白术内酯Ⅰ改善术后肠梗阻大鼠的肠功能、炎症反应、细菌移位及肠道屏障

Atractylenolide I improves intestinal function,inflammatory response,bacterial translocation,and intestinal barrier function in postoperative ileus rats

背景白术内酯Ⅰ(atractylenolideⅠ,AT-1)具有抗炎、抗氧化、抑制肠损伤和改善肠屏障功能的作用,但其对术后肠梗阻(postoperative ileus,POI)的影响和机制并不清楚.目的探讨AT-1对POI大鼠的肠功能、炎症反应、细菌移位及肠道屏障的影响,并探讨初步机制.方法60只泼累格·多雷雄性大鼠,分为5组:对照组、模型组、及低、中、高剂量AT-1干预组,每组12只.用摩擦肠管法构建POI大鼠模型,低、中、高剂量AT-1干预组分别在造模前1 h和造模后1-3 d通过尾静脉注射20、40和80 mg/kg AT-1,每天1次.肠推进实验评估肠推进功能,异硫氰酸酯-葡聚糖法检测肠通透性;苏木精-伊红染色观察小肠组织病理形态;生化分析仪计数外周血白细胞和粒细胞含量,酶联免疫吸附试验法检测血清中白细胞介素(interleukin,IL)-6、IL-1β、肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、内毒素、脂多糖和D-乳酸水平;免疫组化检测肠组织中溶霉菌(Lysozyme)、绒毛蛋白(Villin)、闭锁小带蛋白1(zonula occludens protein 1,ZO-1)和密封蛋白(Claudin)表达分布情况;蛋白质免疫印迹法检测肠组织中核因子κB(nuclear factor kappa-B,NF-κB)相关蛋白表达.结果相比于对照组,模型组小肠绒毛断裂或脱落,隐窝丢失,肠推进功能降低,肠道通透性增加;相比于模型组,AT-1干预组小肠绒毛变长和隐窝增多,肠推进功能增高,肠道通透性降低.免疫组化结果显示,相比于对照组,模型组小肠组织中Lysozyme、Villin、ZO-1和Claudin表达降低,而AT-1干预组上述蛋白表达增高.生化检验结果显示,相比于对照组,模型组外周血中白细胞和中性粒细胞数增多,血清中IL-6、IL-1β、TNF-α、内毒素、脂多糖和D-乳酸水平增高;相比于模型组,AT-1干预组上述血液学指标均降低.蛋白质免疫印迹法检测结果显示,相比于对照组,模型组小肠组织中磷酸化(phospho,p)-P65/P65和磷酸化NF-κB抑制蛋白(phospho-inhibitor of NF-κB,p-IκB)/IκB的表达水平均升高;相比于模型组,AT-1干预组p-P65/P65和p-IκB/IκB的表达水平均降低.且以上结果均具有剂量效应.结论AT-1可有效抑制POI大鼠肠道损伤、炎症反应和NF-κB信号活性,同时维持肠屏障功能的完整性并阻止细菌移位.

BACKGROUND Atractylenolide I(AT-1)exhibits anti-inflammatory and antioxidant properties,as well as the ability to inhibit intestinal injury while improving intestinal barrier function.However,the effect and mechanism of AT-1 on postoperative ileus(POI)remain unclear.AIM To investigate the effects of AT-1 on intestinal function,inflammatory response,bacterial translocation,and intestinal barrier function in rats with POI,and to analyze the underlying mechanism.METHODS Sixty male Sprague-Dawley rats were divided into five groups:Control group,model group,and low-,medium-,and high-dose AT-1 intervention groups,with 12 rats in each group.The POI rat model was established using the frictional intestinal method,and the low,medium,and high-dose AT-1 intervention groups were administered 20,40,and 80 mg/kg of AT-1 via the tail vein 1 h before modeling and once daily for 1-3 d post-modeling.The intestinal propulsion test was utilized for the evaluation of intestinal motility function,while the fluoresceine isothiocyanate-dextran method was employed to assess intestinal permeability.Hematoxylin-eosin staining was conducted to observe the pathological morphology of the small intestine.The levels of white blood cells and neutrophils in peripheral blood were quantified using a biochemical analyzer.Enzyme-linked immunosorbent assay was performed to measure the levels of interleukin(IL)-6,IL-1β,tumor necrosis factorα(TNF-α),endotoxin,lipopolysaccharide,and D-lactate in serum.Immunohistochemistry was applied to detect the expression and distribution of Lysozyme,Villin,zonula occludens protein 1(ZO-1),and Claudin in intestinal tissues.Western blot analysis was used for detecting the expression of nuclear factor kappa-B(NF-κB)related proteins in intestinal tissue.RESULTS Compared to the control group,the model group exhibited disrupted or sloughed intestinal villi,loss of crypts,reduced intestinal motility,and increased intestinal permeability.In contrast to the model group,the AT-1 intervention groups demonstrated elongated villi and increased number of crypts in the small intestine,enhanced intestinal motility,and decreased intestinal permeability.Immunohistochemistry analysis revealed a significant decrease in the expression of Lysozyme,Villin,ZO-1,and Claudin in the small intestinal tissue of the model group compared to the control group.Conversely,the AT-1 intervention groups exhibited an increase in the expression of these proteins.Biochemical tests demonstrated that compared to the control group,the model group displayed elevated levels of white blood cells and neutrophils in peripheral blood as well as increased levels of IL-6,IL-1β,TNF-α,endotoxin,lipopolysaccharide,and D-lactic acid in serum.In contrast,these hematological parameters were significantly reduced in the AT-1 intervention groups when compared to the model group.Western blot analysis revealed that the expression levels of phospho(p)-P65/P65 and phospho-inhibitor of NF-κB(p-IκB)/IκB in the small intestinal tissue of the model group were significantly elevated compared to the control group.In contrast,the intervention groups exhibited reduced expression levels of p-P65/P65 and p-IκB/IκB when compared to the model group.All these findings demonstrated a dose-dependent effect.CONCLUSION AT-1 effectively inhibits intestinal injury,inflammatory response,and NF-κB signaling activity in rats with postoperative ileus(POI),while preserving the integrity of the intestinal barrier function and preventing bacterial translocation.