珠子草素通过调控p38/JNK信号通路抑制肠上皮细胞凋亡保护肠屏障改善克罗恩病样肠炎

doi:10.12122/j.issn.1673-4254.2025.11.21

摘要/Abstract

摘要：

目的探讨天然化合物珠子草素（NIR）对克罗恩病样结肠炎的作用及其分子机制。方法采用2，4，6-三硝基苯磺酸（TNBS）诱导小鼠建立结肠炎模型，随机分为4组：WT组注射生理盐水；WT+NIR组腹腔注射NIR（10 mg/kg，1次/d，注射7 d），TNBS组用2.5% TNBS造模并给予等体积的生理盐水；TNBS+NIR组用2.5% TNBS造模并腹腔注射NIR（10 mg/kg，1次/d，注射7 d），6只/组。用体质量变化、疾病活动指数（DAI）和结肠长度评估NIR的治疗效果。ELISA法和实时定量PCR（qRT-PCR）检测肠黏膜组织炎症因子（IL-6、IL-1β、TNF-α、IL-17A和IL-10）水平。TUNEL染色和Western blotting检测肠上皮细胞凋亡情况及相关蛋白（Bcl-2/Bax）的表达。Western blotting评估紧密连接蛋白（TJ）（ZO-1、Claudin-1）和p38/JNK通路的活化水平，并通过Diprovocim干预实验验证NIR的调控分子机制。结果 NIR干预后TNBS小鼠体质量增加，DAI和组织学炎症评分减低，结肠长度增加（P<0.05）；ELISA和qRT-PCR结果表明NIR可降低促炎因子（IL-6，IL-1β、IL-17A和TNF-α）的蛋白和mRNA水平，上调抗炎因子IL-10表达水平（P<0.05）；TUNEL和Western blotting检测显示NIR可抑制肠上皮细胞凋亡，激活抗凋亡通路（P<0.05）；Western blotting结果证实NIR可上调ZO-1和Claudin-1的表达水平，并下调p38和JNK的磷酸化水平（P<0.05）；Diprovocim干预可衰减NIR对p38/JNK通路的失活作用。结论 NIR可通过调控p38/JNK信号的活化抑制肠上皮细胞凋亡，从而改善小鼠CD样肠炎。

关键词: 克罗恩病, 炎症性肠病, 肠上皮细胞凋亡, p38/JNK, 珠子草素

Abstract:

Objective To investigate the therapeutic effect of the natural compound niranthin on Crohn's disease-like colitis in mice and explore the underlying molecular mechanisms. Methods In a mouse model of colitis induced by 2,4,6-trinitro-benzenesulfonic acid (TNBS), the therapeutic effect of niranthin was evaluated by observing the changes in body weight, disease activity index (DAI), and colon length of the mice. The levels of inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-17A and IL-10) in the intestinal mucosal tissue were detected using ELISA and quantitative real-time PCR (qRT-PCR). TUNEL staining and Western blotting were used to assess intestinal epithelial cell apoptosis and the expressions of Bcl-2 and Bax. The expression levels of tight junction proteins (ZO-1 and claudin-1) and the activation of the p38/JNK signaling pathway were investigated using Western blotting, and diprovocim intervention experiments were conducted to explore the molecular regulatory mechanism of niranthin. Results Niranthin treatment significantly increased body weight of TNBS-treated mice, lowered the DAI and histological inflammation scores, and increased colon length of the mice. The niranthin-treated mouse models showed obviously reduced protein and mRNA levels of IL-6, IL-1β, IL-17A, and TNF-α and upregulated expression of IL-10 in the colon tissue. TUNEL staining and Western blotting demonstrated that niranthin significantly inhibited intestinal epithelial cell apoptosis and activated the anti-apoptotic pathway in the mouse models. Niranthin treatment obviously upregulated the expression levels of ZO-1 and claudin-1 and downregulated the phosphorylation levels of p38 and JNK in the colon tissues of the mice. Diprovocim intervention obviously attenuated the inactivation of the p38/JNK signaling pathway induced by niranthin in the mouse models. Conclusion Niranthin ameliorates TNBS-induced Crohn's disease-like colitis in mice by inhibiting intestinal epithelial cell apoptosis and protecting the integrity of the intestinal barrier via regulating the activation of the p38/JNK signaling pathway.

