DMN and Sirius red were purchased from Sigma-Aldrich (Saint Louis, MO, USA). ApopTag Fluorescein in Situ Apoptosis Detection kit (S7110) was purchased from Chemicon International Inc. (Temecula, CA, USA). For primary antibodies used in immunostaining and western blot, anti-caspase-3 rabbit antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), used at 1:1000 dilution; anti-CD68 mouse antibody purchased from AbD Serotec (Kidlington, UK), used in 1:100 dilution; anti-TNF-a and anti-IL-1β rabbit antibodies from Chemicon Internat. Inc., diluted to 0.2 μg/ml; anti-MIP-1 rabbit antibody from BioVision, Inc. (Milpitas, CA, USA), used to 0.2 μg/ml; anti-a-SMA mouse antibody from Sigma, used at 1:400 dilution; and anti-TIMP-1 and TIMP-2 from Lab Vision, Thermo Scientific, Kalamazoo, MI, USA), diluted to 2 μg/ml. Anti-TGF-β1 was obtained from R&D Systems, Inc. (Minneapolis, MN, USA), used at 1:1000 dilution,anti-HGF rabbit antibody from Enzo Life Sciences, Inc. (Farmingdale, NY, USA), used in 5 μg/ml dilution; anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GADPH) mouse antibody from Kangchen Bio-tech Inc. (Shanghai, CN) diluted by 1:5000; the secondary fluorescence-labeling goat anti-mouse FITC, Cy3 antibody from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA), used at 1:1000 dilution; and labeled goat anti-mouse isotype-specific antibody Alexa Fluor 647 immunoglobulin G (IgG)2a from Molecular Probes (Life Technologies, Grand Island, NY, USA), used at 5 mg/ml.
All procedures using animals were carried out in accordance with the guidelines presented in the “Principles for the Care and Use of Animals in the Field of Physiological Sciences,” published by the Physiological Society of China. The study protocol was approved by an ethics committee of Shanghai University of Traditional Chinese Medicine’s Animal Ethics Committee (Shanghai). Forty male Wistar rats (180-200 g) were housed in an air-conditioned room at 25°C with a 12 h light/dark cycle, were randomized into two groups, a control (n = 10) and a DMN-treated group (n = 30). DMN was administered at 10 mg/kg intraperitoneally to rats for 3 consecutive days each week for 4 weeks ; control rats received equal quantities of physiological saline. At the end of the second week, 3 and 6 rats from the control and DMN-treated groups, respectively, were sacrificed for fibrosis development assessment. The remaining DMN rats were further randomized into 2 groups, a DMN-water (n = 12) and a DMN-HQD group (n = 12). With continued weekly DMN treatment, the rats received a daily administration of water or HQD given intragastrically at 1 ml/100 g. At the end of the fourth week, all animals were sacrificed and liver samples collected for subsequent investigations.
Preparation of HQD
HQD consists of crude slices from Radix Astragali and Radix et Rhizoma Glycyrrhizae mixed in a 6/1 ratio (wt/wt). The herbal medicine was accredited by pharmacognosist and prepared by Shuguang Hospital. Specifically, the medicinal herbs mixture was extracted in boiling water and the resulting aqueous extracts dry-sprayed to obtain a powder and then stored at −20°C. The extract powder was weighed and used for experiments by dissolution in pure water at the desired concentrations.
Liver specimens were preserved in 4% paraformaldehyde, dehydrated in a graded alcohol series, embedded in paraffin blocks, sectioned to 5 μm-thick slices, placed on glass slides, and stained with Sirius red. Fibrosis was graded according to the method by Scheuer as follows: grade 0, normal liver; grade 1, increased collagen without formation of septa (small satellite expansion of portal fields); grade 2, formation of incomplete non-interconnecting septa, from portal tract to central vein; grade 3, complete but thin interconnecting septa, which divide the parenchyma into separate fragments; and grade 4, complete cirrhosis, similar to grade 3 but with thicker septa . Three pathologists blind to the rats' treatment assignments performed pathological examinations. Fibrosis scores were given after thorough examination of three different areas of the tissue slide from each rat.
