| Background: Sepsis is the manifestation of the immune and inflammatory response to infection that may ultimately result in multi organ failure. Despite the therapeutic strategies that have been used up to now, sepsis and septic shock remain a leading cause of death in critically ill patients. Myocardial dysfunction is a well-described complication of severe sepsis, also referred to as septic cardiomyopathy, which may progress to right and left ventricular pump failure. Nevertheless, the precise underlying molecular mechanisms as well as their significance in the pathogenesis of septic cardiomyopathy remain incompletely understood. Our research group has reported that: hydrogen was shown to exert an effective therapeutic role in survival rates of mice with sepsis and in the function of organs. Organs with sepsis which we have studied including: lung and intestinal, except heart. It is proved that mitochondrial dysfunction is the major reason of death in patients with spepsis.Mitochondria has many biological functions, and played a important role in many pathophysiology process. In recent years, many studies reported that mitochondrial dynamics play a important role in sepsis and organ dysfuntion. It may change in the early stage of disease, and influence the prognosis. Therefore, it is of great significance to study mitochondrial dynamics in the mechanism of myocardial dysfunction in sepsis. Previous studies suggest that the effect of hydrogen is closely related to the Nrf2/HO-1 pathway, but the exact mechanism is unclear. Mitochodrial dynamics (fusion/fission) balance is a key factor in maintaining mitochondrial morphology and function, disorder of the balance is important to the pathogenesis of sepsis, this may relate to Nrf2/HO-1 pathway. Therefore, this research hypothesis that hydrogen regulate the mitochondrial dynamic by activating the Nrf2/HO-1 pathway,and protect the heart function of septic mice. This research intends to use wild type and Nrf2-/- mice, use umbilical vein endothelial cell model and animal model of sepsis, to state the mechanism of hydrogen‘s treatment effect in sepsis from mitochondrial dynamic, through detecting the expression of related protein in mitochondrial fusion/fission、autophagy、biosynthesis.The aim of this study is to further discuss the role of Nrf2/HO-1 in this process, and to explicit that hydrogen’s protective effect in sepsis through mitochodrail dynamics by activating the pathway of Nrf2/HO-1.This study will provide the theoretical foudation of hydrogen’s efficacy in sepsis, and lay the foundation for clinical application.Experiment one: To discuss the mechanism of treatment effect of hydrogen in cardiomyopathy induced by sepsis from mitochondrial dynamicObjective To evaluate the effects of hydrogen (H2) on survival rate, heart function and mitochondrial function in sepsis-induced myocardial injury mice. And detect the expression of proteins that related to mitochondrial function.Methods 128 male ICR mice,weighing 20-25 g and aged 6 weeks,were randomly divided into 4 groups (n=32 each): sham operation group (Sham group), Sham+H2 group,CLP group and CLP+H2 group. Sepsis was produced by cecal ligation and puncture (CLP). The mice in the Sham+H2 and CLP+H2 groups inhaled 2% H2 for 1 h starting from 1h and 6 h after Sham and CLP operation. The survival rate was recorded on 1、2、3、5、7 days. At 24h after CLP,the data of Echocardiography was collected. The histopathological changes were observed. The change of cardial mitochondrial respiration control rate (RCR) was tested by clark eletron. ATP synthesis capacity was determined using a bioluminescence technique. And the membrane potential was measured. The expression of Drp1、Mfn2、LC3、PGC-1αprotein was analyzed by Western blot. Results Compared with Sham group,the survival rate、heart function decreased, the myocardial pathological score increased in CLP group and the RCR、ATP and MMP of mitochondrial