Ahmadi Badi Sara, Tavakoli Aval Hananeh, Moradi Hamid Reza, Malek Amin, Seyedi Seyed Amirhesam, Davari Mehdi, Bereimipour Ahmad, Khani Soghra, Khatami Shohreh, Siadat Seyed Davar
Microbiology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Department of Biochemistry, Pasteur Institute of Iran, Tehran, Iran.
Gut Pathog. 2025 Jul 17;17(1):54. doi: 10.1186/s13099-025-00728-x.
liver fibrosis is associated with dysregulated iron homeostasis regulated by the hepcidin-ferroportin axis, and dysbiotic gut microbiota. This study aimed to investigate the preventive and ameliorative effects of live and cell-free supernatant (CFS) forms of Akkermansia muciniphila and Faecalibacterium prausnitzii, as important gut microbiota members, on liver fibrosis by targeting the hepcidin-ferroportin axis in both in vitro and in vivo models.
At the in vitro level, the effects of A. muciniphila and F. prausnitzii on the expression of collagen type I alpha 1 (COL1A1) and ferroportin (SLC40A1) transcripts in hepatic stellate cells (HSCs) were evaluated in transforming growth factor beta (TGFβ)-activated LX-2 cells, a human hepatic stellate cell line. In vivo, male C57BL/6 mice were intraperitoneally (IP) injected with 10% carbon tetrachloride (CCl₄) twice weekly for 6 weeks to establish the liver fibrosis model. Administration of live and CFS forms of A. muciniphila and F. prausnitzii was initiated 10 days before CCl₄ injection and continued until the end of the experiment. Liver injury and fibrosis were assessed using serum markers, hematoxylin and eosin (H&E), and Masson's trichrome staining. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) were used to evaluate the effects of the interventions on gene expression related to the hepcidin-ferroportin axis in liver, colon and brain samples. Additionally, qPCR was used to determine alterations in the relative abundance of key gut microbiota members in fecal samples.
Both A. muciniphila and F. prausnitzii, as well as their CFS, significantly downregulated COL1A1 expression in TGFβ-activated LX-2 cells, accompanied by reduced alpha-smooth muscle actin (α-SMA) protein expression in liver tissue. In vivo, intervention with F. prausnitzii, particularly its CFS, led to a greater induction of hepatic hepcidin and ferroportin expression compared to A. muciniphila and its CFS. Serum liver injury markers (alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH)) and iron levels were markedly improved following treatment with live F. prausnitzii and its CFS. Additionally, F. prausnitzii CFS significantly enhanced hepcidin gene expression in brain tissue, suggesting broader systemic benefits.
We demonstrated that F. prausnitzii and its CFS had greater beneficial potential than A. muciniphila and its CFS in the prevention and amelioration of liver fibrosis, likely through modulation of the hepcidin-ferroportin axis. These findings may support the development of next-generation probiotics and postbiotics for liver injury, which warrants further investigation.
肝纤维化与由铁调素-铁转运蛋白轴调节的铁稳态失调以及肠道微生物群失调有关。本研究旨在通过在体外和体内模型中靶向铁调素-铁转运蛋白轴，研究阿克曼氏黏液菌和普拉梭菌这两种重要的肠道微生物群成员的活菌形式和无细胞上清液（CFS）对肝纤维化的预防和改善作用。
在体外水平，在转化生长因子β（TGFβ）激活的LX-2细胞（一种人肝星状细胞系）中评估阿克曼氏黏液菌和普拉梭菌对肝星状细胞（HSCs）中I型胶原蛋白α1（COL1A1）和铁转运蛋白（SLC40A1）转录本表达的影响。在体内，雄性C57BL/6小鼠每周两次腹腔注射10%四氯化碳（CCl₄），持续6周以建立肝纤维化模型。在CCl₄注射前10天开始给予阿克曼氏黏液菌和普拉梭菌的活菌形式和CFS，并持续至实验结束。使用血清标志物、苏木精和伊红（H&E）以及Masson三色染色评估肝损伤和纤维化。逆转录定量聚合酶链反应（RT-qPCR）和免疫组织化学（IHC）用于评估干预措施对肝脏、结肠和脑样本中与铁调素-铁转运蛋白轴相关基因表达的影响。此外，qPCR用于确定粪便样本中关键肠道微生物群成员相对丰度的变化。
阿克曼氏黏液菌和普拉梭菌及其CFS均显著下调TGFβ激活的LX-2细胞中COL1A1的表达，同时肝脏组织中α平滑肌肌动蛋白（α-SMA）蛋白表达降低。在体内，与阿克曼氏黏液菌及其CFS相比，普拉梭菌尤其是其CFS的干预导致肝脏铁调素和铁转运蛋白表达的诱导作用更强。用普拉梭菌活菌及其CFS治疗后，血清肝损伤标志物（丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）、乳酸脱氢酶（LDH））和铁水平显著改善。此外，普拉梭菌CFS显著增强脑组织中铁调素基因的表达，表明具有更广泛的全身益处。
我们证明，普拉梭菌及其CFS在预防和改善肝纤维化方面比阿克曼氏黏液菌及其CFS具有更大的有益潜力，可能是通过调节铁调素-铁转运蛋白轴实现的。这些发现可能支持开发用于肝损伤的下一代益生菌和后生元，这值得进一步研究。
