3.1.Study design, area, and duration
This an experimental laboratory-based study will carry out in Microecology Laboratory at the College of Basic Medical Sciences, during the period of 2018 to 2020.
Normal pellet and high fat diet will obtain from animal facilities of Dalian Medical University. Streptozocin (STZ), mannitol and lactulose will purchase from Sigma-Aldrich, Inc. As well, Ceftriaxone sodium antibiotics will buy from Youcare Pharmaceutical Group Co,LTD, Beijing, China.
32 adult, male, Wistar rats (weight range 200–250 g), aged 5 weeks will obtain from the experimental animal center of Dalian Medical University. Throughout the whole experiments, all rats will house in standard polypropylene cages (4 rats/cage) and maintain under controlled room temperature (22 ± 2 ?C) and humidity (60 ± 5%) with 12 h light and dark cycle. This study will assume with the approval of the Animal Care Ethics Review Committee of Dalian Medical University, and all the steps will perform in accordance with its particular ethical and professional guidelines and standards. Greatest efforts will attain to minimize animals suffering and harms.
At the first point, all rats will subject to a familiarization period for 2 weeks in order to adapt to a new environment. At this time, all rats will provide normal pellet diet (NPD) with free access to water; and will subject to measurement of fasting blood glucose in the beginning and end of the period in order to evaluate the health status of rats. At the end of a familiarization period, rats will randomly divide into 4 groups (A, B, C, and D; N=8 in each group). Next, type 2 diabetes mellitus will induce in group A and B, then dysbiosis in group A and C, consequently. Finally, LMR test will perform, subsequently animals will scarify and samples collect.
3.5.Induction of Type 2 diabetes using HFD/STZ model
Type 2 diabetes will induce in group A and B as described by Zhang et al. and
Liu et al 65. Diabetic rats groups (A an B) will feed high fat diet (consisting of 22% fat, 48% carbohydrate, and 20% protein with total calorific value 44.3 kJ/kg) for 4 weeks. In contrast, the health groups (C and D) will provide a normal pellet diet (consisting 5% fat, 53% carbohydrate, 23% protein, with total calorific value 25 kJ/kg). After manipulation for 4 weeks, every rat in A and B group will inject intra-peritoneally (IP) with a low dose of STZ (30 mg/kg, dissolved in 0.1 M sodium citrate buffer, pH ¼ 4.4). After 1 week later, fasting blood glucose (FBG) will measure and every rat with FBG less than 16.7 mmol/L will reinject with STZ again (30 mg/kg), while the control rats will give vehicle citrate buffer (pH ¼ 4.4) in a dose volume of 0.25 mL/kg, IP, respectively 65. After 4 weeks of STZ injection, the rats with the fasting blood glucose of ?16.7 mmol/L will be considered diabetic. The additional confirmation will achieve by performing an oral glucose tolerance test (OGTT). The rats were kept feeding on their respective diets until the end of the study.
3.6.Induction of dysbiosis
To establish the dysbiosis model, all rats of group A and C will receive 200mg of ceftriaxone sodium (dissolve in 2mL of normal saline), intra-gastrically through a gavage, once a day with an interval of 24h for 28 days. While the other groups will give 2ml of 0.9% normal saline. The successful of model will asses by denaturing gradient gel electrophoresis (DGGE).
3.7.Denaturing gradient gel electrophoresis (DGGE) profiling
Before the LMR test, DGGE analysis will conduct to evaluate the diversity of flora and to confirm the knockdown of microbiota (Dysbiosis) in ceftriaxone sodium received groups. For DGGE, stool samples will collect from every rat and Bacterial DNA will extract by Fast DNA Spin Kit (E.Z.N.A.® Stool DNA kit, Omega, America). The purity and concentration of the extracted DNA will asses and measure, respectively, using by Nanodrop Photometer 2000 (Thermo Fisher Scientific, USA). PCR will amplify using a pair of prime targeted to V3 and 4 regions of the 16S rRNA gene and PCR product will subject to gel electrophoresis, then the digitized DGGE images will analyze with Quantity One image analysis software (version 4.6.1, Bio-Rad USA). Dysbiosis will judge by the difference in numbers and thickness of bands of extracted DNA of ceftriaxone receiver and non-receiver groups.
3.8.Lactulose mannitol test
Lactulose and mannitol ratio is a useful, simple, non-invasive and a reliable test for estimation of intestinal permeability.13 Directly measures the ability of two non-metabolized sugar molecules to permeate the intestinal mucosa. The degree of intestinal permeability or malabsorption is reflected in the levels of the two sugars recovered in a urine sample collected over the next hours. Under the normal physiological condition, disaccharide due to its large size is restricted from passing across the villous tips. In this test, every rat will fast for 6-8h, and empty its bladder, then will receive orally (through gavage) a dose of 120mg lactulose plus 80mg mannitol dissolve in a 2 mL of distal water. No food will allow until the completion of the test, whereas the water will provide after 3 hours from the ingestion of the test solution. 24h urine will collect in a sterile container, titer, and storage at -80oC until analysis. The concentration of one or more sugar probe present in the urine will measure by enzyme assay, high performance liquid chromatography mass spectrometry (HPLC-MS). 13 The results will express as the ratio of the percentage of the ingested dose excretion in the urine (LMR = % lactulose/% mannitol).
