SIRT1对LPS诱导的坏死性小肠结肠炎小鼠模型的保护作用
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摘要
目的探讨沉默信息调节因子1(SIRT1)在脂多糖(lipopolysaccharide,LPS)诱导的坏死性小肠结肠炎(NEC)小鼠的保护作用。方法 3-4日龄C57BL/6小鼠60只被随机分为三组。对照组大鼠与母鼠同笼,鼠乳喂养,不做任何处理; NEC模型组、SIRT1激动剂干预组大鼠出生24 h与母鼠分开,置于保育箱中,采用鼠乳代用品人工饲养,采用低温缺氧诱导NEC模型。模型制备结束后空腹12 h,处死小鼠采集十二指肠下端至回盲部肠管,观察肠管的弹性、色泽及肠腔有无积气、出血、坏死等病变; HE染色观察肠组织病理变化; ELISA测定肠组织IL-6、IL-10、TNF-α水平; qRT-PCR法检测IL-6、IL-10、TNF-αmRNA表达水平; Western blot检测肠组织SIRT1蛋白表达水平。结果模型组小鼠均出现不同程度的腹胀、排黄绿色稀便、嗜睡、胃滞留、呼吸急促等症状,SIRT1激动剂干预组小鼠各症状较模型组减轻,对照组小鼠无异常情况,模型组死亡7只,干预组无死亡。肠组织病理切片显示,对照组肠道上皮及肠绒毛完整,无血管扩张,模型组黏膜下层出血明显、水肿,黏膜下层和固有层分离,肠绒毛结构不完整,部分脱落、坏死,干预组较模型组损伤较轻。Western blot检测显示,与对照组比较,模型组、干预组小鼠肠组织SIRT1表达显著降低(P <0. 05),与模型组比较,SIRT1激动剂干预组SIRT1水平显著升高(P <0. 05)。ELISA检测显示,与对照组比较,模型组肠组织中IL-6、TNF-α水平显著升高,IL-10水平显著降低(P <0. 05);与模型组比较,干预组肠组织中IL-6、TNF-α、IL-1β水平显著降低,IL-10水平显著升高(P <0. 05)。qRT-PCR验证结果显示,模型组肠组织中IL-6、TNF-αmRNA水平较对照组显著升高,IL-10 mRNA水平显著降低(P <0. 05);与模型组比较,干预组肠组织中IL-6、TNF-αmRNA水平显著降低,IL-10 mRNA水平显著升高(P <0. 05)。结论 SIRT1在LPS诱导的坏死性小肠结肠炎小鼠肠内表达水平降低,SIRT1激动剂干预后其表达水平升高,其可能通过抑制炎症反应保护肠道组织完整。
Objective To investigate the protective effect of SIRT1 on lipopolysaccharide( LPS)-induced necrotizing enterocolitis(NEC) in mice. Methods Sixty C57 BL/6 mice aged 3-4 days were randomly divided into three groups. The mice in NEC model group and SIRT1 agonist group were separated from their mothers at 24 h after birth,and then placed in incubator and artificially reared with rat milk substitutes. At the same time,NEC model was induced by hypoxia-induced hypothermia. The mice in control group were fed in the same cage with their mothers without any treatment. Fasting for 12 h after model preparation,the mice were sacrificed to collect the intestinal tube from the lower duodenum to ileocecum. The elasticity and color of ileocecum,and pneumatosis,hemorrhage,necrosis changes in intestinal cavity were observed. The pathological changes of intestinal tissue were observed by HE staining. The levels of IL-6,IL-10 and TNF-α in intestinal tissue were measured by ELISA. The expression levels of IL-6,IL-10,and TNF-α mRNA were detected by qRT-PCR. SIRT1 protein expression level in intestinal tissue was detected by Western blot. Results The mice in model group showed abdominal distension,yellowish-green loose stool,lethargy,gastric retention,shortness of breath and other symptoms in varying degrees. The symptoms were lighter in SIRT1 agonist group than in model group,and there was no abnormality in control group. Seven mice died in model group and no one in SIRT1 agonist group. The pathological sections of intestinal tissues showed that intestinal epithelium and villi were intact in control group without vasodilatation,however,submucosal hemorrhage and edema were obvious in model group,in which the submucosal and lamina propria were separated and intestinal villus structure was incomplete with some of them being exfoliated and necrotic. The injury in SIRT1 agonist group was lighter than that in model group.Western blot analysis showed that the expression of SIRT1 in intestinal tissue were significantly lower in model group and SIRT1 agonist group than in control group( P < 0. 05). SIRT1 level in SIRT1 agonist group increased significantly compared with model group( P <0. 05). ELISA showed that,compared with control group,the levels of IL-6 and TNF-α in intestinal tissue increased significantly in model group,while the levels of IL-10 decreased significantly( P < 0. 05). Compared with model group,the levels of IL-6,TNF-α,and IL-1β in intestinal tissue decreased significantly in SIRT1 agonist group,while the levels of IL-10 increased significantly( P < 0.05). The results of qRT-PCR showed that the levels of IL-6 and TNF-α mRNA in intestinal tissues were significantly higher in model group than in control group,while the level of IL-10 mRNA was significantly lower( P < 0. 05). Compared with model group,the levels of IL-6 and TNF-α mRNA in intestinal tissues in SIRT1 agonist group were significantly decreased,while the levels of IL-10 mRNA were significantly increased( P < 0. 05). Conclusion The expression level of SIRT1 decreases in intestine of LPS-induced necrotizing enterocolitis mice,and increases after the intervention with SIRT1 agonist. SIRT1 may protect intestinal tissue integrity by inhibiting inflammatory response.
