Carbon tetrachloride (CCl₄)-induced liver fibrosis model and intervention

To establish the liver fibrosis model, mice were intraperitoneally (IP) injected a 10% Carbon tetrachloride (CCl₄) solution in olive oil, twice per week for 6 weeks. Prior to liver fibrosis induction, mice received oral pretreatment with either live and CFS forms of A. muciniphila and F. prausnitzii for 10 consecutive days. This intervention was continued throughout the duration of the experiment following the initiation of CCl₄ injections. To examine the effect of studied bacteria and their CFS on liver fibrosis, a total of eight experimental groups were established, each comprising five mice (n = 5), as follows: CNT group: healthy mice receiving no CCl₄ and no intervention, (2) Olive group: mice receiving intraperitoneal injections of olive oil alone (vehicle control), without bacterial intervention, CCl₄ group: mice receiving intraperitoneal injections of 10% CCl₄ solution without any oral intervention, PBS group: mice receiving CCl₄ injections and oral gavage of phosphate-buffered saline (PBS) as an intervention control, (5) Am group: mice receiving CCl₄ injections and oral gavage with 1 × 10⁹ CFU/200 µL live A. muciniphila, (6) Am-CFS group: mice receiving CCl₄ injections and oral gavage with 200 µL of A. muciniphila derived CSF, Fp group: mice receiving CCl₄ injections and oral gavage with 1 × 10⁹ CFU/200 µL live F. prausnitzii, (8) Fp-CFS mice receiving CCl₄ injections and oral gavage with 200 µL of F. prausnitzii-derived CFS.

At the end of the experiment, all liver fibrotic model mice were sacrificed by cervical dislocation two days after the last CCl₄ injection. Blood was collected for the assessment of serum biochemical markers. Liver, colon, and brain tissues, as well as fecal samples, were isolated from each mouse, snap-frozen in liquid nitrogen, and stored at − 80 °C for further analyses. Liver tissues were also used for histopathological evaluation [9].