3.10.Measurement of body weight
The body weight of every rat will measure at zero time, then weekly using weight balance
3.11.Animals scarifying and Samples collection
Each rat will fast for 6-8h, afterward ip anesthetize using a mixture of ketamine (100 mg/kg) and diazepam (5 mg/kg) , subsequently scarify and samples collect. Jejunum content will collect in sterile 1.5ml microcentrifuge tube and store at -80oC for the subsequent step. While four pieces of every jejunum tissue will cut in 1.5 centimeters and wash in cold phosphate buffer saline. Three pieces will transfer into sterile eppendorf tube and store at -80oC, to use later for the measurement of Na-K ATPase activity , genes expression levels; and anther will fix in 10% neutral buffer formalin and store at room temperature for histopathological examination.
3.12.Na-K ATPase activity
Jejunum tissue will weight, cut, 0.02 M concentration of PBS (pH 7.4) will add and the specimen will homogenize by a homogenizer, then centrifuge for about 20 minutes (2000-3000 rpm). Carefully, supernatant will collect and test for Na-K ATPase activity using Rat Na-K-ATPase ELISA Kits (Shang Hai Lengton Bioscience Co.,LTD, Shang Hai, China) according to manufacturer instructions. The absorbance (OD value) of each well will measure in sequence with a blank air conditioner of zero and a wavelength of 450 nm. All ELISA condition will perform in triplicate. The linear regression equation of the standard curve will calculate according to the concentration of the standard and the corresponding OD value, then the corresponding sample concentration will calculate on the regression equation according to the OD value of the sample.
3.13.Bacterial DNA extraction, and 16sRNA gene sequencing
DNA will be extracted from jejunum content using Qiagen Kits (QIAamp DNA Stool Mini Kit, Qiagen, Germany) according to company protocol. The purity and concentration of the extracted DNA will asses and measure, respectively, using Nanodrop Photometer 2000 (Thermo Fisher Scientific, USA). Then all the extracted DNA samples will store at ?80 ? until further processing.
For DNA sequencing, 16S rRNA gene sequences will amplify from the bulk DNA with the barcoded primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′), which covers V3–V4 regions of the 16S rRNA gene. PCR reactions will perform on an ABI GeneAmp 9700 PCR system (Applied Biosystems, Foster City, CA, United States) with an initial denaturation step at 94? for 5 min;32 cycles of denaturation at 94? for 30 s, annealing at 58.4? for 30 s , and extension at 72? for 30 s; plus a final extension at 72? for 7 min. The 16S amplicons will purify with GeneJET PCR Purification Kit (Thermo Fisher Scientific, Waltham, MA, United States) and DNA library will construct by using TruSeq DNA PCR-Free Library Kit (Illumina Inc., San Diego, CA, United States), then sequence by Hiseq sequencing System (Illumina Inc., San Diego, CA, United States).
3.14.RNA extraction and the measurement of transcription level of targeted genes using quantitative real time PCR
Jejunum tissue will dissect and immerse in RNAlater. Total RNA will extract from Jejunum tissue of every rat using Trizol reagent (Life Technologies, Carlsbad, CA, USA), and the purity and concentration of the extracted RNA will asses and measure, respectively, using by Nanodrop Photometer 2000 (Thermo Fisher Scientific, USA). Then all the extracted RNA samples will store at ?80 ? pending further steps.
The same concentration of RNA will convert to cDNA using a reverse transcription kit (RR036A, PrimeScript™ RM Master Mix; Takara, Kusatsu, Japan). Real-time quantitative polymerase chain reaction (RT-qPCR) will perform by a CFX96™ RT-PCR system (BIO-RAD, Hercules, CA, USA) using SYBR Premix Ex Taq (Takara, Kusatsu, Japan), and 3 pairs of targeted genes primers. The 2???CT method will use to analyze the obtained data. The sequences of the specific primers will be provide by company. Targeted genes (three genes) for RT-qPCR includes tight junction protein , Sodium Glucose transporter 1 (SGLT1) and Glucose transporter 2 (GLUT 2) in jejunum tissue of Wistar rats.
3.15.Western blot technique
The total protein will extract from jejunum tissue of every rat. Equivalent amounts of protein will separate by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferee onto nitrocellulose membranes. Antibodies against tight junction protein , Sodium Glucose transporter 1 (SGLT1) and Glucose transporter 2 (GLUT 2) in jejunum tissue of Wistar rats will use for blotting, and the secondary antibodies conjugated with horseradish peroxidase (HRP, USCN, USA) will employ to show the bands. The immune complexes will detect with a
Western Bright™ ECL Western Blotting HRP Substrate kit and analyzed with image lab software (Bio-Rad, USA).
Gross morphologic changes include the length, and wide of jejunum will record. For histological examination, 10% neutral formalin fixed jejunum tissues will subject to tissue processing, embedding in paraffin, section at 4 micron in size using microtome, stain with haematoxylin and eosin, mount in DPX, then examine using light microscope (Olympus CK20, Japan) by a pathologist, who will be blind to the diagnosis and clinical data. Each sample will process and examine in triplicate using different sections. Morphologic and inflammatory changes of tissues will report.
Comparative analysis of microbiome composition and relative abundance will perform at the phylum, family, genus and species levels. The diversity indices (Chao, Ace, Simon, Shannon, observed OTUs) and PCoA will implement to evaluate the diversity and community structure of gut microbiota, respectively. Statistical analysis will perform using GraphPad Prism 5 (Graph Pad Software, La Jolla, CA, USA) and the results will present in form of figures and tables. The body weights, blood glucose and genes expression levels, mannitol and lactulose quantity, LMR, and Na-K ATPase activity will express as the means ± SD. At 95% level of significant, the statistical difference among comparative data will examine using the suitable statistical tests and a P value less than .05 will consider statistically significant. The correlation between data will assess by Spearman Correlation coefficient (R ), r value 0 as a positive correlation.