Ecliptasaponin A ameliorates DSS-induced colitis in mice by suppressing M1 macrophage polarization via inhibiting the JAK2/STAT3 pathway

doi:10.12122/j.issn.1673-4254.2025.06.19

Abstract

Abstract:

Objective To investigate the effect of ecliptasaponin A (ESA) for alleviating dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD) in mice and the underlying mechanism. Methods Twenty-four male C57BL/6 mice (8-10 weeks old) were equally randomized into control group, DSS-induced IBD model group, and DSS+ESA (50 mg/kg) treatment group. Disease activity index (DAI), colon length and spleen index of the mice were measured, and intestinal pathology was examined with HE staining. The expressions of inflammatory mediators (TNF-α, IL-6, and iNOS) in the colon mucosa were detected using ELISA and RT-qPCR, and intestinal barrier integrity was assessed using AB-PAS staining and by detecting ZO-1 and claudin-1 expressions using immunofluorescence staining and Western blotting. In cultured RAW264.7 macrophages, the effects of treatment with 50 μmol/L ESA, alone or in combination with 20 μmol/L RO8191 (a JAK2/STAT3 pathway activator), on M1 polarization of the cells induced by LPS and IFN-γ stimulation and expressions of JAK2/STAT3 pathway proteins were analyzed using flow cytometry and Western blotting. Results In the mouse models of DSS-induced IBD, ESA treatment significantly alleviated body weight loss and colon shortening, reduced DAI, spleen index and histological scores, and ameliorated inflammatory cell infiltration in the colon tissue. ESA treatment also suppressed TNF‑α, IL-6 and iNOS expressions, protected the goblet cells and the integrity of the mucus and mechanical barriers, and upregulated the expressions of ZO-1 and claudin-1. ESA treatment obviously decreased CD86⁺ M1 polarization in the mesenteric lymph nodes of IBD mice and in LPS and IFN-γ-induced RAW264.7 cells, and significantly reduced p-JAK2 and p-STAT3 expressions in both the mouse models and RAW264.7 cells. Treatment with RO8191 caused reactivation of JAK2/STAT3 and strongly attenuated the inhibitory effect of ESA on CD86⁺ polarization in RAW264.7 cells. Conclusion ESA alleviates DSS-induced colitis in mice by suppressing JAK2/STAT3-mediated M1 macrophage polarization and mitigating inflammation-driven intestinal barrier damage.

Key words: ecliptasaponin A, inflammatory bowel disease, intestinal barrier, macrophage polarization, JAK2/STAT3

Minzhu NIU, Lixia YIN, Tong QIAO, Lin YIN, Keni ZHANG, Jianguo HU, Chuanwang SONG, Zhijun GENG, Jing LI. Ecliptasaponin A ameliorates DSS-induced colitis in mice by suppressing M1 macrophage polarization via inhibiting the JAK2/STAT3 pathway[J]. Journal of Southern Medical University, 2025, 45(6): 1297-1306.

Figures/Tables 8

Fig.1 ESA significantly reduces percentage of M1-type macrophages in RAW264.7 cells induced by LPS and IFN-γ. A: Morphology of macrophages from each group (scale bar=50 μm). B: Percentages of F4/80+CD86+ macrophages detected by flow cytometry. *P<0.05 vs M0 group. #P<0.05 vs M1 group.

Fig.2 Effect of ESA on the disease status of DSS model mice. A: Changes of body weight. B: Changes of DAI scores. WT: Control group; DSS: DSS-induced model group; DSS+ESA: ESA treatment group. *P<0.05 vs WT group. #P<0.05 vs DSS group.

Fig.3 ESA improves pathological phenotype of DSS-induced colitis in mice. A, B: Representative images of mouse colons in each group. C: Changes of spleen index. D: HE staining of the colon tissues. E: AB-PAS staining of colon tissue from each groups. F: Histopathological scores in different groups. *P<0.05 vs WT group. #P<0.05 vs DSS group. (scale bar=100 μm).

Fig.4 Effect of ESA on intestinal mucosal inflammatory mediators in DSS model mice. A-C: Concentrations of proinflammatory cytokines TNF-α, iNOS and IL-6 in the colon tissues detected by ELISA. D-F: The mRNA levels of proinflammatory cytokines TNF-α, iNOS and IL-6 in different groups. *P<0.05 vs WT group. #P<0.05 vs DSS group.

