艾司洛尔对脓毒症肠损伤的保护作用及对自噬蛋白AMPK表达水平的影响

Protective effect of esmolol on sepsis-induced intestinal injury and its mechanism

目的探讨艾司洛尔(ES)对脓毒症大鼠肠损伤的保护作用及其具体作用机制。方法选择18只成年雄性SD大鼠,按随机数字表法分为假手术(Sham)组、脂多糖组(LPS)组和ES干预组(LPS+ES组),每组6只。LPS组、LPS+ES组采用腹腔注射LPS(10 mg/kg)建立脓毒症肠损伤模型;Sham组注射等量等渗盐水。LPS+ES组持续经大鼠左颈内静脉泵入ES注射液(15 mg·kg^(-1)·h^(-1));Sham组、LPS组持续泵入等量等渗盐水。模型构建成功后12 h处死大鼠,并留取血标本及小肠组织。采用酶联免疫吸附法(ELISA)检测肠型脂肪酸结合蛋白(I-FABP)和二胺氧化酶(DAO);苏木素-伊红(HE)染色观察小肠组织病理学变化;ELISA检测白介素-6(IL-6)和白介素-10(IL-10)水平;Western blotting检测Beclin-1、LC3-Ⅱ及p-AMPK的蛋白表达水平。结果与Sham组相比,LPS组光镜下可见小肠黏膜下层、肌层广泛空泡化变性,炎症细胞浸润,绒毛缩短,结构模糊;与LPS组相比,ES干预后黏膜下层、肌层广泛空泡化变性及炎症细胞浸润明显减轻,绒毛变长,病理损伤减轻。LPS组IL-6、IL-10、I-FABP及DAO较Sham组显著升高[两组分别为:(131.00±8.67)pg/mL比(106.40±6.70)pg/mL;(116.62±9.09)pg/mL比(103.23±7.38)pg/mL;(9.61±1.70)ng/mL比(3.91±0.70)ng/mL;(234.30±30.14)ng/mL比(37.49±12.11)ng/mL,P均<0.05];ES干预后IL-6、I-FABP及DAO水平较LPS组明显降低[(117.50±9.00)pg/mL比(131.0±8.67)pg/mL;(5.34±1.10)pg/mL比(9.61±1.70)pg/mL;(147.80±17.07)ng/mL比(234.30±30.14)ng/mL,P均<0.05],而IL-10水平较LPS组明显升高[(129.74±10.94)pg/mL比(116.62±9.09)pg/mL,P<0.05]。与Sham组相比,LPS组小肠组织Beclin-1、LC3-Ⅱ及p-AMPK的蛋白表达水平均明显降低(P<0.05);与LPS组相比,ES干预后Beclin-1、LC3-Ⅱ及p-AMPK的蛋白表达量均明显增加(P均<0.05)。结论ES对脓毒症大鼠急性肠损伤有显著的保护作用,其机制可能是通过促进AMPK介导的自噬及降低炎症反应有关。

Objective To investigate the protective effect and specific mechanism of esmolol(ES)on sepsis-induced intestinal injury in rats.Methods Eighteen adult male SD rats were randomLy divided into three groups according to the random number table method:Sham group(6 rats),lipopolysaccharide(LPS)group(6 rats),and LPS+ES group(6 rats).The sepsis-induced acute intestinal injury rat model was reproduced by intraperitoneal injection of lipopolysaccharide(LPS)(10 mg/kg).The Sham group only received an equivalent amount of saline.In LPS+ES group,esmolol dilution(15 mg·kg^(-1)·h^(-1))was continuously pumped through left internal jugular vein;normal saline was continuously pumped into Sham group and LPS group.The rats were sacrificed when the model was successfully constructed for 12 h.The blood samples and the small intestinal tissues were collected.The diamine oxidase(DAO)and intestinal fatty acid-binding protein(I-FABP)were examined by enzyme-linked immunosorbent assay(ELISA).The intestinal tissue injury was analyzed by HE staining.The concentration of(interleukin-6,IL-6)and(interleukin-10,IL-10)were measured by ELISA.The expression levels of Beclin-1,LC3-Ⅱ,and p-AMPK was detected by Western blotting.Results Compared with Sham group,LPS group showed extensive vacuolization and inflammatory cell infiltration in submucosa and muscularis of small intestine,shortening of villi and blurring of structure under light microscope.After esmolol intervention,extensive vacuolization and inflammatory cell infiltration in submucosa and muscle layer were significantly reduced,villi became longer,and pathological damage was reduced.In LPS group,the levels of IL-6,IL-10,I-FABP and DAO were significantly higher than that of Sham group[(131.00±8.67)pg/mL vs(106.40±6.70)pg/mL;(116.62±9.09)pg/mL vs(103.23±7.38)pg/mL;(9.61±1.70)ng/mL vs(3.91±0.70)ng/mL;(234.30±30.14)ng/mL vs(37.49±12.11)ng/mL,respectively,all P<0.05].The levels of IL-6,DAO,I-FABP in the LPS+ES group were significantly decreased as compared with those in LPS group[(117.50±9.00)pg/mL vs (131.0±8.67)pg/mL;(5.34±1.10)pg/mL vs(9.61±1.70)pg/mL;(147.80±17.07)ng/mL vs(234.30±30.14)ng/mL,respectively,all P<0.05],but the expression of IL-10 was significantly increased in LPS+ES group compared with that in LPS group[(129.74±10.94)pg/mL vs(116.62±9.09)pg/mL,P<0.05].At the same time,compared with LPS group,the levels of Beclin-1,LC3-Ⅱand p-AMPK was significantly decreased(all P<0.05),but the expression of Beclin-1,LC3-Ⅱand p-AMPK protein was significantly higer in LPS+ES group than those in LPS group(all P<0.05).Conclusion The protective effects of esmolol on sepsis-induced intestinal injury may be attributed to the up-regulation of AMPK-mediated autophagy and reduction of inflammatory response.