6-姜烯酚通过调节 CDX2/miR-216a-5p 轴缓解棕榈酸诱导的 Caco-2细胞紧密连接损伤

6-Shogaol Alleviates Palmitic Acid-Induced Tight Junction Damage in Caco-2 Cells by Regulating the CDX2/miR-216a-5p Axis

摘要

棕榈酸（Palmitic acid， PA）会损伤人体肠道上皮细胞紧密连接，导致肠道屏障功能紊乱。我们前研究发现，6-姜烯酚可通过调控miR-216a-5p靶向抑制Toll样受体4（Toll-like receptor 4，TLR4）进而抑制炎症因子，保护肠道细胞免受PA诱导的损伤。然而，6-姜烯酚调控miR-216a-5p表达的具体机制不清楚。本研究采用PA处理人结直肠腺癌Caco-2细胞建立肠道紧密连接损伤模型，通过DNA pulldown实验和双荧光素酶报告基因实验，初步确定尾型同源框转录因子2（Caudal-related homeobox transcription factor 2，CDX2）是miR-216a-5p 的候选调控因子。通过染色质免疫共沉淀-聚合酶链反应（Chromatin immunoprecipitation followed by polymerase chain reaction，ChIP-PCR）实验和电泳迁移率实验（Electrophoretic mobility shift assay，EMSA）实验相结合，证实转录因子CDX2 可以与miR-216a-5p 启动子相结合并促进其转录。最后评估了转录因子CDX2在促炎因子和紧密连接相关蛋白表达中的作用，证明了CDX2是6-姜烯酚调节miR-216a-5p 表达的靶标。本研究首次揭示CDX2 是miR-216a-5p 的直接转录激活因子，为揭示生姜的健康调节作用和相关功能食品的研究提供了参考和依据。

Abstract

Palmitic acid (PA) can damage the tight junction of human intestinal epithelial cells, leading to intestinal barrier dysfunction. Our previous study demonstrates that 6-shogaol can modulate the expression of microRNA miR-216a-5p, leading to the targeted inhibition of the Toll-like receptor 4 (TLR4) signaling pathway. This, in turn, reduces the production of inflammatory factors and protects intestinal cells from PA-induced damage. However, the specific mechanism by which 6-shogaol regulates miR-216a-5p expression remains unclear. This study establishes a model of intestinal tight junction injury by treating human colorectal adenocarcinoma Caco-2 cells with PA. Through DNA pulldown assays and dual-luciferase reporter gene experiments, this study preliminarily identifies Caudal-related homeobox transcription factor 2 (CDX2) as a candidate regulator of miR-216a-5p. By combining chromatin immunoprecipitation followed by polymerase chain reaction (ChIP-PCR) and electrophoretic mobility shift assay (EMSA), this study confirms that the transcription factor CDX2 can bind to the miR-216a-5p promoter and promotes its transcription. Finally, this study evaluates the role of transcription factor CDX2 in regulating the expression of pro-inflammatory factors and tight junction-related proteins, proving that CDX2 is the target through which 6-shogaol regulates miR-216a-5p expression. This study is the first to show that CDX2 is a direct transcriptional activator of miR-216a-5p, providing a reference and basis for revealing the health-regulation effects of ginger and related functional food research.

关键词

6-姜烯酚 / 转录因子 / CDX2 / miR-216a-5p / 紧密连接

Key words

6-shogaol / transcription factor / CDX2 / miR-216a-5p / tight junction

参考文献

[1] Günzel D, Yu A S . Claudins and the modulation of tight junction permeability [J]. Physiological Reviews, 2013, 93(2): 525-569.

[2] Ishimoto H, Oshima T, Sei H, et al. Claudin-2 expression is upregulated in the ileum of diarrhea predominant irritable bowel syndrome patients [J]. Journal of Clinical Biochemistry and Nutrition, 2017, 60(2): 146-150.

[3] Yang W, Guo Y, Lu J, et al. Differential gene expression of tight-junction proteins and their correlation with PRSS8 and prognosis in colorectal cancer [J]. Journal of Clinical Oncology, 2021, 39(15_suppl): e15548.

[4] Wang K, Ding Y, Xu C, et al. Cldn-7 deficiency promotes experimental colitis and associated carcinogenesis by regulating intestinal epithelial integrity [J]. OncoImmunology, 2021, 10(1): 1923910.

[5] Jiang S, Miao Z . High-fat diet induces intestinal mucosal barrier dysfunction in ulcerative colitis: emerging mechanisms and dietary intervention perspective [J]. American Journal of Translational Research, 2023, 5(2): 653-677.

[6] Opie L H, Walfish P G . Plasma free fatty acid concentrations in obesity [J]. The New England Journal of Medicine, 1963, 268: 757-760.

