Preparation of JPQHF
The JPQHF consists of Codonopsis pilosula (Franch.) Nannf (15 g), Astragalus membranaceus (Fisch.) Bunge (15 g), Dioscoreae rhizoma(15 g), Polygonatum sibiricum (15 g),Coptis chinensis Franch (3 g), Scutellaria baicalensis Georgi (9 g), Radix Puerariae (15 g) and Euonymus alatus (Thunb.) Sieb (15 g). The herbs were obtained from Lei-Yun-Shang Pharmacy(Shanghai,China) and were identified by Dr Guanglin Xu from Pharmacy Department of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. The herbs fulfilled the standard requirements of the 2015 version of the Chinese Pharmacopoeia.
JPQHF was prepared by Pharmacy Department of Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. All above eight herbs were submersed in 1000 ml of distilled water and extracted in a ceramic clay pot at 100 ℃ for 30 min with continuously stirred twice. The mixed extract was concentrated at 100 ℃ for 30 min and centrifuged at 13,000 rpm for 30 min at 4 ℃.Then the supernatant was filtered through a sieve until each milliliter contains 1.5 g drug.
Animal groups and treatment
Six-week-old healthy male C57BL/6 mice weighted 18 ± 2 g were acquired from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Shanghai Branch, (Shanghai, China), certification no. SCXK(HU)2017–0011. Mice were housed under controlled conditions: 12 h light/12 h dark cycle, ambient temperature of 23 ± 3 °C and humidity 55 ± 15%. All experimental procedures involving animals were approved by the Animal Experiment Ethics Committee of Shanghai University of Traditional Chinese Medicine, and the approval no.PZSHUTCM 190,823,004.
The mice were randomly divided into normal group (NOR) and the high-fat diet group, and were fed a normal diet (1,010,009; Nanjing Synergy Biology Co., Ltd.; Nanjing, Jiangsu province, China) or the HFD containing 60 kJ % fat (D12492; Research Diets, New Brunswick, NJ, USA) respectively for 12 weeks. After continuous feeding of 12 weeks, the weight of mice in the high-fat diet group was 1.2 times of the normal group were considered as DIO mice. The DIO mice were then divided into three groups according to body weight via SPSS 25.0 (SPSS Inc., Chicago, USA): DIO group (DIO), Metformin group (MET) and JPQHF group (JPQH),and daily treated with oral normal saline, metformin suspension and JPQHF individually. The metformin tablets (Merck Serono, Geneva, Switzerland) was dissolved in physiological saline at a density of 300 mg/kg body weight.
Metabolic phenotypes correlated parameters analysis
The body weight of mice was measured by the same electronic scale at the same time every week (Saturday morning 9:00–11:00).
After administration of 5 weeks, both IPGTT and IPITT were performed after 12 h of fasting. Glucose(1.5 g/kg) or insulin(0.5U/10 g,D134876A,Eli Lilly and Company, Indianapolis, Indiana, U.S.) was administered intraperitoneally respectively. Glucose levels were monitored by glucometer (ACCU-CHEK Active, Roche, Mannheim, Germany) via sampling from the end of the caudal vein at 0, 30, 60, 90 and 120 min after glucose or insulin injection. Finally the area under the curve (AUC) was calculated by the trapezoidal rule.
Levels of serum fasting insulin(FINS) were determined by ELISA(CSB-E05071 m, Wuhan Huamei Biological Engineering Co., Ltd., Wuhan, Hubei province, China).The cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), and low density lipoprotein cholesterol (LDL-C) concentration was tested by the fully automated biochemical analyzer(7020, Hitachi High-Tech, Tokyo, Japan).
The small intestine was separated and fixed in 2% neutral buffered formalin for 8 h, then transferred to 20% glucose, 30% glucose, and embedded with OCT. The tissue specimens were applied to analysis the transposition of GLUT2 using the anti-GLUT2 antibody (Ab54460,1:200, Cambridge, UK) by fluorescence microscope (80i, Nikon, Tokyo, Japan).
Total protein was extracted from small intestine and quantified by BCA assay in accordance with the manufacturers protocol (23,225, Thermo Fisher Scientific, MA, USA). Immunoblotting was performed as described previously . The primary antibodies p-P38 MAPK (4613S,1:1000), P38 MAPK (9212S,1:1000), p-JNK (4668S, 1:1000), JNK (9252S,1:1000), p-ERK1/2(4370S,1:1000), ERK1/2 (4695S,1:1000), PI3K (4249S,1:1000), Akt(3063S,1:1000), p-Akt (4060S, 1:1000), IRS1 (2382S,1:10 00), p-IRS1 (3203S,1:1000), p-IRS2 (4502S,1:1000) and secondary antibody were purchased from Cell Signaling Technology (MA, USA).Chemiluminescent images were acquired by imaging system (5200, Tonan, Shanghai, China) and analyzed using the Protein Array Analyzer plugin for ImageJ.
Enzyme-linked Immunosorbent Assays (ELISA)
Concentrations of apoB48 in small intestinal tissue in serum were evaluated by ELISA (CSB-E16506m, Wuhan Huamei Biological Engineering Co., Ltd., Wuhan, Hubei province, China) according to kit instructions. The OD value was read at 450 nm by a microplate (ELX800, Biotek Instruments, Vermont, USA) after incubation at 37 °C for 20 min.
Separation and digestion of small intestinal tissue were operated under the guidance of instructions(Lamina Propria Dissociation Kit, mouse,130–097-410, Miltenyi Biotec, Bergisch Gladbach, Germany). Intestinal immune cell single cell suspension was placed in blank, compensation control and sample tube. The corresponding cell surface fluorescent antibody was added in compensation control tube, meanwhile, Fc Block, Fixable Viability Stain 510 and cell-surface fluorescent antibodies such as CD45 (557, 659), F4/80 (565, 411), CD11b (552, 850), CD206 (565 250) and CD86 (553,692) which were purchused from Becton, Dickinson and Company (New Jersey, USA) were put into sample tube in turn. Of note, incubating for 10 min at 4 °C in the dark and washing by FBS were needed after the reagent was added at above step. The flow cytometer (FACS Canto II, Becton, Dickinson and Company, New Jersey, USA) and software flow cytometer (V10, Becton, Dickinson and Company, New Jersey, USA) was utilized to detect and analysis the ratio of immune cells in the lamina propria of the small intestine.
Total protein was extracted from small intestine and quantified by BCA assay in adherence to the the manufacturer,s protocol (23,225,Thermo Fisher Scientific, MA, USA). The standard and premixed beads mixture were diluted according to the method provided in the instructions (MTH17MAG-47 K,MILLIPLEX,Merck KGaA, Darmstadt, Germany). 25 μl of distilled premixed beads mixture and 25 μl of sample were added to sample well, while 25 μl standard and 25 μl of cell clesolution were put in each standard well.The 96-well plate was incubated at room temperature for 2 h on the shaker and then was placed on magnetic stand. Biotin-labeled antibody complex and diluted streptavidin-labeled PE were put in each well sequentially. Finally, the beads was detected by Luminex(X-200, Luminex Corporation, Texas, USA). The concentration ratio of cytokine to sample was applied to statistics.
All data were expressed as the means ± SD. One-way ANOVA test was applied for the comparison between groups using SPSS 25.0 (SPSS Inc., Chicago, USA). P < 0.05 was considered to indicate statistical significance.
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