Studies Of Mitochondrial Permeability Transition And Its Regulation During Rat Liver Regeneration

The liver has remarkable ability to regenerate in response to surgical removal , injury and liver diseases. Indeed, the liver regenerative process may be involved in the many factors: cytokines, hormones, the transcription factor, stress and inflammatory responses, cytoskeletal and extracellular matrix modification, regulation of cellcycle entry. However the mechanism of liver regeneration, the initiation and the end of liver regeneration after partial hepatectomy has been uncovered yet. The energy metabolism is significance component element during rat liver regeneration, the oxidative phosphorylation function occure to changes has been reported in rat liver regeneration. It has been suggested that the changes of in the earlier period of mitochondria were potential modulation which triggers liver cell reenter division cycle. In the present study, we established a rat liver regeneration model through 70% partial hepatectomy (PH) to illuminate the possible molecule mechanism involved in this process. After the rats performed PH, the changes of mitochondrial permeability transition was produced during rat liver regeneration, mitochondrial oxidative phosphorylation, cell apoptosis and generation of free radicals may be related to mitochondrial permeability transition, mitochondrial permeability transition pore(PTP) involves a dynamic multiprotein complex formed in the contact site between the inner and outer mitochondrial membrances. The exact molecular composition of the PTP has not been definitively established, it has been known that several proteins such as the voltage-dependent anion channel or prorin (VDAC), the adenine nucleotide carrier, the peripheral-type benzodiazepine receptor (PBR), and apoptosis-regulatory proteins from the Bcl-2 family are its important constituents. The open and close of PTP may has accommodated by the interaction of the constituents together, determining not only whether cells live or die, but also whether death occurs by apoptosis or necrosis. 1. Studies on changes of mitochondrial permeability transition during rat liverregenerationThe liver mitochondria oxidative phosphorylation function occure to meet the significantly change during liver regeneration, the mitochondria are potential modulator which triggers liver cell reenter division cycle.We have found that themitochondrial respiratory control rate(RCR) significantly increased during rat liver regeneration after 70%partial hepatectomy as compared with sham - operated groups , and two peaks in RCR occured at half day and the fourth day after partial hepatectomy , and then reduced to the level of sham - operated groups at the seventh day. The first peak in RCR was due to the increase of respiratory state III(R3) ,the second due to the decrease of repiratory state IV (R4), the repiratory state IV (R4) mainly reflects permeability of the inner membrane. The change of oxidative phosphorylation function reflects the alteration of the mitochondrial membrane structure. Greco et al reported some protein release to cytoplasm from mitochondrial within one day after partial hepatectomy. These findings hint that change in mitochondrial oxidative phosphorylation may be related to mitochondrial permeability transition during rat liver regeneration. Nevertheless the time-phase change of mitochondria permeability transition occurred during rat liver regeneration has not been reported yet. To investigate changes in mitochondrial permeability transition and the mechanism of oxidative phosphorylation during rat liver regeneration, liver regeneration model was produced by 70% partial hepatectomy performed in male SD rats. The rats were killed by decapitation. The livers were removed, weighted and processed for isolation of mitochondria. Determinations of the mitochondrial permeability transition, without or in the presence of different concentrations of Ca2+ induction, were based on the changes of the absorbance of the mitochondrial suspension at 540nm. Results: The mitochondria showed first shrinkage and then swell during early phase (0~24h after PH) and late phase (120~168h after PH) of rat liver regeneration after partial hepatectomy, respectively, as compared with sham-operated groups, i.e. the permeability of mitochondria represented first decreased and then increased. Meanwhile, the Mitochondria isolated from rat liver at 3h, 6h after PH were more resistant to Ca2+-induced permeabilization than other liver regeneration groups and sham-operated groups, While the Mitochondria isolated from rat liver at 24h, 168h after PH were more sensitivity to Ca2+-induced permeabilization than other groups. Cyclosporin-A (CsA) proved to be able to prevent the changes in mitochondrial membrane permeability properties. The remarkable changes of mitochondria permeability transition occurred during rat liver regeneration may be the basis of the alterations of oxidative phosphorylation , and also may be related to the initiation and the end of liver regeneration after partialhepatectomy.2. Studies on oxidant and antioxidant activity during rat liver regenerationIt has been suggested that free radical damage occurs in the early phase of liver regeneration. Hepatocyte mitochondrial oxidative phosphorylation significant decreased in 24 hour after partial hepatectomy, and gradually recovered, and a peak occured at fourth day after partial hepatectomy. The oxidative phosphorylation decrease often was followed abnormal production of reactive oxygen species, which exists evident growth and decline relation. To interpret the changes of mitochondria permeability transition occurred during rat liver regeneration, we studied MDA production, SOD activity, GR activity and GSH levels during rat liver regeneration compared to that of the sham groups. MDA production increase much more during early phase after partial hepatectomy. SOD activity ,GR activity and GSH levels were all significantly lowered, then returned normal when compared to the corresponding controls. Oxidant and antioxidant activity alterations might be related to the mitochondrial permeability transition.3. mRNA Expression of gene involved in PT regulation during rat live regenerationPBR is important regulator site of mitochondrial permeability transition. Hydrophobic profile analysis of the amino acid sequences suggested a putative five transmembrahe structure that has been experimentally confirmed. Subcellular fractionation studies demonstrated that PBR is primarily localized on the outer mitochondrial membrane and concentrated at the outer/inner mitochondrial membrane contact sites. These suggested PBR posses structure that may receive and transmit signal. PBR can interact with other component of the MPTP to increase mitochondrial membrane fluidity. The studies suggested that PBR ligands can activate the PTP and trigger cell death . The Bcl-2 family of proteins, whose members may be anti-apoptotic or pro-apoptotic, Bcl-2 and Bcl-xl, which can inhibit apoptosis. In contrast, many proteins such as Bax> Balu BcI-xSn BadN Bid^ Bik, which they enhance the release of cytochrome c and trigger cell death. Bcl-2 is localized on the outer mitochondrial membrane. Bcl-2/bax increasing promotes cell survival, conversely induces PTP open. To illuminate the regulative mechanism of mitochondrial permeability transition in the liver regeneration, we investigate mRNA expression of PT correlated gene during resection induced live regeneration. Liver regeneration model was produced by 70% partial hepatectomy (PH) performed in male SD rats.