Background

Cinnamicaldehyde(CA)is a key flavor compound in cinnamon essential oil extracted from the stem bark of Cinnamomum cassia in nature[1].CA is widely used in the perfume,pharmacy,and food processing industries due to its antioxidant,anti-microbial,and anti-diabetic properties[2-6].Moreover,it has been reported that CA has chemotherapeutic and anticancer effects through the inhibition of proliferation,inducing apoptosis,and blocking angiogenesis[7-9].Importantly,CA is a safe flavor compound approved by FDA and the‘Flavor and Extract Manufacturers’Association of the United States,suggesting the potential administration of this dietary factor may be achievable within an acceptable safety range for humans and animals[5].The well documented antimicrobial properties and safety of CA have promoted its application to the nutrition of humans and animals.It has been reported that supplementation of essential oils,in which CA is the major component,increases the digestibility of crude protein in weaned pigs[10].The beneficial effects of CA are associated with the enhanced secretion of digestive enzymes,improved nutrient digestion,and enhanced feed intake[11-14].It remains unknown whether CA has any effect on intestinal barrier function,as well as nutrient transport and absorption in humans and pigs.

一般定植20～30 d后待植株扎根土壤，4～6对叶展开后，摘去上位1对叶，留2～3对叶，促使侧茎萌发，提高产量。

The epithelial barrier is formed by the apical plasma membrane and intercellular tight junction(TJ),which provides physical and functional barriers to prevent bacteria,endotoxins and other harmful substances from entering the blood circulation,while also allowing for the absorption of nutrients[15].Diverse physiological or pathological stimuli can regulate the intestinal mucosal-barrier function,which contributes to nutrient transport,absorption,and intracellular homeostasis[16-19].Consistently,dysfunction of TJ proteins has been reported to be associated with increased paracellular permeability,and the development and progression of multiple intestinal disorders[20].

Although CA is known to improve the digestibility of dietary fiber,lipids,and crude protein,the underlying cellular and molecular mechanisms remain largely unknown.We have hypothesized that CA may up-regulate the expression of TJ proteins and amino acid transporters in intestinal epithelial cells,thus contributing to the intestinal barrier function in neonates.This hypothesis was tested in the present study using porcine intestinal epithelial cells(IPEC-1),isolated from neonatal pigs.

Methods

Reagents

Effects of CA on the protein abundance for amino acid transporters

Cell culture

Intestinal porcine epithelial cell line 1(IPEC-1)cells,which were isolated from the jejunum of newborn pigs without access to milk or any food[21],were then cultured in a DMEM-F12 medium supplemented with 5%FBS,insulin(5 μg/mL),transferrin(5 μg/mL),selenium(5 ng/mL),epidermal growth factor(5 μg/L),penicillin(50 μg/mL)and streptomycin(4 μg/mL)as previously described[22].All cell cultures were carried out at 37°C in a humidified incubator containing 5%CO2.

Measurement of transepithelial electrical resistance(TEER)

The tightness of the TJ was assessed by measuring TEER aspreviously　described　[23].Briefly,IPEC-1　Cells(5×104cells per well)were seeded in culture transwells(the membrane area,0.33 cm2;pore size,0.4 μm)which were placed in 24-well culture plates.Cells were incubated with 0,12.5,or 25 μmol/L CA for the indicated time periods.TEER was determined every 12 h by using a Millicell ERS-2 Voltage-Ohm Meter(World Precision Instruments)equippedwith　aSTX01electrodeas described here[24].All values are expressed as percentages of the basal level for the controls.

Monolayer paracellular permeability determination

Paracellular permeability was determined as previously described[25].Briefly,IPEC-1 cells were seeded in culture transwells as for TEER determination.1 mg/mL FITC-dextran(20 kDa)was added to the apical side of the monolayer and the flux of FITC-dextran was determined by serially sampling the basolateral compartment every 12 h.The concentration of FITC-dextran was measured using the SpectraMax M3 Multi-Mode Microplate Reader(Molecular Devices)with excitation and emission wavelengths of 490 and 520 nm,respectively.The permeability of monolayer cells was defined as the amount of FITC-dextran that was transported from the apical side into the basolateral chamber.FITC-dextran concentration was calculated by subtracting　the　fluorescence　value　ofthe　FITC-free medium.

