Protective Effect And Mechanism Of Hydrogen In Oxidative Stress Insulted Enterocytes

Objective: To determine the protective effect and the molecular mechanism of hydrogen in oxidative stress insulted enterocytes.As a result, the current study would provide a new preventive approach and therapeutic targets for the prevention of NEC.Methods: We used human and rat intestinal epithelial cell lines Caco-2 and IEC-6 cells as the objects of study.The cells were divided as 3 groups on the basis of whether dealing with antimycin A and hydrogen: antimycin A(-)/ H2(-);antimycin A(+)/ H2(-);antimycin A(+)/ H2(+),which were control group(antimycin A 0?g/ml,H2 0m M), antimycin A treated group(antimycin A 30?g/ml,H2 0m M)and hydrogen treated group(antimycin A 30?g/ml,H2 0.6m M). We used Western Blot?Real-time PCR?FCM(flow cytometry)?ELISA(enzyme-linked immunosorbent assay)in the intestinal epithelial cell oxidative stress model to test the following indicators.(1) Levels of O2-??H2O2 and ?OH;(2) Mitochondrial membrane potential;(3) MDA and 8-OH-G;(4) Cell apoptoiss and LDH activity;(5) Proinflammatory factors(IL-6?TNF-??IL-1?)?anti-inflammatory factors(IL-10),expressions of Nrf2 and the downstream target genes(HO-1?SOD?Cat?GPx);(6) Expression of phosphorylation-Nrf2 were detected for further study.Results: Compared with the antimycin A treated groups,we found that hydrogen can significantly reduce the oxidative damage induced by antimycin A and the level of oxygen specis(the contents of O2-??H2O2 and?OH were respectively antimycin A treated groups' 0.236 times,0.456 times and 0.124 times),recover the mitochondrial membrane potential(MMP), reduce the contents of oxidative metabolites in the cells,and the cell apoptosis rate(control group 5.8%, antimycin A treated group 60.1±5.89%, hydrogen treated group 23.7±3.48%)and lactate dehydrogenase(LDH) activity in cells also decreased. Besides, hydrogen could reduce the cell apoptosis rate and the activity of LDH in oxidative stress insulted enterocytes induced by menadione and Fenton reaction.At last,hydrogen treatment increased the level of the phosphorylation of transcription factor Nrf2.And compared with the antimycin A treated groups, hydrogen treatment reduced the contents of inflammatory factors significantly. In the antimycin A group, the expression of Nrf2 and its downstream genes, HO-1, SOD, Cat, GPx was increased, and hydrogen treatment significantly increased the expression.Conclusions: The hydrogen can reduce the oxidative damage of Caco-2 cells induced by antimycin A, characterized by clearing the content of free radicals in cells, repair the oxidative damage of mitochondrial membrane potential, reduce the oxidation of metabolites, inhibit apoptosis and decrease cell lactate dehydrogenase activity, the protection functions may realized through the Nrf2-ARE signaling pathway.