关键词: 日本血吸虫, 肝纤维化, 线粒体, 柠檬酸循环, Beclin1

Abstract: Objective To explore the relationship between Beclin1 and mitochondrial function in hepatic fibrosis in schistosomiasis. Methods Forty female C57BL/6 mice were randomly divided into infected group and healthy control group (20 mice in each group). Mice in the infected group were infected with 20 Schistosoma japonicum cercariae by the abdominal patch method. Ten weeks after infection, fresh liver tissues were collected from the two groups. Liver pathology was assessed by HE and Masson staining of liver tissue sections. The relative protein levels of Beclin1, p-Beclin1 and voltage-dependent anion channel protein 1 (VDAC1) in liver were assessed by Western blotting. Fluorescence quantitative PCR was performed to analyze the relative mRNA expression of collagen Col 1a1 and Col 3a1, as well as enzymes involved in the mitochondrial citric acid cycle reaction, including citrate synthase, isocitrate dehydrogenase, alpha ketoglutarate dehydrogenase and malate dehydrogenase. Another part of liver tissues was sectioned for visualizing mitochondrion morphology by electron microscopy. To examine the mitochondria oxygen consumption rate (OCR) using cell energy metabolizer, the mitochondria extracted from liver tissues were added to the culture plates, to which complex V substrate ADP, complex V inhibitor oligomycin, uncoupler FCCP, and antimycin A (an inhibitor for complex Ⅱ plus Ⅲ) were sequentially added, allowing culture for 5 min, then the OCR was detected. Results HE and Masson staining showed that granulomas were formed in mouse liver in the infected group, and obvious fibrosis occurred around the granulomas. Western blotting showed that the protein level of VDAC1 remained unchanged in the infected liver, while Beclin1 (0.65 ± 0.05) and p-Beclin1 (0.78 ± 0.03) were decreased, compared with the control (P < 0.05). The fluorescence quantitative PCR results indicated that the relative transcription levels of Col 1a1 and Col 3a1 mRNA in livers of the infected group were 4.05 ± 0.23 and 2.71 ± 0.14, respectively, which were higher than those of the healthy control group (P < 0.01). The relative transcription levels of citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase mRNA were 0.61 ± 0.03, 0.65 ± 0.01, 0.41 ± 0.03 and 0.55 ± 0.01, respectively, which were lower than those of the healthy control group (P < 0.01). Electron microscopy revealed that in the infected group, the mitochondrial structure was damaged and the ridge was broken. Mitochondrial function test showed that after addition of complex Ⅴ substrate ADP, the OCR of the healthy control group [(335 ± 29) pmol/min] was higher than that of the infected group [(78 ± 23) pmol/min] (P < 0.01); addition of complex Ⅴ inhibitor oligomycin reduced the difference [(80 ± 2) pmol/min in the control group and (31 ± 6) pmol/min in the infected group], although significance of difference still existed (P < 0.05); after addition of uncoupler FCCP, the OCRs of the healthy control and the infected groups increased to (159 ± 4) pmol/min and (42 ± 5) pmol/min(P < 0.01), respectively. After addition of complex Ⅱ plus Ⅲ inhibitor, the OCRs of both groups decreased to zero. Conclusion In the development of liver fibrosis in schistosomiasis, Beclin1 is reduced, the citric acid cycle is restrained, and the mitochondrial OCR further declines. The complex Ⅴ plays an important role in the regulation of mitochondrial activity during liver fibrosis in schistosomiasis.

Key words: Schistosoma japonicum, Liver fibrosis, Mitochondria, Citric acid cycle, Beclin1