JHD preparation
The components of JHD are described in Table 1. To prepare JHD, C. longa L., C. aurantium L., and A. macrocephala Koidz. were distilled for 1–2 h with 60% ethanol three times. The ethanol was filtered and recovered. S. chinensis (turcz.) Baill. was extracted twice with 70% ethanol, and the ethanol was filtered and recovered. S. miltiorrhiza Bge., P. lactiflora pall., A. orientale (sam.) Juzep, and P. lobata (willd.) Ohwi. were extracted with water, and the filtrate was refrigerated and stored for later use. JHD was prepared in the Science and Technology Center of Shanghai University of Traditional Chinese Medicine. The concentration of the crude drug was 0.9 g/mL.
Ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) analysis of JHD
JHD index compounds (puerarin, paeoniflorin, naringin, curcumin, atractylolide, schisandrin a, alisol 23b, and tanshinone) were analyzed using UHPLC-MS. The chromatography method was established on an ultra-fast liquid chromatography (UFLC) system (SHIMADZU, Kyoto, Japan) with an Agilent Eclipse XDB-C18 (4.6.150 mm, 5 µm; Santa Clara, CA, USA) analytical column for chromatographic separation. Mass spectrometric detection was carried out on a 4000 QTRAP (AB SCIEX, Framingham, MA, USA) equipped with an electrospray ionization source. The analysis was performed with a UHPLC-Q/Exactive system (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a quaternary gradient pump, autosampler, and high-resolution mass spectrometry detector. Puerarin, paeoniflorin, naringin, curcumin and atractylolide were analyzed in ESI- mode, schisandrin a, alisol 23b, and tanshinone were analyzed in ESI+ mode.
Animals
Male Sprague-Dawley (SD) rats (n = 30; 10 rats per group) weighing 130–160 g were acquired from Shanghai Bikai Laboratory (Shanghai, China). All animal testing protocols were approved by the Animal Experimental Ethics Committee of Shanghai University of Traditional Chinese Medicine. All rats were kept in a controlled environment at room temperature (25 °C) and for a 12 h light/dark cycle. After 7 d of adapting to laboratory conditions, rats were randomly divided into three groups with each group housed in its own cage.
SD rats were pair-fed a modified Lieber-DeCarli liquid diet (Tables 2 and 3) [19] containing alcohol (model group, n =10; JHD group, n =10) or isocaloric maltose dextrin (control group, n = 10) for six weeks. After three weeks on the respective diets, rats were administrated JHD intragastrically for three weeks. Rats in the JHD group were given JHD. Rats in the control and model groups received equal volumes of double-distilled water. At the end of the experiment, rats were orally administered 10 mg/kg LPS. After 3.5 h, rats were sacrificed and the plasma, liver, and intestinal tissues were collected.
Biochemical assays
The levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (AKP) were measured using corresponding commercial test kits (Nanjing Jianchen Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocols and in accordance with the manufacturer's agreement. Liver tissue (100 mg) was homogenized in 1.5 mL of 1:1 ethanol:acetone (v/v). The liver tissue triglyceride (TG) levels were determined using TG quantification kits (Dongou Bioengineering, Zhejiang, China).
Histopathology
In order to study pathological changes, liver and ileum tissues were fixed in 10% neutral formalin for 48 h. Then, the tissues were embedded in paraffin and sectioned continuously (4 meters thick). The slices were placed on glass slides and stained with hematoxylin and eosin (H&E; Nanjing Jianchen Institute of Bio Engineering, Inc.) and examined under a light microscope (Olympus Medical Systems, Tokyo, Japan). Analyses of liver pathological adjustments were based totally on the percentage of inflammation, necrosis (0 points, 0 foci; 1 point, < 2 foci; 2 points, 2–4 foci; 3 points, > 4 foci; per 200× field), and steatosis (0 points, < 5%; 1 point, 6–33%; 2 points, 34–66%; 3 points, > 66%), which were independently assessed by three examiners [20]. Ileum injury was evaluated by "Chiu" scoring method [21].
