| BACKGROUNDHepatic ischemia/reperfusion injury (HI/RI) remains a primary complication of transplant surgery, and is a major cause of morbidity and mortality in other pathologic conditions such as shock and liver resection. The insult caused by HI/RI is a result of the interplay among different complex mechanisms including cell damage by free radicals, ATP depletion and energy use impairments, calcium overload, inappropriate inflammation and immune reactions, mitochondrial dysfunction.As a novel endogenous gasotransmitter, hydrogen sulfide (H2S) can exert antioxidant, anti-inflammatory and antiapoptic effects through complex signaling systems and has been recognized as a useful tool to induce cytoprotection during the evolution of myocardial infarction. Recently, studies have focused on the possible protective role of H2S in HI/RI despite the fact that H2S is a toxic molecule at higher concentration. As the main source for glycogen synthesis and storage, the liver has its unique pathophysiolgical process during IRI while also shares similarities with myocardial ischemia and stroke. Whether and how H2S induces liver protection is far from clear.In this study, we demonstrated that in the rat after segmental (70%) hepatic ischemia, pretreatment of NaHS, a widely recognized donor of H2S, can protect liver cells against HI/RI by regulating mitochondria function and Akt signaling pathway.PART ONE H2S preconditioning protected liver cells against HI/RI.Histological examinations and oxidation reduction index were determined to evaluate rat liver injuries.Materials and MethodsThe segmental (70%) hepatic ischemia model was induced by clamping the portal vein, hepatic artery, and bile duct of the left and median for1h followed by reperfusion for24h. SD rats were randomly divided into four groups:Sham, I/R (ischemia/reperfusion); IPC (ischemia preconditioning by10min×1short ischemia1h before I/R); NaHS+I/R (NaHS, iv5min before ischemia).The optimal NaHS dose was decided according to the results of histopathologic examinations using HE staining. Apoptotic levels were detected by TUNEL staining. GSH levels were detected as an indicator for hyperoxidation.ResultsThe optimal NaHS dose was25μmol/kg. HE dyeing showed that NaHS preconditioning and IPC are both effective in reducing the damages of liver cells. The liver tissues from the NaHS+I/R group were with a few cytoplasm vacuolization and focal nuclear pyknosis while that from the I/R were characterized by disintegration of hepatic cords, hemorrhage, and severe PMN infiltration. Those in IPC group was in the middle. Suzuki scores in NaHS preconditioning group showed a statistical significance compared with which in I/R group(1.667±0.52VS2.667±0.52, P<0.05). GSH content in NaHS group was significantly higher compared with which in I/R group (P<0.05).ConclusionNaHS (25umol/kg) preconditioning protected liver against ischemia/reperfusion injury which was accompanied by reduced liver damages and higher GSH levels. PART TWO Mitochondria protection mechanisms of H2S preconditioning against hepatic ischemia reperfusion injury EXPERIMENT ONE Mitochondria isolation/identification and mitochondria calcium capacity testMitochondria of the liver cells were isolated by differential centrifugation and the purity was confirmed. Mitochondrial membrane permeability was detected using mitochondria calcium capacity test.Materials and MethodsThe rats were sacrificed24h after reperfusion and liver tissue samples were obtained for subsequent mitochondria isolation by differential centrifugation. The purity of mitochondria was confirmed by the transmission electron microscope. We used the FlexStation II fluorescence detector to test mitochondria calcium capacity which was recognized as an indicator of the mitochondrial membrane permeability.ResultsThe mitochondria purity met the requirements. Mitochondria from the NaHS+I/R group could withstand exogenous calcium pulse (10μM CaCl2/min) about7times and the number for the I/R group was about3-4times. NaHS preconditioning was with lower mitochondrial membrane permeability (198.63nmol/mg±24.86nmol/mg VS150.685nmol/mg±13.04nmol/mg).ConclusionThe differential centrifugation separation method was useful for high-purity mitochondria isolation. HI/RI was followed by impaired mitochondrion’s calcium capacity, increased MPTP sensitivity, and damaged mitochondrial function. NaHS preconditioning could stabilize the mitochondrial membrane potential, reduce MPTP sensitivity, reduce the mitochondria swelling and protect the mitochondrial function against liver ischemia-reperfusion injury. EXPERIMENT TWO PI3K-Akt signaling pathway was involved in the liver-protective role of H2SThe aim is to see whether the protective mechanism was associated with Akt signaling activation.Materials and MethodsThe rats were sacrificed24h after reperfusion and liver samples were harvested. The protein expressions of Akt, p-Akt, Bcl-2, activated Caspase-3, Cyt C were determined by Western blot analysis.ResultsAfter HI/RI, cell apoptosis increased and Akt pathway activation was inhibited with a lower p-Akt/Akt. Bcl-2expression was decreased, and activated Caspase-3and Cyt C expressions were increased after HI/RI. These changes were partly reversed by H2S.Conclusion H2S preconditioning was effective in reducing hepatic ischemia/reperfusion injury and cell apoptosis by activating Akt pathways which included higher expressions of p-Akt, and Bcl-2and lower levels of activated Caspase-3and Cyt C. The protection mechanisms of H2S preconditioning against hepatic ischemia/reperfusion injury may be mediated by activating Akt pathway. 1. H2S preconditioning can protect against hepatic ischemia/reperfusion injury with higher GSH levels and decreased apoptosis.2. H2S preconditioning can increase the mitochondrion’s calcium capacity and reduce MPTP sensitivity which are liver-protective.3. H2S preconditioning can protect liver cells against HI/RI through activating PI3K-Akt pathway which induces p-Akt and Bcl-2expression, inhibits activated Caspase-3and Cyt C expression and reduces apoptosis. |
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