Carbon tetrachloride (CCl4)-induced liver fibrosis rat model and sample collection

The 50 rats were randomly divided into the normal control group (n=6) and the liver fibrosis model group (n=44). The liver fibrosis model group received intraperitoneal injections of 0.15 ml/100 g 50% CCl₄ in olive oil (5:5, v/v) twice weekly for eight weeks. Rats in the normal control group received intraperitoneal injections of the same volume of physiological saline over the same period. Reversal of liver fibrosis was investigated for 4 weeks after the final intraperitoneal injection. Rats were sacrificed at 2, 4, 6, 8, 10 and 12 weeks. Seven rats were sacrificed at each time point. Two rats succumbed without investigator intervention during week 12. All rats from the normal group were sacrificed during week 8.

All rats were euthanized under anesthesia using 1% sodium pentobarbital (50 mg/kg, i.p). Blood samples were collected for liver function tests. In addition, liver specimens were collected, divided into three parts and subjected to the following analyses: i) mRNA extraction using TRIzol for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses; ii) protein expression analysis of fibrotic indices using western blotting; and iii) histological and immunohistochemical analyses.