Key words: Crohn's disease, inflammatory bowel disease, intestinal epithelial cell apoptosis, p38/JNK, Niranthin

陶露, 陈悦, 黄林林, 郑旺, 宋雪, 项平, 胡建国. 珠子草素通过调控p38/JNK信号通路抑制肠上皮细胞凋亡保护肠屏障改善克罗恩病样肠炎[J]. 南方医科大学学报, 2025, 45(11): 2483-2495.

Lu TAO, Yue CHEN, Linlin HUANG, Wang ZHENG, Xue SONG, Ping XIANG, Jianguo HU. Niranthin ameliorates Crohn's disease-like enteritis in mice by inhibiting intestinal epithelial cell apoptosis and protecting intestinal barrier via modulating p38/JNK signaling[J]. Journal of Southern Medical University, 2025, 45(11): 2483-2495.

图/表 9

表1 引物序列

Tab.1 Primer sequences for qRT-PCR in this study

Gene	Primer sequences (5'-3')
TNF-α	F: CACGCTCTTCTGTCTACTGAACTTC
TNF-α	R: CTTGGTGGTTTGTGAGTGTGAGG
IL-1β	F: AATCTCGCAGCAGCACATCAAC
IL-1β	R: AGGTCCACGGGAAAGACACAG
IL-6	F: GAGAGGAGACTTCACAGAGGATACC
IL-6	R: TCATTTCCACGATTTCCCAGAGAAC
IL-10	F: GGACAACATACTGCTAACCGACTC
IL-10	R: GGGCATCACTTCTACCAGGTAAAAC
IL-17A	F: TGGCGGCTACAGTGAAGGC
IL-17A	R: AGGGAGTTAAAGACTTTGAGGTTGAC
GAPDH	F: AACTCCCACTCTTCCACCTTCG
GAPDH	R: TCCACCACCCTGTTGCTGTAG

图1 NIR干预对TNBS诱导小鼠结肠炎症状的影响

Fig.1 Effect of niranthin (NIR) treatment on symptoms of TNBS-induced colitis in mice. A: Body weight changes of the mice. B: DAI score. C: Colon length. D: Comparison of colon length of the mice among the 4 groups. E: Colon inflammation score of the mice. F: HE staining of the colon tissues of the mice in the 4 groups.*P<0.05.

图2 NIR干预对TNBS诱导小鼠结肠炎炎症因子水平的影响

Fig.2 Effect of NIR treatment on inflammatory factor levels in mice with TNBS-induced colitis. A-E: Results of ELISA for detecting the levels of IL-6, IL-1β, IL-17A, TNF-α and IL-10 in the colon tissue of TNBS-induced mice. F-J: qRT-PCR for detecting IL-6, IL-1β, IL-17A, TNF‑α and IL-10 mRNA expressions in the colon tissues of TNBS-induced mice. *P<0.05.

图3 NIR干预对TNBS诱导小鼠结肠炎肠上皮细胞凋亡的影响

Fig.3 Effect of NIR treatment on TNBS-induced apoptosis of intestinal epithelial cells in the mouse models of colitis. A, B: TUNEL staining for detecting apoptosis in TNBS-induced mice with NIR treatment. C: Western blotting for detecting the expression of Bcl-2 and Bax proteins in TNBS-induced mice with NIR treatment. D: Immunofluorescence staining for detecting Bcl-2 expression in TNBS-induced mice with NIR treatment. *P<0.05.

图4 NIR干预对TNBS诱导小鼠结肠炎肠屏障的影响

Fig.4 Effect of NIR treatment on intestinal barrier function in mice with TNBS-induced colitis. A: Immunofluorescence staining for detecting ZO-1 and Claudin-1 expression in TNBS-induced mice with NIR treatment. B: Western blotting for detecting ZO-1 and claudin-1 protein expressions in TNBS-induced mice with NIR treatment. *P<0.05.