Western blot analysis

IPEC-1 cells treated with various concentrations of CA for 24 h were harvested for the analysis of the abundance of TJ proteins,as previously described[24].Equal amounts of proteins(25 μg)were separated on SDSPAGE gels,transferred to polyvinylidene difluoride membranes(Millipore),and then incubated with a primary antibody(1:2,000)overnight at 4°C and then incubated with an appropriate secondary antibody(1:2,000)at 25°C for 1 h.The blots were detected with the Image Quant LAS 4000 mini system(GE Healthcare Bio-sciences AB,Inc.,Sweden)after incubation with the ECL plus system(Amersham Biosciences,Sweden).Chemifluorescence was quantified with the use of the Quantity One software(Bio-Rad Laboratories).All results were normalized to GAPDH and expressed as relative values to those of the control group.

Immunofluorescence assay

此时，老人执拗得像门前的那棵老胡杨树，“这些东西都是团场的文物，我就想着团里找个地方把它们存起来，给后人留点念想，这些东西有了去处，我才能放心搬家”，老人讲起了自己的初衷。环顾四周，老人住在三十年的老土坯房中，可以没有有线电视、可以没有自来水、甚至可以没有电，但却唯独少不了这些老物件、老伙计，在这个问题上，老人绝不妥协，和不远处那棵挨着沙丘长着的胡杨一样。

IPEC-1 cells treated with or without CA were fixed with 4%paraformaldehyde at 37°C for 20 min,and then were incubated with a specific primary antibody against claudin-1,claudin-3,claudin-4,ZO-1,ZO-2 and ZO-3 for 16 h at 4°C.Cells were washed three times with PBS,and then were incubated with an appropriate secondary antibody(1:100)for 1 h at 25°C.Nuclei were stained by using Hoechst 33258(1 μg/mL)for 10 min at 25°C.The distribution of TJ proteins was visualized under a fluorescence microscope(Axio Vert.A1,Zeiss,Germany).

Statistical analysis

Values are expressed as mean±SEM.Data was analyzed by one-way ANOVA and the Student-Newman-Keuls multiple comparisons test,using the SPSS statistical software(SPSS for Windows,version 17.0).P≤0.05 were taken to indicate statistical significance.

Results

Effects of CA on barrier function in the IPEC-1 cell monolayer

As shown,incubation of cells with 25 μmol/L CA led to greater(P＜0.05)TEER at 36-48 h(Fig.1a)when compared with controls.In contrast,no difference was observed between the cells treated with 12.5 μmol/L CA and the control cells at 12-48 h.Consistent with increased TEER,cells incubated with 25 μmol/L CA had reduced(P＜0.05)paracellular permeability,as indicated by FITC-dextran flux at 12-48 h(Fig.1b)when compared with controls.Cells treated with 12.5 μmol/L CA had lowered permeability(P＜0.05),compared with the control cells at 24-48 h.Although both 12.5 and 25 μmol/L CA treatment led to decreased permeability in enterocytes compared with the controls,cells treated with 25 μmol/L CA had lower permeability(P＜0.05)when compared with cells incubated with 12.5 μmol/L CA at 36-48 h.

Fig.1　Effects of CA on intestinal barrier function in IPEC-1 cells.Cells were cultured for 24 h in the absence or presence of 12.5-or 25 μmol/L CA.a TEER and b paracellular permeability were then determined.Values are expressed as means±SEM,n=6.Means at a time point without a common letter differ,P＜0.05.CA,Cinnamicaldehye;IPEC-1,intestinal porcine epithelial cell line 1;TEER,trans-epithelial electrical resistance

Effects of CA on expression of TJ proteins in IPEC-1 cells

Compared with control cells,25 μmol/L CA enhanced(P＜0.05)the abundance of proteins for claudin-4(Fig.2c)and ZO family proteins including ZO-1,ZO-2,and ZO-3(Fig.3).The protein abundance for ZO-2(Fig.3b),instead of other proteins,was enhanced by 12.5 μmol/L CA(P＜0.05)compared with that of the control.The protein abundances for claudin-1(Fig.2a),claudin-3(Fig.2b),and occludin(Fig.2d)were not affected(P＞0.05)by 12.5 or 25 μmol/L CA treatment.