Hepatic hydroxyproline content
Liver tissue (100 mg) was prepared for hydroxyproline (Hyp) determination using to a modified version of a method developed by Jamall . Hyp liver content served as an indirect measure of tissue collagen content, expressed as μg/g wet weight (μg/g).
Western blot analysis
Liver samples were prepared in radio immunoprecipitation lysis buffer containing protease inhibitors (Mini Protease Inhibitor Cocktail cOmplete, Roche Applied Science, Tokyo, JP) and phenylmethylsulfonyl fluoride (Ameresco Inc., Solon, OH, USA). After protein quantification, equal amounts of protein (50 μg/lane) were separated by 10 or 15% polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P transfer membranes (Millipore, Billerica, MA, USA), which were then blocked and exposed to antibodies. The antigens were visualized using an ECL kit for 1 min followed by exposure to Kodak film.
After deparaffinization and dehydration, microwave antigen retrieval was performed for 5 min prior to peroxidase quenching with 3% H2O2 in phosphate buffered saline (PBS) for 15 min. Subsequently, sections were preblocked with 5% bovine serum albumin (BSA) for 30 min and incubated with a primary antibody (anti-HGF, diluted to 5 μg/ml in PBS) overnight at 4°C. A negative control was treated as the other samples except that primary antibodies were replaced with PBS and no staining took place. After washing in PBS, sections were incubated with biotinylated secondary antibody for 30 min and then stained with 3,3’-diaminobenzidine (Vector Laboratories, Inc., Burlingame, CA, USA) for 2–5 min. Slides were finally counterstained with hematoxylin for 2–3 min, mounted, and examined.
Gelatinase activity assay
Liver proteins (50 μg/lane) were electrophoresed in 10% sodium dodecyl sulfate (SDS)-PAGE containing 0.1% gelatin (Sigma-Aldrich), the SDS removed by soaking the gels in buffer containing 50 mM Tris (pH 7.6), 10 mM CaCl2, and 2.5% Triton X-100 (Sinopharm Chemical Reagent Co., Ltd, Shanghai, CN) for 20 min, followed by the same buffer containing 1% Triton X-100. Digestion was allowed to occur at 37°C for 24 h and gels then stained with Coomassie brilliant blue and destained until clear bands became evident.
Liver samples were excised, and immersed immediately in cryomatrix (Tissue-Tek OCT, Sakura Finetek USA, Inc., Torrance, CA, USA), and flash-frozen in liquid nitrogen; sectioned to 5 μm slices, and mounted on slides. Slides were then incubated in 5% BSA for 30 min and followed by incubation with primary antibody (anti-α-SMA, CD68) at 37°C for 1 h. Slides were then washed 3 times with PBS and incubated with secondary antibody for 30 min.
Confocal microscopy for detection of hepatocyte apoptosis detection and KC and HSC interactions
First, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay were performed using a commercially available kit (Chemicon, Internat. Inc.) following the manufacturer’s instructions. then, slides were incubated in 5% BSA for 30 min, and followed by incubation with primary antibody (anti-Heppar) at 37°C for 1 h.washed 3 times with PBS and they were incubated with secondary antibody (Cy3-conjugated affinipure goat anti-mouse) for 30 min.
For double-color labeling of CD68 and α-SMA, liver sections were incubated with monoclonal anti-CD68 for 1 h at room temperature, biotin-conjugated anti-mouse IgG1 (A85-1, BD Pharmingen Inc., San Diego, CA, USA) was then added, and the mixture was incubated for 30 min. The staining reaction was developed with a Cy3-conjugated streptavidin incubation for 30 min and, after washing, the sections were incubated with α-SMA antibody followed by Alexa Fluor 647-labeled goat anti-mouse isotype-specific antibody and then covered with mounting medium.
All results were expressed as mean and standard deviation. Measurement data were analyzed using a one-way analysis of variances (ANOVA, SPSS, Inc., Chicago, IL, USA). Rank data were analyzed with ridit. Groups were compared using ANOVA with Dunnett’s multiple comparison test. Results with p < 0.05 were considered to be statistically significant.