丁酸钠和吲哚-3-丙酸共处理对Caco-2细胞紧密连接以及炎症因子的影响

doi:10.11843/j.issn.0366-6964.2024.11.028

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (11): 5124-5134.doi: 10.11843/j.issn.0366-6964.2024.11.028

丁酸钠和吲哚-3-丙酸共处理对Caco-2细胞紧密连接以及炎症因子的影响

王轲信¹(), 吴娴¹, 龚海洲³, 方巧玉¹, 李向臣¹, 张亚南¹^,²^,*()

1. 浙江农林大学动物科技学院·动物医学院浙江省畜禽绿色生态健康养殖应用技术研究重点实验室动物健康互联网检测技术浙江省工程实验室, 杭州 311300
2. 浙江大学动物科学学院动物分子营养学教育部重点实验室, 杭州 310058
3. 扬州大学动物科学与技术学院, 扬州 225009

收稿日期:2024-01-03 出版日期:2024-11-23 发布日期:2024-11-30
通讯作者: 张亚南 E-mail:2661895207@qq.com;ynzhang1515@163.com
作者简介:王轲信(2003-), 女, 甘肃庆阳人, 本科生, 主要从事动物分子营养研究, E-mail: 2661895207@qq.com
基金资助:
浙江农林大学科研发展基金项目(2023LFR006);浙江大学动物分子营养学教育部重点实验室开放性课题(KLMAN202307);浙江农林大学国家级大学生创新创业训练计划(202310341010，张亚南)

Effects of Co-Treatment of Sodium Butyrate and Indole-3-Propionic Acid on Tight Junctions and Inflammatory Cytokines in Caco-2 Cells

Kexin WANG¹(), Xian WU¹, Haizhou GONG³, Qiaoyu FANG¹, Xiangchen LI¹, Yanan ZHANG¹^,²^,*()

1. Zhejiang Provincial Engineering Laboratory for Animal Health Inspection & Internet Technology, Key Laboratory of Applied Technology on Green-Eco-Healthy Animal Husbandry of Zhejiang Province, College of Animal Science and Technology, College of Veterinary Medicine, Zhejiang A & F University, Hangzhou 311300, China
2. The Key Laboratory of Molecular Animal Nutrition, Ministry of Education, College of Animal Science, Zhejiang University, Hangzhou 310058, China
3. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China

Received:2024-01-03 Online:2024-11-23 Published:2024-11-30
Contact: Yanan ZHANG E-mail:2661895207@qq.com;ynzhang1515@163.com

摘要/Abstract

摘要：

本研究以人克隆结肠腺癌Caco-2细胞系为模型，旨在探究丁酸钠(sodium butyrate, NaB)和吲哚-3-丙酸(indole-3-propionic acid，IPA)共处理对Caco-2细胞紧密连接和炎症因子的影响。使用0.4 mmol·L^-1 NaB和0.1 mmol·L^-1 IPA分别和共处理Caco-2细胞24 h，检测细胞跨膜电阻(TEER)值、细胞紧密连接蛋白表达、炎症因子的基因表达和细胞上清液中炎症因子的分泌水平。结果发现，与对照组相比，NaB和IPA共处理组均显著提高TEER值(P < 0.01)，而NaB和IPA单独处理组TEER值无显著变化(P>0.05)。qPCR和蛋白免疫印迹结果表明，与对照组相比，共处理组显著下调了Claudin-2的mRNA相对表达量(P < 0.01)；与NaB单独处理组相比，共处理组显著上调了Claudin-1的mRNA相对表达量(P < 0.05)，并且极显著促进了Claudin-1、ZO-1和Occludin蛋白的表达(P < 0.001)；与IPA单独处理组相比，共处理组显著上调了ZO-1的mRNA相对表达量(P < 0.05)，并极显著提高了Claudin-1和ZO-1蛋白表达量(P < 0.001)。在炎症因子方面，与对照组相比，NaB和IPA共处理组显著下调炎症因子IL-6、IL-1β、IL-8及TNF-α的mRNA相对表达量(P < 0.05)。ELISA结果显示，与对照组相比，共处理极显著降低了细胞上清液中IL-6、IL-1β、IL-8及TNF-α的分泌量(P < 0.001)。本研究表明，相比于NaB或IPA单独处理，NaB和IPA共处理可增加细胞跨膜电阻值，促进细胞间紧密连接蛋白表达并且降低促炎因子的分泌，降低了肠道通透性。综上，NaB和IPA共处理在增强上皮细胞屏障功能以及降低炎症反应方面具有更强的作用。

关键词: 丁酸钠, 吲哚-3-丙酸, 紧密连接, 炎症因子, Caco-2细胞

Abstract:

