The Effect And Mechanism Of Mitochondrial ROS On Liver Regeneration

Background:As an important digestive gland in the body,the liver plays a critical role in eliminating toxins as well as in the metabolism of nutrients.Moreover,the liver has a strong ability to recover the weight and function after acute or chronic infection or injury.Liver regeneration（LR）process is very complex and the mechanism underlying remains unknown.Elucidating the molecular mechanism will benefit to find the potential intervention targets for liver regeneration,which is great value for clinical treatment of liver diseases.Intracellular reactive oxygen species（ROS）is mainly produced during the oxidative respiration process in mitochondria.As active oxygen-containing molecules in cells,ROS can affect signal transduction pathways to regulate many physiological processes including cellular proliferation.It is generally accepted that the LR process depends on cellular proliferation,so we speculate that mitochondria-derived ROS play a critical role in the regulation of LR.However,the precise molecular mechanism remains unclear.To demonstrate this hypothesis and to make clear the mechanism underlying,the mice LR model induced by 70%partial hepatectomy（PH）were employed to study the role of mitochondrial ROS as well as the molecular targets in LR process.Objective:To study the role of mitochondrial ROS in LR,and clarify the molecular mechanism of mitochondrial ROS regulating LR in the mice LR model induced by PH.Methods:1.The C57 mice LR model were established by classical 70%PH operation.The mice after operation were randomly divided into sham operation group（Sham group）,PH group and PH with intervention group.The mice in the sham group were only opened abdominal cavity without partial hepatectomy,while the mice in the PH group accepted70%PH operation.The mice in PH with intervention group were injected with MitoQ,Mito TEMPO,Mn（Ⅲ）TMPyp,Catalase or Tic10 respectively.2.The sections of liver tissue were stained with HE and immunohistochemistry respectively to observe the pathological changes of liver tissue and cellular proliferation markers during LR after operation.3.The sections of liver tissue were stained with DHE or Mito SOX respectively to observe the level of intracellular ROS or mitochondrial ROS.4.The resected liver tissue and harvested liver tissue after PH were weighed to measure the liver regeneration rate of mice after operation.5.The level of malondialdehyde（MDA）in liver tissue was detected by kit.6.The protein levels of Cyclin D1,PCNA and Ki67 in liver tissue were detected by Western Blot.7.The levels of H₂O₂ and O₂^·-in liver tissue were detected by kits.8.The total mRNA in liver tissue was extracted by kit,then reverse transcribed.The expression of p27 gene was detected by Q-PCR.9.The protein levels of Akt,Erk,FoxO3a as well as their phosphorylated forms,and p27 protein level in liver tissue were detected by Western Blot.Results:1.Both the cellular proliferation and the mitochondrial ROS level in LR showed a trend of"increased at first and then restored",indicating a certain correlation between them.The HE staining and immunohistochemical staining of liver tissue sections detection showed that cellular volume,the accumulation of lipid droplets in cells and the level of cellular proliferation increased at first and then restored in LR.The change of cellular proliferation level was confirmed by the results of Western Blot and regeneration rate.At the same time,the change of cellular ROS level was consistent with that of mitochondrial ROS level indicated by DHE or Mito SOX staining of liver tissue sections and MDA levels,which showed a certain correlation with the cellular proliferation.2.Regulating mitochondrial ROS can regulate LR.The level of mitochondrial ROS increased significantly after PH.The intervention of MitoQ or Mito TEMPO,which were both mitochondrial ROS scavengers,could significantly reduce mitochondrial ROS level.At the same time,the cellular proliferation level showed by the levels of proliferation-related proteins（Cyclin D1,PCNA and Ki67）increased significantly after PH,which was markedly inhibited by the intervention of MitoQ or Mito TEMPO.The level of cellular proliferation and the rate of LR were also inhibited.There results demonstrated that regulating mitochondrial ROS could regulate LR.3.The regulation of LR by mitochondrial ROS is mainly related to H₂O₂.The levels of O₂^·-and H₂O₂ in mice liver tissue showed that H₂O₂ in LR increased first and then restored,but the change of O₂^·-was not significant.With the intervention of Mn（Ⅲ）TMPyp(O₂^·-scavenger)and catalase（H₂O₂ scavenger）,the results showed that the removal of H₂O₂ could reduce the proportion of proliferative cells,indicated by the proliferation-related proteins（Cyclin D1,PCNA and Ki67）after PH,as well as the rate of LR.However,clearing O₂^·-showed no significant effect.In addition,the H₂O₂ level detection showed that mitochondrial ROS scavengers could reduce H₂O₂ after PH.These results indicated that H₂O₂ was the key role of regulating LR by mitochondrial ROS.4.Akt&Erk/FoxO3a/p27 pathway was involved in LR regulation.The level of FoxO3a phosphorylation form（Ser253,Ser294）increased significantly after PH.Akt and Erk are the upstream molecules of the FoxO3a signal pathway.The data of Western Blot showed that the phosphorylation levels of Akt and Erk also increased accordingly.At the same time,the level of FoxO3a in the nucleus decreased significantly after PH,while the level in the cytoplasm increased accordingly.p27 is the downstream target gene of FoxO3a molecule.The results of Q-PCR and Western Blot showed that p27 levels of mRNA and protein both decreased after PH.These results indicated that Akt&Erk/FoxO3a/p27 was involved in regulating LR.5.H₂O₂ regulated LR through Akt&Erk/FoxO3a/p27 pathway.Mn（Ⅲ）TMPyp or catalase was used as O₂^·-scavenger or H₂O₂ scavenger respectively.It was found that scavenging H₂O₂ by catalase reduced the phosphorylation level of Akt,Erk and FoxO3a.At the same time,it increased the level of FoxO3a in nucleus,as well as the transcription and expression level of p27.However,scavenging O₂^·-by Mn（Ⅲ）TMPyp had no obvious effect.In the combined intervention of catalase with Tic10（which could inhibit Akt and Erk to activate FoxO3a）,it was found that catalase or Tic10 could inhibit the change of FoxO3a-related signal pathway induced by PH,including the activation of FoxO3a-related pathway,the inhibition level of cellular proliferation protein（Cyclin D1,PCNA and Ki67）,as well as the rate of LR.However,the combined intervention catalase with Tic10 had no further intervention effect compared with catalase or Tic10 alone.These results suggested that H₂O₂ regulated LR through Akt&Erk/FoxO3a/p27 pathway.Conclusions:1.Mitochondrial ROS are closely related to LR after PH,and regulating mitochondrial ROS can regulate LR.2.H₂O₂ mediates the regulation of LR by mitochondrial ROS.3.Mitochondria-derived H₂O₂ regulates LR through Akt&Erk/FoxO3a/p27 pathway.