Reagents
Lipopolysaccharide (LPS), d-galactosamine (D-GalN) and 1,10-phenanthroline were obtained from Sigma Chemical Co. All other chemicals and solvents were of the highest grade commercially available.
Drug preparation
The powder of CMCS (1.0 kg), were provided from Shanghai Sundise Chinese Medicine Technology Development Co., Ltd, and were extracted successively three times with 6 l of water under reflux, and the combined extract were concentrated under vacuum at 50 °C and then dried by lyophilization to afford the extraction of CMCS (150 g). The end extraction contained 150 mg/ml of CMCS drug. The quantitative analyses of active compounds were determined by high performance liquid chromatography (HPLC), which performed on Waters2695 systerm (Waters Corporation, Milford, USA), equipped with an Waters PDA2996 analyzer. Alliance software was used for the data analysis and the result was shown in Fig. 1.
Animals
Male BABL/c mice weighing 18-22 g, specific-pathogen-free (SPF) level, were provided by Sino-British SIPPR/BK Lab Animals (Shanghai, China). They were housed in a room under controlled temperature (22–25 °C), humidity (46-52 %), and lighting (12-hour artificial light and dark cycle), with free access to tap water and mouse chow. The standard diet pellets contained not less than 20 % protein, 5 % fibers, 3.5 % fats, and 6.5 % ash and vitamins mixture. This experiment was conducted according to the local ethical guidelines (Shanghai University of TCM, Shanghai, China).
Experimental design
All the mice were randomly divided into four experimental groups: normal control (n = 8), model control (n = 12), CMCS treatment (n = 10) and 1,10-phenanthroline treatment group (n = 9). Mice in normal control and model control were administered orally by gavage with distilled water once a day, for four consecutive days at a dose of 4 ml/kg of body weight, respectively. Mice in CMCS treatment group were treated orally with CMCS at a daily dose of 120 mg/kg of body weight at the same time, which was equivalent to 10 times of the 60 kg adult dose. In 1,10-phenanthroline treatment group, mice were intraperitoneally injected with 1,10-phenanthroline twice a day, for four consecutive days at a daily dose of 25 ml/kg of body weight. 1,10-phenanthroline was used as the positive control medicine and its dosage was based on our previous trial experiment. On the fourth day, 1 h after the last intragastrical administration, the normal control group was received intraperitoneal injections of saline at a dose of 2 ml/kg of body weight. The other mice were received intraperitoneal injections of LPS and D-GalN at the dose of 10 μg/kg and 900 mg/kg of body weight respectively [7]. All mice were sacrificed 7 h after the last injection.
Serum levels of liver function
Measurement of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured by using SpectraMax-M5 Multifunctional microplate reader (Molecular Devices, Inc., Sunnyvale, California, USA) according to the manufacture’s instructions. Liver function tests kits were supplied by Nanjing Jian Cheng Bioengineering Institute (Nanjing, China).
Parameters for peroxidative damage in liver
Hepatic homogenates were centrifuged at 3,000 r/min for 20 min at 4 °C. Supernatants were immediately collected and assayed for enzyme activities. Levels of antisuperoxideanion (ASAFR), hydroxyl free radical (·OH), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione S-transferase (GST) were assayed according to the protocols of kits purchased from NanJing Jian Cheng Bioengineering Institute. All these parameters were expressed by gram protein which was determined by the BCA protein assay kit (Pierce, Thermo Scientific, Rockford, USA) according to the manufacturer’s protocol using bovine serum albumin as a standard.
Histopathology
Liver specimens were fixed in 10 % formaldehyde solution and dehydrated in a graded alcohol series, embedded in paraffin blocks, and cut into 4 μm thick slices. Slices for histopathological examination were stained by using standard procedure of hematoxylin and eosin (H&E) and silver, respectively.
TUNEL staining
Fresh liver tissue was fixed in 10 % formaldehyde solution, embedded in paraffin, and sliced into 4-μm sections. Induction of apoptosis was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) (ApopTag Peroxidase In Situ Apoptosis Detection Kit, S7100, Merck Millipore, Chemicon International, Inc, Billerica, MA). The apoptotic cells were stained brown, and nuclei were counterstained with Hematoxylin. TUNEL staining were analyzed with light microscope (Olympus BX40, Japan). To quantify the histologic findings, semi-quantification data for apoptosis in TUNEL staining were determined with the computer-assisted image analysis system.
Besides, some of the liver tissues were embedded in OCT compound and stored at −70 °C. The frozen tissue block was sectioned using a crytome (Leica CM1850, Germany). Ten-micrometer-thick cryostat sections were cut and transferred to poly-l-lysine-coated slides. Cell apoptosis detection was performed with the one step TUNEL kit (Beyotime, Jiangsu, China) according to the manufacturer’s protocol. Briefly, the sections were permeabilized with 0.1 % Triton X-100 for 2 min at 4 °C followed by TUNEL for 1 h at 37°. After washing, tissues were double stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Images were obtained using a confocal microscope (Fluoview FV10i, Olympus, Japan) equipped with the Ultraviolet/Visible light LD laser combination. Photographs were taken with Olympus confocal software.
Liver perfusion and processing for ultrastructural analysis
Livers were thoroughly cleared by perfusion with phosphate-buffered saline (PBS) of room temperature through the portal vein at a flow rate of 3 ml/min. One minute later, 2.5 % glutaraldehyde was perfused for an additional one minute at the same flow rate [8]. Subsequently, livers were carefully removed and quickly immersed in 2.5 % glutaraldehyde for 48 h at 4 °C as described previously [9].
