Hydrogen Gas Inhalation Attenuates Sepsis-induced Liver Injury In A FUNDC1-denpendent Manner

Objective:Sepsis is well recognized to be a kind of systemic inflammatory response syndrome?SIRS?induced by invading microorganisms,however,there is still no effective therapeutic treatment for sepsis up to now.During sepsis,the liver plays a key role in metabolism and immune homeostasis in human body.Acute liver injury happened in early stage of sepsis,which may contribute directly to the disease deterioration and death in patients with sepsis.While reducing liver injury or restoring liver function may lower the morbidity and mortality of septic patients.Mitochondria can provide most of energy for organic life,while mitochondrial dysfunction had involved in pathogenesis of sepsis.In septic patients,mitochondrial dysfunction in vital organs can deprive cellular energy,and then result in multiple-organ failure.Mitophagy,as a selective form of autophagy,is a basic process for controlling mitochondrial quality and quantity at the organelle level and is also a necessary process for maintaining the mitochondrial networks and reprogramming the cell metabolism.Moreover,Fun14 domain-containing protein 1?FUNDC1?is one of newly found mitophagy receptors,localizing at the outer membrane of mitochondria,which can clean up damaged mitochondria in response to mitochondrial stresses and hypoxia.Molecular hydrogen?H₂?,as a special selective antioxidant,can be effectually used for the treatment of more than 70 kinds of diseases up to now.Our previous studies have shown that H₂ gas inhalation or hydrogen-rich saline drinking provided beneficial effects against sepsis and organ damages caused by sepsis,including liver,lung,brain and kidney.However,the underlying mechanism remains unclear.The aim of this research is to discuss the role of FUNDC1-induced mitophagy in the pathogenesis of liver injury caused by sepsis and the mechanism of 2%H₂ gas inhalation in protecting septic mice in a CLP model.Methods:Adult ICR mice were randomly arranged into five groups:Sham group,CLP group,CLP+peptide P group,CLP+H₂ group,CLP+H₂+peptide P group.Septic models were made by cecal ligation and puncture?CLP?.The cell penetrating peptide P?1 mg/kg?,a kind of FUNDC1 inhibitor,was given to mice 24 h through intraperitoneal?i.p.?injection before CLP or Sham operation.The mice in CLP+H₂and CLP+H2+peptide P groups inhaled H2 gas?2%?for 3 hours beginning at 1 h after Sham or CLP surgery.At 1h after Sham or CLP operation,the H₂ gas concentration in mice's arterial and venous blood of different groups were detected after starting H₂ gas inhalation and stopping H₂ gas inhalation,respectively;The survival rates in each group were observed from the 1st day to the 7th day?7 days?after Sham or CLP surgery;At 6 h post-surgery,we executed the mice for obtaining blood samples and liver tissues in each group.Liver slices in different groups were used to do the histological examination with HE staining;Co-Immunoprecipitation was used to show the connection levels between FUNDC1 and LC3BII in mice liver;Mitochondria were also isolated from liver tissues in each group at 6 h post-operation to analyze the respiratory control rate?RCR?.Results:After H₂ inhalation,the H₂ concentration in Sham+H₂ group and CLP+H₂group were gradually increased both in arterial and venous blood in mice.After the end of H₂ inhalation,the H₂ concentration in Sham+H₂ group and CLP+H₂ group were dropped to the base line quickly both in arterial and venous blood;Co-Immunoprecipitation shown the connection levels between FUNDC1and LC3BII in mice livers were enhanced in CLP group,while 2%H₂ inhalation could enhanced this level.Peptide P could effectively inhibit the connection between FUNDC1and LC3BII;According to the expression levels of FUNDC1 and p-18-FUNDC1,at 6 h post-operation,the protein levels of FUNDC1 and p-18-FUNDC1of liver tissues were lower in CLP group compared with Sham group?P<0.05?.However,2%H₂treatment could decrease FUNDC1 and p-18-FUNDC1 levels in CLP+H₂ group compared with CLP group.?P<0.05?;During 7-days'experimental observation,the7-day survival rates in CLP group markedly decreased compared with Sham group?P<0.05?.Compared with CLP group,2%H₂ inhalation could markedly increase the7-day survival rates in CLP+H₂ group?P<0.05?.However,there was no statistical difference between CLP+peptide P group and CLP+H₂+peptide P group?P>0.05?;For the evaluation of liver injury,in CLP group,there were morphologic changes associated with liver injury,and the levels of ALT and AST in CLP group were markedly increased compared with Sham group?P<0.05?.However,injury was significantly attenuated in CLP+H₂ groups compared with CLP group,and 2%H₂inhalation could markedly decrease the levels of ALT and AST?P<0.05?.However,the pathological score and the levels of ALT and AST were all no statistical differences between CLP+peptide P group and CLP+H₂+peptide P group.?P>0.05?,showing that peptide P could inhibit the therapeutic effects of H₂ on liver injury in septic mice;At 6 h post-operation,liver mitochondria were isolated to evaluate their mitochondrial respiration,the respiratory control ratio?RCR?was decreased in CLP group compared with Sham group?P<0.05?,but 2%H₂ treatment could improve the RCR in CLP+H₂ group?P<0.05?.Additionally,there was no statistical change between CLP+peptide P group and CLP+H₂+peptide P group?P>0.05?;The protein levels of autophagy-associated proteins?P62,LC3B II,Tim23 and caspase-1?were analyzed by Western blot.Compared with Sham group,the protein levels of P62 and LC3B?enhanced in CLP group?P<0.05?,while CLP+H₂ group had higher levels of those proteins than CLP group?P<0.05?.Furthermore,CLP group had lower protein level of Tim 23 than Sham group?P<0.05?,while this expression was further decreased in CLP+H₂ group compared with CLP group?P<0.05?.In addition,the expression of caspase-1 was obviously raised in CLP group compared with Sham group?P<0.05?,while the expression level of caspase-1declined after 2%H₂ treatment compared with CLP group?P<0.05?.However,there was no big difference with all these proteins expression between CLP+peptide P group and CLP+H₂+peptide P group?P>0.05?.Conclusion:According to the above results,it reveals that sepsis could markedly decreased the 7-day survival rates,induced liver injury and impaired respiratory function of liver mitochondria.As shown with expression levels of FUNDC1 and autophagy-associated proteins,sepsis could increase the level of FUNDC1-induced mitophaghy.While 2%H₂ inhalation could effectively improve the 7-day survival rates,attenuate liver injury and improve mitochondrial respiratory function,at the same time,2%H₂ treatment could further enhanced the level of FUNDC1-induced mitophaghy.Moreover,peptide P,a FUNDC1 inhibitor,could effectively inhibit the interaction of FUNDC1 and LC3B?,blocking the mitophaghy process which induced by H₂ treatment for sepsis and reverse the protective effects of H₂ on liver injury in septic mice.In conclusion,FUNDC1-induced mitophaghy was involved in the pathogenesis of sepsis;H₂ protects against sepsis-induced liver injury in vivo;H₂can ameliorate liver injuries through the regulation of mitophagy,Peptide P could effectively inhibit the connection between FUNDC1and LC3BII,indicating that FUNDC1 plays a key role in the protective effects of H₂ against sepsis-induced liver injury.