Effects of CA on the intracellular distribution of TJ proteins in IPEC-1 cells

The cellular distributions of TJ proteins were assessed by an immunofluorescence microscope.Treatment with 25 μmol/L CA promoted the localization of claudin-1 and claudin-3(Fig.4a and b)to the plasma membrane without affecting the localization of other TJ proteins,including claudin-4,occludin,ZO-1,ZO-2,and ZO-3,compared to the control cells(Fig.4).In contrast,12.5 μmol/L CA had no effect on the localization of TJ proteins determined in our study,such as claudin-1,claudin-3,claudin-4,occludin,ZO-1,ZO-2,and ZO-3(Fig.4).It should be noted that most of the ZO-3 was located at the nucleus membrane which was not affected by CA exposure(Fig.4g).

Dulbecco’s modified Eagle’s F12 Ham medium(DMEMF12)and fetal bovine serum(FBS)were purchased from Invitrogen　(Carlsbad,CA,USA).Epidermal growth factor was a product of BD Biosciences(Carlsbad,CA,USA).Trypsin/EDTA was procured from Gibco(Carlsbad,CA,USA).Antibodies against occludin,claudin-1,claudin-3,claudin-4,zonula occludens(ZO)-1,ZO-2,and ZO-3 were products of Invitrogen(Carlsbad,CA,USA).Unless indicated,all other chemicals including CA were purchased from Sigma-Aldrich(St.Louis,MO,USA).

Data and material sharing applicable to this article.

Fig.2　Protein abundances for claudin-1(a),claudin-3(b),claudin-4(c),and occludin(d)in IPEC-1 cells.IPEC-1 cells were cultured in the absence or presence of 12.5 or 25 μmol/L CA for 24 h.Cells were collected and protein abundances were analyzed.Values are expressed as means±SEM,n=3.Means without a common letter differ,P＜0.05.CA,Cinnamicaldehyde;IPEC-1,intestinal porcine epithelial cell line 1

Fig.3　Protein abundances for ZO-1(a),ZO-2(b),and ZO-3(c)in IPEC-1 cells.Cells were cultured in the absence or presence of 12.5 or 25 μmol/L CA for 24 h.Cells were collected and protein abundances were analyzed.Values are expressed as means±SEM,n=3.Means without a common letter differ,P＜0.05.CA,Cinnamicaldehye;IPEC-1,intestinal porcine epithelial cell line 1;ZO,zonula occludens

Discussion

In the present study,we have shown that CA,a key flavor compound in cinnamon essential oil,promoted the intestinal mucosal-barrier function as indicated by increased TEER and decreased paracellular permeability.Western　blotanalysis　revealed　thatcells treated with CA had enhanced protein abundances for TJ proteins,such as claudin-4 and scaffolding proteins.Moreover,the protein abundance for amino acid transporters,including rBAT,LAT2,and xCT,which are required for amino acid transport and absorption in enterocytes,were also enhanced by CA treatment.

To test this hypothesis,we first measured TEER,an indicator of intestinal epithelial integrity and permeability of intestinal epithelium.Incubation of the enterocyte with CA led to increased TEER and decreased FITC-dextran flux in intestinal porcine monolayers,suggesting a beneficial effect of CA on barrier function.Epithelial barrier function and paracellular permeability are primarily determined by epithelial TJ proteins[29,30].Transmembrane proteins(e.g.,the claudin family protein,occludin)and peripheral membrane proteins(e.g.,ZO-1,ZO-2 and ZO-3)have been identified as critical components of TJ proteins[31,32].Disruption of epithelial TJ proteins has been reported to be associated with multiple intestinal disorders[29,33].Consistently,restoration of TJ proteins by nutrients or prebiotics can improve mucosal barrier integrity and function in humans and animals[34].We have recently found that dietary supplementation of glutamine prevented weanling stress-induced intestinal-mucosal barrier breakdown by augmenting TJ protein abundance[35],suggesting a functional role for amino acids in regulating mucosal barrier function.

