Effect Of The Hydrogen-rich Electrolyzed Water On The Raw264.7 Macrophages And Its Polarization

Research background and Objectives:One of the important causes of implant failure is peri-implant diseases.Similar to the treatment of periodontitis,the treatment of peri-implant diseases include nonsurgical treatment and surgical treatment.Adjuvant drugs take an important role in both treatment methods.In the recoverable pathological changes of implant mucositis,cells such as macrophages increases while the connective tissue is destroyed,which indicates the close relationship between macrophage and peri-implant disease.As a medical gas,hydrogen can selectively reduce ROS.In addition,it can also participate in the regulation of inflammatory signaling pathways such as NF-κB and TLR4.In terms of oral application,it has been reported in the literatures that hydrogen can relieve inflammation by controlling gingival oxidative stress and reducing the expression of related inflammatory factors.In some animal experiments,the inhibitory effect on alveolar bone resorption can also be seen.The application of hydrogen-rich water greatly reduces the risk and technical threshold of its application.In conclusion,hydrogen-rich water may become a possible family-use biological complication for implant diseases prevention and health care.In the inflammatory regulation,macrophages have the characteristic of differentiating into Ml and M2 phenotypes and exerting their differential functions with the microenvironment under different inflammatory states.Therefore,for exploring the effect of hydrogen on macrophage and its polarization,this project intends to use hydrogen-rich water to treat macrophages in the resting state and in the induced inflammatory state,providing a certain basis for the treatment of related diseases and the application of daily health care.Material and Method:1.The effect of hydrogen-rich electrolyzed water on the cell viability of RAW264.7 macrophage cellsThe concentration titration of the prepared hydrogen-rich electrolyzed water is carried out to clarify the water quality and the optimal treated time.The effect of hydrogen-containing medium and ordinary medium on the proliferation activity of RAW264.7 macrophages was determined by CCK-8,respectively;The motility trend of RAW264.7 macrophages in the two culture media was observed by Transwell migration experiment.2.Effects of hydrogen-rich electrolyzed water on anti-inflammatory ability of RAW264.7 macrophage cellsThe control group and hydrogen-rich electrolyzed water treated group were set up.Relative mRNA expression of IL-6,TNF-α,IL-10 and CD206 was determined by qRTPCR.Induced by LPS,M1 macrophages were constructed.The control group,HEW group and LPS+HEW treated group were set up.The expression of M1(IL-6 and TNFa)and M2(IL-10,CD206)was determined by qRT-PCR and ELISA.The polarized phenotype of macrophages was detected by flow cytometry.Results:1.Determination of hydrogen-rich electrolyzed water concentration and cytotoxicityThe hydrogen concentration of unfiltered hydrogen-rich electrolyzed water decreased gradually until it was difficult to measure at 120 minutes;the initial concentration of sterile-filtered hydrogen-rich electrolyzed water was maintained at 0.8 ppm.It began to decrease gradually after 30min,and disfused at 60min.Compared with the control group,the number of cells migrated in the HEW group was more,but there was no statistical difference.2.Effect of hydrogen-rich electrolyzed water on Raw264.7 macrophages after LPSinduced polarizationAfter 1h of hydrogen-rich electrolyzed water treated,the qRT-PCR found that the relative mRNA expressions of IL-6 and iNOS decreased,the expression of TNF-α was upregulated,and the expressions of L-10 and CD206 are upregulated.The experiment successfully used 100μg/L LPS to induce Ml-type RAW264.7 macrophages after 24h.Beside,after 1 h of HEW treatment,compared with the LPS group,RT-qPCR showed that the relative mRNA expression of pro-inflammatory related IL-6 and TNF-α in the RAW264.7 macrophages in the HEW+LPS group decreased,and inhibited it of IL-10 and CD206 related to inflammation was up-regulated.ELISA showed the contents of IL-6 and TNF-α in the supernatant of the cells were decreased,and the content of IL10 was increased.Flow cytometry showed that the percentage of M1 type RAW267.4 macrophage cells decreased and the percentage of M2 type increased.Conclusions:1.Hydrogen-rich medium has a certain stimulating effect on the proliferation of RAW264.7 macrophages in vitro.2.It is the first time that hydrogen-rich electrolyzed water has been used to strengthen the anti-inflammatory effect of macrophages in a short-term treatment.And it was preliminarily explored that this ability was related to change the polarization of macrophages from M1 type to M2 type,suggesting that hydrogen can start inflammation regulation quickly.