Effects Of Hydrogen-rich Medium On Inflammation Induced By Lipopolysaccharide In Raw264.7Macrophages

Objective:Sepsis as a systemic inflammatory response syndrome was the common complication of burns, trauma and major surgery and also the leading cause of deaths in intensive care patients. While LPS, the main pathogen to induce sepsis, was the major component of gram-negative bacteria. And it’s a potent activator for immune cells (e.g macrophages) in pathogenic bacteria and could provoke a series of pathophysiological responses. Macrophages as the recognizer and the first defense line against the noxious stimuli would play a vital role in the body’s inflammatory response to injury or infection through adjusting activity and release of inflammatory mediator both within and outside cells. Hydrogen was the smallest and simplest molecule but the richest element in nature. It’s a kind of diatomic gas which was colorless, odorless, and tasteless, and certainly had some kind of reducibility. In recent years, hydrogen is shown to have the effects such as anti-oxidation, anti-inflammation and anti-apoptosis in many researches, and has the therapeutic effects in various illnesses.On the basic of CLP model of sepsis, we found H2exerts anti-inflammatory effect, but the mechanism is not clear. In our study, we want to explore and validate the protective effect of hydrogen-rich medium on release of inflammatory factors in Raw264.7macrophages stimulated by LPS.Methods:The Raw264.7murine macrophages were cultured with DMEM medium containing10%FBS, and seeded in6or96-well plates, at3mL/well or200μL/well and2×106cell/mL.Part1:The effect of H2-rich medium on cell viability and apoptosis. The part experiment was divided into four groups:control group; control+H2group; LPS group and LPS+H2group.The macrophages were cultured for24h, it’s changed into normal or different concentration (0.15mmol/L;0.30mmol/L;0.60mmol/L) H2-rich medium to continue to cultute, at the same time LPS(1μg/mL) or same dose PBS was added into medium for another24h, cell viability was processed with MTT, apoptosis were assayed by Annexin V/PI.Part2:The effect of H2-rich medium on inflammatory response in Raw264.7macrophages induced by LPS. the groups were dividedas the part1:c After cultured for24h, cell medium were collected to detect the release of inflammatory factors, including TNF-α, IL-1β, HMGB1and IL-10. Also at different time point (Oh,3h,6h,12h and24h), on basic of0.60mmol/L H2medium, cell medium were collected to detect the release of above inflammatory factors.Part3:The effect of HO-1that H2-rich medium regulated inflammatory response in Raw264.7macrophages induced by LPS.the groups were divided as part1, but the LPS+H2group was cultured only by the concentration of0.6mmol/L. At different time point (0h,3h,6h,12h and24h), cells were harvested to investigate the HO-1activity and protein by western blotting. Based on ZnPP-Ⅸ(20μmol/L),which inhibited HO-1activity, the inflammatory factors were detected by ELISA to observe the effect of HO-1on inflammatory response.Results:Part1:Hydrogen had no harmful effect on macrophages by detecting apoptosis and MTT (P>0.05). LPS induced excessive macrophages apoptosis. But after hydrogen treatment, cell viability was improved (P<0.05) and showed a dose-dependent effect, while apoptosis were ameliorated (P<0.05).Part2:LPS induced increased inflammatory factors release, the peak of pro-inflammatory factors TNF-α and IL-1β release was appeared at6h, which anti-inflammatory factors IL-10and late pro-inflammatory factors was24h. After the treatment of hydrogen-rich medium at the concentration of0.15mmol/L, the release of inflammatory factors was decreased with no statistical differences (P>0.05). At the concentration of0.3mmol/L or0.6mmol/L, the statistical differences appeared (P<0.05).Part3:LPS induced HO-1expression and activity in macrophages, compared with Con group (P<0.05). H2improved the expression and activity of HO-1, which was time-dependent. ZnPP-IX completely reversed the therapeutic effect of H2, it’s shown that Znpp-IX inhibited anti-inflammatory role of H2.Conclusion:Hydrogen-rich medium promoted cell viability, reduced cell apoptosis; H2improved inflammatory response by inhibiting excessive release of pro-inflammatory factors and further increasing anti-inflammatory factor release in a concentration-dependent manner; hydrogen-rich medium regulated the abnormal inflammatory response by improving HO-1activity and protein expression.