Shixiaosan Attenuates Intestinal Epithelial Barrier Injury in TNF-α-stimulated Caco-2 Cells and DSS-induced Colitis Mice

Tianyu She; Bei Huang; Yunlong Gao; Weiling Liu; Wenjing Yan; Qiyi Hu; Xiaoying Tian; Xiaoxiao Zhao; Yina Zhao; Hangyan Qian; Na Jing; Yuan Wang; Dan Yun

doi:10.54097/26jsj240

Authors

Tianyu She Department of Medical Imaging Function, Xi’an Electric Power Central Hospital, Xi’an 710032, Shaanxi, China;
Bei Huang Department of Anorectal, Xi’an Hospital of Traditional Chinese Medicine, Xi’an 710021, Shaanxi, China;
Yunlong Gao The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710004, Shaanxi, China;
Weiling Liu Department of Colorectal Medicine, Affiliated Hospital of Shanxi University of Traditional Chinese Medicine, Taiyuan 030024, Shanxi, China;
Wenjing Yan State Key Laboratory of Active Components and Pharmacodynamics of Natural Medicines, China Pharmaceutical University, Nanjing 210009, Jiangsu, China;
Qiyi Hu Third People’s Hospital of Beichuan Qiang Autonomous County, Mianyang 622750, Sichuan, China;
Xiaoying Tian Department of Medical Imaging Function, Xi’an Electric Power Central Hospital, Xi’an 710032, Shaanxi, China;
Xiaoxiao Zhao Department of Traditional Chinese Medicine, Xi’an Electric Power Central Hospital, Xi’an 710032, Shaanxi, China;
Yina Zhao Department of Medical Imaging Function, Xi’an Electric Power Central Hospital, Xi’an 710032, Shaanxi, China;
Hangyan Qian Department of Nursing, Xi’an Electric Power Central Hospital, Xi’an 710032, Shaanxi, China;
Na Jing Department of Thyroid and Breast Surgery, Xi’an Electric Power Central Hospital, Xi’an 710032, Shaanxi, China;
Yuan Wang People’s Hospital of Yaozhou District, Tongchuan 727100, Shaanxi, China;
Dan Yun Department of Medical Imaging Function, Xi’an Electric Power Central Hospital, Xi’an 710032, Shaanxi, China;

DOI:

https://doi.org/10.54097/26jsj240

Keywords:

Shixiaosan; Colitis; Intestinal mucosa; Tight junctions; NF-κB.

Abstract

Disruption of the intestinal epithelial barrier is implicated in the development and persistence of inflammatory bowel disease. Although Shixiaosan (SXS) has anti-inflammatory activity, its effects on epithelial barrier injury and the signaling events involved remain insufficiently characterized. We examined SXS in Caco-2 cells challenged with TNF-α and in mice with DSS-induced colitis, focusing on RELA/NF-κB signaling and myosin light-chain (MLC) phosphorylation. Cell experiments used SXS at 25–100 μg/mL and assessed viability, transepithelial electrical resistance (TEER), paracellular tracer flux, inflammatory mediators, and tight-junction proteins. Male C57BL/6 mice received 3% DSS for 7 days and regular water for a further 3 days, together with SXS at 1, 2, or 4 g/kg/day (n = 6 per group). Disease activity, tissue injury, serum barrier-related markers, inflammatory mediators, and colonic tight-junction proteins were evaluated. RELA silencing in the cell model was used to examine pathway involvement. SXS showed no obvious cytotoxicity and counteracted the TNF-α-associated loss of viability and TEER, limited paracellular permeability and inflammatory mediator release, and restored ZO-1, occludin, and claudin-1. In mice, treatment attenuated weight loss, disease activity, histological injury, and colon shortening, while lowering serum permeability markers and restoring colonic tight-junction proteins. Reduced phosphorylation of p65, IκBα, and MLC accompanied these protective effects. RELA knockdown also reduced MLC phosphorylation and partly recovered tight-junction protein expression. Together, these findings indicate that SXS limits experimental intestinal barrier injury and inflammation, with RELA/NF-κB-associated MLC phosphorylation implicated in its effects. Direct validation of MLCK remains necessary.