Fig.4　The distributions of TJ proteins claudin-1(a),claudin-3(b),claudin-4(c),occludin(d),ZO-1(e),ZO-2(f),and ZO-3(g)in IPEC-1 cells.Cells were treated as in Fig.3,and immunofluorescence staining was performed to identify the distributions of the proteins.CA,Cinnamicaldehyde;IPEC-1,intestinal epithelial porcine cell line 1.Scale bar,50 μm

CA,a natural compound isolated from the stem bark of Cinnamomum cassia,is widely used in food processing and animal diets due to its antioxidant,antimicrobial,and anti-diabetic attributes[2-4].Studies in pigs,a widely used animal model for various disorders in humans,have demonstrated that CA supplementation enhances nutrient digestibility in pigs[10].It remains largely unknown whether CA supplementation can have any effect on intestinal barrier integrity,thereby improving nutrient transport,absorption,and intracellular homeostasis.

◎过量的牛奶、巧克力、橘子在小儿体内可能产生变态反应，使膀胱膨胀，容量减少，并能促使膀胱平滑肌变得粗糙，产生痉挛。

在系统的WebGIS应用服务软件平台上，用户能够实现对卫星云图、雨量、雨量距平、天气雷达、数值预报、热带气旋、气温、气象传真、干旱监视、人工降水预报等10余类60多种水文气象实时信息产品的监视应用。各类监视产品均为系统在后台已加工处理好的实时产品，以点图（数值分布图）、面图（数据空间插值分级充色图）和数据表、柱状图（折线图）等多种形式展现。其中图像产品充分应用了GIS平台强大的图像展现能力，与行政区域、流域分区、河流、湖泊、水库等地理信息结合，以透明图层、分量级着色、任意缩放漫游的显示方式实现了产品信息与地理信息的形象直观展现。

In the present study,we found that CA regulates the protein abundance and cellular distributions of TJ proteins in intestinal cells.Specifically,the presence of 25 μmol/L CA led to enhanced protein abundances for claudin-4,ZO-1,ZO-2,and ZO-3,which are correlated well with augmented TEER values in IPEC-1 cells(Fig.1).The claudin family proteins and ZO family proteins,play a crucial role in establishing cell-cell contacts and maintaining paracellular permeability[36,37].Recent studies have demonstrated that the reduction of claudin family proteins is strongly associated with intestinal barrier disruption in rodents[38,39].The regulatory effects of CA on the protein abundance of TJ suggest that supplementation with CA might be a preventive strategy to maintain the appropriate function of the intestinal-mucosal　barrier.Another novel finding of our study is that CA treatment led to the distributions of claudin-1 and claudin-3 to the cellular plasma membrane(Fig.4a and b)without affecting their protein abundances(Fig.2a and b).Thus,CA regulates both the abundance and localization of TJ proteins in enterocytes.Considering that the disruption of TJ is caused by various stresses and pathogens in pigs[40],supplementation of CA may provide an effective nutritional strategy to alleviate mucosal barrier dysfunction.At present,the underlying mechanisms responsible for this effect remain incompletely understood[31].More research involving our IPEC-1 cell model is required to answer this question.

In addition to providing physical and functional barriers to prevent the entry of bacteria,endotoxins,and other harmful substances from entering the blood circulation,appropriate amounts of TJ proteins maintains the integrity of the intestinal epithelium and,therefore,are　also required for the absorption of nutrients[15].Amino acids released from the hydrolysis of dietary proteins and peptides in the lumen of the small intestine are transported across cell membranes by a complex system of multiple amino acid transporters[26-28].A number of transporters have been identified on the apical surface of the mammalian small intestine that are responsible for the intestinal absorption of amino acids[26,41,42].The defective intestinal uptake of amino acids leads to　alterations　in　plasma　amino　acids,growth retardation,and the Hartnup disorder[26].We have found that CA increased protein abundances for amino acid transporters,including LAT2,rBAT,and xCT in porcine enterocytes.This is the first study showing that this flavor compound has the ability to　up-regulate　the　expression　of　amino　acid transporters in enterocytes.The enhanced protein abundance　for　amino　acids　transporters　might promotethetransportand　absorption　ofamino acids,which,in turn,stimulates　protein synthesis and contributes to the growth performance of pigs observed in previous studies[43,44].