图5 NIR干预对LPS诱导Caco-2细胞凋亡的影响

Fig.5 Effect of NIR treatment on LPS-induced apoptosis in Caco-2 cells. A, B: TUNEL staining for detecting LPS-induced apoptosis of Caco-2 cells with NIR treatment. C: Western blotting for detecting the expression of Bcl-2 and Bax proteins in LPS-induced Caco-2 cells with NIR treatment. D: Immunofluorescence staining for detecting Bcl-2 expression in LPS-induced Caco-2 cells with NIR treatment. *P<0.05.

图6 NIR干预对LPS诱导Caco-2细胞TJ蛋白表达的影响

Fig. 6 Effect of NIR treatment on expressions of tight junction proteins in LPS-induced Caco-2 cells. A: Immunofluorescence staining showing ZO-1 and claudin-1 expressions in LPS-induced Caco-2 cells with NIR treatment. B: Western blotting for detecting ZO-1 and claudin-1 protein expressions in LPS-induced Caco-2 cells with NIR treatment. *P<0.05.

图7 NIR干预可调控p38/JNK信号

Fig. 7 NIR treatment modulates p38/JNK signaling. A: Western blotting for detecting p-p38 and p-JNK protein expressions in TNBS-induced mice with NIR treatment. B: Western blotting for detecting p-p38 and p-JNK protein expressions in LPS-induced Caco-2 cells with NIR treatment. C: Western blotting for detecting p-p38 and p-JNK protein expressions in the colon tissues of the mice with NIR and diprovocim treatment. *P<0.05.

图8 Diprovocim干预对NIR治疗Caco-2细胞中凋亡和TJ蛋白表达的影响

Fig. 8 Effects of diprovocim intervention on apoptosis and expressions of tight junction proteins in NIR-treated Caco-2 cells. A: TUNEL staining for detecting apoptosis in NIR-treated Caco-2 cells by Diprovocim treatment. B: Western blotting for detecting the expression of Bcl-2 and Bax proteins in NIR-treated Caco-2 cells with diprovocim treatment. C, D: Immunofluorescence staining for detecting Bcl-2, ZO-1 and Claudin-1 expression in NIR-treated Caco-2 cells with diprovocim treatment. *P<0.05.