Transmission electron microscopy
A small section of the ileum tissue was fixed for at least 4 h in Karnovsky solution (1% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.4). After fixation, the tissue was washed extensively in Veronal acetate buffer (90 mM, pH 6.0), stained with uranylmagnesium acetate (0.5%) in equilibration buffer (at 0 °C for 60 min), washed again, dehydrated, and embedded in paraffin. Then sections were rapidly cut into 0.5 mm cubes with a diamond knife and stained with uranyl acetate and lead citrate for viewing underneath a 200 CX TEM at 80 kV. The ultrastructure of TJs in tissues were evaluated by high magnification photography (11,500×).
Lipopolysaccharide assay
Blood was collected in a pyrogen-free, heparin-pretreated tube and centrifuged at 500 × g at 4 °C for 15 min. The LPS plasma ranges were assessed using a chromogenic kinetic limulus amebocyte lysate assay kit (Associates of Cape Cod, East Falmouth, MA, USA) following the manufacturer’s instructions.
Western blot analysis
An equal amount of protein (30 μg/well) was extracted from each sample, separated by 6–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a nitrocellulose membrane, and blocked for 1 h with a blocking buffer. The membrane was incubated with the appropriate primary antibody (Table 4) at 4 °C. The membrane was then washed three times with phosphate-buffered saline (PBS), then incubated with the appropriate secondary antibody (anti-rabbit IgG H&L). Finally, enhanced chemiluminescence (ECL) exposure was performed. The HP Scanjet XPA scanner was used to scan and save the exposure results, and the FR-980 bioelectrophoresis image analysis system was used to analyze the density integration of each band. Finally, the target western blot density integration and the interior reference blot density integration were calculated. These ratios were then used for quantification.
Immunofluorescence staining
Ileum tissue was embedded in OCT medium (Sakura Finetek, Torrance, CA, USA) and frozen rapidly in liquid nitrogen. Briefly, 8-μm thick cryostat slices were fixed in cold acetone for 15 min, rinsed three times for 5 min each in PBS, then permeabilized in 0.2% Triton X-100 (in PBS) for 5 min. After rinsing in PBS for 5 min, sections were blocked with 3% horse serum for 1 h at 25 °C, then incubated with either anti-occludin (1:100) or anti-ZO-1 (1:50) antibodies overnight at 4 °C. After rinsing three times for 6 min each in PBS, FITC goat anti-rabbit IgG (1:100) was added and incubated at room temperature for 30 min. The sections were then imaged with a laser scanning confocal microscope (OLYMPUS-FV10i, Olympus Corporation).
Quantitative reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was extracted in accordance with the manufacturer's instructions (catalog number: E928KA9723, Sangon Biotech, Shanghai, China), and the RNA concentration was measured. Reverse transcription was carried out in accordance with manufacturer’s instructions. The concentration of the synthesized cDNA was measured and the cDNA was stored at –20 °C. Specific primers for amplifying target genes and endogenous actin (synthesized by Shanghai Shenggong Co., Shanghai, China) are provided in Table 5.
The two-step PCR procedure was as follows: one cycle of pre-denaturation at 95 ℃ for 10 sec, followed by 40 cycles of denaturation at 75 ℃ for 5 sec and amplification at 60 ℃ for 20 sec. The final products were confirmed using 1.5% agarose gel electrophoresis and melt curve analysis. Relative quantitative analysis was carried out using the double standard curve method. Calculation and analysis were carried out using the software program in the Rotor-Gene RG-3000 (Gene Co., Hong Kong, China).
Statistical analysis
Comparisons between groups were performed using one-way analysis of variation (ANOVA). Dunnet's multiple comparison post-tests were used to compare the means of different groups. The results are expressed as mean ± standard deviation. SPSS 24.0 (IBM, Armonk, NY, USA) and GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA) were used for analysis, and P < 0.05 was considered statistically significant.