The Protective Effects And Mechanism Research Of Hydrogen Gas On Intestinal Barrier Dysfunction In Severe Septic Mice

Background:Sepsis is the systemic inflammatory response syndrome caused by infectious or highly suspicious infectious factors, is the common but lethal complication of severe trauma、burn、inflammation、shock and critical surgery, about 50% septic patient could progress to severe sepsis and septic shock along with organ injury and evoke irreformable circulatory failure and MODS/MOF, which make 50% above of clinical septic patients die. Intestine is one of the easily affected organs in the development of sepsis, also the motor organ in septic progression, intestinal epithelial injury induced intestinal barrier dysfunction plays an important role in the SIRS-Sepsis-MODS course, therefore, the protection of the intestinal barrier function is of great clinical importance to sepsis treatment and prognosis. Rho protein and its main downstream effectors, Rho related coiled-coil protein kinase(ROCK) and mammalian diaphanous related formin 1(m Dia1) play an important role on the maintaining of intestinal normal cellular morphology, polarity and epithelial junctions, moreover, Rho protein signaling pathway also plays a key role on the regulation of intestinal barrier function in sepsis. As a new type of medical gas, hydrogen has anti-inflammation,anti-apoptosis and signaling pathways regulating role, besides of characteristized selectively antioxidant effect, which make hydrogen become a new direction of sepsis basic research.Our previous studies have found that hydrogen gas inhalation has concentration- and time-dependent effects in the treatment of sepsis, 2% hydrogen gas inhalation could obviously increase the survival rate and alleviate the lung、kidney、liver and other important internal organs injury in severe septic mice. Cecal ligation and puncture(CLP) was employed in this subject to reproduce severe septic mice model, the protective role of hydrogen gas on intestinal barrier dysfunction was observed and the key role of Rho-ROCK/m Dia signaling pathway in the hydrogen treatment on intestinal barrier dysfunction was preliminary discussed in severe sepsis, which aimed to support strong theoretical basis for application of hydrogen gas in clinical treatment of sepsis and open a new field of vision of the biological effects of hydrogen.severe septic mice Objective: To observe the effects of hydrogen gas inhalation on the survival rate and intestinal injury in severe septic mice, and expound the hydrogen’s protective effects on intestinal injury based on inflammatory response、oxidative stress and apoptosis. Methods: Adult male ICR mice, aged 6~8 weeks and weighed 20 to 25 g, were randomly divided into 4 groups: Sham group, Sham+hydrogen inhalation group(Hydrogen control group),severe sepsis group(Model group) and severe sepsis+hydrogen inhalation group(Hydrogen treatment group). Severe sepsis was reproduced by CLP. Mice in Hydrogen control group and Hydrogen treatment group were received inhalation of 2% hydrogen for 1 h at 1 h and 6 h after sham or CLP operation, respectively. The 7 d survival rate of mice in four groups was observed. The ileum tissues were obtained to evaluate the intestinal histopathological changes and scores by HE staining, to detect the intestinal proinflammatory cytokines(TNF-α、IL-6、HMGB1)、anti-inflammatory cytokine(IL-10) level,myeloperoxidase( MPO) activity,antioxidases(SOD、CAT) activity, oxidative products(MDA、8-iso-PGF2α) content and active caspase3 activity at 12h、24h and 48 h after sham or CLP operation. Results:1.Hydrogen inhalation treatment obviously increased the 7-day survival rate of severe septic mice. 2. Hydrogen inhalation treatment obviously alleviated intestinal histopathological injury and decreased the intestinal histopathological scores in severe septic mice at different times after CLP operation. 3.Hydrogen inhalation treatment significantly decreased the intestinal TNF-α、IL-6 and HMGB1 level, MDA、8-iso-PGF2α concentration and MPO、active caspase3 activity, also increased the intestinal IL-10 level and SOD、CAT activity in severe septic mice at different times after CLP operation. Conclusion: Hydrogen inhalation treatment could effectively improve the intestinal injury and increase the survival rate of severe septic mice, and its protective effect may due to the hydrogen’s ability to inhibit intestinal inflammatory response、oxidative stress and apoptosis. PART 1:Protective effects of hydrogen gas inhalation on intestinal injury inPART 2:The influences and mechanisms of hydrogen inhalation on intestinal epithelial barrier function in severe septic mice Objective: Rho protein is an important middle signaling molecule in the process ofintracellular signal transduction, Rho protein and its downstream effectors could regulate the intercellular tight juntion and adherens junction to modulate intestinal epithelial barrier function and permeability. This study was designed to prove the improved effects of hydrogen inhalation treatment on intestinal barrier dysfunction in severe septic mice and detect the effect of hydrogen gas on the activity of intestinal Rho protein signaling pathway. Methods: Adult male ICR mice, aged 6~8 week and weighed 20 to 25 g, were randomly divided into 4 groups: Sham group, Sham+hydrogen inhalation group(Hydrogen control group),severe sepsis group(Model group) and severe sepsis+hydrogen inhalation group(Hydrogen treatment group). Severe sepsis was reproduced by CLP and mice in Hydrogen control group and Hydrogen treatment group were received inhalation of 2% hydrogen for 1 h at 1 h and 6 h after sham or CLP operation, respectively. At 24 h after sham or CLP operation, the followed items were done to evaluate the intestinal barrier function: the FITC-dextran passing rate was measured to evaluate intestinal permeability; the blood、liver、spleen and kidney were obtained to bacterial culturing to test bacterial translocation; the ultrastructure of intestinal epithelial cell and intercellular junctions were observed by using transmission electron microscope(TEM);the expression and distribution of intestinal epithelial tight junction proteins and adherens junction proteins were detected by Western blot and immunofluorescence. At 24 h after sham or CLP operation, the ileum tissues were obtained to evaluate the intestinal Rho protein activity by GST Pull-Down assay and the expression and distribution of intestinal ROCK1 and m Dia1 were detected by immunohistochemistry. Results:1.Hydrogen inhalation treatment could obviously decreased the serum FITC-dextran concentration、the colony-forming unit counts in bacterial culturing medium of blood、liver、spleen and kidney in severe septic mice and obviously improved the ultrastructure histopathological injury of intestinal epithelia and intercellular junctions, also the expression and distribution of intestinal tight junction proteins and adherens junction proteins. 