丁酸钠对新生大鼠回肠黏膜屏障的保护作用

Sodium butyrate improves ileal epithelial barrier function in neonatal rats

目的 探讨丁酸钠在活体新生SD大鼠肠屏障功能中的保护作用,建立丁酸钠对活体肠黏膜作用的量效关系。方法 将出生后9 d龄的新生SD大鼠按照随机数字表法分为5组,其中4组使用丁酸钠(sodium butyrate,NaB),浓度分别为20 mmol/L、40 mmol/L、80 mmol/L、160 mmol/L等4组,对照组使用0.9%生理盐水;经回肠灌注不同浓度的NaB作用1 h后取回肠组织,在尤斯灌注系统(Ussing Chamber)上测定跨上皮电阻(transepithelial electrical resistance,TER)值,苏木精-伊红(HE)染色观察病理学及炎症损伤情况,实时荧光定量聚合酶链反应(RT-qPCR)法检测紧密连接蛋白(Occludin、ZO-1)的mRNA表达量,Western blot法检测Occludin、ZO-1蛋白的表达水平。结果 尤斯灌注系统测得对照组在30 min、60 min、120 min时的相对TER值(各个时间点测得的TER值除以0 min测得TER值得到的百分比值)分别为(87.36±1.88)、(85.98±0.99)、(101.03±1.47),20 mmol/L组分别为(83.45±2.97)、(107.24±5.35)、(101.44±3.96),40 mmol/L组分别为(126.07±3.48)、(140.54±3.64)、(100.50±4.64),80 mmol/L组分别为(102.24±4.60)、(134.55±7.80)、(106.76±8.82),160 mmol/L组分别为(84.80±2.97)、(78.16±2.89)、(67.11±4.07);使用不同浓度NaB作用30 min,与对照组相比,40 mmol/L组的TER值明显增加(P<0.05),作用1 h时,40 mmol/L组与80 mmol/L组的TER值明显高于对照组、20 mmol/L及160 mmol/L组(P<0.05),但二者的差异无统计学意义(P>0.05)。使用不同浓度NaB作用1 h后,根据损伤分级评分(0~5分)结果,160 mmol/L组(2.16±0.75)分,较对照组(0.33±0.51)分损伤明显(P<0.01),余各组损伤不明显(P>0.05)。40 mmol/L组及80 mmol/L组肠黏膜组织Occludin蛋白mRNA(1.80±0.31、2.13±0.90)表达量较对照组(1.01±0.21)上调,差异具有统计学意义(P<0.05),蛋白表达水平(0.58±0.04、0.73±0.01)较对照组(0.24±0.01)明显升高,差异具有统计学意义(P<0.05);而ZO-1蛋白mRNA(1.36±0.24、1.45±0.58)表达量较对照组(1.01±0.11)上调不显著,差异无统计学意义(P>0.05),蛋白表达水平(1.46±0.04、1.34±0.07)较对照组(1.07±0.05)亦明显升高,差异具有统计学意义(P<0.05);160 mmol/L组Occludin和ZO-1蛋白mRNA(1.27±0.47、0.92±0.25)表达量较对照组的差异无统计学意义(P>0.05),而蛋白表达水平(0.47±0.01、2.90±0.04)均高于对照组,差异具有统计学意义(P<0.01)。结论 NaB对活体新生SD大鼠肠黏膜的作用具有浓度依赖性,40 mmol/L或80 mmol/L的NaB能增强肠黏膜屏障的功能,这可能与肠黏膜组织Occludin和ZO-1蛋白的表达增加有关。

Objective To evaluate the effect of sodium butyrate(NaB)on the protection of intestinal barrier function and establish the effect of NaB on intestinal mucosa in neonatal rats.Methods The 9-day-old neonatal rats were randomly divided into 5 groups.The serial concentrations of NaB were set at 20,40,80 and 160 mmol/L.And saline was selected as a normal control.Intestinal tissue was harvested after an ileal infusion of different concentrations of NaB for 1 hour.Transepithelial electrical resistance(TER)was measured on a Ussing chamber and pathological condition and inflammatory injuries were observed by hematoxylin-eosin stain.The expressions of tight junction proteins Occludin and ZO-1 mRNA were detected by real-time quantitative polymerase chain reaction(RT-qPCR),Occludin and ZO-1 protein levels were detected by Western blot.Results(a)The value of TER(relative to initial value%)of control group at 30/60/120 min was(87.36±1.88),(85.98±0.99),(101.03±1.47)and 20 mmol/L group(83.45±2.97),(107.24±5.35),(101.44±3.96),40 mmol/L group(126.07±3.48),(140.54±3.64),(100.50±4.64),80 mmol/L group(102.24±4.60),(134.55±7.80),(106.76±8.82)and 160 mmol/L group(84.80±2.97),(78.16±2.89),(67.11±4.07)respectively.After acting with different concentrations of NaB for 30 min,as compared with control group,TER value of 40 mmol/L group increased markedly(P<0.05),At 1 h,TER value of 40/80 mmol/L group was significantly higher than those of control and 20/160 mmol/L groups(P<0.05).However,no significant inter-group difference existed(P>0.05);(b)After reacting with different concentrations of sodium butyrate for 1 h,histologic injury scores was significantly higher in 160 mmol/L group than that in control group[(2.16±0.75)vs(0.33±0.51),P<0.01],other groups were not significant(P>0.05);(c)40/80 mmol/L group Occludin mRNA expression(1.80±0.31,2.13±0.90)was higher than control group(1.01±0.21)(P<0.05)while protein level(0.58±0.04,0.73±0.01)also increased as compared with control group(0.24±0.01)(P<0.05).And ZO-1 mRNA expression(1.36±0.24,1.45±0.58)was not significantly higher than that of control group(1.01±0.11)(P>0.05).However,the level of protein(1.46±0.04,1.34±0.07)was significantly higher than that of saline group(1.07±0.05)(P<0.05).In addition,160 mmol/L group Occludin and ZO-1 mRNA expression(1.27±0.47,0.92±0.25)were insignificant(P>0.05)while the protein expression levels(0.47±0.01,2.90±0.04)were higher than that of control group(P<0.01).Conclusions NaB affects on intestinal barrier function in a time/dose-dependent manner.And 40/80 mmol/L sodium butyrate enhances intestinal mucosal barrier through the up-regulations of Occludin and ZO-1.