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References

[1] Ahmad, R., Sorrell, M. F., Batra, S. K., Dhawan, P., & Singh, A. B. (2017). Gut permeability and mucosal inflammation: Bad, good or context dependent. Mucosal Immunology, 10(2), 307–317. https://doi.org/10.1038/mi.2016.88. DOI: https://doi.org/10.1038/mi.2016.128

[2] An, J.-H., Li, Q., Bhang, D.-H., Song, W.-J., & Youn, H.-Y. (2020). TNF-α and INF-γ primed canine stem cell-derived extracellular vesicles alleviate experimental murine colitis. Scientific Reports, 10(1), 2115. https://doi.org/10.1038/s41598-020-59051-4 DOI: https://doi.org/10.1038/s41598-020-58909-4

[3] Awad, W. A., Hess, C., & Hess, M. (2017). Enteric pathogens and their toxin-induced disruption of the intestinal barrier through alteration of tight junctions in chickens. Toxins, 9(2), 60. https://doi.org/10.3390/toxins9020060 DOI: https://doi.org/10.3390/toxins9020060

[4] Barbara, G., Barbaro, M. R., Fuschi, D., Palombo, M., Falangone, F., Cremon, C., Marasco, G., & Stanghellini, V. (2021). Inflammatory and microbiota-related regulation of the intestinal epithelial barrier. Frontiers in Nutrition, 8, 718356. https://doi.org/10.3389/fnut.2021.718356 DOI: https://doi.org/10.3389/fnut.2021.718356

[5] Breugelmans, T., De Man, J., De Winter, B., & Smet, A. (2019). P097 Intestinal barrier dysfunction in association with fibrosis during experimental acute and chronic colitis in mice. Journal of Crohn’s and Colitis, 13(Suppl. 1), S134–S136. https://doi.org/10.1093/ecco-jcc/jjy222.214 DOI: https://doi.org/10.1093/ecco-jcc/jjy222.221

[6] Capaldo, C. T., Powell, D. N., & Kalman, D. (2017). Layered defense: How mucus and tight junctions seal the intestinal barrier. Journal of Molecular Medicine, 95(9), 927–934. https://doi.org/10.1007/s00109-017-1557-x DOI: https://doi.org/10.1007/s00109-017-1557-x

[7] Chen, C., Tao, T., Wen, C., He, W.-Q., Qiao, Y.-N., Gao, Y.-Q., Chen, X., Wang, P., Chen, C.-P., Zhao, W., Chen, H.-Q., Ye, A.-P., Peng, Y.-J., & Zhu, M.-S. (2014). Myosin light chain kinase (MLCK) regulates cell migration in a myosin regulatory light chain phosphorylation-independent mechanism. Journal of Biological Chemistry, 289(41), 28478–28488. https://doi.org/10.1074/jbc.M114.574435 DOI: https://doi.org/10.1074/jbc.M114.567446

[8] Chen, S., Zhang, Y., Niu, X., Mohyuddin, S. G., Wen, J., Bao, M., Yu, T., Wu, L., Hu, C., Yong, Y., Liu, X., Abd El-Aty, A. M., & Ju, X. (2021). Coral-derived endophytic fungal product, butyrolactone-I, alleviates LPS induced intestinal epithelial cell inflammatory response through TLR4/NF-κB and MAPK signaling pathways: An in vitro and in vivo studies. Frontiers in Nutrition, 8, 748118. https://doi.org/10.3389/fnut.2021.748118 DOI: https://doi.org/10.3389/fnut.2021.748118

[9] Lyu, S.-C., Zhong, G.-Q., Shi, R.-J., Sun, Y., Li, J., Li, M.-S., & Chen, Y. (2025). Diamine oxidase as a biomarker for depression and disease activity in inflammatory bowel disease: A cross-sectional observational study. World Journal of Psychiatry, 15(8), 106971. https://doi.org/10.5498/wjp.v15.i8.106971 DOI: https://doi.org/10.5498/wjp.v15.i8.106971

