DHA对细菌脂多糖诱导的孕鼠炎症和胎鼠生长受限的作用

EFFECT OF DHA ON LIPOPOLYSACCHARIDE-INDUCED INFLAMMATION IN PREGNANT MICE AND GROWTH RESTRICTION IN FETAL MICE

目的探讨二十二碳六烯酸(docosahexaenoic acid,DHA)通过调节肠道紧密连接蛋白拮抗细菌脂多糖(lipopolysaccharide,LPS)诱发的孕鼠炎症和胎儿生长受限的影响及机制。方法模型建立:ICR孕鼠分为对照、DHA、LPS和DHA+LPS四组,每组11只孕鼠,DHA和DHA+LPS组全孕期(GD0-17)给予DHA(300mg/kg)灌胃处理,其余两组灌以等量的玉米油;LPS和DHA+LPS组在GD15-17灌胃1 h后腹腔注射LPS(100μg/kg),其余两组给予等量的生理盐水处理,GD18取材时测量胎鼠、胎盘发育指标。机制探索:分组同上,每组6只孕鼠,DHA和DHA+LPS组在GD0-15给予DHA(300 mg/kg)灌胃处理,其余两组灌以等量的玉米油;LPS和DHA+LPS组在GD15灌胃1小时后腹腔注射LPS(100μg/kg),其余两组给予等量的生理盐水处理,LPS注射1h后收集孕鼠血清及小肠组织。ELISA检测孕鼠血清中IL-6、TNF-α及DAO的水平;Westernblot、qPCR和IHC法检测母鼠小肠上皮紧密连接相关蛋白的表达及分布情况。qPCR方法检测孕鼠小肠组织中炎性因子和紧密连接相关蛋白的基因表达水平。结果DHA预处理可使LPS所致胎儿生长受限发生率从61%下降至42%(P<0.05),每窝胎鼠生长受限的只数由(6.373±0.706)下降至(4.0±0.289)(P<0.05)。LPS组孕鼠血清中IL-6、TNF-α和DAO以及小肠组织Il-1β、Il-6、Tnf-α和Kc mRNA水平均较对照组明显升高(P<0.01),DHA预处理均可使其降低(P<0.05)。LPS组肠道紧密连接相关蛋白ZO-1、Occludin、Claudin-1在蛋白和基因水平上表达较对照组均明显降低(P<0.01),DHA+LPS组各指标水平均高于LPS组(P<0.05)。结论DHA预处理可能通过增加孕鼠肠道上皮紧密连接蛋白的表达,降低肠道通透性,缓解体内炎症水平达到拮抗胎儿生长受限的作用。

Objective To investigate the effect of docosahexaenoic acid(DHA)on lipopolysaccharide(LPS)-induced inflammation and fetal growth restriction in pregnant mice by modulating the expression of intestinal tight junction proteins.Methods The ICR pregnant mice were divided into control,DHA,LPS,and DHA+LPS groups.Eleven pregnant mice were in each group.The DHA and DHA+LPS groups were treated with DHA(300mg/kg)by gavage during the whole gestation period(GDO-17),and the remaining two groups were treated with an equal amount of corn oil.The LPS and DHA+LPS groups were treated with LPS(100μg/kg)by intraperitoneal injection 1 hour after gavage.The fetal mice and placental development indexes were measured at the time of GD18.The serum and small intestine tissues of pregnant mice were collected 1 h after last LPS injection.The levels of IL-6,TNF-α,and DAO in the serum of pregnant mice were measured by ELISA.The expression and distribution of tight junction-related proteins in the small intestinal epithelium of pregnant mice were detected by Western blot,qPCR,and IHC.qPCR was used to detect the gene expression levels of inflammatory factors and tight junction-related proteins in small intestinal tissues of pregnant mice.Results DHApretreatmentdecreased the incidence of LPS-induced fetal growth restriction from 61%to 42%(P<0.05),and the number of fetuses with growth restriction per litter decreased from(6.373±0.706)to(4.0±0.289)(P<0.05).The levels of IL-6,TNF-α,and DAO in the serum and IL-1β,IL-6,TNF-α,and KC mRNA in small intestinal tissues of pregnant mice in the LPS group were significantly higher than those in the control group(P<0.01),and all of them were reduced by DHA pretreatment.The expression of intestinal tight junction-related proteins zo-l,claudin-1,and occludin was significantly lower in the LPS group compared with the control group at both protein and mRNA levels(P<0.01),and the levels in the DHA+LPS group were higher than those in the LPS group(P<0.05).Conclusion DHA pretreatment may antagonize fetal growth restriction induced by LPS exposure via increasing the expression of intestinal epithelial tight junction proteins in pregnant mice,reducing intestinal permeability,and alleviating the level of inflammation in vivo.