丁酸对哺乳期大鼠肠黏膜屏障发育的作用

Effects of butyric acid on development of intestinal mucosal barrier in lactating rats

目的探讨丁酸对SD大鼠肠黏膜屏障的作用情况,为防治坏死性小肠结肠炎等肠道疾病提供实验依据。方法以生后7d(P7)的SD新生鼠为实验对象,采用随机数字表法,选取实验组(12只)、生理盐水对照组(8只)和空白对照组(8只)大鼠。实验组给予丁酸,生理盐水对照组给予生理盐水,连续灌胃早晚各1次;空白对照组不做任何处理。10d后取幼鼠近端结肠组织,利用尤氏灌注系统(Ussing Chamber)检测肠黏膜跨上皮电阻(transepithelial electrical resistance,TER)值;WesternBlot检测紧密连接蛋白ZO-1、Occludin及Claudin-1的表达情况;免疫组织化学法检测近端结肠组织ZO-1和Occludin的表达及分布情况(OD值)。结果尤氏灌注系统发现丁酸组在0、30、60、90、120min的TER值分别为(70.65±5.03)Ω·cm^2、(72.40±5.58)Ω·cm^2、(68.84±5.02)Ω·cm^2、(64.64±4.64)Ω·cm^2和(58.95±6.23)Ω·cm^2,生理盐水对照组分别为(63.56±4.28)Ω·cm^2、(59.31±4.07)Ω·cm^2、(51.73±4.42)Ω·cm^2、(45.28±5.18)Ω·cm^2和(42.09±4.06)Ω·cm^2,空白对照组分别为(59.99±4.21)Ω·cm^2、(56.64±3.71)Ω·cm^2、(51.53±4.00)Ω·cm^2、(48.01±7.40)Ω·cm^2和(45.41±10.00)Ω·cm^2。丁酸组各时间点TER值与生理盐水对照组及空白对照组比较,差异均有统计学意义(P<0.001);生理盐水对照组与空白对照组比较,差异无统计学意义(P>0.05)。丁酸组紧密连接(TJ)蛋白Occludin及Claudin-1的表达(1.03±0.05、2.02±0.21)较生理盐水对照组(0.74±0.08、1.06±0.09)及空白对照组(0.73±0.05、1.04±0.06)增加,且组间比较,差异均有统计学意义(P<0.001);而生理盐水对照组与空白对照组间差异无统计学意义(P>0.05)。组织学免疫组织化学检测发现丁酸组肠上皮细胞间ZO-1及Occludin的表达(0.22±0.04、0.26±0.04)较生理盐水对照组(0.16±0.02、0.21±0.02)及空白对照组明显增加(0.16±0.01、0.21±0.02),且组间比较,差异均有统计学意义(P<0.05);而生理盐水对照组与空白对照组间差异无统计学意义(P>0.05)。结论在哺乳期肠黏膜屏障模型中,丁酸增加紧密连接蛋白的表达,能较快促进肠黏膜组织结构与功能发育成熟。

Objective To explore the effect of butyric acid on intestinal mucosal barrier in rats and to provide experimental rationales for preventing and treating such an intestinal disease as necrotizing enterocolitis. Methods Sprague-Dawley (SD) neonatal rats at 7 days after birth (P7) were used as experimental subjects.Three groups of experiment (12 rats), saline control (8 rats) and blank control (8 rats) were randomized by a number table.The experiment group received butyric acid while saline control group normal saline continuously administered once or twice a day.Blank control group had no treatment.After 10 days, proximal colonic tissue was harvested and transepithelial electrical resistance (TER) detected by Ussing’s chamber.The tight junction proteins ZO-1, Occludin and Claudin-1 were detected by Western blot.And the expressions of ZO-1 and Occludin in proximal colonic tissue was detected by immunohistochemistry (OD value). Results The TER values of butyric acid group at 0, 30, 60, 90 and 120 min were (70.65±5.03)Ω·cm^2,(72.40±5.58)Ω·cm^2,(68.84±5.02)Ω·cm^2,(64.64±4.64)Ω·cm^2 and (58.95±6.23)Ω·cm^2, saline control group (63.56±4.28)Ω·cm^2,(59.31±4.07)Ω·cm^2,(51.73±4.42)Ω·cm^2,(45.28±5.18)Ω·cm^2 and (42.09±4.06)Ω·cm^2, blank control group (59.99±4.21)Ω·cm^2,(56.64±3.71)Ω·cm^2,(51.53±4.00)Ω·cm^2,(48.01±7.40)Ω·cm^2 and (45.41±10.00)Ω·cm^2.The TER values of butyric acid group were significantly different from those of saline control and blank control groups (P<0.001). No significant difference existed between saline control and blank control groups (P>0.05). The expressions of Occludin and Claudin-1 were significantly higher in butyric acid group than those in saline control group (0.74±0.08, 1.06±0.09) and blank control group (0.73±0.05, 1.04±0.06). The increases and differences were statistically significant (P<0.001). No significant difference existed between saline control and blank control groups (P>0.05). Histological immunohistochemistry showed that the expressions of ZO-1 and Occludin in intestinal epithelial cells of butyric acid group (0.22±0.04, 0.26±0.04) were significantly higher than those of saline control group (0.16±0.02, 0.21±0.02) and blank control group (0.16±0.01, 0.21±0.02). And the differences were statistically significant (P<0.05). No significant difference existed between saline and blank control groups (P>0.05). Conclusions In lactating intestinal mucosal barrier model, butyrate up-regulates the expression of tight junction protein and promotes the development of intestinal mucosal structure and function.