[10] Chen, Y., Li, X., Sun, R., Yang, F., Tian, W., & Huang, Q. (2024). Screening and experimental validation of diagnostic gene in ulcerative colitis with anti-TNF-α therapy. IUBMB Life, 76(7), 451–463. https://doi.org/10.1002/iub.2867 DOI: https://doi.org/10.1002/iub.2807

[11] Dmytriv, T. R., Storey, K. B., & Lushchak, V. I. (2024). Intestinal barrier permeability: The influence of gut microbiota, nutrition, and exercise. Frontiers in Physiology, 15, 1380713. https://doi.org/10.3389/fphys.2024.1380713 DOI: https://doi.org/10.3389/fphys.2024.1380713

[12] Engevik, A. C., Krystofiak, E. S., Kaji, I., Meyer, A. R., Weis, V. G., Goldstein, A., Coutts, A. W., Melkamu, T., Saqui-Salces, M., & Goldenring, J. R. (2021). Recruitment of polarity complexes and tight junction proteins to the site of apical bulk endocytosis. Cellular and Molecular Gastroenterology and Hepatology, 12(1), 59–80. https://doi.org/10.1016/j.jcmgh.2021.02.007 DOI: https://doi.org/10.1016/j.jcmgh.2021.01.022

[13] Feng, Z., Wei, Y., Zhang, Z., Li, M., Gu, R., Lu, L., Liu, W., & Qin, H. (2024). Wheat peptides inhibit the activation of MAPK and NF-κB inflammatory pathways and maintain epithelial barrier integrity in NSAID-induced intestinal epithelial injury. Food & Function, 15(2), 823–837. https://doi.org/10.1039/D3FO02942A DOI: https://doi.org/10.1039/D3FO03954D

[14] Ficek, J., Wyskida, K., Ficek, R., Wajda, J., Klein, D., Witkowicz, J., Rotkegel, S., Spiechowicz-Zatoń, U., Kocemba-Dyczek, J., Ciepał, J., Więcek, A., Olszanecka-Glinianowicz, M., & Chudek, J. (2017). Relationship between plasma levels of zonulin, bacterial lipopolysaccharides, D-lactate and markers of inflammation in haemodialysis patients. International Urology and Nephrology, 49(4), 717–725. https://doi.org/10.1007/s11255-017-1530-8 DOI: https://doi.org/10.1007/s11255-016-1495-5

[15] Fu, R., Wang, L., Meng, Y., Xue, W., Liang, J., Peng, Z., Meng, J., & Zhang, M. (2022). Apigenin remodels the gut microbiota to ameliorate ulcerative colitis. Frontiers in Nutrition, 9, 1062961. https://doi.org/10.3389/fnut.2022.1062961 DOI: https://doi.org/10.3389/fnut.2022.1062961

[16] Han, J., Li, J.-H., Bai, G., Shen, G.-S., Chen, J., Liu, J.-N., Wang, S., & Liu, X.-J. (2017). Acanthopanax senticosus polysaccharides-induced intestinal tight junction injury alleviation via inhibition of NF-κB/MLCK pathway in a mouse endotoxemia model. World Journal of Gastroenterology, 23(12), 2175–2184. https://doi.org/10.3748/wjg.v23.i12.2175 DOI: https://doi.org/10.3748/wjg.v23.i12.2175

[17] Iyer, N., & Corr, S. C. (2021). Gut microbial metabolite-mediated regulation of the intestinal barrier in the pathogenesis of inflammatory bowel disease. Nutrients, 13(12), 4259. https://doi.org/10.3390/nu13124259 DOI: https://doi.org/10.3390/nu13124259

[18] Kang, J., Zhou, Y., Zhu, C., Ren, T., Zhang, Y., Xiao, L., & Fang, B. (2022). Ginsenoside Rg1 mitigates porcine intestinal tight junction disruptions induced by LPS through the p38 MAPK/NLRP3 inflammasome pathway. Toxics, 10(6), 285. https://doi.org/10.3390/toxics10060285 DOI: https://doi.org/10.3390/toxics10060285

