Restraint Stress in Hypertensive Rats Activates the Intestinal Macrophages and Reduces Intestinal Barrier Accompanied by Intestinal Flora Dysbiosis
Colonic Barrier Function Assessment
Colonic tissue was opened along the mesenteric border and mounted in Ussing chambers (Physiologic Instruments, San Diego, CA) with an 0.5 cm2 exposing area of the tissue surface to 5 mL of V O2/V CO2 95:5 Krebs-glucose (10 mM) at 37°C. After adapted for 20 minutes, tetrodotoxin (TTX, 0.1 μM, Shanghai Aladdin Biochemical Technology Co., Ltd., Shanghai, China) and indomethacin (Indo, 1μM, Sigma-Aldrich Corp., St. Louis, MO, USA) were added to the basal side, respectively, to inhibit enteric neurons and prostaglandin synthase and sections let they reached a steady-state level for at least 30 minutes before each experiment began.
The paracellular pathway and transcellular pathway were measured as the flux of 4-kDa FITC-dextran (FD-4; 0.5 mg/mL; Sigma-Aldrich Corp., St. Louis, MO, USA) and horseradish peroxidase (HRP; 0.5 mg/mL; Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), respectively. FD-4 and HRP were added to the luminal side, and the samples of 400 μL were collected from the basolateral per 20 min for 2 h and replaced by an equal volume of the corresponding buffer. The concentration of FD-4 was measured using fluorescence at emission 528 nm and excitation 485 nm. 3.3′,5,5′-Tetramethylbenzidine (TMB) was used to detect HRP at absorbance 405 nm.18
Data were expressed as mean±SEM using Prism 8.0, and nonparametric Wilcoxon (two-group comparisons) or Kruskal–Wallis test (multi-group comparisons) was used to determine the significance between different treatment groups. N was the number of animals in each experiment. The statistical differences between control and treatment groups were analyzed using Student’s paired or unpaired t-test when appropriate.
The correlation among plasma cytokine and hormone concentrations for all y-WKY, y-WKY-S, y-SHR, and y-SHR-S animals were determined using Pearson correlation coefficients and p-values were compared between groups with paired Mann-Whitney’ test. Correlation matrices also were displayed as schematic correlograms.19,20 Because each of these cytokines and hormone measurement range widely, the multiple change of its level relative to the reference level was used for the following dynamic analysis, which the dynamics were fitted on the change folds along the time line through locally weighted scatter plot smoothing (LOWESS). The bandwidth (the most important parameter) determined by trial and error is 0.3. All statistical analyses were performed in Stata/SE 12 and open source procedure R 3.2 (https://www.r-project.org/).21