Materials and reagents
LPS (Escherichia coli 055:B5) was purchased from Sigma Chemical Co. (St. Louis, USA). Foetal bovine serum and Dulbecco Modified Eagle Mediumas (DMEM) was purchased from Gibco (New York, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Fluorescent probe dichlorofluorescin diacetate (DCFH-DA) for ROS dection were purchased from Applygen (Beijing, PR China). Mouse TNF-α, IL-6, IL-8, IL-10 and IL-1β enzyme-linked immuno sorbent assay (ELISA) kits were purchased from Neobioscience (Shenzhen, PR China). RAW264.7 cells were purchased from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, PR China). Assay kits for NO, T-SOD, T-AOC, GSH-PX, GSH, lysozyme (LZM), acid phosphatase (ACP), MPO, MDA dection were purchased from the Institute of Nanjing Jiancheng Bioengineering (Nanjing, PR China). RNAiso Plus Kit, PrimeScript® RT reagent Kit (Perfect Real Time) and SYBR® Premix Ex TaqTM II Kit (Perfect Real Time) used in RT-qPCR were purchased from Takara Biotechnology (Dalian, PR China). Cell culture dishes and plates (12 mm polycarbonate membrane, 0.4 μm pore size, 1.12 cm2 surface area) were purchased from Corning (New York, USA). iNOS (D6B6S), COX-2 (D5H5), MAPKs Family Antibody Sampler Kit, phospho-MAPKs Family Antibody Sampler Kit, c-JUN (60A8), phospho-c-JUN (Ser 3), AMPKα (D5A2), phospho-AMPKα (40H9), β-Actin (13E5) and GADPH (14C10) were obtained from Cell Signaling Technology (Beverly, USA).
Herbal extract preparation
Polygonum hydropiper L. was purchased from Tai Hua pharmaceutical Co. Ltd. The plant material was authenticated by Professor Renbin Huang, School of Pharmacy, Guangxi medical University. The voucher specimen (2010–125) has been deposited at the Herbarium of Faculty of Pharmacy, Guangxi medical University for future reference. The extraction and purification of FNP were carried out as followed. Briefly, the air-dried whole plant (500 g) was ground to a powder and extracted with ultrasonic in a buffered solution(Hac-NaAc pH 4.8,1.5 l) added with cellulase and pectinase (0.25 % W/V respectively) at 50 °C for 1.5 h. Then the pH value was adjusted to 9.0 with sodium carbonate solution and the enzyme was inactivated at 85 °C water bath for 15 min, and then macerated with ethanol for 24 h. The extracts were filtered and residue were extracted by maceration in ethanol. The combined filtrate was evaporated under reduced pressure to obtain a crude extract (yield 52.3 %) and successively treated with petroleum ether (40–60 °C), chloroform, ethyl acetate and normal butanol. The normal butanol fraction yield was 11.96 %. The normal butanol fraction was added into column loading-treated XDA-8 macroreticular resin for adsorption for 24 h, and then washed with water and 20 % ethanol, respectively, to get rid of polar impurities. FNP in the column was eluted with 75 % ethanol, and the eluting solution was dried in vacuum condition until yellow powders were achieved.
Quality control of FNP
Approximately 100 mg of FNP were transferred to a 10 mL volumetric flask and diluted with methanol to volume. The test sample was finally filtered through a 0.45 μm membrane filter before analysis. The total flavonoids content was determined using a colorimetric method as previously described but with slightly modified [25]. Briefly, 1.0 mL of NaNO2 solution (5 %, w/v), 1.0 mL of AlCl3 solution (10 %), and 4.0 mL NaOH solution (1.0 mol/L) were mixed with the same volume of the test sample. The final volume was adjusted to 25 mL with methanol (80 %, v/v). The mixture was allowed to stand for 5 min and the absorbance was measured at 510 nm against the same mixture, without the sample as a blank. Rutin was used as a reference standard and the total flavonoids content was expressed as rutin. The calibration curve (Y = 0.0011X + 0.00125 where X was absorbance value of sample, and Y was sample concentration) was ranged from 100 to 500 μg/mL (R2 = 0.9992). The contents of three markers (rutin, quercetin and quercitrin) in the extract samples were determined by High Performance Liquid Chromatography (HPLC) using followed chromatographic condition: Waters e2695 separation module and Waters 2998 photodiode array detector were controlled by EMPOWER™ 2 chromatography data sofeware (Waters Co. Ltd. USA). Waters X Bridge™ C18 HPLC column (4.6 × 250 mm 5 μm, Waters Co. Ltd. USA). Column temperature was kept constantly at 30°C. UV detection wavelength was 254 nm. The injection volume was 10 μL. The isocratic mobile phase consisted of acetonitrile-0.3 % phosphoricacid (27:73, v/v) was used to elute for 40 min and the flow rate was kept at 1.0 mL/min.