Objective To investigate the protective effect of SIRT1 on lipopolysaccharide( LPS)-induced necrotizing enterocolitis(NEC) in mice. Methods Sixty C57 BL/6 mice aged 3-4 days were randomly divided into three groups. The mice in NEC model group and SIRT1 agonist group were separated from their mothers at 24 h after birth,and then placed in incubator and artificially reared with rat milk substitutes. At the same time,NEC model was induced by hypoxia-induced hypothermia. The mice in control group were fed in the same cage with their mothers without any treatment. Fasting for 12 h after model preparation,the mice were sacrificed to collect the intestinal tube from the lower duodenum to ileocecum. The elasticity and color of ileocecum,and pneumatosis,hemorrhage,necrosis changes in intestinal cavity were observed. The pathological changes of intestinal tissue were observed by HE staining. The levels of IL-6,IL-10 and TNF-α in intestinal tissue were measured by ELISA. The expression levels of IL-6,IL-10,and TNF-α mRNA were detected by qRT-PCR. SIRT1 protein expression level in intestinal tissue was detected by Western blot. Results The mice in model group showed abdominal distension,yellowish-green loose stool,lethargy,gastric retention,shortness of breath and other symptoms in varying degrees. The symptoms were lighter in SIRT1 agonist group than in model group,and there was no abnormality in control group. Seven mice died in model group and no one in SIRT1 agonist group. The pathological sections of intestinal tissues showed that intestinal epithelium and villi were intact in control group without vasodilatation,however,submucosal hemorrhage and edema were obvious in model group,in which the submucosal and lamina propria were separated and intestinal villus structure was incomplete with some of them being exfoliated and necrotic. The injury in SIRT1 agonist group was lighter than that in model group.Western blot analysis showed that the expression of SIRT1 in intestinal tissue were significantly lower in model group and SIRT1 agonist group than in control group( P < 0. 05). SIRT1 level in SIRT1 agonist group increased significantly compared with model group( P <0. 05). ELISA showed that,compared with control group,the levels of IL-6 and TNF-α in intestinal tissue increased significantly in model group,while the levels of IL-10 decreased significantly( P < 0. 05). Compared with model group,the levels of IL-6,TNF-α,and IL-1β in intestinal tissue decreased significantly in SIRT1 agonist group,while the levels of IL-10 increased significantly( P < 0.05). The results of qRT-PCR showed that the levels of IL-6 and TNF-α mRNA in intestinal tissues were significantly higher in model group than in control group,while the level of IL-10 mRNA was significantly lower( P < 0. 05). Compared with model group,the levels of IL-6 and TNF-α mRNA in intestinal tissues in SIRT1 agonist group were significantly decreased,while the levels of IL-10 mRNA were significantly increased( P < 0. 05). Conclusion The expression level of SIRT1 decreases in intestine of LPS-induced necrotizing enterocolitis mice,and increases after the intervention with SIRT1 agonist. SIRT1 may protect intestinal tissue integrity by inhibiting inflammatory response.