Fig.5 ESA improves gut barrier disruption in mice with DSS-induced colitis. A: Immunofluorescence staining showing expression of ZO-1 and claudin-1 proteins in the colon tissues. B, C: Relative expression levels of ZO-1 and claudin-1 proteins in colon mucosa detected by Western blotting. *P<0.05 vs WT group. #P<0.05 vs DSS group (scale bar=100 μm).

Fig.6 Effect of ESA on M1 macrophages in the mesenteric lymph nodes of DSS model mice. The percentages of F4/80+CD86+ macrophages in mesenteric lymph nodes were detected by flow cytometry. *P<0.05 vs WT group. #P<0.05 vs DSS group.

Fig.7 ESA suppresses JAK2/STAT3 pathway activation by blocking its phosphorylation in vitro and in vivo. A, B: Relative expression levels of JAK2, p-JAK2, STAT3, and p-STAT3 proteins in mouse colon tissue detected by Western blotting (*P<0.05 vs WT group; #P<0.05 vs DSS group). C, D: Relative expression levels of JAK2, p-JAK2, STAT3, p-STAT3 proteins in RAW264.7 cells detected by Western blotting (*P<0.05 vs M0 group. #P<0.05 vs M1 group).

Fig.8 JAK2/STAT3 signaling pathway is involved in ESA-mediated inhibition of M1-type macrophage polarization of RAW264.7 cells. A, B: Relative expressions of JAK2, p-JAK2, STAT3 and p-STAT3 proteins detected by Western blotting in RAW264.7 cells. C: Morphology of the macrophages (×400). D: Proportion of F4/80+CD86+ cells in each group. *P<0.05 vs ESA group (Scale bar=50 μm).