Fig.5　Protein abundances for rBAT(a),xCT(b),LAT2(c),and EAAT3(d)in IPEC-1 cells.Cells were cultured in the absence or presence of 12.5 or 25 μmol/L CA for 24 h.Cells were collected and protein abundances for amino acid transporters were analyzed.Values are expressed as means±SEM,n=3.Means without a common letter differ,P＜0.05.CA,Cinnamicaldehyde;IPEC-1,intestinal porcine epithelial cell line 1;ZO,zonula occludens

Conclusions

In summary,studies with porcine enterocytes have revealed that CA improved the intestinal epithelial barrier integrity,as indicated by increased TEER and decreased paracellular permeability.This beneficial effect of CA is accompanied by enhanced distribution　of　specific　TJ　proteins　in　intestinal epithelial cells.Importantly,the protein abundance for amino acid transporters was enhanced by CA.Further studies with animal model are needed to validate this beneficial effect of CA on intestinal barrier function observed in the present study.Supplementation with CA might be a potential nutritional strategy to improve the intestinal mucosal barrier function and nutrient absorption in neonatal piglets.

创新的前提是创造，创造是指产生原来没有的东西。如果原来就有的东西，你再做出来，这是重复。重复和创造是两种活动。针对人的主体性，我曾专门写过题为《论建设和创造的主体性》的文章，区分了重复的主体性和创造的主体性。在重复性的活动中，人也有主体性，但仅仅重复已有的活动，还不是最高境界。最高的主体性应该是创造的主体性，这是人的创造力的体现。

Abbreviations

二是人与自然和谐的场景显现。在水生态文明建设上，聊城以河道生态修复为重点，侧重滨水景观建设，营造绿色生态长廊，提升区域的景观品位，改善城区生态环境。市城区现有水面面积为13km2，占城区建成区面积的18.8%。建成区绿化覆盖面积为30.84km2，绿化覆盖率达到44.08%，绿化长度与水体岸线长度比为96%，水土流失治理率达98%。通过加大排污许可控制力度、实施入河排污口人工湿地水质净化工程、河道治理等措施，全市生活污水集中处理率达到91.02%，城区水域水质有了明显改善，目前城区80%以上的水体清澈、无杂物，满足水质标准。

This work was supported the National Natural Science Foundation of China(31572410,31572412,31625025),the 111 Project(B16044),the Program for New Century Excellent Talents in University(NCET-12-0522),the Agriculture and Food Research Initiative Competitive Grant from the USDA National Institute of Food and Agriculture(No.2014-6701521770),and Texas A&M AgriLife Research(H-8200).

Acknowledgements

三余爷爷过世后，小先生接过了他的衣钵。小先生是三余爷爷的长孙，奔七之年，正是做风水这一行的盛年。他初中毕业，就跟在爷爷后面学着给人家看风水。先是在爷爷的监管下每天练毛笔字，之后就跟在爷爷后面拿罗盘，勘察现场。那时就有人喊他小先生了，后来不知怎么小先生又改行做了缝纫师傅。多年不见，日前偶然在老家的一个酒席上相遇，才知道他早已从做缝纫转回看风水了。

Not applicable.

Availability of data and materials

The active transport of amino acids is the major mechanism for their uptake into enterocytes[26-28].We determined the protein abundance for the following amino acid transporters,EAAT3(high-affinity glutamate transporter),LAT2(arginine and leucine transporter),rBAT(basic amino acid transporter),and xCT(acidic amino acid transporter)in IPEC-1 cells by Western blot analysis.The protein abundance for rBAT(Fig.5a)and LAT2(Fig.5c)in IPEC-1 cells were enhanced(P＜0.05)by both 12.5-and 25 μmol/L CA,compared with the control cells.In contrast,25 μmol/L CA increased(P＜0.05)the protein abundance for xCT in the intestinal epithelial cells,but 12.5 μmol/L CA had no effect(Fig.5b).The protein abundance for EAAT3 was not affected(P＞ 0.05)by either 12.5 or 25 μmol/L CA(Fig.5d),compared with the control cells.

