Materials
Primary anti-P-Akt and AKT antibodies were purchased from Signalway Antibody (USA), ZO-1 and Occludin (02902) antibodies were purchased from ProteinTech (USA). The Biotinylated Goat Secondary Antibody (G23303) was purchased from Servicebio (Wuhan, China). Beta-actin (#8457) weas purchased from Cell Signaling Technology (Cambridge, MA, USA). The mouse GLP-1 ELISA Kit and 5-HT ELISA Kit were purchased from cloud-clone (Wuhan, China). The mouse IL-6 ELISA kit and IL-10 ELISA kit were purchased from Dakewe Biotech Co. The CFX96 real-time PCR instrument was from Bio-Rad (USA), and the Powerlab 8/35 data acquisition and analysis system were from AD Instruments (Australian).
Extraction of herbal components
Dried Artemisia capillaris, Astragalus propinquus, Phellodendron chinense, Salvia miltiorrhiza, Poria cocos, and Anemarrhena asphodeloides (Shengsheng Traditional Chinese medicine Yinpian, Shaanxi) were extracted individually under reflux using deionized water for 1 h and the extraction were repeated once. The extracts were filtered, centrifuged to remove undissolved particles and freeze-dried into powder. The extracts were mixed in a certain proportion and dissolved in normal saline and stored at 4℃.
Animals and animal treatment
Male C57 mice (4 - 6weeks old) were purchased from the Experimental Animal Center of the Fourth Military Medical University and housed in cages under controlled temperature (24 ± 2℃) and 12 h light/12 h dark cycles with free access to water and chow. All animal procedures were approved by the Laboratory Animal Center and Animal Care and Welfare Ethics Committee of the Fourth Military Medical University.
Mice were randomly divided into the blank control group, the high-fat control group and the high-fat drug group (26 g/ kg per day, 13 g· kg-1-day -1 and 6.5 g· kg-1-day -1) (n = 10). The blank control group was given normal diet and the high fat control group were given high-fat diet (Research Diets, USA). After 8 weeks, 6.5 g/kg, 13 g/kg and 26 g/kg herbal extracts were given daily by gavage to the high-fat drug group while the high-fat control group was given normal saline. Dietary patterns remained the same during that time. In the early experiments, it was found that there was no significant difference between the high-dose group and the middle-dose group, while the therapeutic effect of the low-dose group was relatively weak. Therefore, after comprehensive consideration, we finally selected the middle-dose group (13g/kg) for subsequent experiments.
After 12 weeks of the dietary treatments, mice were euthanized by cervical dislocation. Orbital blood was collected, and plasma was obtained by centrifugation at 3,000 rpm/min for 10 min at room temperature. The intestine was dissected, measured, divided into its different portions (large intestine, small intestine and colon). The liver and fat were also harvested. Tissues were rinsed with PBS containing protease inhibitors, frozen in liquid nitrogen, and then stored at −80°C until further analysis.
Small intestine motor function experiment
This experiment studied the improvement effect of the drug on small intestine motor ability of mice. The contractility of the small intestine of mice under the same pressure and different drugs was measured by Power Lab polysomnography. Acetylcholine (ACH, Sigma, USA) excites the gut, whereas atropine (Sigma, USA) would inhibit this effect. By the ability of the gut to respond to ACH and atropine we assessed the motor function of the gut. The small intestine was cut into several pieces, put in good physiological salt buffer. Selected the intestinal segment with the best spontaneous peristalsis, fixed it on the pressure sensor, gave 0.5 g pressure, let the intestine adapt to the buffer for a period of time, then added 10-3 mM atropine into the buffer, then recorded the maximum contraction of the mouse intestine and then replaced the buffer. When intestine movements were stable, added the 10-4mM ACH into buffer, and waited for the waveform to stabilize, determined the maximum diastolic value of the mouse intestine, and changed to a new buffer to continue the next group of experiments.
Fecal microbiome transplantation (FMT)
To eliminate as much bacteria in the gut of SPF mice as possible, penicillin (Sigma-Aldrich, 2000 U/ml) and streptomycin (Sigma-Aldrich, 2 mg/ml), which were typical spectrum antibiotics, dissolved in sterile distilled water in the final concentrations described. The mixed solution weas administered to mice (500 μl per mouse) by oral gavage lasted for 3 days. Feces were collected from formula-treated mice under high fat diet and stored at -80℃ until use. The feces were dissolved in 1.5 ml PBS homogenized, and then filtered through a 100-μm nylon mesh strainer. The filtered contents were resuspended with PBS in a total volume of 2 ml, which 200 μl per mouse was administered to obesity mice by oral gavage.