Animals
The study protocol was approved by the Ethics Committee of Guangxi University and was performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals. Male special pathogen free KM mice were obtained from the Laboratory Animals Center of Guangxi Medical University (Certificate No. 2011–002). After three days of acclimatization, 50 mice were randomly divided into five groups of 10 mice each. Mice in the LPS alone group were injected intraperitoneally with 17 mg/kg (body weight) of Escherichia coli LPS (Serotype 055:B5, Sigma) in phosphate-buffered saline (PBS) [26], while control mice were injected with only PBS (10 mL/kg). The animals in FNP50, FNP100 and FNP200 group were orally administrated with diverse dose FNP (50 mg/kg, 100 mg/kg or 200 mg/kg) for 3 days before instillation of LPS. All mice were euthanized at 24 h after LPS or PBS instillation, and the samples were collected for subsequent analysis.
Mortality and measurement of TNF-α values
The mortality of mice was recorded for 24 h after the LPS challenge in each treated group. Homogenates of murine liver and intestine were prepared using physiological saline (1:9, w/v ratio) and kept on an ice bath. The homogenates were then centrifuged at 1,000 g at 4 °C for 15 min, and the supernates were collected. Blood samples were collected and the TNF-α levels in serum and supernates was analyzed using a cytokine-specific ELISA kit according to the manufacturer’s protocol.
Biochemical analysis
Homogenates of murine small intestine were prepared using physiological saline (1:9, w/v ratio) and kept on an ice bath. The homogenates were then centrifuged at 1,000 g at 4 °C for 15 min, and the supernates were collected and analyzed for MPO activities and MDA levels. Homogenates of murine liver were prepared using physiological saline (1:9, w/v) and kept on an ice bath. The homogenates were then centrifuged at 1,000 g at 4 °C for 15 min, and the supernates were collected and analyzed for T-SOD, T-AOC and GSH-PX activities. Blood samples were collected and the serum were then centrifuged at 1,000 g at 4 °C for 15 min, and the supernates were collected and analyzed for levels of LZM, ACP and GSH.
Quantification of messenger RNA (mRNA) in lung of mice
Total RNA was extracted from lung tissues by using RNAiso Plus Kit, and RNA was reverse transcribed to cDNA using reverse transcription and amplified by PCR with PrimeScript® RT reagent Kit according to the manufacturer’s instructions. Relative expression of TNF-α、IFN-α、IFN-γ and IL-2 were measured with a SYBER green detection system by using CFX96 Real-Time PCR Detection System (Bio-Rad, USA), Each reaction was run in triplicate and the cycle threshold (CT) values for each mRNA were subtracted from that of β-actin mRNA averaged and converted from log-linear to linear term. The primer sequences used were as follows: TNF-α (NM_013693): forward: 5'-AAGACCTCTATGCCAACACAGT-3' and reverse: 5'-TTTACTCAGTGCCAGAAGCTGGA-3’; IL-2 (NM_008366.3): forward: 5'-CCCAGGATGCTCACCTTCA-3' and reverse: 5'-CCGCAGAGGTCCAAGTTCA-3’; IFN-α (NM_010502.2): forward: 5'-CTGTGCTTTCCTGATGGTCCTG-3’ and reverse: 5'-GGAATCCAAAGTCCTTCCTGTCCT-3'; IFN-γ (NM_00801778): forward: 5'-GCTTTGCAGCTCTTCCTCATG-3' and reverse: 5'-CTTCCACATCTATGCCACTTGAG-3'; β-actin (NM_007393.3): forward: 5'-AAGACCTCTATGCCAACACAGT-3' and reverse: 5'-TTTACTCAGTGCCAGAAGCTGGA-3'.
Histopathology
To characterize the histological alterations, the lungs, ileum and colon of the mice were excised and fixed in 10 % neutral buffered formalin. The tissue samples were dehydrated with graded alcohol, embedded in paraffin, and the sections stained with hematoxylin and eosin were examined by light microscopy.
Cell culture and treatments
Mouse macrophage RAW 264.7 cells were cultured in DMEM supplemented with 10 % heat-inactivated fetal bovine serum,100 units/mL penicillin sodium, 100 μg/mL strepto-mycin and 2 mmol/L glutamine at 37 °C under a humidified atmosphere of 5 % CO2. In all experiments, cells were allowed to acclimate for 24 h before any treatment. Cells were incubated with or without FNP that was added 1 h prior to LPS treatment.