decreased. The expression of Drp1、LC3、PGC-1α protein were elevated in CLP group ,but the expression of Mfn2 increased (P<0.05). Compared with CLP group,the survival rate、heart function increased, the myocardial pathological score decreased in CLP group and the RCR、ATP and MMP of mitochondrial increased. The expression of Drp1 decreased,and the expression of LC3、PGC-1α protein increased again. The expression of Mfn2 increased (P<0.05).Conclusion H2 treatment could decrease the level of cardial mitochondrial fission,and improve the cardial mitochondrial function and heart function in sepsis-induced myocardial injury in mice.Experiment two: To discuss the effect of mitochondrial dynamic in hydrogen’s treatment in sepsis from cell levelObjective To investigate the effects of hydrogen rich medium on mitochondrial function and Drp1、LC3 expression in lipopolysaccharides(LPS)- treated HUVCEs .Methods Cultured HUVECs were randomly divided into 4 groups: sham group, H2 group, LPS group and LPS+H2 group. Sham group received no treatment, H2 group and LPS+H2 group were cultured with hydrogen-saturated medium, and LPS group and LPS+H2 group was stimulated with lOug/mL of LPS at the same time. Then, cell proliferation activity was detected by MTT. ATP content was obtained by ATP assay kit. Mitochondrial membrane potential was monitored by JC-1 dye. Mitochondria in HUVECs were visualized with Mitotracker orange .The levels of Drp1、LC3 expression were tested by Western blot at 2h, 8h and 24h after LPS treatment.Results Compared with sham group, cell proliferation activity, mitochondrial membrane potential and ATP content was significantly decreased in LPS group at 2h,8h and 24h (P<0.05). Compared with LPS group, cell proliferation activity,mitochondrial membrane potential and ATP content was significantly increased in LPS+H2 group 2h, 8h and 24h (P<0.05). Compared with sham group, the length of mitochondria was significantly decreased in LPS group (P<0.05). Compared with LPS group, mitochondrial membrane potential was longer in LPS+H2 group.Furthermore, western blot showed that compared with sham group, the expression of Drpl and LC3 was up-regulated in LPS group at 2h, 8h and 24h (P<0.05) and compared with LPS group, the expression of Drp1 was down-regulated in LPS+H2 group at 2h, 8h and 24h (P<0.05). But the expression of LC3 increased again.Conclusion H2 can alleviate the LPS-induced mitochondrial dysfunction in HUVECs via supressing Drpl expression and up-regulating LC3 expression.Experiment three: To discuss the effect of Nrf2/HO-1 pathway in the mechanism of treatment effect of hydrogen induced by mitochondrial dynamic in sepsisObjective: To investigate mitochondrial function and expression of related protein in Nrf2 knockout mice established as sepsis model and wild type sptic mice treated with HO-1 inhibitor.Method:Part 1: 16 ICR were randomly divided into 2 groups (n=8): WT/CLP and WT/CLP+H2. Nrf2 knockout mice 16 were randomly divided into 2 groups (n=8):KO/CLP and KO/CLP+H2. Sepsis was produced by cecal ligation and puncture(CLP). The mice in the WT/CLP+H2 group and KO/CLP+H2 group inhaled 2% H2 for 1h starting from 1h and 6h after CLP operation.All mice were put to death and achieve the hearts which were used to extract mitochondria, and then RCR, ATP, MMP were measured. The expression of Drp1、Mfn2、LC3、PGC-1α were detected by westernblot.Part 2: 32 male ICR mice, weighing 20-25 g and aged 6 weeks, were randomly divided into 4 groups (n=8 each): CLP group, CLP+H2 group, CLP+ZNPPIX group,CLP+H2+ZNPPIX group. Sepsis was produced by cecal ligation and puncture(CLP). The mice in the CLP+H2 and CLP+H2+ZNPPIX groups inhaled 2% H2 for 1h starting from 1h and 6 h after CLP operation. ZNPPIX (40mg/kg) was injected in the abdominal cavity of the mice in CLP+ZNPPIX group and CLP+H2+ZNPPIX group before operation.Part 3: Cultured HUVECs were randomly divided into 4 groups: LPS group,LPS+H2 group, LPS+ZNPPIX