References 44

1	Glassner KL, Abraham BP, Quigley EMM. The microbiome and inflammatory bowel disease[J]. J Allergy Clin Immunol, 2020, 145(1): 16-27. doi：10.1016/j.jaci.2019.11.003
2	Ordás I, Eckmann L, Talamini M, et al. Ulcerative colitis[J]. Lancet, 2012, 380(9853): 1606-19. doi：10.1016/s0140-6736(12)60150-0
3	Xu L, He B, Sun Y, et al. Incidence of inflammatory bowel disease in urban China: a nationwide population-based study[J]. Clin Gastroenterol Hepatol, 2023, 21(13): 3379-86. doi：10.1016/j.cgh.2023.08.013
4	Wangchuk P, Yeshi K, Loukas A. Ulcerative colitis: clinical biomarkers, therapeutic targets, and emerging treatments[J]. Trends Pharmacol Sci, 2024, 45(10): 892-903. doi：10.1016/j.tips.2024.08.003
5	Saez A, Herrero-Fernandez B, Gomez-Bris R, et al. Pathophysiology of inflammatory bowel disease: innate immune system[J]. Int J Mol Sci, 2023, 24(2): 1526. doi：10.3390/ijms24021526
6	Zhou X, Li WY, Wang S, et al. YAP aggravates inflammatory bowel disease by regulating M1/M2 macrophage polarization and gut microbial homeostasis[J]. Cell Rep, 2019, 27(4): 1176-89. e5. doi：10.1016/j.celrep.2019.03.028
7	Krausgruber T, Blazek K, Smallie T, et al. IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses[J]. Nat Immunol, 2011, 12(3): 231-8. doi：10.1038/ni.1990
8	Pan XH, Zhu Q, Pan LL, et al. Macrophage immunometabolism in inflammatory bowel diseases: From pathogenesis to therapy[J]. Pharmacol Ther, 2022, 238: 108176. doi：10.1016/j.pharmthera.2022.108176
9	Zhang K, Guo J, Yan W, et al. Macrophage polarization in inflammatory bowel disease[J]. Cell Commun Signal, 2023, 21(1): 367. doi：10.1186/s12964-023-01386-9
10	Ruan S, Xu L, Sheng Y, et al. Th1 promotes M1 polarization of intestinal macrophages to regulate colitis-related mucosal barrier damage[J]. Aging: Albany NY, 2023, 15(14): 6721-35. doi：10.18632/aging.204629
11	Guo HR, Guo H, Xie Y, et al. Mo3Se4 nanoparticle with ROS scavenging and multi-enzyme activity for the treatment of DSS-induced colitis in mice[J]. Redox Biol, 2022, 56: 102441. doi：10.1016/j.redox.2022.102441
12	Yang Y, Ma Q, Wang Q, et al. Mannose enhances intestinal immune barrier function and dextran sulfate sodium salt-induced colitis in mice by regulating intestinal microbiota[J]. Front Immunol, 2024, 15: 1365457. doi：10.3389/fimmu.2024.1365457
13	You XY, Xue Q, Fang Y, et al. Preventive effects of Ecliptae Herba extract and its component, ecliptasaponin A, on bleomycin-induced pulmonary fibrosis in mice[J]. J Ethnopharmacol, 2015, 175: 172-80. doi：10.1016/j.jep.2015.08.034
14	Han J, Lv W, Sheng H, et al. Ecliptasaponin A induces apoptosis through the activation of ASK1/JNK pathway and autophagy in human lung cancer cells[J]. Ann Transl Med, 2019, 7(20): 539. doi：10.21037/atm.2019.10.07
15	Xi FM, Li CT, Han J, et al. Thiophenes, polyacetylenes and terpenes from the aerial parts of Eclipata prostrata[J]. Bioorg Med Chem, 2014, 22(22): 6515-22. doi：10.1016/j.bmc.2014.06.051
16	Ge SM, Wu SH, Yin Q, et al. Ecliptasaponin A protects heart against acute ischemia-induced myocardial injury by inhibition of the HMGB1/TLR4/NF-κB pathway[J]. J Ethnopharmacol, 2024, 335: 118612. doi：10.1016/j.jep.2024.118612
17	Ge ST, Yang YT, Zuo LG, et al. Sotetsuflavone ameliorates Crohn’s disease-like colitis by inhibiting M1 macrophage-induced intestinal barrier damage via JNK and MAPK signalling[J]. Eur J Pharmacol, 2023, 940: 175464. doi：10.1016/j.ejphar.2022.175464
18	Lei X, Zou F, Tang X, et al. CD3D silencing alleviates diabetic nephropathy via inhibition of JAK/STAT pathway[J]. FASEB J, 2024, 38(21): e70169. doi：10.1096/fj.202401879r
19	Wirtz S, Popp V, Kindermann M, et al. Chemically induced mouse models of acute and chronic intestinal inflammation[J]. Nat Protoc, 2017, 12(7): 1295-309. doi：10.1038/nprot.2017.044
20	Chougule PR, Sangaraju R, Patil PB, et al. Effect of ethyl gallate and propyl gallate on dextran sulfate sodium (DSS)-induced ulcerative colitis in C57BL/6J mice: preventive and protective[J]. Inflammopharmacology, 2023, 31(4): 2103-20. doi：10.1007/s10787-023-01254-5
21	Kobayashi T, Siegmund B, Le Berre C, et al. Ulcerative colitis[J]. Nat Rev Dis Primers, 2020, 6: 74. doi：10.1038/s41572-020-0205-x
22	Le Berre C, Honap S, Peyrin-Biroulet L. Ulcerative colitis[J]. Lancet, 2023, 402(10401): 571-84. doi：10.1016/s0140-6736(23)00966-2
23	Dai N, Haidar O, Askari A, et al. Colectomy rates in ulcerative colitis: a systematic review and meta-analysis[J]. Dig Liver Dis, 2023, 55(1): 13-20. doi：10.1016/j.dld.2022.08.039