引文
[1]王功僚,韦巍.新生儿坏死性小肠结肠炎病因及发病机制研究进展[J].中外医学研究,2015,13(16):150-151.
[2]刘亚丽,许丽,魏克伦.早产及低出生体重儿输血相关坏死性小肠结肠炎发病机制与防治研究进展[J].山东医药,2016,56(19):97-99.
[3]Chowning R,Radmacher P,Lewis S,et al.A retrospective analysis of the effect of human milk on prevention of necrotizing enterocolitis and postnatal growth[J].J Perinatol,2016,36(3):221-224.
[4]刘斌,阎静江,李晓霞.新生鼠坏死性小肠结肠炎动物模型建立方法改进[J].中国现代医生,2015,53(35):22-25.
[5]Wang N,Zhang F,Yang L,et al.Resveratrol protects against L-arginine-induced acute necrotizing pancreatitis in mice by enhancing SIRT1-mediated deacetylation of p53 and heat shock factor 1[J].Int J Mol Med,2017,40(2):427-437.
[6]王玲玲,金芳,温博贤,等.坏死性小肠结肠炎晚发和长病程动物模型的建立[J].当代医学,2018,24(8):1-5.
[7]Warner BB,Deych E,Zhou Y,et al.Gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants:a prospective case-control study[J].Lancet,2016,387(10031):1928-1936.
[8]Abdullah F,Zhang Y,Camp M,et al.Necrotizing enterocolitis in 20,822 infants:analysis of medical and surgical treatments[J].Clin Pediatr(Phila),2010,49(2):166-171.
[9]Emami CN,Petrosyan M,Giuliani S,et al.Role of the host defense system and intestinal microbial flora in the pathogenesis of necrotizing enterocolitis[J].Surg Infect(Larchmt),2009,10(5):407-417.
[10]Liu L,Liu C,Zhang Q,et al.SIRT1-mediated transcriptional regulation of SOX2 is important for self-renewal of liver cancer stem cells[J].Hepatology,2016,64(3):814-827.
[11]Tian Y,Ma J,Wang W,et al.Resveratrol supplement inhibited the NF-κB inflammation pathway through activating AMPKα-SIRT1 pathway in mice with fatty liver[J].Mol Cell Biochem,2016,422(1-2):75-84.
[12]李晶,周伟,袁伟明,等.不同浓度脂多糖对大鼠肠上皮细胞增殖和白细胞介素-6、白细胞介素-1β及肿瘤坏死因子-α分泌的影响[J].中华实用儿科临床杂志,2015,30(7):490-493.
[13]刘素佳,杨长仪,陈涵强.新生儿坏死性小肠结肠炎相关炎性标志物研究进展[J].中国新生儿科杂志,2015,30(6):468-471.
[14]Patel RM,Knezevic A,Shenvi N,et al.Association of red blood cell transfusion,anemia,and necrotizing enterocolitis in very lowbirth-weight infants[J].JAMA,2016,315(9):889-897.
[15]Wang Y,Bi Y,Chen X.Histone deacetylase SIRT1 negatively regulates the differentiation of interleukin-9-producing CD4(+)T cells[J].Immunity,2016,44(6):1337-1349.
[16]Gao J,Zhou R,You X,et al.Salidroside suppresses inflammation in a D-galactose-induced rat model of Alzheimer’s disease via SIRT1/NF-κB pathway[J].Metab Brain Dis,2016,31(4):771-778.
[17]Li Z,Sheng L.Significance of dynamic evolution of TNF-α,IL-6and intestinal fatty acid-binding protein levels in neonatal necrotizing enterocolitis[J].Exp Ther Med,2018,15(2):1289-1292.
[18]Lodha A,Howlett A,Ahmed T,et al.The role of interleukin-6and interleukin-8 circulating cytokines in differentiating between feeding intolerance and necrotizing enterocolitis in preterm infants[J].Am J Perinatol,2017,34(13):1286-1292.
[19]Simon JM,Davis JP,Lee SE,et al.Alterations to chromatin in intestinal macrophages link IL‐10 deficiency to inappropriate inflammatory responses[J].Eur J Immunol,2016,46(8):1912-1925.
[20]Bell JA,Jerome JP,Plovanich-Jones AE,et al.Outcome of infection of C57BL/6 IL-10(-/-)mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up-and down-regulated in vivo[J].Microb Pathog,2013,54(1):1-19.