[7] Bonhomme C, Duluc I, Martin E, et al. The Cdx2 homeobox gene has a tumour suppressor function in the distal colon in addition to a homeotic role during gut development [J]. Gut, 2003, 52(10): 1465-1471.

[8] Saad R S, Ghorab Z, Khalifa M A, et al. CDX2 as a marker for intestinal differentiation: Its utility and limitations [J]. World Journal of Gastrointestinal Surgery, 2011, 3(11): 159-166.

[9] Zhu H T, Luo J, Peng Y, et al. Nitazoxanide protects against experimental ulcerative colitis through improving intestinal barrier and inhibiting inflammation [J]. Chemico-Biological Interactions, 2024, 395: 111013.

[10] Funakoshi S, Kong J, Crissey M A, et al. Intestine-specific transcription factor Cdx2 induces E-cadherin function by enhancing the trafficking of E-cadherin to the cell membrane [J]. American Journal of Physiology, 2010, 299(5): G1054-67.

[11] Boyd M, Hansen M, Jensen T G, et al. Genome-wide analysis of CDX2 binding in intestinal epithelial cells (Caco-2) [J]. Journal of BiologicaI Chemistry, 2010, 285(33): 25115-25125.

[12] Mukohyama J, Koizumi M, Yamashita K, et al. Knockdown of CDX2 induces microRNA-221 up-regulation in human colon cancer cells[J]. Anticancer Research, 2024, 44(8): 3553-3556.

[13] Taucher V, Mangge H, Haybaeck J . Non-coding RNAs in pancreatic cancer: challenges and opportunities for clinical application [J]. Cell Oncol (Dordr), 2016, 39(4): 295-318.

[14] Liu A M, Poon R T, Luk J M . MicroRNA-375 targets Hippo-signaling effector YAP in liver cancer and inhibits tumor properties [J]. Biochemical and Biophysical Research Communications, 2010, 394(3): 623-627.

[15] Chandra Mangalhara K, Manvati S, Saini S K, et al. ERK2-ZEB1-miR-101-1 axis contributes to epithelial-mesenchymal transition and cell migration in cancer [J]. Cancer Letters, 2017, 391: 59-73.

[16] Salem M, O'Brien J A, Bernaudo S, et al. miR-590-3p promotes ovarian cancer growth and metastasis via a novel FOXA2-versican pathway [J]. Cancer Research, 2018, 78(15): 4175-4190.

[17] Piché M E, Tchernof A, Després J P . Obesity phenotypes, diabetes, and cardiovascular diseases [J]. Circulation Research, 2020, 126(11): 1477-1500.

[18] Tanaka K, Arita M, Sakurai H, et al. Analysis of chemical properties of edible and medicinal ginger by metabolomics approach [J]. BioMed Research International, 2015, 2015(1): 671058.

[19] Prasad S, Tyagi A K . Ginger and its constituents: role in prevention and treatment of gastrointestinal cancer [J]. Gastroenterology Research and Practice, 2015, 2015(1): 142979.

[20] Kamaruddin M S H, Chong G H, Mohd Daud N, et al. Bioactivities and green advanced extraction technologies of ginger oleoresin extracts: A review [J]. Food Research International, 2023, 164: 112283.

[21] Schwertner H A, Rios D C . High-performance liquid chromatographic analysis of 6-gingerol, 8-gingerol, 10-gingerol, and 6-shogaol in ginger-containing dietary supplements, spices, teas, and beverages [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2007, 856(1-2): 41-47.

[22] Foudah A I, Shakeel F, Yusufoglu H S, et al. Simultaneous determination of 6-shogaol and 6-gingerol in various ginger (Zingiber officinale Roscoe) extracts and commercial formulations using a green RP-HPTLC-Densitometry method [J]. Foods, 2020, 9(8): 1136.

[23] Kou X, Li X, Rahman M R, et al. Efficient dehydration of 6-gingerol to 6-shogaol catalyzed by an acidic ionic liquid under ultrasound irradiation [J]. Food Chemistry, 2017, 215: 193-199.

[24] Han Q, Yuan Q, Meng X, et al. 6-Shogaol attenuates LPS-induced inflammation in BV2 microglia cells by activating PPAR- γ [J]. Oncotarget, 2017, 8(26): 42001-42006.

[25] Wu H, Hsieh M-C, Lo C-Y, et al. 6-Shogaol is more effective than 6-gingerol and curcumin in inhibiting 12-O-tetradecanoylphorbol 13-acetate-induced tumor promotion in mice [J]. Molecular Nutrition & Food Research, 2010, 54(9): 1296-1306.

[26] Pan M-H, Hsieh M-C, Hsu P-C, et al. 6-Shogaol suppressed lipopolysaccharide-induced up-expression of iNOS and COX-2 in murine macrophages [J]. Molecular Nutrition & Food Research, 2008, 52(12): 1467-1477.

