2.1 Experimental animals
A total of 120 healthy and clean male Sprague–Dawley rats (90–120 g) were purchased from the Laboratory Animal Center of Xi’an Jiao Tong University (Xi’an, Shaanxi, China), acclimated to the research laboratory for five days before experiments, and maintained in a light-controlled room (12 h light/dark cycle) at an ambient temperature of 25 °C with free access to water and standard chow. The experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996, Nat. Acad. Press) and approved by the Xi’an Jiao Tong University (XJTU20121210009) Institutional Animal Care and Use Committee (IACUC).
2.2 Experimental materials
Seven crude herbs and polyene phosphatidylcholine (Sanofi, China Pharmaceutical Co., Ltd) were purchased from the pharmacy of the First Affiliated Hospital of Xi’an Jiao Tong University. Lipopolysaccharide (LPS from E. coli 055:B5) was purchased from Sigma–Aldrich Chimie Company (Fallavier, France). Apoptosis detection kit was obtained from Biosynthesis Biotechnology Co., Ltd (Beijing, China). Carbon tetrachloride analytical reagent (AR) was purchased from Chemical Reagent Branch of Tianjin Zonghengxing Industrial and Trading Co., Ltd. Olive oil AR was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Rat endotoxin quantitative detection kit was procured from Beijing Jinshanchuan Technology Development Co., Ltd. Isoflurane was purchased from Forane, Abbott Laboratories, Abbott Park, IL, USA. The cardiac pacing wires were obtained from Medtronic, Minneapolis, MN, USA. The aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TB), albumin (ALB), and prothrombin time (PT) commercial kits were purchased from Sigma–Aldrich, USA.
2.3 Experimental methods
2.3.1 Reagent preparation
1) CCL4 oil solution preparation: A volume of 40 mL CCL4 was solubilized in 60 mL olive oil; and 40% CCL4 oil solution was prepared and stored at room temperature.
2) XCHD (Su et al., 2014): It consisted of seven crude herbs (herbs implementing standards: Pharmacopoeia of the People’s Republic of China 2015 first edition). These herbs were mixed and decocted three times (100 g/1000 mL the first time; 100 g/400 mL the second time; 100 g/400 mL the third time) with water for 30 min, as described previously. The decoction was filtered through the gauze; the filtrate was concentrated as 4.45 g/mL.
Table 1. Herbs of XCHD
Chinese name
|
Botanical name
|
Lot no.
|
Dosage (g/day)
|
Chaihu
|
Radix Bupleurum scorzonerifolium Willd.
|
20160408; Shannxi, China
|
12
|
Huangqin
|
Scutellaria baicalensis Georgi
|
C3022004001; Shanxi, China
|
9
|
Banxia
|
Pinellia ternata (Thunb.) Makino
|
343200401; Gansu, China
|
9
|
Renshen
|
Panax ginseng C. A. Mey
|
343200401; Jilin, China
|
9
|
Zhigancao
|
honey-fried Radix Glycyrrhiza uralensis Fisch
|
20160301; Gansu, China
|
6
|
Shengjiang
|
Zingiber officinale Roscoe
|
C0512005001; Yunnan, China
|
6
|
Dazao
|
Ziziphus jujuba Mill
|
20160112; Xinjiang, China
|
9
|
2.3.2 Protocol for surgery
After an overnight fast, under anesthesia with the inhalation of 1.5–2.0% isoflurane, the hair was shaved, the skin on top of the corresponding position was cut open, and then one pair of cardiac pacing wires was implanted in the serosal surface of the ileum about 4 cm proximal to the ileocecum for ileum slow-wave recording. The distance between the two electrodes in the pair was 0.3 cm. After the isolated lead wires were tunneled and externalized on the rat’s neck, buprenorphine (0.05 mg/kg) and cefazolin (30 mg/kg) was administered for 2 days to alleviate postoperative pain and prevent infection, respectively. The rats were housed individually to avoid the wires and tubes from being chewed off by other rats. The experiments were initiated after the rats were completely recovered from the surgery, usually 7 days after the operation.
2.3.3 Protocol for the making of CCL4-induced ACLF model (Figure 1)
After 7 days of conventional adaptive feeding, 110 rats were used to establish the model. Then, 10 rats were used as the normal control group. During the initial 4-week treatment, 40% CCL4 olive oil solution was injected subcutaneously at 1.5 mL/kg body weight every three days. Then, in the following two weeks, the 40% CCL4 olive oil solution was injected subcutaneously at 2 mL/kg body weight every three days. After the establishment of the cirrhotic rat model, lipopolysaccharide (LPS)- and D-galactosamine (D-Gal)-induced ACLF models were constructed. On day 45, the rats were injected LPS at a dose of 10 mg/kg and D-Gal at a dose of 700 mg/kg intraperitoneally, which induce acute liver failure based on chronic liver injury.
The normal control rats were administered physiological saline at the time points same as in the model rats. The animal behavior was closely monitored during the modeling.