group and LPS+H2+ZNPPIX group. All the 4 groups were stimulated with LPS. LPS+H2 group and LPS+H2+ZNPPIX group were cultured with hydrogen-saturated medium, and LPS+H2+ZNPPIX group was stimulated with lOug/mL of ZNPPIX at the same time. Then, ATP content was obtained by ATP assay kit. Mitochondrial membrane potential was monitored by JC-1 dye.Mitochondria in HUVECs were visualized with Mitotracker orange .The levels of Drp1、LC3 expression were tested by Western blot.Result:Part1: Compared with WT/CLP group, RCR、ATP and MMP increased in WT/CLP+H2 group, the expression of Drp1 decreased, the expression of HO-1、PGC-1α、LC3 and Mfn2 increased. Compared with WT/CLP group, RCR、ATP and MMP in KO/CLP group decreased, the expression of Drpl increased, HO-1、PGC-1α、LC3 and Mfn2 decreased(P<0.05). The mitochondrial function and protein expression didn’t change in KO/CLP+H2 group(P>0.05). Compared with WT/CLP+H2 group,RCR、ATP and MMP in KO/CLP and KO/CLP+H2 group decreased. The expression of HO-1、PGC-1α、LC3 and Mfn2 decreaed in KO/CLP and KO/CLP+H2 group,Drp1 increased(P<0.05).Part 2: Compared with CLP group, RCR, ATP, MMP in CLP+H2 group increased.Expression of HO-1、Mfn2、LC3、PGC-1α increased, and Drpl decreased (P<0.05),RCR、ATP、MMP in CLP+ZNPPIX group decreased, expression of Drpl increased,expression of HO-1、Mfn2、LC3、PGC-1α decreased (P<0.05). The mitochondrial function didn’t change in CLP+H2+ZNPPIX, either for protein expression (P>0.05).Compared with CLP+H2 group, RCR、ATP、MMP in CLP + ZNPPIX group decreased, the expression of Drpl increased, and the expression of HO-1、Mfn2、PGC-1α、LC3 decreased (P<0.05).RCR、ATP、MMP in CLP+H2+ZNPPIX group decreased, the expression of Drpl increased, and the expression of HO-1、Mfn2、PGC-1α、LC3 decreased (P<0.05).Part 3: Compared with LPS group, maximal respiration rate、mitochondrial membrane potential and ATP content was significantly decreased in LPS+ZNPPIX group (P<0.05), and there was no change in LPS+H2+ZNPPIX group. Compared with LPS+H2 group, maximal respiration rate、mitochondrial membrane potential and ATP content was significantly decreased in LPS+ZNPPIX group and LPS+H2+ZNPPIX group(P<0.05). Compared with LPS group, the length of mitochondria was significantly decreased in LPS+ZNPPIX group. There was no change in LPS+H2+ZNPPIX group. Compared with LPS+H2 group, the length of mitochondria decreased in LPS+ZNPPIX group and LPS+H2+ZNPPIX group. Furthermore, western blot showed that compared with LPS group, the expression of LC3 was up-regulated in LPS+H2 group (P<0.05), LC3 was downregulated in LPS+ZNPPIX group, but did’t change in LPS+H2+ZNPPIX group. Compared with LPS group, the expression of Drp1 was down-regulated in LPS+H2 group, Drp1 was up-regulated in LPS+ZNPPIX group, but didn’t change in LPS+H2+ZNPPIX. Compared with LPS+H2 group, the expression of LC3 decreased in LPS+ZNPPIX group and LPS+H2+ZNPPIX group, but the expression of Drpl increased.Conclusion: ZNPPIX reversed the therapeutic effect of hydrogen. And the therapeutic effects of hydrogen disappeared when Nrf2 has been knocked out. So the therapeutic effects of hydrogen in sepsis were induced by the improving of mitochondrial function through Nrf2/HO-1 pathway.The improving of mitochondrial function was induced by hydrogen through Nrf2/HO-1 pathway, thereby realize the theraputic effect in septic mice.Summary1. 2% H2 treatment could increase the survival rate and improve the heart function of septic mice though improving mitochondrial function.2. H2 can alleviate the LPS-induced mitochondrial dysfunction in HUVECs, then the activity of cell proliferation increases.3. Through the study of Nrf2 knock-out mice and inhibitor ZNPPIX treated mice and cell, we can speculate that H2 regulate the mitochondrial dynamic balance by activating Nrf2/HO-1 pathway. And H2 plays an important role in sepsis treatment by improving the mitochondrial function. |
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