2.Hydrogen inhalation treatment significantly decreased the intestinal Rho activity and ROCK1 expression, and obviously up-regulated the intestinal m Dia1 expression.Conclusion: Hydrogen inhalation treatment could effectively improved the intestinal barrier dysfunction of severe septic mice, the mechanism maybe related to the hydrogen’s ability to regulate intestinal Rho protein signaling pathway to improved the intercellular junctions of intestinal epithelia.PART 3:The mechanisms of Rho protein signaling pathway in hydrogen treatment on intestinal barrier dysfunction in severe septic mice Objective: ROCK and m Dia both involved in the reorganization of actin cytoskeleton, which may play different even opposing role on maintaining normal intestinal epithelial form、polarity and intercellular junctions. This study was designed to investigate the detailed effects of Rho protein and its downstream effectors ROCK1 and m Dia1 in the hydrogen gas treatment on intestinal barrier dysfunction in severe septic mice. Methods: Adult male ICR mice, aged 6~8 week and weighed 20 to 25 g, were randomly divided into 4 groups: severe sepsis group(Model group), severe sepsis +Rho inhibitor group(Rho inhibitor group),severe sepsis+ROCK inhibitor group(ROCK inhibitor group) and severe sepsis+hydrogen inhalation group(Hydrogen treatment group). Severe sepsis was reproduced by CLP.Mice in Rho inhibitor group and ROCK inhibitor group were received ip.100μg/kg C3 exoenzyme and 5mg/kg Y-27632,respectively, and 5m L/100 g of normal saline were used in Model group and Hydrogen treatment group at the same time. Mice in Hydrogen treatment group were received inhalation of 2% hydrogen for 1 h at 1 h and 6 h afte CLP operation. The 7-day survival rate of mice in four groups was observed. At 24 h after CLP operation, the intestinal TNF-α、IL-6、HMGB1 and IL-10 level, MPO、SOD、CAT and active caspase3 activity and MDA、8-iso-PGF2α content were measured. At 24 h after CLP operation,the FITC-dextran passing rate was measured; the blood、liver、spleen and kidney were obtained to bacterial culturing to test bacterial translocation. At 24 h after CLP operation, the ileum tissues were obtained to evaluate the intestinal histopathological changes and scores by HE staining; the ultrastructure of intestinal epithelial cell and intercellular junctions were observed by using TEM; the expression and distribution of intestinal epithelial tight junction proteins and adherens junction proteins were detected by Western blot andimmunofluorescence. On the basis of experimental grouping, the expression of intestinal ROCK1 and m Dia1 were detected by Western blot. Results:1.The 7-day survival rate of mice in Model group and Rho inhibitor group was 0;the 7-day survival rate in ROCK inhibitor group and Hydrogen treatment group were 40% and 50%,respectively. 2. ROCK inhibitor and Hydrogen inhalation treatment could significantly decrease the intestinal proinflammatory cytokines TNF-α、IL-6、HMGB1 level, oxidative products MDA、8-iso-PGF2α content and the intestinal MPO and active caspase3 activity, and increase the activity of SOD and CAT in severe septic mice at 24 h after CLP operation,moreover, the intestinal anti-inflammatory cytokine IL-10 level was increased in Hydrogen treatment group; Rho inhibitor made no difference on aboved indexes in severe septic mice. 3. ROCK inhibitor and Hydrogen treatment could significantly decrease the serum FITC-dextran concentration, the colony-forming unit counts in bacterial culturing medium of blood、liver、spleen and kidney, and improved intestinal injury、histopathological scores, ultrastructure changes and the expression and distribution of intestinal tight junction proteins and adherens junction proteins in severe septic mice at 24 h after CLP operation; Rho inhibitor made no difference on aboved intestinal barrier functional indexes in severe septic mice. 4. ROCK inhibitor and Hydrogen treatment could significantly down-regulated the expression of intestinal ROCK1 expression and up-regulated the expression of intestinal m Dia1 in severe septic mice at 24 h after CLP operation; Rho inhibitor treatment obviously down-regulated the intestinal ROCK1 and m Dia1 expression in severe septic mice. Conclusion: Hydrogen inhalation treatment could up-regulate m Dia1 expression and down-regulate ROCK1 expression to regulate Rho protein signaling pathway to improve intestinal barrier dysfunction, sequentially inhibit intestinal inflammatory response、oxidative stress and apoptosis to ameliorate intestinal pathological injury, which ultimately may improve the septic process and increase the survival rate of mice with severe sepsis.Summary 1. 2% hydrogen inhalation treatment could regulate intestinal Rho protein signaling pathway to improve intestinal epithelial barrier dysfunction in severe septic mice, thedetailed mechanism is that hydrogen gas could enhance the stability of intestinal intercellular junctions to improve intestinal barrier dysfunction through up-regulating m Dia1 expression and down-regulating ROCK1 expression in severe septic mice, sequentially inhibit intestinal inflammatory response、oxidative stress and apoptosis, which may ameliorate intestinal pathological injury and improve the septic process and prognosis. 2.2% hydrogen treatment could exert curative effect and have no significant side effects in the process of sepsis treatment in this study, supporting that hydrogen gas might be potentially a clinically feasible approach for sepsis with high security.