Studies On Mitochondrial Permeability Transition Related Factors During Rat Liver Regeneration

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 discovered yet. Energy metabolism plays an important role in many cellular processes, including rat liver regeneration. Mitochondria not only provide the necessary energy for life activities, but also take part in many courses of signal transduction. It has been suggested that mitochondrial permeability transition (PT) played an important role in these processes, which is also meaningful to liver regeneration. In our previous study on the mitochondrial PT, we found that the mitochondria showed first shrinkage (3～6h after PH) and then swell during early phase (24h after PH). The late phase (120～168h after PH) also represented a process with the permeability of mitochondria first decreased and then increased. These findings imply that mitochondrial PT may be involved in the initiation and stop signal of liver regeneration. PT is based on mitochondrial permeability transition pore (PTP), a dynamic multiprotein complex between the inner and outer mitochondrial membrances. The exact molecular composition of the PTP has not been definitely established. PTP is characterized as reversibly open/closing. The abnormal opening of this pore will lead to mitochondrial membrane potential breakdown and apoptosis agents releasing. PTP is linked exterior and interior of mitochondria, of which function depends on PTP open/closing. PTP open/closing is regulated by ROS (Reactive oxygen species), Ca²⁺, peripheral-type benzodiazepine receptor (PBR) and so on. PBR is an important component of PTP, regulating on the mitochondria-induced apoptosis. Diazapam-binding inhibitor (DBI) is the endogenous ligand of PBR, which can replace the binding of Diazapam and PBR. DBI exist in many species, and the interaction of DBI and PBR can regulate mitochondrial PT and induce cell apoptosis, DBI can bind acyl-CoA and transport it to mitochondrial matrix which can regulate energy metabolism and biosynthesis of steroid in mitochondria.ROS is an important factor in regulation of PT, and about 2～5% oxygen consumption of the mitochondrial is used in ROS production.It is mainly produced by the electronic,spilled from respiratory chain complexes I and III,directly reacts with oxygen; Opening of mitochondria PTP can induce mitochondrial ROS. It was reported that radicals and mitochondrial function have an evident rise and fall relationship during the early stage of liver regeneration. This indicates that the researches on the change of ROS and mitochondrial PT during liver regeneration, will contribute to the understanding of the possible mechanism in regulation the hepatic mitochondrial PT.Ca²⁺ is another important factor that can induce PT. It is reported that the concentration of Ca²⁺ in mitochondria changes during liver regeneration. When cytoplasm Ca²⁺ run into the mitochondrial matrix, mitochondrial concentration increased, which lead to the mitochondrial membrane potential changes, and then adjust the opening of PTP. Calcium increase induced ROS production, so that increased mitochondrial membrane permeability. Meanwhile Ca²⁺ can also regulate the functions of cell and mitochondria through mitochondrial PTP. Such as acceleration in mitochondrial respiration, electronic leakage caused by increased superoxide production. So, studies on the change of Ca²⁺ in mitochondria during the liver regeneration process, will contribute to the understanding of possible adjustment mechanism of mitochondrial PT.1 Expression of PBR and DBI during liver regenerationTo investigate the expression of PBR and DBI during rat liver regeneration and to explore their relationship with mitochondrial PT. Dynamic changes of PBR and DBI mRNA expression were measured with real-time RT-PCR method and PBR protein expression were measured with immunohistochemistry method.The mRNA expression of PBR was increased at 6h and 2d in hepatectomy group and significant difference was found when compared with that of sham operated group (P <0.01),while other time groups were no significant difference when compared with that of sham operated group at the same periods; Meanwhile,the mRNA expression of DBI was significantly decreased during early phase (3h and 6h after PH ) (P < 0.01)and was significantly increased at 2d (P < 0.05) during rat liver regeneration when compared with that in sham operated and normal groups at the same periods, while other time groups were no significant difference when compared with that of sham operated group.The PBR protein positive cell number was significantly decreased during early phase (6h and 12h after PH ) (P < 0.01) and was significantly increased at 3d(P < 0.01) when compared with that of normal group, while other time groups were no significant difference when compared with that of normal group at the same periods; the PBR protein average gray value was significantly increased at 6h (P < 0.05) and was significantly decreased at 12h, 2d, and 3d (P <0.01 or P <0.05), while other time group was no significant difference when compared with that of normal group at the same periods.We conclusion that the changes in expression PBR and DBI may be positive correlation to mitochondrial PT, the changes in subcellular distribution of PBR may be association with liver cells proliferation and mitochondria induced apoptosis during rat liver regeneration.2 Changes of free radical concentration in hepatic cell during liver regenerationROS is anther factor that can induce PT, so we detect the changes of MDA and NO by method of TBA and nitric acid disoxidation respectively.Our results showed that: the production of MDA was increased in 3h, 6h and 24h (P <0.05) and 2d (P <0.01) after PH compared to that of SH group.The production of NO went through the same change as MDA in control of SH group expect that the production of NO was increased in 7d after PH.We may conclude from these datas that the increase of ROS in early phase may be association with mitochondrial PT during the same phase. The changes of NO and MDA in the intermediate phase and the changes of NO in the late phase may be association with mitochondria induced apoptosis during rat liver regeneration.3 Changes of Ca²⁺ concentration To explore the regulatory mechanism of PT, we investigated the Ca²⁺ concentration in mitochondria during liver regeneration.We found that concentration of Ca²⁺ was elevated in 12h and 7d (P <0.05) and 24h,2d (P <0.01) after PH compared to that of SH group.Our data reflected that the increase of Ca²⁺ concentration may positively correlated to PT, the increase of NO in late phase (7d after PH) may be association with the increase of Ca²⁺ concentration.Conclusion:On the basis of our results after PH, the changes in expression PBR and DBI may be positive correlation to mitochondrial PT, the changes in subcellular distribution of PBR may be association with liver cells proliferation and mitochondria induced apoptosis during rat liver regeneration.We may conclude from the increase of ROS in early phase may be association with mitochondrial PT during the same phase. The changes of NO and MDA in the intermediate phase and the changes of NO in the late phase may be association with mitochondria induced apoptosis during rat liver regeneration. The increase of Ca²⁺ concentration may positively correlated to PT, the increase of NO in late phase (7d after PH) may be association with the increase of Ca²⁺ concentration.