24	Asano K, Takahashi N, Ushiki M, et al. Intestinal CD169⁺ macrophages initiate mucosal inflammation by secreting CCL8 that recruits inflammatory monocytes[J]. Nat Commun, 2015, 6: 7802. doi：10.1038/ncomms8802
25	Goddard ZR, Searcey M, Osbourn A. Advances in triterpene drug discovery[J]. Trends Pharmacol Sci, 2024, 45(11): 964-8. doi：10.1016/j.tips.2024.10.003
26	Martin LBB, Kikuchi S, Rejzek M, et al. Complete biosynthesis of the potent vaccine adjuvant QS-21[J]. Nat Chem Biol, 2024, 20(4): 493-502. doi：10.1038/s41589-023-01538-5
27	Hirata K, Helal F, Hadgraft J, et al. Formulation of carbenoxolone for delivery to the skin[J]. Int J Pharm, 2013, 448(2): 360-5. doi：10.1016/j.ijpharm.2013.03.045
28	Chen Y, Hu Z, Li R, et al. The effects of carbenoxolone against experimental autoimmune encephalomyelitis in a mouse model[J]. Neuroimmunomodulation, 2020, 27(1): 19-27. doi：10.1159/000505333
29	Kou RW, Li ZQ, Wang JL, et al. Ganoderic acid a mitigates inflammatory bowel disease through modulation of AhR activity by microbial tryptophan metabolism[J]. J Agric Food Chem, 2024, 72(32): 17912-23. doi：10.1021/acs.jafc.4c01166
30	Stronati L, Palone F, Negroni A, et al. Dipotassium glycyrrhizate improves intestinal mucosal healing by modulating extracellular matrix remodeling genes and restoring epithelial barrier functions[J]. Front Immunol, 2019, 10: 939. doi：10.3389/fimmu.2019.00939
31	Hong GU, Lee JY, Kang H, et al. Inhibition of osteoarthritis-related molecules by isomucronulatol 7-O-β-d-glucoside and ecliptasaponin a in IL-1β-stimulated chondrosarcoma cell model[J]. Molecules, 2018, 23(11): E2807. doi：10.3390/molecules23112807
32	De Schepper S, Verheijden S, Aguilera-Lizarraga J, et al. Self-maintaining gut macrophages are essential for intestinal homeostasis[J]. Cell, 2019, 176(3): 676. doi：10.1016/j.cell.2019.01.010
33	Tao Q, Liang Q, Fu Y, et al. Puerarin ameliorates colitis by direct suppression of macrophage M1 polarization in DSS mice[J]. Phytomedicine, 2024, 135: 156048. doi：10.1016/j.phymed.2024.156048
34	Westendorp BF, Büller NVJA, Karpus ON, et al. Indian hedgehog suppresses a stromal cell-driven intestinal immune response[J]. Cell Mol Gastroenterol Hepatol, 2018, 5(1): 67-82.e1. doi：10.1016/j.jcmgh.2017.08.004
35	Huang B, Chen Z, Geng L, et al. Mucosal profiling of pediatric-onset colitis and IBD reveals common pathogenics and therapeutic pathways[J]. Cell, 2019, 179(5): 1160-76.e24. doi：10.1016/j.cell.2019.10.027
36	Xue C, Yao Q, Gu X, et al. Evolving cognition of the JAK-STAT signaling pathway: autoimmune disorders and cancer[J]. Signal Transduct Target Ther, 2023, 8(1): 204. doi：10.1038/s41392-023-01468-7
37	Huang B, Lang X, Li X. The role of IL-6/JAK2/STAT3 signaling pathway in cancers[J]. Front Oncol, 2022, 12: 1023177. doi：10.3389/fonc.2022.1023177
38	Lu MN, Xie KG, Lu XZ, et al. Notoginsenoside R1 counteracts mesenchymal stem cell-evoked oncogenesis and doxorubicin resistance in osteosarcoma cells by blocking IL-6 secretion-induced JAK2/STAT3 signaling[J]. Investig New Drugs, 2021, 39(2): 416-25. doi：10.1007/s10637-020-01027-9
39	Salas A, Hernandez-Rocha C, Duijvestein M, et al. JAK-STAT pathway targeting for the treatment of inflammatory bowel disease[J]. Nat Rev Gastroenterol Hepatol, 2020, 17(6): 323-37. doi：10.1038/s41575-020-0273-0
40	Huang XL, Lin R, Liu H, et al. Resatorvid (TAK-242) ameliorates ulcerative colitis by modulating macrophage polarization and T helper cell balance via TLR4/JAK2/STAT3 signaling pathway[J]. Inflammation, 2024, 47(6): 2108-28. doi：10.1007/s10753-024-02028-z
41	Wang Y, Song X, Xia Y, et al. Complanatuside A ameliorates 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis in mice by regulating the Th17/Treg balance via the JAK2/STAT3 signaling pathway[J]. FASEB J, 2024, 38(10): e23667. doi：10.1096/fj.202301127rr
42	罗雪菲, 王伟, 赵晓芳, 等. 基于网络药理学探讨桃莲绞复方增强全成分肿瘤细胞疫苗抗结直肠癌作用分子机制[J]. 中草药, 2021, 52(2): 459-68. doi：10.7501/j.issn.0253-2670.2021.02.020
43	Xiao QP, Luo L, Zhu XY, et al. Formononetin alleviates ulcerative colitis via reshaping the balance of M1/M2 macrophage polarization in a gut microbiota-dependent manner[J]. Phytomedicine, 2024, 135: 156153. doi：10.1016/j.phymed.2024.156153
44	Xiao QP, Huang JQ, Zhu XY, et al. Formononetin ameliorates dextran sulfate sodium-induced colitis via enhancing antioxidant capacity, promoting tight junction protein expression and reshaping M1/M2 macrophage polarization balance[J]. Int Immuno-pharmacol, 2024, 142: 113174. doi：10.1016/j.intimp.2024.113174

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