Objective: To explore the mechanism of the intestinal barrier damage caused by Blastocystis hominis infections in rats.
Methods: Thirty SD rats were randomly divided into the control group, and the 1-, 3-, 6- and 9-week-infection groups, of 6 rats in each group. Rats in each infection group were orally infected with B. hominis trophozoites at a density of 2 × 108 parasites per rat, and the control group was given an equal volume of phosphate buffered saline solution. The 7-hour urine samples were collected 1, 3, 6 and 9 weeks post-infection for the measurement of the intestinal permeability. Then, rats were sacrificed using the cervical dislocation method, and the cecum specimens were collected for the detection of the intestinal epithelial cell permeability. The expression of tight junction-related Occludin and Claudin - 1 genes and apoptosis-related Bcl - 2 and Bax genes was quantified in cecum epithelial cells using the real-time fluorescent quantitative PCR (qPCR) assay, and cell apoptosis was detected in the rat cecum using the TdT-mediated dUTP nick-end labeling (TUNEL) assay.
Results: The median urinary lactolose to mannitol ratios were 0.29, 0.72, 0.44, 0.46 and 0.38 in the control group, and the 1-, 3-, 6- and 9-week-infection groups, respectively, and the difference was statistically significant (H = 12.09, P < 0.05). B. hominis invasion and epithelial injury were observed in intestinal epithelial cells of rats infected with B. hominis, and transmission electron microscopy displayed the destruction of tight junctions between intestinal epithelial cells. The relative expression of Occludin, Claudin-1, Bcl-2 and Bax genes was 1.04, 0.62, 0.71, 0.68 and 0.96; 1.03, 0.61, 0.63, 0.76 and 0.86; 1.08, 0.70, 0.75, 0.74 and 1.03; and 1.00, 1.57, 1.33, 1.35 and 1.10 in the control group and the 1-, 3-, 6- and 9-week-infection groups, respectively, and all differences were statistically significant (F = 2.86, 2.85, 3.37 and 4.45, all P values < 0.05). The median number of positive staining cells were 1.00, 13.00, 9.00, 3.50 and 1.00 in rat cecum specimens in the control group, and the 1-, 3-, 6- and 9-week-infection groups, respectively, and the difference was statistically significant (H = 22.95, P < 0.01).
Conclusions: B. hominis infection may cause an increase in the rat intestinal permeability through triggering the apoptosis of intestinal epithelial cells to destroy the tight junction between intestinal epithelial cells, thereby destroying the intestinal barrier function.
［摘要］ 目的 探讨大鼠感染人芽囊原虫后引起肠道屏障损伤的机制。方法 将 30 只SD大鼠随机分为对照组及感染 1、3、6、9 周组 (感染组), 每组 6 只。各感染组大鼠经口感染人芽囊原虫 2 × 108 个/鼠, 对照组大鼠经口灌胃等体积磷酸盐 缓冲液。分别于感染后 1、3、6、9 周收集 7 h 大鼠尿液检测肠道通透性, 随后采用颈椎脱臼法处死大鼠, 取盲肠组织检测 肠道细胞通透性。采用实时荧光定量PCR技术检测大鼠盲肠细胞紧密连接跨膜蛋白基因Occludin、Claudin-1 及凋亡相 关基因Bcl-2 和Bax 表达水平; 以原位末端标记法 (TdT-mediated dUTP nick-end labeling, TUNEL法) 检测盲肠细胞凋亡。结果 对照组及感染 1、3、6、9 周组大鼠尿液中乳果糖与甘露醇排出率比值中位数分别为 0.29、0.72、0.44、0.46 和 0.38, 差 异有统计学意义 (H = 12.09, P < 0.05) 。感染组大鼠肠上皮细胞可见虫体入侵并出现上皮损伤, 透射电镜下可见细胞间 紧密连接破坏。对照组及感染 1、3、6、9 周组大鼠盲肠组织中 Occludin 基因相对表达量分别为 1.04、0.62、0.71、0.68 和 0.96, Claudin-1 基因相对表达量分别为 1.03、0.61、0.63、0.76 和 0.86, Bcl-2 基因相对表达量分别为 1.08、0.70、0.75、0.74 和 1.03, Bax 基因相对表达量分别为 1.00、1.57、1.33、1.35和1.10, 差异均有统计学意义 (F = 2.86、2.85、3.37 和 4.45, P 均< 0.05) ; 盲肠组织凋亡染色阳性细胞数中位数分别为 1.00、13.00、9.00、3.50 个和 1.00个, 差异有统计学意义 (H = 22.95, P < 0.01) 。结论 人芽囊原虫感染可能通过引发大鼠肠道上皮细胞凋亡而破坏细胞间紧密连接, 导致肠道通透性增高, 进而破坏肠道屏障功能。.
Keywords: Apoptosis; Blastocystis hominis; Intestinal barrier; Intestinal epithelial cell; Intestinal permeability; Rat; Tight junction.