environment before baseline testing. The nociceptive behavior was monitored 1 day before surgery and 10, 14, 17, 21, 24, 28, 31, 35 and 42 days after surgery. The stimulus with Von Frey filaments, ranging from 0.6 to 26 g, was applied to the plantar surface of the hind paw. The paw withdrawal threshold was accessed via the up-down method using the Chaplan formula .
Following the final behavioral testing, rats were anesthetized and perfused through the ascending aorta first with isotonic saline and then with fresh 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The rat was decapitated and the lumbar spinal cords (L4-L6) were removed immediately, immersed continuously in the 4% paraformaldehyde at 4 °C overnight. The spinal cord tissue was dehydrated with ethanol gradient, embedded in paraffin, and then sliced at a thickness of 4 μm. Paraffin-processed tissues were deparaffinized in xylene and rehydrated with a graded alcohol solution. The sections were placed in 0.01 M citrate buffer (pH 6.0) and heated in a microwave oven for hot repair antigen [39, 40]. These sections were incubated with goat serum at 37 °C for 20 min and then with a mixture of rabbit-anti-Iba-1 monoclonal antibody and mouse-anti-GFAP monoclonal antibody at 4 °C overnight. After three washes with PBS, the sections were incubated with secondary antibody conjugated with Alexa-488 or 647 for 1 h in the dark. Ultimately, followed by three washes with PBS twice for 5 min each, the sections were counterstained with DAPI and examined under a confocal microscope.
Origin 8 was used for the plotting of the data and statistical analysis. Non-linear Logistic regression was used to plot and analyze concentration-response curves and to obtain IC50. For the analyses of qRT-PCR data, immunoblotting data, von Frey test and quantification of immunofluorescence, an unpaired Student t-test was used for comparisons between two groups. Data are presented as mean ± SEM. P-value summary is mentioned on the bar of each figure. # P < 0.05; ## P < 0.01; ### P < 0.001 versus the control/sham group; * P < 0.05; ** P < 0.01; *** P < 0.001 versus the LPS/CCI group. ns, not significant. P < 0.05 was considered statistically significant in all analyses.