Glucose tolerance tests in mice
The mice that had to be treated began fasting the day before and could not stop drinking water. After 16 hours, each mouse in each group was marked, removed from the cage, and the fasting blood glucose was measured with a glycemeter (Sigma, USA). The measured value was taken as the blood glucose value at 0 min. These steps were performed as carefully as possible to prevent the mice from excessive fright. After the mice were undisturbed for 30 minutes, the weight was determined, and glucose was injected intraperitoneally at 0.01 ml/g and the timing were started. Blood samples were taken at 15, 30, 60, 90 and 120 minutes to detect blood glucose levels. At the end of the experiment, feed was added to each cage and the experimental results were analyzed by software.
Insulin tolerance tests in mice
After the mice had fasted for 4 hours, each mouse in each group was marked, removed from: the cage and the fasting blood glucose was measured with a glycemeter. The measured value was taken as the blood glucose at 0 min. After the mice were undisturbed for 30 min, the weight was determined and the mice were intraperitoneally injected with insulin at a dose of 0.01 ml/g. The timing was started. The blood glucose of each mouse was measured at 15, 30, 45 and 60 min.
Quantitative PCR
For quantitative PCR analysis, mice tissues were homogenized respectively and RNA was extracted using the Trizol Reagent (Tiangen Biotech, China) according to the manufacturer’s protocol. Briefly, 40 mg colonic tissues of each sample added 1mL Trizol Reagent. In order to fully homogenize, the colon tissue was homogenized in a mechanical homogenizer until no large tissue mass was observed. Lithium chloride method was used to remove all polysaccharides including residual DSS contaminants. cDNA was synthesized with Reverse Transcriptase kits (Takara, Japan). Real-time PCR was performed using SYBR Green (Vazyme, China), and data were acquired in the ABI fluorescence quantitative PCR apparatus (7500, USA) and analyzed using the comparative Ct method. The primers were listed in Table 1. Target-gene transcription of each sample was normalized to the respective levels of β-actin.
Western blot analysis
A 1-cm section of colon tissue was immediately frozen in liquid nitrogen and stored at -80℃ before homogenization in cell extraction buffer (Thermo-Fisher Scientific, NSW, Australia) supplemented with 1 mM Phenylmethanesulfonyl fluoride (PMSF) (Sigma, USA) and protease inhibitor cocktail (Sigma, USA). The total protein was obtained and quantified by a BCA procedure. The wasolated proteins were separated by SDS-PAGE electrophoresis. Transferred onto NC membranes and blocked with 5% skim milk in Tris buffer saline-Tween 20 (TBST) and washed three times for 5 min each time. Then incubated overnight at 4℃ with respective antibodies. After washed with TBST three times for 15 min, the membranes were incubated with HRP-conjugated secondary antibodies (1:10000) for 1 h at room temperature. Then, the membrane was washed in TBST and detected by the ECL chemiluminescence detection system (MIniChemi 610, China).
16S rRNA gene amplicon sequencing
PCR amplification of the bacterial 16S rRNA genes V4–V5 region was performed using the forward primer 515F (5’-GTGCCAGCMGCCGCGGTAA-3’) and the reverse primer 907R (5’-CCGTCAATTCMTTTRAGTTT-3’). Sample-specific 7-bp barcodes were incorporated into the primers for multiplex sequencing. The PCR reactions contained 5 μl of Q5 reaction buffer (5×), 5 μl of Q5 HighFidelity GC buffer (5×), 0.25 μl of Q5 High-Fidelity DNA Polymerase (5 U/μl), 2 μl (2.5 mM) of dNTPs, 1 μl (10 uM) of each primer, 2 μl of DNA Template, and 8.75 μl of ddH2O. Thermal cycling consisted of an initial denaturation at 98 °C for 2 min, followed by 25 cycles consisting of denaturation at 98 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final extension of 5 min at 72 °C. PCR amplicons were purified using Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and paired-end 2×300 bp sequencing was performed using the Illlumina MiSeq platform with MiSeq Reagent Kit v3 at Shanghai Personal Biotechnology Co., Ltd (Shanghai, China)
Statistical analysis
Statistical analysis data were expressed as means ± SEM. Statistical analysis and significance analysis were performed using one-way ANOVA. Post-mortem testing was performed using GraphPad Prism software version 6.0 (GraphPad Software, USA). P-values <0.05 were considered statistically significant.