基于TLR4/Myd88/NF-κB通路探讨大黄牡丹汤对术后早期炎性肠梗阻大鼠的干预机制

Exploration on Intervention Mechanism of Dahuang Mudan Decoction on Early Postoperative Inflammatory Intestinal Obstruction in Rats Based on TLR4/Myd88/NF-κB Pathway

目的:观察大黄牡丹汤对术后早期炎性肠梗阻模型大鼠小肠推进率、炎症因子、Toll样受体4/髓系分化因子88/核转录因子-κB(Tlr4/Myd88/Nfκb)通路基因和蛋白表达的影响。方法:将40只大鼠按照随机数字表法分为正常对照组、模型对照组、大黄牡丹汤0.63、1.25、2.5 g/kg组,每组各8只大鼠。除正常对照组外,其余各组均按照肠管表面反复擦拭法进行造模。造模成功后,正常对照组和模型对照组给予相同体积的生理盐水,药物组分别给予对应浓度的大黄牡丹汤药液,连续7 d灌胃,于第8 d处死大鼠。HE染色比较各组大鼠回盲部组织的病理学改变,采用美兰染色法测量小肠推进率,Elisa法测定炎症因子、RT-PCR、Western Blot法检测回盲部组织中Tlr4/Myd88/Nfκb的基因和蛋白表达。结果:与正常对照组比较,模型对照组大鼠小肠推进率明显下降,血清TNF-α、IL-6含量显著升高(P<0.01),回盲部Tlr4/Myd88/Nfκb通路的基因、蛋白表达显著上调(P<0.01),病理切片观察发现模型对照组肠绒毛炎性肿胀明显。与模型对照组相比,大黄牡丹汤各组小肠推进率升高,血清TNF-α、IL-6含量明显降低(P<0.05);大黄牡丹汤2.5 g/kg可降低大鼠回盲部组织中Tlr4/Myd88/Nfκb通路的基因和蛋白表达水平,且呈剂量依赖性(P<0.05或P<0.01),大黄牡丹汤各组肠绒毛炎性浸润得到不同程度改善,且其改善程度与浓度呈正相关。结论:大黄牡丹汤可减轻大鼠术后肠黏膜损伤程度,降低炎症因子含量,其机制可能与抑制Tlr4/Myd88/Nfκb信号通路的基因和蛋白表达有关。

Objective:To explore the effects of Dahuang Mudan Decoction on propulsive intestinal motility,inflammatory factors,and gene and protein expression in Toll-like receptor-4/myeloid differentiation factor 88/nuclear factorκB(TLR4/MyD88/NF-κB)pathway of rats with early postoperative inflammatory intestinal obstruction.Methods:Forty rats were randomly divided into the blank control group,model group,and low-,medium-,and high-dose(0.63,1.25,and 2.5 g/kg)Dahuang Mudan Decoction groups,with eight rats in each group.Rats in all groups except for the blank control group were modeled via the repeated wiping of intestinal surface.After successful modeling,rats in the medication groups were intragastrically administered with Dahuang Mudan Decoction at the corresponding doses,whie those in the blank control group and model group with an equal volume of normal saline for seven days.On the 8 th day,the rats were sacrificed and the ileocecal junction were harvested for observing the histopathological changes after HE staining,followed by the determination of propulsive intestinal motility by methylene blue staining.The inflammatory factors like tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)were detected by ELISA,and the gene and protein expression levels in the TLR4/MyD88/NF-κB pathway were assayed by RT-PCR and Western blotting.Results:Compared with the blank control group,the model group exhibited significantly decreased propulsive intestinal motility,increased serum TNF-αand IL-6(P<0.01),up-regulated protein and gene expression levels in the TLR4/MyD88/NF-κB pathway of ileocecal junction(P<0.01),and obvious inflammatory swelling of intestinal villi.Compared with the model group,Dahuang Mudan Decoction at each dose significantly enhanced the propulsive intestinal motility,lowered the contents of serum TNF-αand IL-6(P<0.05),improved the inflammatory infiltration of intestinal villi to varying degrees in a dose-dependent manner.Dahuang Mudan Decoction at 2.5 g/kg down-regulated the protein and gene expression levels in the TLR4/MyD88/NF-κB pathway of ileocecal junction(P<0.01 or P<0.05).Conclusion:Dahuang Mudan Decoction alleviates the intestinal mucosal injury in rats after operation and reduces the contents of inflammatory factors,which may be related to its inhibition of protein and gene expression levels of the TLR4/MyD88/NF-κB pathway.