This study aims to investigate the effects of co-treatment of sodium butyrate and indole-3-propionic acid on the expression of tight junction-related proteins and inflammatory factors in Caco-2 cells. Caco-2 cells were administrated with 0.4 mmol·L^-1 sodium butyrate and 0.1 mmol·L^-1 indole-3-propionic acid each and together for 24 h; Then the transmembrane resistance (TEER) value, expression of tight junction related proteins, gene expression and secretion level of inflammatory cytokines were measured. The results showed that, compared with control group, the co-treatment of sodium butyrate and indole-3-propionic acid significantly increased the TEER value in Caco-2 cells (P < 0.01), while no significant changes were observed in sodium butyrate or indole-3-propionic group (P>0.05); In addition, the results by qPCR and Western blot showed that the relative expression of Claudin-2 mRNA was significantly down-regulated in the co-treatment group compared with the control group (P < 0.01); Compared with sodium butyrate group, the relative mRNA expression of Claudin-1 was significantly up-regulated in the co-treatment group (P < 0.05), and the protein expression of Claudin-1, ZO-1 and Occludin was highly significantly increased in the co-treatment group(P < 0.001); Compared with the indole-3-propionic acid group, the relative mRNA expression of ZO-1 was significantly up-regulated in the co-treatment group (P < 0.05), and the protein expression of Claudin-1 and ZO-1 was highly significantly increased (P < 0.001). For inflammatory cytokines, the relative mRNA expression levels of IL-6, IL-1β, IL-8 and TNF-α were significantly down-regulated in the co-treatment group (P < 0.05); Meanwhile, the results of ELISA showed that the co-treatment group highly significantly suppressed the secretion of IL-6, IL-1β, IL-8 and TNF-α in the cell supernatant compared with the control group (P < 0.001). In conclusion, compared with sodium butyrate or indole-3-propionic acid, the co-treatment of sodium butyrate and indole-3-propionic acid increased the TEER value, promoted the expression of tight junction proteins, suppressed the secretion of pro-inflammatory cytokines, and reduced intestinal permeability. Overall, these results suggested that the co-treatment of sodium butyrate and indole-3-propionic acid has a stronger role in enhancing intestinal epithelial cell barrier function and inhibiting inflammatory response.

Key words: sodium butyrate, indole-3-propionic acid, tight junction, inflammatory cytokines, Caco-2 cells

中图分类号:

S816

王轲信, 吴娴, 龚海洲, 方巧玉, 李向臣, 张亚南. 丁酸钠和吲哚-3-丙酸共处理对Caco-2细胞紧密连接以及炎症因子的影响[J]. 畜牧兽医学报, 2024, 55(11): 5124-5134.

Kexin WANG, Xian WU, Haizhou GONG, Qiaoyu FANG, Xiangchen LI, Yanan ZHANG. Effects of Co-Treatment of Sodium Butyrate and Indole-3-Propionic Acid on Tight Junctions and Inflammatory Cytokines in Caco-2 Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5124-5134.