Funding

花样游泳又叫水上芭蕾、同步花样游泳，它被视为奥运会最美项目，常使整场的观众看得如痴如醉。因为花样游泳是全方位观赏性很强的项目，无论是运动员的连贯性动作，还是节奏性和优美程度都要做得惟妙惟肖。

CA:Cinnamic aldehyde;DMEM-F12:Dulbecco’s modified Eagle’s F12 Ham medium;EGF:Epidermal growth factor;FBS:Fetal bovine serum;HRP:Horseradish peroxidase;ITS:Insulin-transferrin-selenium;IPEC-1:Intestinal porcine epithelial cells-1;TEER:Trans-epithelial electrical resistance;TJ:Tight junction;ZO:Zonula occludens

Authors’contributions

盖挖逆作法是在基坑施工过程中将结构板作为支撑结构的一种基坑施工方法。由于结构板刚度较大，基坑安全优势较明显，因此能节省大量的临时混凝土支撑，以及节约施工工期，但施工难度较明挖方案较高。本站采用盖挖逆作法施工时，需要重点考虑的问题包括：①施工过程中围护结构与主体结构的安全问题；②结构柱形式的选择及定位；③主体结构防水层的设计和施工；④针对如此大面积的基坑，如何安全快速出土的问题。

ZLW and GW designed research;KJS,YL,and RJW conducted research;ZLW,ZL.Dai,and GW analyzed data;KJS,and ZLW wrote the paper.ZLW and GW had primary responsibility for final content.All authors read and approved the final manuscript.

人民一直是社会主义事业建设的主体力量，习总书记在十九大报告中强调要依靠人民群众创造历史伟业。这一人民群众的范畴内蕴含着为社会主义建设贡献力量的所有集体，即依靠他们进行社会主义政治、经济、文化、生态、社会建设。新时代下，社会主义现代化经济体系的建立、社会主义民主政治的发展、社会主义文化的繁荣兴盛、社会治理的创新以及美丽中国的建设，都离不开人民群众主体性和能动性的发挥。因而，重视并坚持人民主体地位是中国特色社会主义事业持续推进的应有之义。

国防科技工业虽然包括民口配套企业，同时也具有经济职能，但一直以来，军工企业都是以满足国家军工生产需求为主要目标，而对于民用产品的研发与生产却并不重视，这使得军工企业本身的经济效益普遍较差，对于国民经济发展的推动作用也非常之小。在这样的情况下，不断增长的民用产品市场需求一直得不到良好的补充，社会经济的发展自然也就受到了很大的限制，而军民融合发展对于军工企业民用产品的生产与研发给予了更高的重视，自然也就能够使国防科技工业在经济方面职能充分发挥出来，满足民用产品市场需求，进而为社会经济的发展提供有力支持。由此可见，军工企业的军民融合发展实际上是社会经济不断发展这一背景下的必然趋势。

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

二是用层级式的学习，用与优秀人员之间的差距，让阿姨从急功近利式的节奏中慢下来，帮助她们认真充电，做好储备。

BSA323S电子分析天平，赛多利斯科学仪器（北京）有限公司；RVA-TecMaster快速粘度仪，瑞典波通仪器公司；DHR-1流变仪，美国 TA仪器公司；X'Pert3 Powder10300 X射线衍射仪，荷兰帕纳特有限公司；CT3质构仪，美国Brookfield公司。

Ethics approval and consent to participate

Not applicable.

Author details

1State Key Laboratory of Animal Nutrition,College of Animal Science and Technology,China Agricultural University,Beijing 100193,China.2DadHank(Chengdu)Biotech Corp,Sichuan,China.3Department of Animal Science,Texas A&M University,College Station,TX 77843,USA.4Department of Animal Nutrition and Feed Science,China Agricultural University,Beijing 100193,China.

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