Effects And Mechanisms Of Exogenous Hydrogen Sulfide On Brain Mitochondria After Cardiopulmonary Resuscitation

[Objective] Mitochondrial dysfunction plays a critical role in brain injury after cardiac arrest and cardiopulmonary resuscitation (CPR). Recent studies demonstrated that hydrogen sulfide (H2S) donor compounds preserve mitochondrial morphology and function during ischemia-reperfusion injury. In this study, we sought to explore the effects of sodium hydrosulfide (NaHS) on brain mitochondria after cardiac arrest and resuscitation and the potential mechanisms.[Methods]Male Sprague-Dawley rats were randomly divided into three groups:(1) Sham group:rats received the same procedures except cardiac arrest;(2) CPR group:rats were subjected to6minutes cardiac arrest induced by transcutaneous electrical epicardium stimulation and then resuscitated successfully. Rats received vehicle (0.9%NaCl,1.67ml/kg)1minute before the start of CPR intravenously, followed by a continuous infusion of vehicle (5ml/kg/h) for3h;(3) CPR+NaHS group:rats were subjected to6minutes cardiac arrest induced by transcutaneous electrical epicardium stimulation and then resuscitated successfully. Rats received NaHS (0.5mg/kg)1minute before the start of CPR intravenously, followed by a continuous infusion of NaHS (1.5mg/kg/h) for3h. Neurologic deficit scoring (NDS) were performed24h after CPR. After that, cortex samples were collected for assessments immediately.①Nissl stain was employed to observe the neurons damage in the brain from the three groups.②Mitochondrial damage in the three groups was studied by transmission electron microscopy.③Cortex single cell suspension were prepared immediately at24h after CPR. Intracellular ROS, Ca2+concentration and mitochondrial membrane potential (MMP) in the three groups were evaluated by flow cytometry.④After the isolation of cortex mitochondria, mitochondrial permeability transition pore (mPTP) opening was detected by a monochromator microplate reader. The expression of cytochrome c (cyt c) in mitochondria and cytosol were detected by Western blot.⑤The ATP levels in the three groups were evaluated by a monochromator microplate reader.⑥Real time PCR was employed to evaluate the content of mitochondrial DNA (mtDNA) in all three groups.⑦The expression of peroxisome proliferator-activated receptor-y coactvator-la (PGC-la), nuclear respiratory factor-1(NRF-1) and nuclear factor erythroid2-related factor2(Nrf-2) were detected using Western blot.[Results]①In CPR and CPR+NaHS groups, the NDS were decreased after CPR (P<0.01), while rats treated with NaHS revealed an improved neurological outcome (P<0.01).②Compared with Sham group, neurons and mitochondria were damaged both in CPR and CPR+NaHS groups. However, NaHS treatment reduced the damage.③When compared with Sham group, intracellular ROS and Ca2+concentration were both increased in CPR and CPR+NaHS groups (P<0.01), while the MMP was both decreased (P<0.01). Compared with CPR group, intracellular ROS and Ca2+concentration were both decreased (P<0.01) and MMP level was elevated after NaHS treatment (P<0.05).④When compared with Sham group, the opening of brain mPTP was increased both in CPR and CPR+NaHS groups (P<0.01). Compared with CPR group, NaHS exhibited inhibition effect on mPTP opening (P<0.01).⑤Compared with Sham group, cyt c distribution were observed both in CPR and CPR+NaHS groups (P<0.01), while NaHS therapy reduced the release of cyt c from mitochondria into cytosol (P<0.01).⑥When compared with Sham group, the ATP level were both decreased in CPR and CPR+NaHS groups (P<0.01). Compared with CPR group, ATP level was elevated in CPR+NaHS group (P<0.01).⑦When compared with Sham group, the mtDNA contents were both increased in CPR and CPR+NaHS groups (P<0.01). Compared with CPR group, the mtDNA contents were even more in CPR+NaHS group (P<0.05).⑧When compared with sham group, the expressions of PGC-la, NRF-1, Nrf-2were both increased in CPR and CPR+NaHS groups (for NRF-1:P<0.05, for others:P<0.01). Compared with CPR group, the expression of PGC-la, NRF-1, Nrf-2were further increased in CPR+NaHS group (P<0.01). [Conclusions] The results of the present study suggested that administration of NaHS1min prior CPR and followed by a continuous infusion ameliorated neurological dysfunction24h after CPR, possibly through preservation of mitochondrial function as well as by promoting mitochondrial biogenesis.