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参考文献 33

1	PETERSON L W , ARTIS D . Intestinal epithelial cells: regulators of barrier function and immune homeostasis[J]. Nat Rev Immunol, 2014, 14 (3): 141- 153. doi: 10.1038/nri3608
2	SLIFER Z M , BLIKSLAGER A T . The integral role of tight junction proteins in the repair of injured intestinal epithelium[J]. Int J Mol Sci, 2020, 21 (3): 972. doi: 10.3390/ijms21030972
3	GASALY N , DE VOS P , HERMOSO M A . Impact of bacterial metabolites on gut barrier function and host immunity: a focus on bacterial metabolism and its relevance for intestinal inflammation[J]. Front Immunol, 2021, 12, 658354. doi: 10.3389/fimmu.2021.658354
4	KAŹMIERCZAK-SIEDLECKA K , MARANO L , MEROLA E , et al. Sodium butyrate in both prevention and supportive treatment of colorectal cancer[J]. Front Cell Infect Microbiol, 2022, 12, 1023806. doi: 10.3389/fcimb.2022.1023806
5	HUANG C , SONG P X , FAN P X , et al. Dietary sodium butyrate decreases postweaning diarrhea by modulating intestinal permeability and changing the bacterial communities in weaned piglets[J]. J Nutr, 2015, 145 (12): 2774- 2780. doi: 10.3945/jn.115.217406
6	ZHAO H B , JIA L , YAN Q Q , et al. Effect of Clostridium butyricum and butyrate on intestinal barrier functions: study of a rat model of severe acute pancreatitis with intra-abdominal hypertension[J]. Front Physiol, 2020, 11, 561061. doi: 10.3389/fphys.2020.561061
7	WANG H B , WANG P Y , WANG X , et al. Butyrate enhances intestinal epithelial barrier function via up-regulation of tight junction protein Claudin-1 transcription[J]. Dig Dis Sci, 2012, 57 (12): 3126- 3135. doi: 10.1007/s10620-012-2259-4
8	HUANG X Y , OSHIMA T , TOMITA T , et al. Butyrate alleviates cytokine-induced barrier dysfunction by modifying Claudin-2 levels[J]. Biology (Basel), 2021, 10 (3): 205.
9	MA N , MA X . Dietary amino acids and the gut-microbiome-immune axis: physiological metabolism and therapeutic prospects[J]. Compr Rev Food Sci Food Saf, 2019, 18 (1): 221- 242. doi: 10.1111/1541-4337.12401
10	JIANG H , CHEN C Y , GAO J . Extensive summary of the important roles of indole propionic acid, a gut microbial metabolite in host health and disease[J]. Nutrients, 2022, 15 (1): 151. doi: 10.3390/nu15010151
11	JENNIS M , CAVANAUGH C R , LEO G C , et al. Microbiota-derived tryptophan indoles increase after gastric bypass surgery and reduce intestinal permeability in vitro and in vivo[J]. Neurogastroenterol Motil, 2018, 30 (2): e13178. doi: 10.1111/nmo.13178
12	LI J J , ZHANG L , WU T , et al. Indole-3-propionic acid improved the intestinal barrier by enhancing epithelial barrier and mucus barrier[J]. J Agric Food Chem, 2021, 69 (5): 1487- 1495. doi: 10.1021/acs.jafc.0c05205
13	QIU Y Q , MA X Y , YANG X F , et al. Effect of sodium butyrate on cell proliferation and cell cycle in porcine intestinal epithelial (IPEC-J2) cells[J]. In Vitro Cell Dev Biol Anim, 2017, 53 (4): 304- 311. doi: 10.1007/s11626-016-0119-9
14	LAN H , ZHANG L Y , HE W , et al. Sinapic acid alleviated inflammation-induced intestinal epithelial barrier dysfunction in lipopolysaccharide-(LPS-) treated Caco-2 cells[J]. Mediators Inflamm, 2021, 2021, 5514075.
15	MA Y H , WANG Q M , YU K , et al. 6-Formylindolo(3, 2-b)carbazole induced aryl hydrocarbon receptor activation prevents intestinal barrier dysfunction through regulation of Claudin-2 expression[J]. Chem Biol Interact, 2018, 288, 83- 90. doi: 10.1016/j.cbi.2018.04.020
16	HE S S , GUO Y H , ZHAO J X , et al. Ferulic acid ameliorates lipopolysaccharide-induced barrier dysfunction via MicroRNA-200c-3p-mediated activation of PI3K/AKT pathway in caco-2 cells[J]. Front Pharmacol, 2020, 11, 376. doi: 10.3389/fphar.2020.00376
17	KUO W T , ODENWALD M A , TURNER J R , et al. Tight junction proteins occludin and ZO-1 as regulators of epithelial proliferation and survival[J]. Ann N Y Acad Sci, 2022, 1514 (1): 21- 33. doi: 10.1111/nyas.14798
18	RUSSO E , GIUDICI F , FIORINDI C , et al. Immunomodulating activity and therapeutic effects of short chain fatty acids and tryptophan post-biotics in inflammatory bowel disease[J]. Front Immunol, 2019, 10, 2754. doi: 10.3389/fimmu.2019.02754
19	FU Y F , LYU J , WANG S S . The role of intestinal microbes on intestinal barrier function and host immunity from a metabolite perspective[J]. Front Immunol, 2023, 14, 1277102. doi: 10.3389/fimmu.2023.1277102
20	PARADA VENEGAS D , DE LA FUENTE M K , LANDSKRON G , et al. Short chain fatty acids (SCFAs)-mediated gut epithelial and immune regulation and its relevance for inflammatory bowel diseases[J]. Front Immunol, 2019, 10, 277. doi: 10.3389/fimmu.2019.00277
21	张秋玉, 卞中博, 孙小蝶, 等. 丁酸钠通过自噬途径降解HIF-1α抑制结直肠癌细胞生长[J]. 现代肿瘤医学, 2022, 30 (5): 762- 767.
	ZHANG Q Y , BIAN Z B , SUN X D , et al. Sodium butyrate inhibits the growth of colorectal cancer cells by promoting autophagic degradation of HIF-1α[J]. Journal of Modern Oncology, 2022, 30 (5): 762- 767.
22	MARTIN-GALLAUSIAUX C , MARINELLI L , BLOTTIōRE H M , et al. SCFA: mechanisms and functional importance in the gut[J]. Proc Nutr Soc, 2021, 80 (1): 37- 49. doi: 10.1017/S0029665120006916
23	张笑添, 车昌燕, 姚步月, 等. miR-92a/Dickkopf相关蛋白1介导丁酸钠调控Wnt/β-catenin信号通路抑制结肠癌细胞增殖[J]. 中国生物化学与分子生物学报, 2023, 39 (12): 1743- 1752.
	ZHANG X T , CHE C Y , YAO B Y , et al. Sodium butyrate inhibits colon cancer proliferation through miR-92a/DKK1-mediated inhibition of the Wnt/β-catenin pathway[J]. Chinese Journal of Biochemistry and Molecular Biology, 2023, 39 (12): 1743- 1752.
24	PARADIS T , BōGUE H , BASMACIYAN L , et al. Tight junctions as a key for pathogens invasion in intestinal epithelial cells[J]. Int J Mol Sci, 2021, 22 (5): 2506. doi: 10.3390/ijms22052506
25	CHELAKKOT C , GHIM J , RYU S H . Mechanisms regulating intestinal barrier integrity and its pathological implications[J]. Exp Mol Med, 2018, 50 (8): 1- 9.
26	GANAPATHY A S , SAHA K , SUCHANEC E , et al. AP2M1 mediates autophagy-induced CLDN2 (Claudin 2) degradation through endocytosis and interaction with LC3 and reduces intestinal epithelial tight junction permeability[J]. Autophagy, 2022, 18 (9): 2086- 2103. doi: 10.1080/15548627.2021.2016233
27	冯燕海. 紧密连接蛋白Claudin-2研究进展[J]. 重庆医学, 2018, 47 (5): 697- 699.
	FENG Y H . Research progress of tight junction protein Claudin-2[J]. Chongqing Medicine Journal, 2018, 47 (5): 697- 699.
28	MIAO W , WU X J , WANG K , et al. Sodium butyrate promotes reassembly of tight junctions in Caco-2 monolayers involving inhibition of MLCK/MLC2 pathway and phosphorylation of PKCβ2[J]. Int J Mol Sci, 2016, 17 (10): 1696. doi: 10.3390/ijms17101696
29	SEGAIN J P , RAINGEARD DE LA BLETIERE D , BOURREILLE A , et al. Butyrate inhibits inflammatory responses through NFκB inhibition: implications for Crohn's disease[J]. Gut, 2000, 47 (3): 397- 403. doi: 10.1136/gut.47.3.397
30	GAO J , XU K , LIU H N , et al. Impact of the gut microbiota on intestinal immunity mediated by tryptophan metabolism[J]. Front Cell Infect Microbiol, 2018, 8, 13. doi: 10.3389/fcimb.2018.00013
31	VENKATESH M , MUKHERJEE S , WANG H W , et al. Symbiotic bacterial metabolites regulate gastrointestinal barrier function via the xenobiotic sensor PXR and Toll-like receptor 4[J]. Immunity, 2014, 41 (2): 296- 310. doi: 10.1016/j.immuni.2014.06.014
32	CAPALDO C T , NUSRAT A . Cytokine regulation of tight junctions[J]. Biochim Biophys Acta, 2009, 1788 (4): 864- 871. doi: 10.1016/j.bbamem.2008.08.027
33	KORSTEN S G P J , VROMANS H , GARSSEN J , et al. Butyrate protects barrier integrity and suppresses immune activation in a Caco-2/PBMC Co-culture model while HDAC inhibition mimics butyrate in restoring cytokine-induced barrier disruption[J]. Nutrients, 2023, 15 (12): 2760. doi: 10.3390/nu15122760