MTT assay for testing cell viability
The examination of cytotoxicity induced by FNP was performed by MTT assay [27]. RAW 264.7 cells were mechanically scraped, plated at a density of 2 × 105 cells /mL into 96-well plates and incubated in a 37 °C, 5 % CO2 incubator overnight. FNP was dissolved in Dimethyl Sulphoxide (DMSO), and the DMSO concentrations in all assays was not exceed 0.1 %. After overnight incubation, the cells were treated with diverse concentrations of FNP (20–400 μg/mL) in the presence or absence of LPS (1 μg/mL) according to the experimental design. After 20 h, 20 μL of 5 mg/mL MTT was added to each well and the cells were further incubated for an additional 4 h. MTT was removed and cells were lysed with 150 μL/well DMSO. The optical density was measured at 570 nm on a microplate reader.
Measurement of NO production
After pre-incubation of RAW264.7 cells (1 × 106 cells/mL) for 18 h, cells were pre-treated with FNP (20–80 μg/mL) for 1 h and were further incubated with LPS (1 μg/mL) for 12 h or 24 h. The inhibitory effect of FNP on NO production was determined by Griess reagent as described previously [28].
Measurement of ROS levels
After pre-incubation of RAW264.7 cells (1 × 106 cells /mL) for 18 h, the cells were pre-treated with FNP (20, 40 or 80 μg/mL) for 1 h and were further incubated with LPS (1 μg/mL) for 12 h or 24 h. The inhibitory effect of FNP on ROS production was determined by fluorescent probe DCFH-DA as described previously [29].
Measurement of inflammatory cytokine values
To investigate the effect of FNP on inflammatory cytokine levels from LPS-treated RAW 264.7 cells, The pre-incubated cells (1 × 106 cells /mL) were pretreated with 20, 40 or 80 μg/mL of FNP for 1 h prior to 1 h treatment with 1 μg/mL LPS at 37 °C, 5 % CO2 incubator. 24h later post LPS treatment, cell-free supernatants were collected and stored at −20 °C until assayed for cytokine levels. The concentrations of TNF-α, IL-6, IL-8, IL-10 and IL-1β in the supernatants of RAW 264.7 cell culture were determined by a cytokine-specific ELISA kit, according to the manufacturer’s instructions.
Western blot analysis
RAW 264.7 cells (2 × 105 cells/mL) were plated in 24-well plates and pretreated with 20 μg/ mL or 80 μg/ mL of FNP for 2 h or 4 h and then stimulated with 1 μg/mL of LPS for 1 h. After incubation, the cells were collected and washed twice with cold PBS. The washed cell pellets were resuspended in extraction lysis buffer (1:10 10 × RIPA Buffer(CST,USA),1:10 cOmplete ULTRA EDTA-free protease inhibitor cocktail tablets (Roche, Swiss), 1:10 PhosSTOP Phosphatase Inhibitor Cocktail Tablets (Roche, Swiss)) and maintained on ice for 30 min. The lysates were centrifuged (12,000 g at 4 °C) for 5 min to obtain the cytosolic fraction and the protein concentrations were determined using a BCA™ protein assay kit (Cwbiotech, China) according to the manufacturer’s instructions. Aliquots of the cell extracts (50 μg of protein) were separated on 10 % SDS-polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane (Roche, Swiss) with a glycine transfer buffer [192 mM glycine, 25 mM Tris–HCl (pH 8.8), 10 % methanol (v/v)]. The membrane was incubated overnight with specific primary antibody(CST,USA) at 4 °C after blocking the nonspecific site with 5 % bovine serum albumin (BSA). The membrane were washed with Tween 20/Tris buffered saline [TTBS, 20 mM Tris–HCl buffer, pH 7.6, containing 137 mM NaCl and 0.05 % (v/v) Tween 20] and incubated an additional 60 min with a peroxidase-conjugated secondary anti-mouse antibody(1:5000 CST,USA) at room temperature. The membranes were washed with TTBS twice and the immunoactive proteins were detected using an enhanced chemiluminescence (ECL) Western blotting detection kit (Merck Millipore, Germany).
Statistical analysis
All experimental data in this study was represented as mean ± SD. All statistical comparisons were conducted using oneway ANOVA followed by Tukey’s test and analyzed using SPSS 16.0 for Windows. Difference was considered statistically significant when P < 0.05, whereas less than 0.01 were considered extremely significant (P < 0.01).