2.3.3 Method of administration
During modeling, 110 rats were randomly divided into model control group with 30 rats and four treatment groups with 20 rats in each group: polyene phosphatidylcholine (100 mg/kg; PP) group, high-dose (44.5 g/kg; HXCHD), middle-dose (26.5 g/kg; MXCHD), and low dose (8.5 g/kg; LXCHD) Xiao Chai Hu decoction. In the model and control groups, rats were given an equivalent volume of water by gavage daily.
2.3.3.1 Specimen collection and preparation
On day 44, five rats were selected randomly in the ACLF model group, and their liver histopathology (included) was detected to determine the cirrhosis. After an acute attack, 0.6 mL blood samples withdrawn at 2 h, 10 h, and 18 h were collected from the inner canthus in all rats to detect the serum biochemical levels. After 47 days of treatment, the colon slow-wave was measured. Then, all the surviving rats were sacrificed for specimen collection. One part of the liver tissue was cut and embedded in paraffin for hematoxylin and eosin (H&E) and Masson’s trichrome staining.
2.3.3.2 Analysis of liver disease progression
1) Serum levels of liver functionality measurements
The serum was obtained by centrifugation of blood samples at 1500 ×g for 10 min at 4 °C and stored at room temperature for 1 h and then at 20 °C until further analysis. The activity of AST, ALT, TB, ALB, and PT was measured using commercially available kits (Sigma–Aldrich), according to the manufacturer’s instructions.
2) Survival rate and rat-adapted model of end-stage liver disease (MELD) score (Said et al., 2004)
Based on the literature, the rats’ MELD was adapted as follows: Rat-adapted MELD score = 0.957×Loge (creatinine mg/dL) + 0.378×Loge (bilirubin mg/dL) + 1.120×Loge ( INR) + 0.643. MELD is a scale system to score the severity of liver disease and predict death. A high MELD score indicates a high probability of death.
3) H&E staining, Masson’s trichrome staining, and image analysis
The H&E staining and Masson’s trichrome staining revealed hepatic lobule structures and hepatic cords. In Masson staining, blue color indicated hyperplastic collagen fibers. Five low-power (×40) fields were randomly assessed for each slice. The ratio of the area of hepatic hyperplastic collagen fibers (AHHCF%) was calculated by Image Pro-Plus 6.
2.3.3.3 Mechanisms of XCHD involving endotoxin-induced hepatocyte apoptosis
1) Detection of serum endotoxin and TNF-α levels with two-step double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).
According to the manufacturer’s description 2.5–80 pg/mL was used in ELISA to detect and quantitatively analyzed the serum endotoxin and TNF-α levels of rats in all the groups. A volume of 100 μL samples was dispensed in each well in 96-well plates after pre-coating with rat TNF-α/endotoxin antibody for 30 min at 37 ℃. Then, a protein-specific biotinylated antibody was incubated for 2 h at 37 ℃. Between each reaction, the unbound proteins were washed away from the wells. Then, substrate solutions A and B were added to each well, and the reaction incubated for 20 min at 37 ℃. The absorbance was measured at 450 nm in a microplate reader, and the concentration data expressed as a mean value.
2) Detection of hepatocyte apoptosis by terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL)
According to the manufacturer’s instructions, TUNEL method was used to detect cellular apoptosis on the liver tissue. The sections were fixed in ethanol-acetic acid (2:1), incubated with proteinase K (100 μg/mL), rinsed in PBS, incubated in 3% H2O2, and washed with phosphate-buffered saline (PBS) for 10 min, followed by permeabilization (0.1% Triton X-100, 0.1% sodium citrate) for 5 min. Subsequently, the sections were washed and incubated in TUNEL reaction mixture. Converter-POD with 0.02% 3,3’-diaminobenzidine (DAB) was used for visualization, and Mayer’s hematoxylin was used for counter-staining. TUNEL-positive cells were dyed as the yellow nucleus. The liver cells were counted under high-power (×400) field. Five high-power fields were examined in each case, and the number of TUNEL-positive cells was counted. Finally, 500 cells were counted in each field, and the apoptotic index (AI%) of each group was calculated.
2.3.3.4 Mechanisms of XCHD involving gastrointestinal motility
After overnight fasting, each rat was fed 2 g of solid dry food at the end of the experiment. A volume of 1.5 mL phenol red (0.5 mg/mL) was mixed with 1.5% methylcellulose was delivered by gavage after 10 min. Then, the rat was euthanized, the content of the stomach and the small intestine was collected for measuring the gastric emptying (GE) (Lin et al., 2018) and small intestinal transit (SIT) (Ohno et al., 2006), using a previously established method.
The percentage of GE was described as the ratio between the amount of undigested food in the stomach and 2 g of solid dry food. The small intestine was cut into 10 equal segments. The SIT was assessed using the geometric center based on the amount of phenol red in each of the segments (Scarpignato et al., 1980).
2.4 Statistical methods
SPSS16.0 statistical analysis software package (IBM, Armonk, NY, USA) was utilized. Continuous data were expressed as mean ± standard. One-way analysis of variance (ANOVA) was performed, and q test (Student–Newman–Keuls test, S-N-K) was used for pairwise comparisons. Kaplan–Meier method was used to calculate the survival rate. Two-sided P-values < 0.05 were considered statistically significant.