Animals
A total of 30 male SPF KM mice and 30 female SPF KM mice (18-22 g) were obtained from Chengdu Dashuo Biotechnology Co., Ltd. (Chengdu, China). According to the Declaration of Helsinki, all animals received humane care, which was promulgated in 1964 and amended in 1996 [10]. All experimental protocols were approved by the Animal Care and Use Committee of Chengdu Municipal Hospital of Traditional Chinese Medicine, Chengdu, China (approval no. SCXK (Chuan) 2015-30). The animals were kept in a sterile animal room, which includes individually ventilated cages, and the feed and drinking water were subjected to autoclaving during the test.
Modeling and treatment
After adaptation to feeding, 12 mice were randomly selected as a blank group, and the remaining mice were made into a dyspepsia model. A blank group was given a regular diet, and the other groups were fed a self-made high-protein, high-calorie diet (from soy flour, fish pine, flour, milk powder in a ratio of 2:1:1:1) and thoroughly mixed with water. It is made into a biscuit shape and dried. At the same time, feed 50% milk 2 mL·(kg)-1, free feeding, and drinking for 7 days, causing the spleen deficiency model. The body weight, abdominal circumference, and average food intake and feces of each mouse were measured and recorded on days 2, 5, and 8. Compared with the blank group, food intake and feces were significantly reduced, abdominal fullness and abdominal circumference increased, and the spleen deficiency food was successfully simulated. After successful modeling, the 48 mice were randomly divided into a model group (administered saline 50 ml/kg/day, n=12), an MMF group (LSQ group, administered 4.8 g/kg/day, n=12), a Jianweixiaoshi group (JWXS group, administered 2.88 g/kg/day, n=12), a Domperidone group (DP group, administered 0.006 g/kg/day, n=12), and a blank group (administered saline 50 ml/kg/day, n=12).
Sample collection
On the seventh day of administration, the mice were fasted and deprived of water. After 24 h, Take the second feces of stress defecation in mice under strict aseptic conditions and quickly transfers them to a sterile cryotube. The cryotubes were stored in a -80°C refrigerator for later use. Before the mice were sacrificed, blood was taken from the eyeballs. Then centrifuged at 3000 rpm for 15 min, and the serum was collected and stored in the refrigerator at -70 ℃ for later use. Follow the instructions of the three kits to determine the contents of gastrin, cholinesterase, and nitric oxide in serum.
Determination of gastrin, cholinesterase and nitric oxide in serum
Take frozen mouse sera and test gastrin, cholinesterase, and nitric oxide according to the instructions.
DNA extraction and purification
The mice fecal bacterial genome total DNA was extracted using a soil DNA kit according to the kit standard according to the manufacturer's instructions, and the extracted genomic DNA was detected using 2% agarose gel electrophoresis and a super differential spectrophotometer (Thermo Fisher). The purified DNA extract was stored at -80 °C until use.
MetaVxTM Library Preparation and Illumina MiSeq Sequencing
Take the feces of the mouse, extract the DNA, and use 0.8% agarose gel electrophoresis to detect the DNA. The purified genomic DNA was amplified by polymerase chain reaction (PCR) according to experimental instructions. 515F (5`-GTGYCAGCMGCCGCGGTAA-3`) and 806R (5`-GGACTACHVGGGTWTCTAAT-3`) were used as primers [11,12]. Perform PCR product detection, purification, and quantification on the test sample. Use the TruSeq DNA PCR-Free Sample Preparation Kit to construct the library. After the quantified library has been quantified and the library is qualified, it is sequenced using the PE250 mode of the MiSeq 3000 platform PE300 of Chengdu Luoning Biotechnology Co., Ltd. The original offline data obtained by sequencing is spliced. Filter to get the high-quality target sequence required for subsequent analysis.
The library was constructed using the TruSeq DNA PCR-Free Sample Prep Kit (Illumina, FC-121-3001/3003). After the constructed library was qualified and tested by the library, it was sequenced using the MiSeq 3000 platform PE300 mode sequencing, sequencing kit. Use the Hiseq Rapid SBS Kit v2 (Illumina, FC-402-4023 500 Cycle). DNA samples were quantified using a Qubit 2.0 Fluorometer (Thermo Scientific). V4 hypervariable regions of microbial 16S rDNA were selected for generating amplicons and following taxonomy analysis. Synthesis of specific primers with Barcode for the 16S rRNA gene in fecal DNA extracts PCR amplification, the corresponding sequences of primers are 515F (5`-GTGYCAGCMGCCGCGGTAA-3`) and 806R (5`-GGACTACHVGGGTWTCTAAT-3`), respectively. Each 25 μL system included 1x PCR buffer, 1.5 mM MgCl2, 0.4 μM dNTPs forward and reversed primers of 1.0 μM each, 0.5 U KOD-Plus-Neo enzyme (TOYOBO) and 10 ng template. The PCR procedure consisted of starting at 94 ℃for 1 min and then 30 cycles (denaturation at 94 ℃ for 20 s, annealing at 54℃ for 30 s and extension at 72 ℃ for 30 s), and finally at 72 ℃for 5 min. Three replicates were performed for each sample. After the end of the PCR, the PCR products of all the same samples were mixed and subjected to electrophoresis detection. A recovery kit recovered the PCR products, and the target DNA fragment was eluted with TE buffer. PCR was mixed with 1/6 volume of 6×loading buffer and detected by agarose gel electrophoresis. Take the strip for recycling and recycle the QIAquick Gel Extraction Kit (QIAGEN).
Data analysis
Based on Usearch (http://drive5.com/uparse/) software, OTUSE algorithm [13] is used to perform OTU clustering at 97% consistency level, and the highest frequency sequence in each OTU is selected as the representative one. Annotated analysis was performed using the UCLUST classification [14] and the SILVA database (Rlease_123 http://www.arb-silva.de/). Representative sequences were subjected to multiple alignments using PyNAST. Use FastTree[15] to build a phylogenetic tree. Each sample was homogenized and resampled, with the least amount of data in the sample. Community composition analysis was performed using the R language [16] for various data transformations. Use the ggplot[17] package to the plot. Differential species analysis uses the Python LEfSe package. Random forest analysis uses the R language randomForest package. The metastatic analysis is performed using the R language. Correlation analysis uses the cor.test function of R's stats package. Use the R language psych and corrplot package to analyze the relationship between the promotion of digestion and decreased gastric relaxation and bacterias. The analysis results were statistically significant, with P<0.05. Data were expressed as mean ± standard deviation (M ± S), basic statistical analysis and charting were performed using SPSS Statistics v17.0 statistical analysis software, one-way ANOVA, and LSD and Dunnett's T3 method were used. After the two-two comparative analysis, those who do not conform to the normal distribution use the rank-sum test. P < 0.05 was considered statistically significant.