Abstract: Objective The aim of this experiment was to investigate the effects of hepatocyte-specific cell cycle inhibitor protein P21 (P21) overexpression on transdifferentiation of biliary epithelial cells (BEC) into mature hepatocytes in mice with choline-deficient and ethionine -supplemented (CDE) -induced chronic liver injury. Methods We established a mice with rosa-stop^flox/flox-EGFP by biliary epithelial cells lineage tracing via Krt19-Cre^ERT, and the mice were injected with the adeno-associated virus serotype 8 encoding Cdkn1a under the control of the CAG promoter(AAV-CAG-P21), a virus which infected only hepatocytes. The overexpression of P21 and the bile duct epithelial cell tracing model were determined by Western blot and immunohistochemistry. Then, the male Krt19-Cre^ERT and Rosa-stop^flox/flox-EGFP mice injected with AAV-CAG-P21(experiment group) and AAV-CAG-MCS(control group) were fed with CDE diet for 2 weeks. The liver tissues were collected for liver damage and cell proliferation analysis. The mice received 2 weeks normal diets after CDE diet-induced liver injury to observe biliary epithelial cells transdifferentiation into hepatocytes, and the liver tissues were collected from all mice and analyzed by immunohistochemistry and immunofluorescence. Results The mice with P21 overexpression in hepatocytes from CDE diet-fed mice developed severe liver injury with pale liver surface, decreased significantly the ratio of liver weight/body weight from (4.5±0.1)% to (5.2±0.2)% (P＜0.05) and increased serum AST levels from (175.9±11.4) U/L to (385.4±12.7) U/L (P＜0.05), ALT levels from (214.8±23.5) U/L to (423.8±32.4) U/L (P＜0.05) and bilibubin levels from (10.5±0.9) μmol/L to (21.3±1.4) μmol/L (P＜0.05); the immunohistochemistry results showed an impaired hepatocyte proliferation in mice with P21 overexpression; the hepatocytes were arrested at G1/S transition-phase with decreased Ki67⁺ hepatocytes from (8.2±1.5)% to (0.1±0.1)% , with lower S phase marker CyclinA2 expression from (3.2±0.7)% to (0.1±0.2) and similar CyclinD1 (G1 phase marker) expression level [(43.2±3.5) % to (42.0±2.8)%]; importantly, we observed the differentiation of biliary epithelial cells into hepatocytes as clones in liver parenchyma with 2 weeks' diet recovery; the lineage tracing and immunofluorescence analysis showed the new hepatocytes were derived from cholangiocytes with GFP, and they had all features of mature functional hepatocytes expressing albumin, CYP3A4, CYP2E1, and CYP2D6, and so on. Conclusion During the chronic procedure of liver injury, the overexpression of cell cycle inhibitor P21 in hepatocytes aggravates the liver injury and impaires hepatocyte proliferation, which triggers a stimulus for BECs to differentiate into mature functional hepatocytes.

Key words: Hepatocyte, Biliary epithelial cells, Cell cycle inhibitor protein P21, Transdifferentiation, Mice