The Molecular Mechanisms Of Hydrogen Gas Treatment On Acute Lung Injury In Septic Mice Through Proteomics Technology

Objective: Sepsis is considered to be the life-threatening multiple organ that is triggered by the microbial infection in clinical practice.When sepsis occurs,the most severely affected organ is lung.Our previous studies have indicated that a low concentration(2 %)of H2 can effectively ameliorate the organ function including acute lung injury and the survival rate in septic mice.The mechanism may be closely related to the anti-inflammatory effects,antioxidant effects,antiapoptotic effects and the signaling pathways such as NF-κB and Nrf2/HO-1.However,the detailed mechamism of H2 treatment still remains unclear.Proteomic,a powerful screening technology for the global evaluation of protein expression in a particular physiological or pathological condition,is helpful to completely explain the mechanism of H2 alleviating ALI in septic mice.Herein,this study used quantitative proteomics approaches of i TRAQ labeling coupled with LC-MS/MS to explore the molecular mechanism of H2 treatment on ALI in septic mice,which might lay scientific foundation for developing the new strategy for the treatment of sepsis.Methods: Adult male ICR mice,aged 6 weeks and weighed 20-25 g,were randomly divided into four groups as follows: sham,sham+H2,sepsis and sepsis+H2.Cecal ligation and puncture(CLP)operation was performed to induce sepsis.The mice in Sh+H2 group and S+H2 group were given H2 treatment for 1 h at the 1st h and 6th h.The 7-day survival rate was observed,and at the 24 th h after operation.At the 24 th h after CLP,the mice of all groups were deeply anesthetized and killed.Lung tissue were then harvested and quickly frozen by liquid nitrogen and stored at-80 oC for i TRAQ-based quantitative proteomic approach to screen the differentially expressed proteins related to H2 protection against sepsis.Functional enrichment analysis such as GO function annotation,KEGG pathway enrichment analysis and STRING protein-protein interation network were performed.The expression level of differentially expressed proteins were measured by Western Blot to confirm the proteomic results.Results: During the 7-day survival rate observation,no mice died in sham and sham+H2 groups.However,the 7-day survival rate in sepsis and sepsis+H2 groups decreased significantly to 10% and 50%,respectively(P<0.05).After the CLP operation,the mice in sepsis and sepsis+H2 groups were observed to have several significant physiopathologic changes such as messy hair,loose stool,reduced physical activity and increased respiratory rate.Compared with sepsis group,the 7-day survival rate in septic mice significantly improved in sepsis+H2 group(P <0.05).At the 24 th h after CLP or sham operation,the mice lung tissue in each group were disposed and used for the proteomics analysis.5688 proteins were identified,of which 4472 proteins were quantifiable.Based on the pairwise comparison in four groups,a total of 192 proteins were identified and considered to be closely related to the mechanism of H2 alleviating ALI in septic mice.For the GO function annotation analysis,the top ten significantly enriched molecular function terms are transporter activity,endopeptidase regulator activity,serine-type endopeptidase inhibitor activity,oxygen binding,tetrapyrrole binding,peptidase inhibitor activity,heme binding,oxygen transporter activity and enzyme inhibitor activity.Among the top ten terms,there are five significantly enriched molecular function terms about oxygen transport.The top ten significantly enriched cellular component terms are extracellular region,extracellular region part,hemoglobin complex,extracellular matrix,sarcomere,contractile fiber part,myofibril,contractile fiber,blood microparticle and extracellular space.Among the top ten cellular component terms,there are four extracellular terms and four terms about muscle fiber.The top ten significantly enriched biological process terms are oxygen transport,tetrapyrrole metabolic process,negative regulation of hydrolase activity,heart contraction,striated muscle contraction,response to metal ion,prophyrincontaining compound metabolic process,muscle contraction and muscle system process.There are four biological process terms closely related to muscle contraction.Clusters of proteins about ribosome proteins,myosin and troponin related proteins,collagen and adhesion related proteins,coagulation related proteins were found in STRING protein-protein interaction networks.At the 24 th h after CLP or sham operation,compared with sham group,the expression levels of Sema 7A,OTULIN and MAP3K1 were increased in sepsis group and Transferrin’s expression level decreased(P<0.05).Compared with sepsis group,the expression levels of Sema 7A,OTULIN and MAP3K1 were decreased in sepsis+H2 group and Transferrin’s expression level increased(P<0.05).The expression tendency of above proteins obtained from western blot were in good agreement with our proteomics results.Conclusion: 2 % H2 inhalation can significantly improve the 7-day survival rate in septic mice.192 differentially expressed proteins including Sema 7A,TRF,OTULIN and MAP3K1 were identified by i TRAQ labeling coupled with LC-MS/MS quantitative proteomics technology.These proteins take part in biochemical metabolic processes such as oxygen transport,muscle contraction,protein synthesis,cytoskeleton,microcirculation and vascular endothelial barrier,coagulation function and signaling pathways such as PI3K-Akt signaling pathway,which maybe play key roles in the molecular mechanism of H2 treatment on ALI in septic mice.