参考文献 33

[1]	Dolinger M, Torres J, Vermeire S. Crohn’s disease[J]. Lancet, 2024, 403(10432): 1177-91. doi：10.1016/s0140-6736(23)02586-2
[2]	Loftus EV Jr, Panés J, Lacerda AP, et al. Upadacitinib induction and maintenance therapy for Crohn’s disease[J]. N Engl J Med, 2023, 388(21): 1966-80. doi：10.1056/nejmoa2212728
[3]	Honap S, Jairath V, Danese S, et al. Navigating the complexities of drug development for inflammatory bowel disease[J]. Nat Rev Drug Discov, 2024, 23(7): 546-62. doi：10.1038/s41573-024-00953-0
[4]	Wang L, Song X, Zhou YQ, et al. Sclareol protected against intestinal barrier dysfunction ameliorating Crohn’s disease-like colitis via Nrf2/NF-B/MLCK signalling[J]. Int Immunopharmacol, 2024, 133: 112140. doi：10.1016/j.intimp.2024.112140
[5]	Zhou L, Zhu L, Wu X, et al. Decreased TMIGD1 aggravates colitis and intestinal barrier dysfunction via the BANF1-NF-κB pathway in Crohn’s disease[J]. BMC Med, 2023, 21(1): 287. doi：10.1186/s12916-023-02989-2
[6]	Xu S, Peng YF, Yang K, et al. PROTAC based STING degrader attenuates acute colitis by inhibiting macrophage M1 polarization and intestinal epithelial cells pyroptosis mediated by STING-NLRP3 axis[J]. Int Immunopharmacol, 2024, 141: 112990. doi：10.1016/j.intimp.2024.112990
[7]	Tan G, Huang C, Chen J, et al. HMGB1 released from GSDME-mediated pyroptotic epithelial cells participates in the tumorigenesis of colitis-associated colorectal cancer through the ERK1/2 pathway[J]. J Hematol Oncol, 2020, 13(1): 149. doi：10.1186/s13045-020-00985-0
[8]	Liu J, Di B, Xu LL. Recent advances in the treatment of IBD: targets, mechanisms and related therapies[J]. Cytokine Growth Factor Rev, 2023, 71: 1-12. doi：10.1016/j.cytogfr.2023.07.001
[9]	Veyrard P, Nancey S, Roblin X. Editorial: 5-ASA in IBD patients on biologics-' stop or continue'[J]? Aliment Pharmacol Ther, 2021, 54(6): 843-4. doi：10.1111/apt.16541
[10]	Sridhar A, Bakke I, Gopalakrishnan S, et al. Tofacitinib and budesonide treatment affect stemness and chemokine release in IBD patient-derived colonoids[J]. Sci Rep, 2025, 15(1): 3753. doi：10.1038/s41598-025-86314-2
[11]	Biologic therapy for inflammatory bowel disease: real-world comparative effectiveness and impact of drug sequencing in 13 222 patients within the UK IBD BioResource[J]. J Crohns Colitis, 2024, 18(6): 790-800. doi：10.1093/ecco-jcc/jjad212.1168
[12]	Ota R, Karasawa D, Oshima M, et al. Asymmetric total synthesis of four bioactive lignans using donor–acceptor cyclopropanes and bioassay of (-)- and (+)-niranthin against hepatitis B and influenza viruses[J]. RSC Adv, 2022, 12(8): 4635-9. doi：10.1039/d2ra00499b
[13]	Harikrishnan H, Jantan I, Alagan A, et al. Modulation of cell signaling pathways by Phyllanthus amarus and its major constituents: potential role in the prevention and treatment of inflammation and cancer[J]. Inflammopharmacology, 2020, 28(1): 1-18. doi：10.1007/s10787-019-00671-9
[14]	Chowdhury S, Mukherjee T, Mukhopadhyay R, et al. The lignan niranthin poisons Leishmania donovani topoisomerase IB and favours a Th1 immune response in mice[J]. EMBO Mol Med, 2012, 4(10): 1126-43. doi：10.1002/emmm.201201316
[15]	Harikrishnan H, Jantan I, Haque MA, et al. Anti-inflammatory effects of hypophyllanthin and niranthin through downregulation of NF-κB/MAPKs/PI3K-Akt signaling pathways[J]. Inflammation, 2018, 41(3): 984-95. doi：10.1007/s10753-018-0752-4
[16]	Tan G, Huang CY, Chen JY, et al. Gasdermin-E-mediated pyroptosis participates in the pathogenesis of Crohn’s disease by promoting intestinal inflammation[J]. Cell Rep, 2021, 35(11): 109265. doi：10.1016/j.celrep.2021.109265
