Studies On Mechanism Of Mitochondrial Stress And P53Translocation Mediated Hepatic Cells Apoptosis

【Background】Many studies indicate that oxidative stress associated with multiple hepatopathies, suchas, alcoholic hepatitis, viral hepatitis, drug-induced liver injury, and liver cancer. Hepaticcells apoptosis is the base of liver injury, hence, to study it is required for treatment andprevention of hepatic damage and cancer. As a central stress sensor, p53is responding tomultiple insults, including oxidative stress to induce cell death. Upon to oxidative stress,p53expression increases and causes apoptosis. However, it is still unclear that in whichway p53participates in cell apoptosis. Recent study reported that p53might play animportant role in mitochondrion, but what relation to apoptosis was unclear. As theworkshop to product energy, liver mitochondria are not only generating abundant ROS, butalso are attacking the main target by ROS. Therefore, elucidating the influence of p53andmitochondria stress to apoptosis during oxidative stress mediated hepatic damage hasparamount importance.Glucose oxidase (glucose oxidase), is able to specifically catalytic β-D-glucose reactingwith oxygen, which generates glucose acid and hydrogen peroxide. Since GOX sub-strates(glucose and oxygen) are abundant in organisms, the GOX-system can be considered an ideal model for physiological H2O2release. Here, in order to observe how p53andmitochondria stress influenced hepatic apoptosis, glucose oxidase was used to induceoxidative stress in HepG2cells and mice liver.【Aims】In vivo and in vitro researches, GOX was used to sustain H2O2release, which caninduce the hepatic cells oxidative stress. We detected the ROS abundant in cell andmitochondria level; the change of mitochondrial permeability and mitochondrialmembrane potential; and the p53mitochondrialtranslocation. At last, we explored theinfluence of p53and mitochondria stress to apoptosis. The details are as following.(1) To investigate if there are mitochondria oxidative stress and p53mitochondrialtranslocation, and then explore the relationship between them.(2) To study the change of mitochondrial permeability and mitochondrial membranepotential after the increasing of mitochondrial ROS.(3) To explore the way p53participating in hepatic cells apoptosis under the conditionof mitochondrial stress.【Methods】HepG2cell lines and Babl/c mice are study models of in vivo in vitro experiments,which are divided into the following groups:(A) control group;(B) GOX group;(C)PFT+GOX group;(D) CsA+GOX group. Here, glucose oxidase was used to induceoxidative stress in hepatic cells and mice liver. PFT and CsA were also administrated toinhibit the expression of p53and permeability transition pore opening, separately. So wecan study the mechanism of how p53and mitochondria oxidative stress participates inROS induced hepatic cells apoptosis.(1) EX vivo studies: HepG2cell lines were exposed to Glucose Oxidase to generateROS. Immunofluorescence was used to study ROS signal regulates the expression andSubcellular localization of p53. Mitochondrial superoxide anion was measured by afluorescent probe, named Mito-SOXTM. MnSOD is the primary mitochondrial antioxidantenzyme and is essential for scavenging superoxide anion, so we also detected its expression. In addition, mitochondrial membrane potential, mitochondrial permeabilitytransition, the release of cytochrome c into the cytosol, and the function of mitochondriawere also detected. We also extracted the proteins of mitochondria, cytoplasm, and nuclear,and then analyzed the apoptosis related protein, such as, cytochrome c, bax, caspase-9,caspase-3, and p53. Redox related indexes, such as, MnSOD, GSH, and MDA.(2) In vivo studies: Based on the study of cytology, Babl/c mice were injected400U/kgGOX with or without PFT and CsA, in vena caudalis for3consecutive days, Physiologicalsaline was used as control. The main observation indexes are as following: After thecompletion of the experimental treatment, mice liver was removed. We detected the redoxrelated indexes of mitochondria; p53subcellular localization, mitochondrial membranedamage, and the liver tissue apoptosis. The methods are the same as the cytologicalexperiment.【Results】(1) GOX had a dramatic inhibition on HepG2cell viability in a dose and timedependent manner, accompanying with the intracellular ROS and MDA increasing, andthe GSH content decreasing. At the same time, MnSOD protein expression reduced, andMito-SOXTMstaining result indicated a significant rise of superoxide anion, whichconformed mitochondria were under oxidative stress state.(2) Upon exposure to GOX, the level of p53increasing dramatically, and plenty of p53was detected in mitochondrial, while the level of nucleus and cytoplasmic p53has noobvious change.(3) GOX treatment can trigger mitochondrial permeability transition, collapse ofmitochondrial membrane potential, induce cytochrome c releasing from mitochondrial tocytoplasm, recruit Bax to mitochondria and activate the caspase cascade reaction.Meanwhile, TUNNEL and FITC-Annexin V/PI staining results indicated that GOXinduced abundant cells apoptosis.(4) Animal experiment showed that GOX would induce MDA and ROS level increaseand GSH level decrease in mitochondria, p53enter into mitochondria and MnSOD protein expression reduce. Meanwhile, abundant cells apoptosis in mice liver.(5)PFT and CsA processing can not only suppress p53translocated to mitochondria,but also reduce the mitochondrial stress and cell apoptosis in HepG2cells and livers ofmice.【Conclusions】(1)Mitochondrial p53, rather than nucleus p53, is a critical regulating factor inoxidative stress-induced hepatic cells apoptosis.(2)The decrease of mitochondrial membrane potential and increase membranepermeability may is the main mechanisms of p53transferred to mitochondria. Whilemitochondrial p53can in turn influence mitochondrial oxidative stress by regulatingMnSOD expression.(3)P53participates in cell apoptosis regulation in two ways. One is upregulating ROSlevel in mitochondria, triggering mitochondrial permeability transition, and inducing thecollapse of mitochondrial membrane potential. Then cytochrome c will release frommitochondrial to cytoplasm, and activate the caspase cascade reaction. Another is torecruit Bax to mitochondria, and the changing mitochondrial permeability transition.In conclusion, GOX can induce oxidative stress in hepatic cells, trigger p53increasing dramatically and mitochondrial translocation. While mitochondrial p53can inturn influence mitochondrial oxidative stress, mitochondrial permeability transition, whichwill result in release of the apoptosis factors and cell apoptosis. Here, we revised previousopinions about p53mainly works in the nucleus, and provides a new mechanism while p53regulating cell apoptosis.