Drugs
XSLJZ decoction was prepared by the Pharmaceutical Department of Xiyuan Hospital in accordance with good manufacturing practices, and included eight Chinese medicinal herbs (Table 1) at a mass ratio of 6:12:12:4:6:5:4:5. All herbs were purchased from qualified suppliers in China and identified as eligible medicinal material. The pure extracts of the components were prepared. The components were dissolved in sterile water. High-dose (11.2g/kg), middle-dose (5.6g/kg) and low-dose (2.8g/kg) of XSLJZ decoction were administered to the rats. Mosapride (1.6mg/kg Lunambett Pharmaceuticals Co. Ltd., China) was administered to rats in the Mosapride group. Meanwhile, the rats in the model and control group received 1mL/100g sterile water daily by oral gavage for 14 consecutive days.
Table 1 Components of the XSLJZ decoction
English name
|
Chinese name
|
Latin name
|
Weight (g)
|
Part used
|
Codonopsis pilosula
|
Dang Shen
|
Codonopsis pilosula (Franch.) Nannf.
|
6
|
Roots
|
Rhizoma atractylodis
macrocephalae
|
Bai zhu
|
Atractylodes macrocephala Koidz.
|
12
|
Rhizome
|
Poria cocos
|
Fu Ling
|
Poria cocos (Schw. ) Wolf.
|
12
|
Sclerotia
|
Liquorice
|
Gan Cao
|
Glycyrrhiza uralensis Fisch.
|
4
|
Roots
|
Pinellia ternata
|
Ban xia
|
Pinellia ternata (Thunb.) Breit.
|
6
|
Tuber
|
Tangerine Peel
|
Chen Pi
|
Citri Reticulatae Pericarpium
|
5
|
Pericarp
|
Costustoot
|
Mu Xiang
|
Radix Aucklandiae
|
4
|
Roots
|
Fructus Amomi
|
Sha Ren
|
Amomum villosum
|
5
|
Fruit
|
Animals
Healthy seven‑day‑old male Sprague-Dawley rats (12-15g) were purchased from SPF Biotechnology Co., Ltd., Beijing, China. The rats were housed in cages maintained on a 12-h light/dark cycle with the room temperature of 22-24℃ and a humidity of 60%-70%. Animal procedures were approved by the Committee on Animal Care and Use of the Institute of Xiyuan Hospital, China Academy of Chinese Medical Sciences, and conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publications No. 85-23, revised 1996).
Generation of functional dyspepsia model
The FD model was established by neonatal gastric irritation combined with modified multiple platform method(MMPM). Previous studies revealed that the model rats exhibited delayed gastric emptying and VH, which are considered to be the major pathophysiological features of FD[19, 20]. As shown in Fig. 1, ten-day old rat pups were randomly divided into 2 groups, including the IA-treated group (n = 40) and control group (n=8). The IA-treated group received 0.2mL of 0.1% iodoacetamide (IA) in 2% sucrose by oral gavage daily for 6 days. The control group received 0.2 ml of 2% sucrose only[21]. All the rats were fed normally until 42 days old when IA-treated rats were randomly divided into five groups (n = 8, each group): model group, mosapride group, low-dose XSLJZ group, middle-dose XSLJZ group, and high-dose XSLJZ group. Then, the 42-day-old IA-treated rats were exposed to MMPM. In brief, the rats were placed on top of the platforms (6.5cm in diameter) inside a water tank. The tanks were filled with water at room temperature until 1 cm of the platform. Exposure to the MMPM began at 17:00, with a duration of 14 hours per day for 14 days. After modeling, rats received 1ml/100g of each drug or sterile water daily by oral gavage for 14 days. At the end of treatment, rats were anesthetized with 1% pentobarbital sodium, and the gastric antrum was excised for the following experiments.
Body weight and food intake
The body weight and food intake of rats were detected before and after the drug treatment. After 12 h of fasting, the rats were housed individually. The food was provided for 24 h, and the food consumption was calculated.
