miR-431调控CaMK Ⅳ信号通路参与坏死性小肠结肠炎症的机制

miR-431 participates in the occurrence of NEC intestinal inflammation by regulating the CaMK Ⅳ signaling pathway

[目的]探讨miR-431调控CaMKⅣ信号通路对新生大鼠坏死性小肠结肠炎(NEC)的影响。[方法]将新生SD雄性大鼠32只随机分为对照组、模型组、阴性对照组、miR-431 antagomir组,每组8只。通过苏木素-伊红(HE)染色对各组大鼠进行肠组织病理评分;酶联免疫吸附法(ELISA)检测各组IL-6、TNF-α表达水平;实时荧光定量逆转录聚合酶链反应(qRT-PCR)检测各组miR-431、CaMKⅣmRNA的表达水平;蛋白印迹(Western blot)检测各组CaMKⅣ蛋白及pCaMKⅣ蛋白的表达水平。[结果]对照组肠组织结构清晰,上皮完整,腺体规则排列,无黏膜层及黏膜下层充血水肿及炎症细胞浸润;模型组及阴性对照组肠组织坏死严重,腺体排列紊乱,黏膜下层或固有层有重度分离,黏膜下层和肌层有严重水肿,绒毛崩解分离和缺血坏死,存在大量炎症细胞浸润,IL-6、TNF-α、miR-431、CaMKⅣmRNA及蛋白、pCaMKⅣ蛋白表达水平显著升高(P<0.05);miR-431 antagomir组肠道组织充血水肿情况较模型组减轻,腺体排列稍显紊乱,肠黏膜及黏膜下层可见轻度水肿,有少量炎症细胞浸润,IL-6、TNF-α、miR-431、CaMKⅣmRNA及蛋白、pCaMKⅣ蛋白表达水平较模型组和阴性对照组显著降低(P<0.05)。[结论]敲低miR-431的表达可减轻NEC新生大鼠的炎症水平,改善其肠组织的损伤情况,其机制可能与miR-431低表达抑制CaMKⅣ信号通路活化有关。

[Objective]To explore the effect of miR-431 regulating CaMK Ⅳ signaling pathway on necrotizing enterocolitis(NEC)in neonatal rats.[Methods]Newborn male SD rats were randomly divided into control group, model group, negative control group, miR-431 antagomir group.Hematoxylin-eosin(HE)staining was used to intestinal histopathological score.The expression levels of IL-6 and TNF-α in each group were determined by enzyme-linked immunosorbent assay(ELISA).Real-time fluorescence quantitative reverse transcription polymerase chain reaction(qRT-PCR)was used to detect the expression levels of miR-431 and CaMK Ⅳ mRNA.The expression levels of CaMK Ⅳ protein and pCaMK Ⅳ protein were detected by Western blot.[Results]In the control group, the intestinal tissue structure was clear, the epithelium was intact, the glands were regularly arranged, and there was no congestion, edema and inflammatory cell infiltration in the mucosa and submucosa.The model group and the negative control group had severe intestinal tissue necrosis, disordered gland arrangement, severe separation of the submucosa or lamina propria, severe edema in the submucosa and muscle layer, disintegration and separation of villi and ischemic necrosis, a large number of inflammatory cell infiltration, IL-6,TNF-α,miR-431,CaMK Ⅳ mRNA and protein, and pCaMK Ⅳ protein expression levels were significantly increased(P<0.05).The congestion and edema of the intestinal tissue in the miR-431 antagomir group was less than that in the model group.The gland arrangement was slightly disordered.There was mild edema in the intestinal mucosa and submucosa with a small amount of inflammatory cell infiltration, IL-6,TNF-α,miR-431,CaMK Ⅳ mRNA and protein, and pCaMK Ⅳ protein expression levels were significantly lower than those of the model group and the negative control group(P<0.05).[Conclusion]Knocking down the expression of miR-431 can reduce the inflammatory level of NEC newborn rats and improve the damage of their intestinal tissues, and the mechanism may be related to the inhibition of CaMK Ⅳ signaling pathway activation by the low expression of miR-431.