[19] Li, L., Huang, J., Feng, L., Xu, L., Lin, H., Liu, K., Li, X., & Wang, R. (2024). Altechromone A ameliorates inflammatory bowel disease by inhibiting NF-κB and NLRP3 pathways. Marine Drugs, 22(9), 410. https://doi.org/10.3390/md22090410 DOI: https://doi.org/10.3390/md22090410

[20] Liu, Z., Jiang, J., Yang, Q., Xiong, Y., Zou, D., Yang, C., Xu, J., & Zhan, H. (2016). MicroRNA-682-mediated downregulation of PTEN in intestinal epithelial cells ameliorates intestinal ischemia–reperfusion injury. Cell Death & Disease, 7(4), e2210. https://doi.org/10.1038/cddis.2016.115 DOI: https://doi.org/10.1038/cddis.2016.84

[21] Luo, Y., Yu, X., Zhao, P., Huang, J., & Huang, X. (2022). Effects of resveratrol on tight junction proteins and the Notch1 pathway in an HT-29 cell model of inflammation induced by lipopolysaccharide. Inflammation, 45(6), 2449–2464. https://doi.org/10.1007/s10753-022-01684-3 DOI: https://doi.org/10.1007/s10753-022-01704-2

[22] Macura, B., Kiecka, A., & Szczepanik, M. (2024). Intestinal permeability disturbances: Causes, diseases and therapy. Clinical and Experimental Medicine, 24(1), 232. https://doi.org/10.1007/s10238-024-01446-7 DOI: https://doi.org/10.1007/s10238-024-01496-9

[23] Morovic, P., Gonzalez Moreno, M., Trampuz, A., & Karbysheva, S. (2024). In vitro evaluation of microbial D- and L-lactate production as biomarkers of infection. Frontiers in Microbiology, 15, 1406350. https://doi.org/10.3389/fmicb.2024.1406350 DOI: https://doi.org/10.3389/fmicb.2024.1406350

[24] Nwako, J., Riedman, A. M., Singh, S., Edwards, K., & McCauley, H. (2025). Enteroendocrine cells regulate barrier integrity and protect against DSS-induced outcomes. Inflammatory Bowel Diseases, 31(Suppl. 1), S46. https://doi.org/10.1093/ibd/izae299.094 DOI: https://doi.org/10.1093/ibd/izae282.108

[25] Okumura, R., & Takeda, K. (2025). The role of the mucosal barrier system in maintaining gut symbiosis to prevent intestinal inflammation. Seminars in Immunopathology, 47(1), 2. https://doi.org/10.1007/s00281-024-01062-0 DOI: https://doi.org/10.1007/s00281-024-01026-5

[26] Olaru-Stavila, C., Steinmann, S. M., Mester, P., Müller, M., Tcaciuc, E., & Gülow, K. (2026). The gastrointestinal barrier—Mechanisms of barrier dysfunction in liver cirrhosis and spontaneous bacterial peritonitis. Biomedicines, 14(5), 1084. https://doi.org/10.3390/biomedicines14051084 DOI: https://doi.org/10.3390/biomedicines14051084

[27] Sánchez-Alcoholado, L., Ordóñez, R., Otero, A., Plaza-Andrade, I., Laborda-Illanes, A., Medina, J. A., Ramos-Molina, B., Gómez-Millán, J., & Queipo-Ortuño, M. I. (2020). Gut microbiota-mediated inflammation and gut permeability in patients with obesity and colorectal cancer. International Journal of Molecular Sciences, 21(18), 6782. https://doi.org/10.3390/ijms21186782 DOI: https://doi.org/10.3390/ijms21186782

[28] Scalavino, V., Piccinno, E., Bianco, G., Schena, N., Armentano, R., Giannelli, G., & Serino, G. (2022). The increase of miR-195-5p reduces intestinal permeability in ulcerative colitis, modulating tight junctions’ expression. International Journal of Molecular Sciences, 23(10), 5840. https://doi.org/10.3390/ijms23105840 DOI: https://doi.org/10.3390/ijms23105840

