Cell-free supernatant of Clostridium butyricum induces mitochondrial apoptosis and suppresses NF-κB pathway in colorectal cancer cells

IF 3.1 4区医学 Q3 IMMUNOLOGY

Immunobiology Pub Date : 2026-03-01 Epub Date: 2026-03-06 DOI:10.1016/j.imbio.2026.153172

Miao Liu , Shuping Liu , Wenjie Xie , Guang Li , Tao Deng

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Abstract

Background

Clostridium butyricum (CB), a butyrate-producing probiotic, exhibits antitumor potential in colorectal cancer (CRC). This study investigated the direct effects and mechanisms of CB-secreted metabolites on CRC cells.

Methods

HT-29 cells were treated with serial dilutions (5–40%) of sterile-filtered CB supernatant (CBS), using E. coli DH5α supernatant as control. Cell viability and proliferation were assessed by CCK-8 and Ki-67 immunofluorescence. Apoptosis was evaluated by Annexin V/PI flow cytometry. Mitochondrial membrane potential (ΔΨm) was monitored via JC-1 staining. Apoptosis-related and NF-κB pathway proteins were analyzed by Western blot.

Results

The CBS notably suppressed HT-29 cell viability and proliferation as concentration and treatment duration increased, with an IC₅₀ of 18.84% (95% CI: 16.2% to 21.5%) at 48 h. CBS exposure markedly increased apoptotic cell proportion. Mechanistically, CBS induced ΔΨm loss, cytosolic cytochrome c translocation, and activation of caspase-9 and -3. Concurrently, CBS inhibited NF-κB pathway activation, downregulated Bcl-2, and upregulated Bax.

Conclusion

CB culture supernatant inhibits growth and induces apoptosis in HT-29 cells through mitochondrial dysfunction and NF-κB suppression.

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无细胞丁酸梭菌上清诱导结直肠癌细胞线粒体凋亡并抑制NF-κB通路

背景丁酸梭菌（clostridium butyricum， CB）是一种产生丁酸的益生菌，在结直肠癌（CRC）中显示出抗肿瘤的潜力。本研究探讨了cb分泌代谢物对结直肠癌细胞的直接作用及其机制。方法以大肠杆菌DH5α上清液为对照，用连续稀释（5-40%）的CB上清液（CBS）处理sht -29细胞。采用CCK-8和Ki-67免疫荧光法检测细胞活力和增殖能力。Annexin V/PI流式细胞术检测细胞凋亡。JC-1染色检测线粒体膜电位（ΔΨm）。Western blot检测凋亡相关蛋白和NF-κB通路蛋白。结果随着浓度和处理时间的增加，CBS明显抑制HT-29细胞的活力和增殖，在48 h时IC₅0为18.84% （95% CI: 16.2%至21.5%）。CBS暴露显着增加了凋亡细胞的比例。在机制上，CBS诱导ΔΨm丢失，胞浆细胞色素c易位，以及caspase-9和-3的激活。同时，CBS抑制NF-κB通路激活，下调Bcl-2，上调Bax。结论cb培养上清通过线粒体功能障碍和抑制NF-κB抑制HT-29细胞生长，诱导细胞凋亡。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Immunobiology 医学-免疫学

CiteScore

5.00

自引率

3.60%

发文量

108

审稿时长

55 days

期刊介绍： Immunobiology is a peer-reviewed journal that publishes highly innovative research approaches for a wide range of immunological subjects, including • Innate Immunity, • Adaptive Immunity, • Complement Biology, • Macrophage and Dendritic Cell Biology, • Parasite Immunology, • Tumour Immunology, • Clinical Immunology, • Immunogenetics, • Immunotherapy and • Immunopathology of infectious, allergic and autoimmune disease.