促红细胞生成素对坏死性小肠结肠炎TLR4、NF-κB及IL-6变化的影响

Effect of erythropoietin on dynamic changes of TLR4,NF-κB,and IL-6 in rat model with necrotizing enterocolitis

目的观察促红细胞生成素(EPO)对新生大鼠坏死性小肠结肠炎(NEC)模型TLR4、NF-κB及IL-6动态变化的影响,探讨EPO对NEC的保护作用及机制。方法 90只3日龄SD大鼠随机分为3组(每组30只):正常对照组、NEC模型组和EPO干预组。采用鼠代乳品人工喂养、缺氧冷刺激建立新生大鼠NEC模型。EPO干预组采用代乳品中添加EPO(0.1U/ml)人工喂养,同时行缺氧冷刺激。正常对照组鼠乳喂养,不进行任何干预。各组于造模后24、48、72h分别处死10只大鼠切取末端回肠3cm,HE染色后观察肠组织病理学变化并进行组织损伤评分,组织学评分≥2分确定为NEC。分别采用荧光定量PCR和ELISA法检测肠组织TLR4 mRNA、NF-κB mRNA和IL-6的动态变化。结果 NEC模型组TLR4 mRNA在24h出现高表达,峰值在48h和72h略有下降,各时间点表达值均较正常对照组明显升高(P<0.01)。EPO干预组24h出现高表达,但随后迅速下降,48h和72h表达值明显低于NEC模型组(P<0.01)。NEC模型组NF-κB mRNAI、L-6表达值在48h和72h明显高于正常对照组(P<0.01);EPO干预组在48h也出现高表达,72h明显低于NEC模型组(P<0.01)。48h内各组动物均未出现组织病理损伤,72h后NEC模型组和EPO干预组出现组织损伤,NEC模型组评分(2.83±0.56)明显高于EPO干预组(1.53±0.35,P<0.01)。结论新生大鼠NEC模型中先天免疫应答启动早于获得性免疫应答,炎症反应早于肠道组织损伤。经肠道给予生理浓度的EPO可下调TLR4表达,抑制炎性介质的产生,从而减轻肠道组织损伤。

Objective To explore the protective effect and the mechanism of erythropoietin（EPO） on dynamic expressions of TLR-4,NF-κB,and IL-6 in the intestinal tissue of neonatal rat model with necrotizing enterocolitis（NEC）.Methods A total of 90 SD rats（3 days old） were divided randomly into 3 groups（30 each） as follows： normal control group,NEC group,and EPO group.The NEC rat model was established by feeding the subjects with rat-milk substitutes after separation from the mother rats.The subjects were then exposed to hypoxia and cold stress.The EPO-treated rats were fed with the substitute of rat-milk containing EPO（0.1U/ml）,and were exposed to hypoxia and cold stress as well.The rats in the control group were raised by their mothers and received no intervention.Ten rats from each group were sacrificed at 24,48,and 72h,respectively.The last 3cm of the terminal ileum was excised and the histological changes of the intestinal tissues were scored after H-E staining.Scores ≥2 were considered NEC.Fluorescence quantitative PCR was used to evaluate the dynamic levels of TLR4 mRNA and NF-κB mRNA.ELISA was used to determine the expression of IL-6.Results The TLR4 mRNA expression in the NEC group increased at 24h,peaked at 48h,and slightly decreased at 72h.The expressions in all time points increased significantly compared with those in the control group（P0.01）.The rats in the EPO group expressed high levels of TLR4 mRNA at 24h.However,the expression rapidly declined later,and was significantly lower at 48 and 72h compared with those in the NEC group（P0.01）.The expressions of NF-κB mRNA and IL-6 were significantly higher in the NEC group than in the control group at 48 and 72 h（P0.01）.The expressions considerably increased in the EPO group at 48h,then declined at 72h,and were significantly lower than those in the NEC group（P0.01）.No apparent pathological change was found in all groups at the time points of 24 and 48h.However,tissue lesion was observed in the NEC and EPO groups at 72 h,and the histological score was significantly higher in the NEC group（2.83±0.56） than in the EPO group（1.53±0.35,P0.01）.Conclusions In the intestinal tract of the neonatal rat with NEC,innate immune response initiates earlier than the acquired immune response.Inflammatory response appears ahead of the tissue lesion,implying that the inflammatory response induces lesion.The enteral administration of human milk with a physiologic concentration of EPO may downregulate the TLR4 mRNA expression and inhibit the release of inflammatory mediators,thereby reducing intestinal tissue injury.