[29] Shi, D., Cui, Y., Liang, H., Wei, Q., Huang, J., & Cao, J. (2026). Gut microbiota-derived tryptophan metabolite indole-3-carboxaldehyde enhances intestinal barrier function via aryl hydrocarbon receptor/AMP-activated protein kinase signaling activation. Animal Bioscience, 39(1), 250225. https://doi.org/10.5713/ab.25.0225 DOI: https://doi.org/10.5713/ab.25.0225

[30] Slifer, Z. M., & Blikslager, A. T. (2020). The integral role of tight junction proteins in the repair of injured intestinal epithelium. International Journal of Molecular Sciences, 21(3), 972. https://doi.org/10.3390/ijms21030972 DOI: https://doi.org/10.3390/ijms21030972

[31] Wang, K., Wang, H., Lou, W., Ma, L., Li, Y., Zhang, N., Wang, C., Li, F., Awais, M., Cao, S., She, R., Fu, Z. F., & Cui, M. (2018). IP-10 promotes blood–brain barrier damage by inducing tumor necrosis factor alpha production in Japanese encephalitis. Frontiers in Immunology, 9, 1148. https://doi.org/10.3389/fimmu.2018.01148 DOI: https://doi.org/10.3389/fimmu.2018.01148

[32] Wang, X., Zhang, R., Gu, L., Zhang, Y., Zhao, X., Bi, K., & Chen, X. (2015). Cell-based screening identifies the active ingredients from traditional Chinese medicine formula Shixiao San as the inhibitors of atherosclerotic endothelial dysfunction. PLOS ONE, 10(2), e0116601. https://doi.org/10.1371/journal.pone.0116601 DOI: https://doi.org/10.1371/journal.pone.0116601

[33] Wang, X., Zhao, X., Gu, L., Zhang, Y., Bi, K., & Chen, X. (2014). Discrimination of aqueous and vinegary extracts of Shixiao San using metabolomics coupled with multivariate data analysis and evaluation of anti-hyperlipidemic effect. Asian Journal of Pharmaceutical Sciences, 9(1), 17–26. https://doi.org/10.1016/j.ajps.2014.01.002 DOI: https://doi.org/10.1016/j.ajps.2013.11.008

[34] Wei, X., Gao, M., Sheng, N., Yao, W., Bao, B., Cheng, F., Cao, Y., Yan, H., Zhang, L., Shan, M., & Chen, P. (2023). Mechanism investigation of Shi-Xiao-San in treating blood stasis syndrome based on network pharmacology, molecular docking and in vitro/vivo pharmacological validation. Journal of Ethnopharmacology, 301, 115746. https://doi.org/10.1016/j.jep.2022.115746 DOI: https://doi.org/10.1016/j.jep.2022.115746

[35] Xiao, Y.-T., Yan, W.-H., Cao, Y., Yan, J.-K., & Cai, W. (2016). Neutralization of IL-6 and TNF-α ameliorates intestinal permeability in DSS-induced colitis. Cytokine, 83, 189–192. https://doi.org/10.1016/j.cyto.2016.04.011 DOI: https://doi.org/10.1016/j.cyto.2016.04.012

[36] Yue, D., Zheng, Z., Fan, W., Zhu, L., Lin, D., Lu, M., Ji, W., Cao, P., Sun, X., & Hu, C. (2022). Effect of Shixiao San on inflammatory factors and pain in rats with endometriosis. Journal of Ethnopharmacology, 290, 115121. https://doi.org/10.1016/j.jep.2022.115121 DOI: https://doi.org/10.1016/j.jep.2022.115121

[37] Zhang, B., Wei, X., Ding, M., Luo, Z., Tan, X., & Zheng, Z. (2022). Daidzein protects Caco-2 cells against lipopolysaccharide-induced intestinal epithelial barrier injury by suppressing PI3K/AKT and P38 pathways. Molecules, 27(24), 8928. https://doi.org/10.3390/molecules27248928 DOI: https://doi.org/10.3390/molecules27248928