[2]刘亚丽,许丽,魏克伦.早产及低出生体重儿输血相关坏死性小肠结肠炎发病机制与防治研究进展[J].山东医药,2016,56(19):97-99.
[3]Chowning R,Radmacher P,Lewis S,et al.A retrospective analysis of the effect of human milk on prevention of necrotizing enterocolitis and postnatal growth[J].J Perinatol,2016,36(3):221-224.
[4]刘斌,阎静江,李晓霞.新生鼠坏死性小肠结肠炎动物模型建立方法改进[J].中国现代医生,2015,53(35):22-25.
[5]Wang N,Zhang F,Yang L,et al.Resveratrol protects against L-arginine-induced acute necrotizing pancreatitis in mice by enhancing SIRT1-mediated deacetylation of p53 and heat shock factor 1[J].Int J Mol Med,2017,40(2):427-437.
[6]王玲玲,金芳,温博贤,等.坏死性小肠结肠炎晚发和长病程动物模型的建立[J].当代医学,2018,24(8):1-5.
[7]Warner BB,Deych E,Zhou Y,et al.Gut bacteria dysbiosis and necrotising enterocolitis in very low birthweight infants:a prospective case-control study[J].Lancet,2016,387(10031):1928-1936.
[8]Abdullah F,Zhang Y,Camp M,et al.Necrotizing enterocolitis in 20,822 infants:analysis of medical and surgical treatments[J].Clin Pediatr(Phila),2010,49(2):166-171.
[9]Emami CN,Petrosyan M,Giuliani S,et al.Role of the host defense system and intestinal microbial flora in the pathogenesis of necrotizing enterocolitis[J].Surg Infect(Larchmt),2009,10(5):407-417.
[10]Liu L,Liu C,Zhang Q,et al.SIRT1-mediated transcriptional regulation of SOX2 is important for self-renewal of liver cancer stem cells[J].Hepatology,2016,64(3):814-827.
[11]Tian Y,Ma J,Wang W,et al.Resveratrol supplement inhibited the NF-κB inflammation pathway through activating AMPKα-SIRT1 pathway in mice with fatty liver[J].Mol Cell Biochem,2016,422(1-2):75-84.
[12]李晶,周伟,袁伟明,等.不同浓度脂多糖对大鼠肠上皮细胞增殖和白细胞介素-6、白细胞介素-1β及肿瘤坏死因子-α分泌的影响[J].中华实用儿科临床杂志,2015,30(7):490-493.
[13]刘素佳,杨长仪,陈涵强.新生儿坏死性小肠结肠炎相关炎性标志物研究进展[J].中国新生儿科杂志,2015,30(6):468-471.
[14]Patel RM,Knezevic A,Shenvi N,et al.Association of red blood cell transfusion,anemia,and necrotizing enterocolitis in very lowbirth-weight infants[J].JAMA,2016,315(9):889-897.
[15]Wang Y,Bi Y,Chen X.Histone deacetylase SIRT1 negatively regulates the differentiation of interleukin-9-producing CD4(+)T cells[J].Immunity,2016,44(6):1337-1349.
[16]Gao J,Zhou R,You X,et al.Salidroside suppresses inflammation in a D-galactose-induced rat model of Alzheimer’s disease via SIRT1/NF-κB pathway[J].Metab Brain Dis,2016,31(4):771-778.
[17]Li Z,Sheng L.Significance of dynamic evolution of TNF-α,IL-6and intestinal fatty acid-binding protein levels in neonatal necrotizing enterocolitis[J].Exp Ther Med,2018,15(2):1289-1292.
[18]Lodha A,Howlett A,Ahmed T,et al.The role of interleukin-6and interleukin-8 circulating cytokines in differentiating between feeding intolerance and necrotizing enterocolitis in preterm infants[J].Am J Perinatol,2017,34(13):1286-1292.
[19]Simon JM,Davis JP,Lee SE,et al.Alterations to chromatin in intestinal macrophages link IL‐10 deficiency to inappropriate inflammatory responses[J].Eur J Immunol,2016,46(8):1912-1925.
[20]Bell JA,Jerome JP,Plovanich-Jones AE,et al.Outcome of infection of C57BL/6 IL-10(-/-)mice with Campylobacter jejuni strains is correlated with genome content of open reading frames up-and down-regulated in vivo[J].Microb Pathog,2013,54(1):1-19.