Butyric Acid Protects Intestinal Epithelial Barrier from Injury Induced by TNFα
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摘要:
目的 探讨丁酸对TNFα所致肠上皮屏障损伤的保护作用。 方法 用CCK-8细胞活力检测探索TNFα对Caco2发挥损伤作用的最佳浓度和时间,并以此为基础探索在TNFα发挥损伤作用最佳作用时间附近丁酸对Caco2细胞的保护情况,随后探索TNFα和丁酸共同作用于Caco2细胞时的最佳时间和浓度,并检测Caco2细胞单层上皮屏障的FITC-dextran渗透率,紧密连接ZO-1和Occludin的mRNA表达情况及免疫荧光观察TNFα和丁酸共同作用后细胞的生长情况及ZO-1和Occludin在Caco2中的表达和分布。 结果 100 ng/mL的TNFα刺激48 h能明显降低Caco2的细胞存活率(P < 0.0001);丁酸作用于Caco2细胞48 h,0.2 mM/L的丁酸能明显提高Caco2的细胞存活率(P < 0.0001)。当TNFα和丁酸共同作用时,与TNFα干预组相比,丁酸可以明显降低TNFα所致的肠上皮单层屏障的FITC-dextran渗透率(P < 0.0001),提高ZO-1(P < 0.01)和Occludin(P < 0.01)的表达及稳定其在Caco2细胞中的分布。 结论 丁酸可以减轻TNFα所致的肠上皮屏障损伤,为进一步明确丁酸在溃疡性结肠炎治疗中作用机制提供实验基础。 Abstract:Objective To explore the protective effect of butyric acid on intestinal epithelial barrier damage induced by TNF α. Methods CCK-8 cell viability assay was used to determine the optimal concentration and time of TNFα in exerting damaged effects on Caco2 damage, and based on this, the protective effect of butyric acid on Caco2 cells was explored around the optimal time when TNFα exerted damage. Subsequently, the optimal time and concentration of TNFα and butyric acid acting on Caco2 cells were explored, and the FITC-dextran permeability of the Caco2 cell monolayer epithelial barrier, the mRNA expression of ZO-1 and Occludin, and the growth of cells after TNFα and butyric acid co-treatment were detected, as well as the expression and distribution of ZO-1 and Occludin in Caco2 cells were observed by immunofluorescence. Results TNFα, at a concentration of 100 ng/mL significantly decreased the cell viability of Caco2 after 48 hours of stimulation (P < 0.0001); butyrate at a concentration of 0.2mM/L significantly increased the cell viability of Caco2 after 48 hours of treatment (P < 0.0001). When TNFα and butyrate were co-administered, compared with the TNFα intervention group, butyrate significantly decreased the FITC-dextran permeability of the intestinal epithelial monolayer barrier caused by TNFα (P < 0.0001), and increased the expression of ZO-1 (P < 0.01) and Occludin (P < 0.01), and stabilized their distribution in Caco2 cells. Conclusion Butyrate can alleviate TNFα-induced damage to intestinal epithelial barrier, providing experimental basis for further elucidating the mechanism of butyrate in the treatment of ulcerative colitis. -
Key words:
- Caco2 /
- Ulcerative colitis /
- TNF α /
- Butyric acid /
- ZO-1 /
- Occludin