Von Frey Test
The pain threshold in response to Von Frey filament stimulation was measured, as described previously[22, 23]. After acclimation for 30 minutes prior to testing, each rat was placed in a chamber on a platform with 10mm grids of iron wires. A series of Von Frey filaments were applied to the central part of the plantar surface of right hind-paw with the force in ascending gram (1 g, 1.4 g, 2 g, 4 g, 6 g, 8 g, 10 g, 15 g, and 26 g). Each stimulus was repeated 3 times following a 5-min interval. The mechanical pain threshold (MPT) was defined as the lowest force in grams required until the rat withdrew its hind-paw. The mean was calculated for each rat’s pain threshold. The lower the value, the higher the sensitivity of rats to mechanical stimulation.
Hematoxylin-eosin (HE) staining
Gastric antrum tissues were embedded in paraffin after fixation in 10% formalin and cut into 4μm-thick sections. Then, the sections were stained with HE and photographed by a microscope.
Immunohistochemistry (IHC)
Embedded gastric antrum samples were deparaffinized, and antigen retrieved was performed. The sections were treated with 3% H2O2 for 30 min, followed by 5% bull serum albumin (BSA) for 20 min. Then, the sections were incubated overnight at 4°C with the following antibodies: polyclonal rabbit anti-NGF (1:300, Abcam), polyclonal rabbit anti-GFAP(1:200, Proteintech), polyclonal rabbit anti-VR1 (1:100, Abcam). After washing, the sections were incubated with Goat Anti-Rabbit horseradish peroxidase (HRP)-conjugated secondary antibody for 50 min. Diaminobenzidine (DAB) was employed to detect the immuno-complex, and hematoxylin was used for nuclear counterstaining. After dehydration through an ascending alcohol gradient and clarification with xylene, the sections were visualised under a microscope, and images were acquired using a LEICA camera. At least 5 fields per rat and 5-6 rats per group were analysed. Mean integrated optical density (MOD) was calculated using Image Pro Plus version 6.0 analysis software.
Immunofluorescence (IF)
Embedded gastric antrum samples were deparaffinized, antigen retrieved was performed, and the sections were blocked. The sections were incubated overnight at 4°C with polyclonal rabbit anti-NGF (1:300, Abcam) and polyclonal rabbit anti-GFAP (1:200, Proteintech). Following three 5-min washing, the sections were then incubated
with a FITC- labeled goat anti-rabbit antibody and a Cyanine3-labeled goat anti-rabbit
secondary antibody (1:300, Abcam) at room temperature for 60 min. The nuclei were stained with DAPI (Genepool). Images were captured at 400× under a fluorescent microscope (Olympus, Japan).
Western blot analysis
Gastric antrum tissues were homogenized in ice-cold RIPA buffer containing 1 mM PMSF (Servicebio, China), and protein concentration was quantified using a BCA protein assay kit (Servicebio, China). Equal amount of total proteins (50 μg) was loaded on 10% SDS-PAGE gels, and then transferred to PVDF membranes (PALL, USA), The PVDF membranes were blocked with 1×Tris-buffered saline Tween (TBST) containing 5% non-fat milk at room temperature for 1h. Membranes were incubated overnight at 4˚C with the following primary antibodies: anti-GFAP (1:1900, Proteintech), anti-NGF (1:1800, Abcam), anti-TrkA (1:1200, Abcam), anti-Akt (1:2000, Proteintech), anti-VR1 (1:1700, Abcam) and anti-β-actin (1:1500, Abcam). After washing three times with TBST, the membranes were incubated with the following secondary antibodies: Goat anti‑rabbit IgG (1:2800, Abcam) or goat anti‑mouse IgG (1:3200, Abcam) at room temperature for 2h. The protein bands were visualized using enhanced chemiluminescence substrate (Pierce, USA) and the protein levels were quantified with Image J software.
Statistical analysis
SPSS 19.0 software was used for all statistical analyses. All values were presented as mean ± SD. One way analysis of variance (ANOVA) was used to test the statistical significance among the groups. p < 0.05 was considered statistically significant.