基因名称 Gene name	引物序列(5'→3') Primer sequence(5'→3')	参考文献 Reference
Occludin	F: GGGCATTGCTCATCCTGAAG R: GCCTGTAAGGAGGTGGACTT	[12]
ZO-1	F: TTCACGCAGTTACGAGCAAG R: TTGGTGTTTGAAGGCAGAGC	[12]
Claudin-1	F: CCAGGTACGAATTTGGTCAGG R: TGGTGTTGGGTAAGAGGTTGT	[14]
Claudin-2	F: CCTAAACCACAAGGCTAAGGGCTA R: GGTGTCTTCCTCAGTACCCCACA	[15]
IL-6	F: TGGCAGCCTTCCTGATTTCT R: AATTTCTGTGTTGGCGCAGT	[12]
IL-1β	F: GAATGACGCCCTCAATCAAAGT R: TCATCTTGGGCAGTCACATACA	[14]
IL-8	F: CCTGAACCTTCCAAAGATGGC R: TTCACCAGGCAAGTCTCCTCA	[14]
TNF-α	F: CTGCCTGCTGCACTTTGGAG R: ACATGGGCTACAGGCTTGTCACT	[12]
β-Actin	F: TGCAGAAAGAGATCACCGC R: CCGATCCACACCGAGTATTTG	[16]

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丁酸钠和吲哚-3-丙酸共处理对Caco-2细胞紧密连接以及炎症因子的影响

Effects of Co-Treatment of Sodium Butyrate and Indole-3-Propionic Acid on Tight Junctions and Inflammatory Cytokines in Caco-2 Cells

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