Cell culture and treatment
Caco‐2 cells(Shanghai Cell Bank, Shanghai, China) were seeded on 24‐well 12‐mm polyester Transwell filters (Corning, NY, USA) with 0.4μm pore size at a concentration of 2×105 cells per transwell. Cells were grown in DMEM supplemented with 10% FBS, and cultured for 21 days until they formed a differentiated monolayer. For treatment, JZL184 was added to the upper chamber of the transwell cultures.
In vitro measurement of FITC-Dextran permeability
To determine the effect of JZL184, the dextran permeability was measured after JZL184(1 umol/L) treatment for 24 hours. After washing the cells, DMEM was dispensed into each filter in the apical and basolateral chamber, fluorescein isothiocyanate (FITC)‐dextran (4 kDa; 3mg/mL) was added to the upper chamber without medium change. Aliquots were withdrawn from the lower chambers after 4 hours, fluorescence values were compared with those of serial dilutions of known FD4 concentrations. Measurements were carried out in triplicate.
Male Wistar rats (200–230 g) were obtained from the Experimental Animal Center of Shandong University (Jinan, Shandong, China). Animals were housed in an animal facility that was maintained at 22°C with an automatic 12-hour light/dark cycle. All experiments were approved by the Shandong Provincial Hospital Committee on Use and Care of Animals. The researcher was blinded to details of animal treatments.
Establishment of rat model of water avoidance stress and experimental protocol
The WAS protocol is a well-established model of chronic stress considered to represent moderate psychological stress. Chronic stress was induced as follows: Rats were placed on a plastic platform in the middle of a tank filled with water (25°C) to 1 cm below the height of the platform. The animals were maintained on the platform for 1 hour per day for 10 consecutive days. The selective MAGL inhibitor JZL184 (Cayman Europe, Talin, Estonia) was prepared in a mixture of saline/ethanol/Tween-80 and administered at a dosage of 10 mg/kg by intraperitoneal (i.p.) injection during chronic stress, then twice daily until sacrifice. The vehicle alone was administered as a control. Antagonists of the CB1 and CB2 receptors; SR141716A and AM630, respectively (Tocris Bioscience, Bristol, UK); were administered once daily at 1 mg/kg in the same vehicle as JZL184. Rats were divided into four groups, with 4 rats in each group, according to the intervention method, denoted the control group, JZL184 group, JZL184 and SR141716A group, and JZL184 and AM630 group.
In vivo measurement of FITC-Dextran permeability
In vivo intestinal permeability was assessed on the first day after completion of the 10 days of WAS. Rats were administered 400 mg/kg of 4 kDa fluorescein isothiocyanate-dextran (FD4) in phosphate-buffered saline (PBS) pH 7.4 by gavage, blood was collected by heart puncture after 4 hours and transferred to ethylenediaminetetraacetic acid-coated tubes. Samples were centrifuged 1,000 g for 5 minutes, to separate the serum and stored at −80°C until analysis. To determine FD4 levels, we diluted 25 μL serum in 175 μL of PBS in a 96-well black-wall microplate and fluorescence values were compared with those of serial dilutions of known FD4 concentrations. Measurements were carried out in triplicate.
The rats were sacrificed with Carbon dioxide for further use. We dissected out colorectal tissues (approximately 10 cm from the anus) of rats. The dissected colorectal segments were reversed inside-out and washed with cold PBS. The mucosal layers were scraped off and collected in PBS, and the resulting mixture was centrifuged at 1,000 g for 5 minutes, then stored at −80°C refrigerator.
The prepared mucosal layers were extracted in radioimmunoprecipitation buffer containing 1 mmol/L phenylmethanesulfonylfluoride, 10 mg/mL leupeptin and 10 mg/mL aprotinin. The extract was centrifuged at 12,000 g for 20 minutes. Samples containing equal amounts of protein (20 mg) were separated by 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membranes (Millipore, USA). Blots were probed with mouse antibodies against occludin (dilution 1:5000; Invitrogen, USA), rabbit antibodies against claudin-1, (dilution 1:1000; Invitrogen, USA), rabbit antibodies against claudin-2 (dilution 1:1000; Invitrogen), mouse antibodies against claudin-5 (dilution 1: 200; Invitrogen) and with rabbit anti-β-actin antibody (1: 5000, Cell Signaling, USA) or mouse anti-β-actin antibody (1:5000, Cell Signaling) as a loading control. Experiments were carried out in triplicate and β-actin protein levels were analyzed as a control for equal protein loading.
Tissues were homogenized in CHCl3 (5 mL), and deuterated standards (2-arachidonoylglycerol-d5, 200 pmol) were added. Then MeOH (5 mL) and H2O (2.5 mL) were added and the lipids were extracted by vigorous mixing, and the organic layer was recovered and dried under nitrogen. The resulting lipid fraction was pre-purified by solid-phase extraction over silica, and 2-AG was eluted using ethyl acetate-acetone (1:1, v/v). The resulting lipid fraction was analyzed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) using an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific; Waltham, MA, USA) coupled to an Accela HPLC system (Thermo Fisher Scientific). Analytes were separated using a C-18 Supelguard precolumn and a Supelcosil LC-18 column. The mobile phases A and B were MeOH-H2O-acetic acid (75:25:0.1, v/v/v) and MeOH-acetic acid (100:0.1, v/v). The gradient used was as follows: 100% A to 100% B in 15 minutes (at 0.5 mL/minute), followed by 10 minutes at 100% B and subsequent re-equilibration at 100% A. Mass spectrometry analysis in positive ion mode was performed with an atmospheric pressure chemical ionization source. Capillary and APCI vaporizer temperatures were 250 and 400°C, respectively. We quantified 2-AG by isotope dilution using the respective deuterated standards. Calibration curves were generated as described, and data were normalized to tissue sample weight.
We used SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) for all statistical analysis. Data are expressed as the mean ± standard deviation. The two-tailed Student’s t-test and one-way analysis of variance were used to evaluate differences between experimental and control groups. Statistical significance was accepted at P < 0.05.