Effects And Mechanisms Of Hydrogen Treatment Of Rheumatoid Arthritis

Background and PurposesRA（Rheumatoid arthritis）is the systemic autoimmune disease with chronic synovial inflammation as its special clinical expression.It is a disease of high morbidity–0.5-2.0%in Occident and 0.32-0.38%in China.It is,meanwhile,a disease with perplexing pathogenesisaffectedbyvariousfactorsleadingtounbalancedorganism immunoregulation and excessive inflammatory response that result in destruction of joint structure.OS（Oxidative stress）is closely related with the morbidity of RA,attaching comparatively great influence on its pathomechenism.ROS（Reactive oxygen species）and RNS（Reactive nitrogen species）,harmful free radicals in organisms generated during metabolism,are the main causes of oxidative injury of RA.OS can raise levels of such markers of oxide as MDA（malondialdehyde）,8-OHdG（8-hydroxy-2’-deoxyguanosine）and protein carbonyl,activate signaling pathways of NF-κB,and alter the redox state of GSH（glutathione）to enter nucleus by inactivating IκB（inhibitor-κB）.H₂（Hydrogen）is colorless,odorless gas extensively existing in nature.It is selectively antioxidative,so that able to neutralize ROS and RNS selectively.However,the mechanism behind its selective antioxidation remains ambiguous,so does the regulation effect of its upstream moleculars.At present,some factors are believed to play vital roles in the antioxidative stress of hydrogen,like NF-κB（Nuclear factor kappa B-κB）correlated with inflammation,MAPKs（Mitogen-activated protein kinase）participating in cell proliferation and apoptosis,and chemokines TGF-β1（Transforming growth factorβ1）reinforcing fibroblasts.Signaling pathways Keap1/Nrf2/ARE are important for organisms in antioxidative stress,a hot issue of the research field in recent years.Selective antioxidation of hydrogen are made into effect by Nrf2,whose concrete mechanism,i.e.the interaction channels between H₂ and Nrf2,is the key and difficult point in hydrogen medical research field.Hydrogen is applied into the treatment of RA for sake of its selective antioxidation.It is reported by foreign scholars that RA patients,who had received hydrogen treatment,performed possitively in DAS28（Disease activityscore-28）and blood inflammation index.Specifics like antioxidation effects of hydrogen on FLS（Fibroblast-like synoviocytes）,the proliferation and apoptosis of FLS and the influences exerted by signaling pathways of relevant signals have not been covered yet.Besides,the signaling pathways by which hydrogen affects FLS and whether or not hydrogen is related with signaling factors like MAPK and NF-κB are still unclear.Furthermore,the significant antioxidative signaling pathway of hydrogen in OS diseases,i.e.Nrf2,should be further studied for whether it participates the inhibition of RA diseases or not.Contents and MethodsWe established CIA models by multipoint hypodermic injection of type（Ⅱ）exogenous emulsive collagen into the backs and tails of mice.The models were evaluated by the morphological changes of their foots and tails.The alleviation of RA diseases by hydrogen in CIA mice models was evaluated by AIscore,morphological and pathological observation.We processed rheumatoid synovium fibroblasts MH7A with hydrogen peroxide solution to create an in-vitro OS environment on hydrogen-rich mediu.Effects of hydrogen on OS reaction of FLS were judged by observing cell proliferation,expression of antioxidative stress system（SOD and GSN）and DNA oxidative injury index.Meanwhile,we detected MAPK,NF-KB and TGF-β1 to assess MAPK pathway and the possible effects of TGF-β1 on the OS reaction of FLS.We also processed fibroblasts isolated from children’s skin cells in the same way.We observed cell proliferation,detected cell apoptosis and death by Annexin V/PI,inspected the generation of reactive oxygen by DHE（Dihydroethidium）and DCFH-DA（2’,7’-Dichlorodihydrofluorescein diacetate）,and analyzed oxidation products MDA,expression of antioxidative stress system（SOD and GSN）,DNA oxidative injury index（8-OHdG）by Elisa method.As a result,we can judge the effects of hydrogen on OS reaction of fibroblasts.In the meantime,we analyzed the expression of protein Nrf2,CATA andγ-GCS to verify the possible effects of signaling pathway Nrf2 on the OS reaction of fibroblasts.Results and ConclusionsWe got the following results in the research:1.Pathologic results denoted no indication of synovial cell proliferation,inflammatory cell infiltration,pannus formation or bone erosion of normal mice in the control group;above symptoms were obvious in the CIA model group;the treatment group processed with hydrogen peroxide solution merely expressed mild symptoms.Pathologic score showed that scores of the treatment group were much lower than that of the CIA model group.2.By visual inspection,we found that a part of mice in the CIA model group presented forefeet swelling on the 30th day（an early stage of the disease）that spread to hindfeet later.Such symptoms deteriorated to the worst on the 40th day（an interim of the disease）.The swelling in the treatment group,however,was postponed and less severe compared with that of the CIA model group.AI（Arthritis index）of mice in the CIA model group started to soar on the 14th day（i.e.an early stage of the disease）to its summit on the 21th day,then gradually fell.The time of AI rise in the treatment group was in parallel with the CIA model group,while the magnitude was more moderate apparently.3.Test results of MH7A cell viability by MTT method were as follow:Compared with the control group,cell proliferation was activated after processed by H2O2.The proliferation rate accelerated as time went by.On the contrary,cell proliferation was intensely inhibited with the interference of hydrogen.4.It was found through Elisa test method that 8-OHDG of rheumatoid synovium fibroblasts increased after hydrogen peroxide solution processing,meanwhile SOD and GSH of antioxidation system decreased.The content of 8-OHDG was diminished but that of SOD and GSH was multiplied after adding hydrogen-rich mediu.It was also found that hydrogen inhibited the expression of MAPK,NF-κB and TGF-β1.5.Fibroblasts were of high apoptosis occurrence after hydrogen peroxide solution（H2O2）processing,as was revealed by the comparison between the OS model group and the others 24 hours after the surgery.The spontaneous apoptosis was at a low level in the hydrogen group in contrast with the group processed by H2O2.6.Reactive oxygen level of fibroblasts was largely improved by H2O2 processing according to the reactive oxygen test,while that was repressed by hydrogen processing.As was denoted by Elisa test,8-OHDG and MDA increased after H2O2 processing,yet SOD and GSH decreased.Hydrogen-rich mediu could diminish the content of 8-OHDG and multiply that of SOD and GSH.7.It was found through western blot test that Nrf2 andγ-GCS as well as CATA of fibroblasts dropped after adding H2O2.On the other hand,Nrf2 andγ-GCS returned to their norms,and CATA level regained after adding hydrogen.Basing on above research results,we come to the conclusions that hydrogen can effectively inhibit RA diseases by suppressing excessive organism OS reaction.It can activate antioxidation system within organisms and retain the expression of MAPK,NF-κB and TGF-β1.It simultaneously activates the Nrf2 signaling pathway.Therefore,the mechanism by which hydrogen regulates the OS of RA is rather complicated.