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人鼻病毒(human rhinovirus,HRV)是由Pelon等[1]于19世纪50年代首次从普通感冒患者中分离出,属于小角鼻病毒科的ssRNA病毒,是一种小型非包膜病毒,直径约27 nm,主要根据VP4/VP2衣壳蛋白基因序列分为HRV-A、HRV-B、HRV-C三大类,目前一共发现有171个血清型,是引起儿童呼吸道感染的最主要病原体之一。近年来国内外许多学者对不同国家、不同地区的HRV的流行病学作了相关研究,但针对昆明地区的研究较少。因此,本研究重点了解昆明地区急性下呼吸道感染(acute lower respiratory tract infection,ALRTI)住院患儿的HRV感染情况,并随机抽取部分阳性标本,测定VP4/VP2基因片段,进行基因分型。
1. 资料与方法
1.1 一般资料
1.1.1 研究对象
收集2019年1月至2019年12月在昆明市儿童医院住院的ALRTI病例共2260例,其中男1398例,女862例,男女比例为1.6∶1.0。纳入标准:(1)年龄29 d~14岁的患儿;(2)符合《诸福棠实用儿科学》(第8版)[2]ALRTI诊断标准;(3)其他临床资料完整,如姓名、年龄、发病日期、临床症状、HRV检测结果等。排除标准:既往有免疫缺陷性疾病、慢性肺部疾病、先天性心脏病的患儿。
1.1.2 标本及临床资料收集
在患者住院24 h内采集鼻咽抽吸物或痰液,将标本装入专用痰液收集器放-70°留存待检。病例资料的收集由经过培训的临床医师完成。样本的采集过程及临床资料的获取均具备知情同意书。
1.2 方法
1.2.1 核酸提取
使用Geneaid公司的Viral Nucleic Acid Extraction Kit VR100V 试剂盒提取核酸,严格按照说明书进行操作。
1.2.2 病毒检测
使用Bio-Rad公司的iScript TM Cdna Synthesis Kit逆转录试剂盒进行逆转录反应。将之前提取的核酸作为模板,采用Tiangen公司的TaqDNA聚合酶以及PCR反应体系,巢式PCR扩增HRV-5′NCR基因,引物设计及基因扩增条件参考文献[3]。选取HRV-5′NCR阳性标本扩增VP4/VP2基因片段,VP4/VP2引物设计参考文献[4]。
1.2.3 结果观察
PCR产物经2%琼脂糖凝胶电泳,全自动凝胶成像仪观察结果。
1.2.4 核苷酸序列测定及HRV分型
VP4/VP2基因扩增产物经电泳观察,阳性扩增产物由擎科生物公司进行纯化和测序。将测序结果采用NCBI网站上的Nucleotide Blast进行比对区分亚型。
1.3 统计学处理
采用SPSS 23.0软件进行统计学分析,计数资料用率(%)表示,采用χ2检验,P < 0.05为差异有统计学意义。
2. 结果
2.1 HRV感染的性别分布
检出HRV阳性患儿共147例,感染率为6.50%,其中男性患儿90例,占阳性患儿中的61.22%,女性患儿57例,占阳性患儿中的38.78%。
2.2 HRV感染年龄分布
< 1岁、1~2岁、3~5岁及≥6岁患儿的HRV检出率分别为6.70%、6.88%、7.14%、3.92%。不同年龄ALRTI患儿HRV检出率,差异无统计学意义(χ2 = 3.240,P = 0.356),见表1。
表 1 不同年龄ALRTI患儿HRV检出情况[n(%)]Table 1. HRV detection in children with ALRTI in different age groups [n(%)]年龄(岁) < 1 1~2 3~5 ≥6 χ2 P RV检测结果 阳性 72(6.704) 40(6.885) 25(7.143) 10(3.922) 3.24 0.356 阴性 1002(93.296) 541(93.115) 325(92.857) 245(96.078) 合计 1074(100.000) 581(100.000) 350(100.000) 255(100.000) 2.3 HRV感染季节分布
不同季节病毒检出率存在差异,以夏季最高,其次为秋季(χ2= 37.674,P < 0.05),见 表2。
表 2 不同季节ALRTI患儿HRV检出情况[n(%)]Table 2. HRV detection in children with ALRTI in different seasons[n(%)]季节 春(3月~5月) 夏(6月~8月) 秋(9月~11月) 冬(12月~2月) χ2 P RV检测结果 阳性 33(5.622) 56(10.000) 49(8.673) 9(1.642) 37.674 P < 0.001 阴性 554(94.378) 504(90.000) 516(91.327) 539(98.358) 合计 587(100.000) 560(100.000) 565(100.000) 548(100.000) 2.4 HRV感染的分型
随机抽取40份阳性标本进行基因分型,结果23份为HRV-A型,5份为HRV-B型,12份为HRV-C型。其中HRV-A型最多,占57.50%(23/40);其次为HRV-C型,占30.00%(12/40);HRV-B型最少,仅占12.50%(5/40)。部分凝胶电泳结果,见图1。
2.5 HRV检测阳性与HRV检测阴性患儿的临床表现比较
HRV检测阳性与HRV检测阴性患儿的临床表现在发热、咳嗽方面,差异无统计学意义(P > 0.05),但在喘息方面,差异有统计学意义( P < 0.05),见 表3。
表 3 HRV检测阳性与HRV检测阴性患儿的临床表现比较[n(%)]Table 3. Comparison of clinical manifestations of children with positive HRV test and negative HRV test [n(%)]临床表现 HRV检测 合计 P 阳性 阴性 发热 有 62(42.177) 704(33.318) 766(33.894) 0.301 无 85(57.823) 1409(66.682) 1494(66.106) 咳嗽 有 20(13.605) 331(15.665) 351(15.531) 0.712 无 127(86.395) 1782(84.335) 1 909(84.469) 喘息 有 23(15.646) 104(4.922) 127(5.619) 0.030 无 124(84.354) 2 009(95.078) 2133(94.381) 3. 讨论
