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Based on the cellular proliferation tracing technique (ProTracer), this study explored the role of rifampicin (RIF) in promoting liver regeneration in a mouse model with zone-specific liver injury. The ProTracer system enables in vivo recording of cell proliferation by labeling proliferating cells with GFP fluorescence. Carbon tetrachloride (CCl4)-induced injury and bile duct ligation (BDL) models were established, followed by RIF or vehicle treatment. Liver function was assessed by measuring serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bile acids (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL). Liver injury area was evaluated by H&E staining, while whole-tissue fluorescence imaging was used to assess liver regeneration. Immunofluorescence co-staining was performed to quantify proliferating hepatocytes, identify cell types, and analyze zonal distribution. RNA sequencing was conducted to identify key differentially expressed genes and signaling pathways, with Western blot used for protein-level validation. In vitro validation was performed using the HepG2 hepatocyte cell line. Results showed that RIF significantly reduced serum ALT and AST levels in CCl4-induced liver injury and decreased TBA, TBIL, and DBIL levels in the BDL-induced liver injury, effectively reducing the area of liver damage. Whole-tissue fluorescence imaging revealed enhanced GFP signals and increased proliferating cells in the RIF-treated group. Immunofluorescence analysis indicated that RIF primarily promoted hepatocyte proliferation, with a marked increase in proliferating cells in zone 2 of the liver lobule (approximately 2.5-fold), and also significant increased in zones 1 and 3. RNA-Seq and Western blot analyses demonstrated upregulation of cell cycle and proliferation-related genes such as the Mcm family, Ccnd1, Ccna2, and Mki67, with enrichment in the cell cycle pathway. In vitro experiments showed that RIF treatment enhanced HepG2 cell viability and increased the proportion of Ki67-positive cells. In conclusion, RIF promotes hepatocyte proliferation and enhances liver regeneration following zone-specific liver injury by activating cell cycle signaling pathways. Animal experiments were conducted with approval from the Animal Ethics Committee of Binzhou Medical University (approval number: 2025-2018).
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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