For transmission electron microscope (TEM), several 1-mm3 cubes were harvested from the liver, washed three times in PBS, and fixed in aqueous 1 % osmicacid, 1 % potassium hexacyanoferrate (III) Red prussiate of potash for 1 h. After another three times washes, blocks were dehydrated through a graded series of 30–100 % ethanol, 100 % propylene oxide, and infiltrated for 1 h in a 1:1 mixture of propylene oxide: Polybed 618 epoxy resin (Shanghai Resin Factory Co. Ltd., Shanghai, China). After several changes of 100 % resin over 24 h, blocks were embedded in molds and cured at 37 °C overnight, followed by additional hardening at 65 °C for 48 h. Ultrathin (60 nm) sections were collected onto 200-mesh copper grids, stained with 2 % uranyl acetate in 50 % methanol for 10 min and 1 % lead citrate for 7 min, respectively. Sections were photographed with a Tecniai-12 transmission electron microscope (Philips, Amsterdam, The Netherlands).
For scanning electron microscopy (SEM), the 3-mm-thick fragments were cut from the fixative, washed three times with PBS, and then immersed in aqueous 1 % Osmicacid for 2 h. After three times washes in PBS, slices were dehydrated through a graded series of 30–100 % ethanol. Before critical point drying, washing with absolute ethanol was necessary. Slices were mounted onto aluminum stubs, sputter coated with gold/palladium, and viewed in a XL-30 scanning electron microscope (Philips, Amsterdam, the Netherlands) at 20.0 kV [10].
Immunofluorescence staining
Frozen tissues embedded in OCT compound were cut into ten-micrometer thick and fixed in 4 % formaldehyde (Dingguo, Shanghai, China). After washing, sections were incubated with primary antibody for 1 h at 37 °C in a moist chamber. Rabbit anti-human von Willebrand Factor antibody (ab6994) was purchased from Abcam and used as the primary antibody at a dilution of 1:200 to examine the integrity of endothelial cells. To visualize the primary antibodies, cells were stained with Cy3-conjugated secondary antibodies. After washing, cells were double stained with DAPI to visualize the nuclei. Images were obtained using a confocal microscope and photographs were taken with Olympus confocal software. The semi-quantification data for vWF protein level in the liver tissue were determined with the computer-assisted image analysis system.
Liver MMP-2/9 activities assay
MMPs activities in liver tissue were analyzed by gelatin zymography and in situ fluorescent zymography respectively. MMPs zymography assay was modified as previously described [11]. Briefly, liver tissues were homogenized and supernatant was aliquoted according to protein concentrations determined as the same procedures mentioned above. Aliquots (30 μg protein/lane) of liver tissue were prepared by dilution in the zymogram sample buffer (5 ×) containing 0.4 mol/l Tris, pH 6.8, 5 % SDS, 20 % glycerol and 0.03 % bromphenol blue, separated with electrophoresis in 10 % SDS-PAGE containing 1 mg/mL gelatine as a substrate under non-reducing conditions. Afterwards, gels were washed in the reaction buffer containing 50 mM Tris–HCl, 5 mM CaCl2, 1 μM ZnCl2 and 2.5 % Triton-X 100 (pH 7.5). The reaction buffer was changed to a fresh one, and gels were incubated at 30 °C for 18 h. Gelatinolytic activity was visualized by staining the gels with 0.1 % Coomassie brilliant blue G-250, destained with 30 % methanol/20 % acetic acid water and destained with 30 % methanol/10 % acetic acid respectively. Clear zones in the background of blue staining exhibit the presence of gelatinase activities. The intensity of the bands was scanned and assayed by Furi Gel Image software (Furi, Shanghai, China).
Fluorescent in situ zymography of liver section was performed according to Ben Wielockx’s methods [12] with modifications. Briefly, 1 mg/ml fluorescein-conjugated gelatine (Molecular Probes, USA) solution is diluted 1: 10 in the agarose-containing solution. Liver sections were mounted to the glass slide with the gelatin agarose mixture and incubated with 40 μg/ml in 0.5 mol/l Tris–HCl (pH 7.6), 50 mmol/l CaCl2 and 1.5 mol/l NaCl for 6 h at 37 °C. Sections were washed three times with water, and subsequently Nuclei was counterstained by adding Hoechst (Beyotime, Haimen, China). Gelatinase activity in situ was visualized using Olympus fluorescent microscopy.
Western blot analyses
Western blot analyses were performed essentially as described [13]. Snap-frozen liver tissues were homogenized in RIPA lysis buffer containing 150 mM NaCl, 1 % NP-40, 0.1 % SDS, 50 mM Tris–HCl pH 7.4, 1 mM EDTA, 1 mM PMSF and 1× complete mini (Roche). Lysates were centrifuged at 10,000 g at 4 °C for 15 min to separate cytosolic-enriched supernatant from nuclei- and membrane-enriched pellets. Protein concentrations were determined using BCA protein assay kit mentioned above. Equal amount of proteins were separated by 10 % SDS gel electrophoresis (SDS-PAGE) under denaturing and non-reducing condition, and transferred to nitrocellulose membrane. Even transfer was confirmed by staining with 0.2 % Ponceau Red S in 3 % trichloroacetic acid. Membranes were blocked with 5 % nonfat milk in TBST (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1 % Tween 20) at room temperature for 1 h, incubated with primary antibody ICAM-1(1:1,000; Santa Cruz Biotechnology, CA) and VCAM-1 (1:1,000; Santa Cruz Biotechnology, CA) at 4 °C overnight, respectively. After washing with TBST, blots were incubated with horseradish-coupled secondary antibody in wash buffer. Signals were developed with the immunoreactive bands were visualized using ECL kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer’s instructions and quantified using a ChemiDoc image analyzer (Bio-Rad, Hercules, CA).
Statistical analysis
All data were expressed as mean ± standard deviation (SD). Differences between the groups were assessed by nonparametric One-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc tests. Statistical significance was taken at the p < 0.05 level.