[27] Moon M, Kim H G, Choi J G, et al. Corrigendum to "6-Shogaol, an active constituent of ginger, attenuates neuroinflammation and cognitive deficits in animal models of dementia" [BBRC 449 (2014) 8-13] [J]. Biochem and Biophys Research Communication, 2020, 521(2): 545.

[28] Ouyang F, Li B, Wang Y, et al. Attenuation of palmitic acid-induced intestinal epithelial barrier dysfunction by 6-shogaol in Caco-2 cells: the role of MiR-216a-5p/TLR4/NF- κB axis [J]. Metabolites, 2022, 12(11): 1028.

[29] Sui H, Imamichi T . A DNA pull-down assay with diversity forms of competitor for detecting or evaluating Protein-DNA interactions [M]. SIMOES-COSTA M. DNA-Protein Interactions: Methods and Protocols. New York, NY; Springer US. 2023: 1-10.

[30] Homma T, Ishibashi D, Nakagaki T, et al. Persistent prion infection disturbs the function of Oct-1, resulting in the down-regulation of murine interferon regulatory factor-3 [J]. Scientific Reports, 2014, 4: 6006.

[31] Artursson P, Karlsson J . Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells [J]. Biochemical and Biophysical Research Communications, 1991, 175(3): 880-885.

[32] Kawashima Y, Watanabe E, Umeyama T, et al. Optimization of data-independent acquisition mass spectrometry for deep and highly sensitive proteomic analysis [J]. International Journal of Molecular Sciences, 2019, 20(23): 5932.

[33] Fornes O, Castro-Mondragon J A, Khan A, et al. JASPAR 2020: update of the open-access database of transcription factor binding profiles [J]. Nucleic Acids Res, 2020, 48(D1): D87-D92.

[34] Cani P D, Bibiloni R, Knauf C, et al. Changes in gut microbiota control metabolic endotoxemia-induced inflammation in High-Fat Diet-Induced obesity and diabetes in mice [J]. Diabetes, 2008, 57(6): 1470-1481.

[35] Gulhane M, Murray L, Lourie R, et al. High fat diets induce colonic epithelial cell stress and inflammation that is reversed by IL-22 [J]. Scientific Reports, 2016, 6(1): 28990.

[36] Gori M, Altomare A, Cocca S, et al. Palmitic acid affects intestinal epithelial barrier integrity and permeability in vitro [J]. Antioxidants, 2020, 9(5): 417.

[37] Al-Sadi R, Ye D, Dokladny K, et al. Mechanism of IL-1β -Induced increase in intestinal epithelial tight junction permeability1 [J]. The Journal of Immunology, 2008, 180(8): 5653-5661.

[38] Fan H, Wu J, Yang K, et al. Dietary regulation of intestinal stem cells in health and disease [J]. International Journal of Food Sciences and Nutrition, 2023, 74(7): 730-745.

[39] Luettig J, Rosenthal R, Lee I-F M, et al. The ginger component 6-shogaol prevents TNF- α -induced barrier loss via inhibition of PI3K/Akt and NF- κB signaling [J]. Molecular Nutrition & Food Research, 2016, 60(12): 2576-2586.

[40] Suzuki T . Regulation of the intestinal barrier by nutrients: The role of tight junctions [J]. Animal Science Journal, 2020, 91(1): e13357.

[41] Coskun M . The role of CDX2 in inflammatory bowel disease [J]. Dan Med J, 2014, 61(3): B4820.

[42] Ilie-Petrov A C, Cristian D A, Grama F A, et al. Evaluation of the immunohistochemical scoring system of CDX2 expression as a prognostic biomarker in colon cancer [J]. Diagnostics (Basel), 2024, 14(10): 1023.

[43] Verzi M P, Shin H, He H H, et al. Differentiation-specific histone modifications reveal dynamic chromatin interactions and partners for the intestinal transcription factor CDX2 [J]. Developmental Cell, 2010, 19(5): 713-726.

[44] Calon A, Gross I, Lhermitte B, et al. Different effects of the Cdx1 and Cdx2 homeobox genes in a murine model of intestinal inflammation [J]. Gut, 2007, 56(12): 1688-1695.

[45] Verzi M P, Shin H, San Roman A K, et al. Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding [J]. Molecular and Cellular Biology, 2013, 33(2): 281-292.

[46] Bhat A A, Sharma A, Pope J, et al. Caudal homeobox protein Cdx-2 cooperates with Wnt pathway to regulate claudin-1 expression in colon cancer cells [J]. PloS One, 2012, 7(6): e37174.

[47] Zhu H T, Luo J, Peng Y, et al. Nitazoxanide protects against experimental ulcerative colitis through improving intestinal barrier and inhibiting inflammation [J]. Chem Biol Interact, 2024, 395: 111013.

基金资助

山东省重点研究发展计划项目(2023TZXD070)

国家自然科学基金面上项目(32372332)

山东省自然科学基金项目(ZR2022MC004)