Effects And Mechanisms Of Hydrogen-Rich Medium Modulating Lipopolysaccharide-Induced Human Intestinal Epithelial Barrier Dysfunction In Vitro

Objective: Sepsis is characterized by a severe inflammatory response to infection. The gastrointestinal epithelium forms the boundary between the body and external environment and provides a selective permeable barrier achieved by tight junction(TJ) and adherens junction(AJ) structures. Recently, H2 has been demonstrated to effectively ameliorate intestinal injury and MODS in septic mice. The purpose of this study aimed at researching the effects of molecular hydrogen on endotoxin-induced intestinal barrier dysfunction in vitro and the mechanisms involved.Methods: Caco2 cells were cultured to passage 28-35 in culture medium. Part 1: To establish intestinal epithelial models in vitro. At first, cells were treated with LPS in different concentrations(1, 10,100 ?g/ml and 1 mg/ml) for different time points(0, 3, 6, 12, 24, 48 h) to measure the transepithelial electrical resistance(TER) and FITC-dextran flux(PE). Then Caco2 cells treated 100 ?g/ml or 1 mg/ml LPS were measured by CCK-8 assay to detect cell viability. After these, Cells were divided into 4 groups: control group, hydrogen-rich medium(H2) group, LPS group and LPS + H2 group. Cells were cultured in hydrogen-rich medium in H2 group and LPS + H2 group. Meanwhile, 100 ?g/ml LPS was simultaneously added into LPS group and LPS + H2 group. TER and PE from each group were measured at 24 h; The expression levels of occludin and E-cadherin were respectively detected at 6, 12 and 24 h Western blot analysis; The morphological structures of occludin and E-cadherin were observed at 24 h by immunofluorescence staining. Part 2: The levels of activated Ras homolog family member A(Rho A) in each group of Part 1 were measured at 24 h by GST pull-down assay. Cells were randomly divided into 5 groups: control group, LPS group, LPS + H2 group, LPS + H2 + CN03 group and LPS + C3 exoenzyme(LPS + C3) group. The treatment methods of control group, LPS group and LPS + H2 group were in line with Experiment One. 2.5?g/ml C3 exoenzyme was added to LPS + C3 group 1 h before incubation, 1 ?g/ml CN03 added to LPS + H2 + CN03 group 3 h before incubation. TER and PE as well as expressions and structures of occludin and E-cadherin in each group were measured.Part 3: Expressions of Mammalian diaphanous-related formin1(m Dia1) in each group of Part 1 were detected at 24 h. Caco2 were transfected to establish si RNA m Dia1 mutant. Cells were divided into 5 groups: control group, LPS group, si RNA m Dia1 group, LPS + H2 group, si RNA m Dia1 + LPS + H2 group. The treatment of control group, LPS group, LPS+H2 group were in line with Part 1. TER, PE and expressions and structures of occludin and E-cadherin in each group were measured.Results: Part 1: 100 ?g/ml and 1 mg/ml LPS changed TER and PE. 100 ?g/ml LPS caused no cytotoxicity. Compared with control group, LPS group showed that besides the increased PE, others were all decreased; the structures of occludin and E-cadherin were disrupted. Compared with LPS group, LPS + H2 group showed that, besides the decreased PE, others were all dropped. Part 2: LPS activated Rho A and H2 lightened its activity. Compared with LPS group, LPS + H2 group and LPS + C3 group showed that, besides the decreased PE, others were all dropped; the disrupted structures of occludin and E-cadherin were partly reversed. Compared with LPS + H2 group, LPS + H2 + CN03 group showed that, besides the increased PE, others were all decreased; the structures of occludin and E-cadherin were disrupted. Part 3: LPS upregulated m Dia1 and H2 downregulated m Dia1. Compared with control group, LPS group and si RNA m Dia1 group showed that, besides the increased PE, others were all decreased; the structures of occludin and E-cadherin were disrupted. Compared with LPS + H2 group, LPS + H2 + CN03 group showed that, besides the increased PE, others were all decreased; the structures of occludin and E-cadherin were disrupted.Conclusion: Hydrogen-rich medium can attenuate LPS-causedgut barrier dysfunction in vitro via regulating Rho A-m Dia1 signaling, thereby lightening the down-regulated expressions and reversing disrupted structure changes of occludin and E-cadherin.