[17]	Spencer DM, Veldman GM, Banerjee S, et al. Distinct inflammatory mechanisms mediate early versus late colitis in mice[J]. Gastroenterology, 2002, 122(1): 94-105. doi：10.1053/gast.2002.30308
[18]	Rath HC, Herfarth HH, Ikeda JS, et al. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats[J]. J Clin Invest, 1996, 98(4): 945-53. doi：10.1172/jci118878
[19]	Morin MD, Wang Y, Jones BT, et al. Diprovocims: A New and Exceptionally Potent Class of Toll-like Receptor Agonists[J]. J Am Chem Soc, 2018, 140(43):14440-54. doi：10.1021/jacs.8b09223
[20]	Wei ZY, Ni X, Cui H, et al. Engeletin attenuates the inflammatory response via inhibiting TLR4-NFκB signaling pathway in Crohn’s disease-like colitis[J]. J Ethnopharmacol, 2025, 336: 118733. doi：10.1016/j.jep.2024.118733
[21]	Mehandru S, Colombel JF. The intestinal barrier, an arbitrator turned provocateur in IBD[J]. Nat Rev Gastroenterol Hepatol, 2021, 18(2): 83-4. doi：10.1038/s41575-020-00399-w
[22]	Odenwald MA, Turner JR. The intestinal epithelial barrier: a therapeutic target[J]? Nat Rev Gastroenterol Hepatol, 2017, 14(1): 9-21. doi：10.1038/nrgastro.2016.169
[23]	Huang S, Xie Z, Han J, et al. Protocadherin 20 maintains intestinal barrier function to protect against Crohn’s disease by targeting ATF6[J]. Genome Biol, 2023, 24(1): 159. doi：10.1186/s13059-023-02991-0
[24]	Safari F, Sharifi M, Talebi A, et al. Alleviation of cholestatic liver injury and intestinal permeability by lubiprostone treatment in bile duct ligated rats: role of intestinal FXR and tight junction proteins claudin-1, claudin-2, and occludin[J]. Naunyn Schmiedeberg’s Arch Pharmacol, 2023, 396(9): 2009-22. doi：10.1007/s00210-023-02455-z
[25]	Pan Z, Huang J, Hu T, et al. Protective effects of selenium nanoparticles against bisphenol A-induced toxicity in porcine intestinal epithelial cells[J]. Int J Mol Sci, 2023, 24(8): 7242. doi：10.3390/ijms24087242
[26]	Yin M, Shen Z, Yang L, et al. Protective effects of CXCR3/HO-1 gene-modified BMMSCs on damaged intestinal epithelial cells: Role of the p38-MAPK signaling pathway[J]. Int J Mol Med, 2019, 43(5): 2086-102.
[27]	Zeng ZW, Shi YM, Cai YH, et al. PHLDA1 protects intestinal barrier function via restricting intestinal epithelial cells apoptosis in inflammatory bowel disease[J]. Exp Cell Res, 2024, 443(1): 114322. doi：10.1016/j.yexcr.2024.114322
[28]	Geng Z, Zuo L, Li J, et al. Ginkgetin improved experimental colitis by inhibiting intestinal epithelial cell apoptosis through EGFR/PI3K/AKT signaling[J]. FASEB J, 2024, 38(14): e23817. doi：10.1096/fj.202400211rr
[29]	Xu Q, Liu M, Chao X, et al. Stevioside improves antioxidant capacity and intestinal barrier function while attenuating inflammation and apoptosis by regulating the NF-κB/MAPK pathways in diquat-induced oxidative stress of IPEC-J2 cells[J]. Antioxidants: Basel, 2023, 12(5): 1070. doi：10.3390/antiox12051070
[30]	Xing Z, Li X, He J, et al. OLFM4 modulates intestinal inflammation by promoting IL-22⁺ILC3 in the gut[J]. Commun Biol, 2024, 7(1): 914. doi：10.1038/s42003-024-06601-y
[31]	Xiao L, Zhang WH, Huang Y, et al. Intestinal ischemia-reperfusion induces the release of IL-17A to regulate cell inflammation, apoptosis and barrier damage[J]. Exp Ther Med, 2022, 23(2): 158. doi：10.3892/etm.2021.11081
[32]	Chu C, Ru H, Chen Y, et al. Gallic acid attenuates LPS-induced inflammation in Caco-2 cells by suppressing the activation of the NF-κB/MAPK signaling pathway[J]. Acta Biochim Biophys Sin: Shanghai, 2024, 56(6): 905-15.
[33]	Tao H, Bao Z, Fu Z, et al. Chlorothalonil induces the intestinal epithelial barrier dysfunction in Caco-2 cell-based in vitro monolayer model by activating MAPK pathway[J]. Acta Biochim Biophys Sin: Shanghai, 2021, 53(11): 1459-68. doi：10.1093/abbs/gmab125