| The remnant liver has remarkable ability to regenerate in response to surgical removal, injury and hepatic diseases. To study the molecular mechanism will help us to diagnose and treat many kinds of hepatic diseases, to improve liver transplantation. However, the molecular mechanism of liver regeneration, especially the mechanism of priming and termination after partial hepatectomy hasn't been covered yet. Energy metabolism plays the important role during rat liver regeneration. Mitochondria not only provide the necessary energy for life activities, but also take part in many courses of signal transduction. Recently, it has been suggested that mitochondrial permeability transition (MPT) played the important role of activities such as proliferation, tumor, apoptosis. injury and aging, etc.Recently, it has been found that the MPT has some remarkable changes during the period of liver regeneration, especially in the early phase of liver regeneration (0-24 h after PH). In 1998 Greco M found that some mitochondrial matrix enzymes were released into cytosol, and Guerrieri F found that mitochondrial ultrastructure had significant changes in the early phase after partial hepatectomy in 2002. Our findings in 2004 suggested that mitochondrial permeability had increased both in the early and later phases during liver regeneration process. However, the relationship between these changes and liver regeneration is still not clear. For the reasons described above, by typical liver regeneration model and the influence of cyclosporin-A ( CsA, the specific inhibitor of the mitochondrial transition pore ) during the whole liver regeneration, to study the relationship between MTP and liver regeneration will help us to clarify the molecular mechanism of liver cell regeneration.1. The changes of liver proliferation and mitochondrial ultrastructure during liver regenerationTo understand liver proliferation and the relationship between liver regeneration and MPT, we observed liver regenerative index (LRI) and mitochondrialultrastructure in vivo during the whole period. The findings showed that the liver volume and weight increased significantly at 24 h after PH, and achieved the peak at 168 h after PH, and LRI in CsA group decreased significantly at 72 h compared with PH group.Mitochondrial ultrastructure changed remarkably at 24 h after PH, showed the conspicuous swelling, an ultrasturctural index of increased membrane permeability, a reduction in the number of cristae, and a rarefied, even vaculoed, matrix. And at 72 h showed the midrange swelling, at other times, showed little changes. Moreover, the permeability of mitochondrial increased at 0 h, 3 h. 6 h in CsA group, and decreased at 24 h and 72 h compared with PH group, which was possibly induced the toxic effect to live cells in the early phase and that mitochondrial permeability was prevented partly by CsA in the late phase.2. The changes of MDA (malondialdehyde )during liver regeneration Reactive oxygen species and antioxidant have the important roles in the course of cell regeneration and differentiation, we determined the changes of MDA in liver tissue during liver regeneration. The results showed that the content of MDA increased at 3 h after PH. and achieved the peak at 24 h , then decreased, and returned normal level at 120 h. In addition, the content of MDA in CsA group increased significantly during the period (0-120 h) significantly compared with PH group.3. The changes of enzyme activities of mAST and GDH in mitochondria and cytosol during liver regeneration.Are there any proteins released from mitochondria when mitochondrial ultrastructure changed during liver regeneration? We observed the changes of enzyme activities of mitochondrial matrix : AST (aspartate aminotransferase) and GDH (glutamic acid dehydrogenase). The results showed that the enzyme activities of AST and GDH in cytosol increased at 3 h after PH, and achieved the peak at 24 h, then decreased gradually, and returned normal level at 120 h. The enzyme activities in mitochondria decreased simultaneously. In CsA group, compared with PH group, the enzymes activities of mAST and GDH increased significantly at 0 h and 3 h, decreased significantly at 24 h, 48 h and 72 h. These changes were coincident withmitochondrial ultrastructure during liver regeneration, which suggested that MPT was related to the releasing of mitochondrial proteins during liver regeneration.4. The changes of activities of caspase-3 and caspase-8 in liver during liver regeneration.To understand the relationship between cell apoptosis and MPT during liver regeneration, we observed the changes of activities of caspase-3 and caspase-8 in liver during this process. The results showed that the activity of caspase-3 increased briefly at 3 h and 6 h after PH, then increased significantly at 72 h. and achieved the peak at 168 h. In CsA group the activity of caspase-3 achieved the peak at 3 h, increased significantly compared with PH and SH group, decreased significantly in period (72-168 h) compared with PH group, but still higher than SH group. The activity of caspase-8 increased significantly at 0 h in CsA group compared with PH and SH group, and increased simultaneously at 3 h in three groups, but the values of PH group is much higher than SH group. At 168 h in PH group, the activity of caspase-8 increased significantly compared with CsA and SH group. There are not any remarkable changes among 3 groups during liver regeneration exepct at 0 h. 3 h and 168 h. The results suggested that the changes of the activities of caspase-3 in the early period was possibly induced by the stress after surgery and extrinsic apoptosic pathway, and the rise in later period was mainly induced mitochondrial-mediated pathway.5. Proteins profile released from mitochondria during liver regeneration Recent studies demonstrated that mitochondrial ultrastructure and proteins released from mitochondrial matrix changed significantly at 24 h after PH. But proteins profile released from the mitochondrial matrix is still not clear. After incubated mitochondria in vitro, we observed the difference of proteins released at 24 h after PH by means of 2-dimensional gel electrophoresis and mass spectrometry. We found that there were about 60 proteins, and there were 3 proteins expressed significantly compared with SH group: calreticulin (AA 1-399), Tpr_predicted protein and Arginase I. Among them, the function of Tprjpredicted protein is still not clear, the other two proteins are related to the synthesis of DNA and protein.Conclusion:1. The changes of mitochondrial ultrastructure during liver regeneraion has something to do with the occurrence and development of liver regeneration, especially, at 24 h after PH, the changes of mitochondrial ultrastructure and liver regenerative index is possibly related to the initiation of liver regeneration after PH.2. The changes of free redials in liver and activities of mitochondria martix enzyme is accordance with the changes of mitochondrial ultrastructure during liver regeneration, which suggests that the changes of MDA and proteins released are both related to MPT.3. The significant changes of the cell apoptosis in the early period is possibly induced by the stress after surgery and extrinsic apoptosic pathway, and the rise in the late period is possibly induced by mitochondrial-mediated pathway.4. By means of CsA interefered with liver regeneration, we found that many kinds of changes above have something to do with MPT during liver regeneration.5. After incubated mitochondria in vitro at 24 h after PH. by means of 2-D gel and mass spectrometry. we found that there were 3 proteins expressed significantly compared with SH group: calreticulin (AA 1-399). Tpr_predicted protein and Arginase I.
|
PDF Full Text Request