关键词: 肝损伤, 转录组测序, Ca²⁺信号通路, 线粒体钙稳态

Abstract: [Objective] The aim of this study was to screen the genes and key signaling pathways associated with the development of carbon tetrachloride （CCl₄）-induced liver injury, analysing the dynamic changes in the expression of genes related to calcium ion （Ca²⁺） transport during liver injury. [Method] Thirty 8-week-old male C57BL/6J mice were randomly divided into control and model groups, with 15 mice in each group. The control group was given a single intraperitoneal injection of saline at a dose of 0.1 mL/（10 g·BW）, and the model group was given a single intraperitoneal injection of a mixture of CCl₄ as well as olive oil （CCl₄∶olive oil = 1∶4, V/V） at a dose of 0.1 mL/（10 g·BW） to establish a model of CCl₄-induced acute liver injury. The viability of ALT and AST in the serum of mice in the model group was detected by ELISA on days 2, 5 and 7 after modelling. Subsequently, the histopathological changes of the liver were observed using HE staining and Masson staining. Three biological replicates of liver tissue samples were selected from each of the control group （Group C）, the group on the 2^nd day after modelling （Group D2）, and the group on the 5^nd day after modelling （Group D5） to construct cDNA libraries for transcriptome sequencing, and the obtained data were assembled and functionally annotated. Afterwards, DESeqR package （1.10.0） was used to screen the significantly differentially expressed genes, and KOBAS software was employed to perform GO functional annotation and KEGG pathway enrichment analysis of the significantly differentially expressed genes involved in the Ca²⁺ signalling pathway. [Result] Compared with the control group, ALT and AST activity in serum of mice in the model group on the 2^nd day after liver injury were highly significant （P<0.01） increased. On the 2^nd day after liver injury, the structure of hepatic lobules in the model group was destroyed. Large necrosis and collagen deposition were formed around the hepatic portal vein, the arrangement of hepatocytes was disordered, and inflammatory cell infiltration could be seen. Extensive necrosis with large collagen deposits formed around the hepatic portal vein. Hepatocyte arrangement was disorganized and inflammatory cell infiltration could be observed. Transcriptome sequencing results showed that the total number of differentially expressed genes in group D2 compared with group C was 6 954, and the number of up-regulated and down-regulated genes was 3 577 and 3 377, respectively. The total number of differentially expressed genes in group D5 compared with group C was 6 028, and the number of up- and down-regulated genes was 3 202 and 2 826, respectively. The total number of differentially expressed genes in the group D5 compared to the group D2 was 6 657, with 3 200 upregulated and 3 457 downregulated genes, respectively. In addition, there were a total of 1 462 co-varying differentially expressed genes among the three groups. GO function enrichment analysis showed that the GO entries enriched for differentially expressed genes between group D2 and group C were mainly involved in metabolism, signal transduction, Ca²⁺ transport, etc. The GO entries enriched for differentially expressed genes between group D5 and group C were mainly related to immune response and intracellular signal transduction. The GO entries of differentially expressed genes enriched between group D5 and group D2 mainly involved metabolic processes and signal transduction regulation. KEGG pathway enrichment analysis showed that the signaling pathways mainly involved in the differentially expressed genes between group D2 and group C included peroxisome, fatty acid degradation, etc. The differentially expressed genes between group D5 and group C mainly involved signaling pathways such as chemokine signaling and cholesterol metabolism. The signaling pathways mainly involved in the differentially expressed genes between group D5 and group D2 include fatty acid degradation, peroxisome, etc. Compared with group C, the expression of Mcub, which is a mitochondrial calcium unidirectional transport protein gene, and Itpr3, which is an endoplasmic reticulum inositol triphosphate receptor 3 gene, increased dramatically in group D2. Expression of Mcub and the voltage-gated calcium channel protein gene Cacna1i increased dramatically in the group D5. The expression of Cacna1i was dramatically increased in the group D5 compared with the group D2. [Conclusion] Gene expression profiles of liver tissues differed before and after the onset of CCl₄-induced liver injury and at different times after the onset of liver injury. The differentially expressed genes involved multiple signaling pathways related to hepatocyte function. In the early stage of liver injury, mitochondria rapidly intervene in the regulation of calcium homeostasis. Meanwhile, mitochondria and Ca²⁺ channels on the cell membrane play a key role in the subsequent regulation of cellular functions.

Key words: liver injury, transcriptome sequencing, calcium ion signaling pathway, mitochondrial calcium homeostasis