HRV是引起儿童呼吸道感染的最主要病原体之一,在世界各地都会引起呼吸道疾病。来自巴西[5]、芬兰[6]、俄罗斯[7]等多地均有类似报道,检出率分别是35.4%、55%、41.7%、26%、10.7%。本组ALRTI患儿HRV检出率为6.50%,与青海相近[8],比北京(34.0%)[9]低。不同地区HRV的检出率不同,可能与地域不同、纳入标准不同、研究方法不同、检测方法不同有关。
本组 < 1岁、1~2岁、3~5岁患儿的HRV检出率相似,≥6岁稍低,但不同年龄ALRTI患儿HRV检出率没有明显差异( P > 0.05)。但国内外不同学者的研究结果不一致。赵梦川 [10]等的研究显示河北省在1 ~3 岁年龄组HRV检出率最高,而法国的Freymuth等[11]的研究显示 < 6月(34.2%)及6月~2岁(37.8%)患儿的检出率相似,2~5岁(19.5%)患儿检出率稍低。还有研究指出在儿童中年龄较小的患儿更容易感染RV,急性下呼吸道感染住院的RV阳性儿童比RV阴性儿童年龄更小,早产儿童被发现特别容易受到RV感染 [6]。本研究的对象主要纳入的是ALRTI患儿,而上诉研究纳入的研究对象与本研究不一致,考虑研究结果不同与此有关。
HRV感染全年均有发生,但最常见于秋季和春季[6]。自20世纪60年代开始,研究者对温带气候中RV感染的流行病学进行纵向研究,发现HRV感染率在初秋达到高峰,在春季出现较小的高峰[12],这与笔者的研究结果一致。但不同地区HRV流行高峰存在差异,有研究表明上海地区HRV流行高峰在冬季[13],而兰州地区HRV流行高峰为春季、秋季和冬季三个季节[14]。不同地区HRV流行高峰存在差异考虑与不同地区气候不同、人体免疫力不同有关。
本研究显示在昆明地区ALRTI患儿中HRV-A型最多,其次为HRV-C型,HRV-B型最少。这与我国兰州地区Jin Y[14]、重庆地区Lu QB[15]、意大利Piralla等[16]的研究结果相似,说明昆明地区HRV流行趋势与国内外基本一致。
有研究[17]表明儿童在HRV感染后,可以出现不同的临床表现,从轻微的普通感冒和急性中耳炎扩展到严重的喘息。喘息性疾病(细支气管炎、反复喘息或哮喘加重)常与HRV有关,婴儿期HRV引起的喘息可能是儿童哮喘的第一个症状[6]。本研究结果显示HRV感染后的患儿更容易出现喘息的临床表现,与之前的研究结果一致[17]。出现此现象的原因,考虑与支气管上皮细胞在HRV感染后嗜酸性粒细胞的募集、诱导血管生成和促炎介质的产生有关[18]。
本研究初步了解2019年昆明地区ALRTI住院患儿HRV感染的流行病学特征,为临床监测及疾病防控补充基础数据。但现有研究观察时间短、病例数少,有待延长研究时间、纳入更多病例进行进一步分析。且12%~22%的无症状人群可检出HRV感染[19],故在以后的研究中应注意加入对昆明地区无症状人群HRV的监测,有利于更准确、更有效的了解HRV的流行病学特征。
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图 4 TNFα和丁酸单独或共同作用48 h对Caco2细胞紧密连接蛋白的mRNA表达的影响
A:TNFα(100 ng/mL)和丁酸(0.2 mM/L)单独或共同作用48 h对Caco2细胞ZO-1的mRNA表达的影响;B:TNFα(100 ng/mL)和丁酸(0.2 mM/L)单独或共同作用48 h对Caco2细胞Occludin的mRNA表达的影响;ns P > 0.05,* P < 0.05,** P < 0.01,***P < 0.001。
Figure 4. The mRNA expression of tight junction proteins in Caco2 cells after exposure to TNF α or butyric acid for 48 h
图 5 TNFα和丁酸和作用48h后Caco2细胞ZO-1表达和分布的变化(免疫荧光染色,放大倍数为×800,DAPI标记细胞核为蓝色,ZO-1染为绿色)
A:对照组;B:丁酸干预组;C:TNFα和丁酸干预组;D:TNFα干预组
Figure 5. The expression and distribution of ZO-1 on Caco2 cells after exposure to TNF α and Butyric acid for 48 hours (immunofluorescence staining,magnification at ×800 ,DAPI labeled nucleus as blue,ZO-1 stained as green)
图 6 丁酸和TNFα作用48 h后Caco2细胞Occludin表达和分布的变化(免疫荧光染色,放大倍数为800倍,DAPI标记细胞核为蓝色,Occludin染为绿色)
A:对照组;B:丁酸干预组;C:TNFα和丁酸干预组;D:TNFα干预组
Figure 6. The expression and distribution of Occludin on Caco2 cells after exposure to TNF α and Butyric acid for 48 hours (immunofluorescence staining,magnification at ×800 ,DAPI labeled nucleus as blue,Occludin stained as green)
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