Research On Regulation And Mechanism Of Hydrogen On Lps-induced In Inflammatory Polarization Of Mouse Bone Marrow Derived Macrophages

ObjectAtherosclerosis（AS）is a crucial pathological basis of cardiovascular and cerebro-vascular diseases.Inflammation is the main reason for plaque rupture.Macrophages,as the main cells of innate immune response and the main source of inflammatory factors,are the main components in the development of atherosclerosis.Previous studies have shown that the classical activated M1 macrophages mainly exist in progressive plaques,while the alternative activated M2 macrophages dominate in the plaques that tend to be stable.Hydr-ogen has prominant anti-inflammatory and antioxidant effect.Our research group has pr-eviously proved that hydrogen-rich saline can reduce the susceptibility of atherosclerotic plaques and increase their stability,yet the specific mechanism has not been clarified.This topic mainly explores the effect of hydrogen on M1 macrophages polarization in athero-sclerosis and its possible molecular mechanism.Materials and MethodAnimal:Apo E knockout(Apo E^-/-)mice bought from Vital River Company,Beijing,China.Wild type（WT）mice were obtained from the Atherosclerosis Institute of Shandong First Medical University.All experimental mice were C57BL/6 genetic background.Exp-erimental animals were housed in a temperature and humidity controlled room with a 12/12h light-dark cycle.Eight-week-age male mice born in the same litter with similar weight were used in this experiment.All experiments were approved by the Experimental Animal Ethics Committee of Shandong First Medical University and followed the National Animal Care and Use Guidelines.Animal experiments:The relationship between effect of hydrogen on aortic root pla-que area,lipid deposition and macrophages polarization in aortic root plaque was inve-stigated.The hydrogen-rich saline group includes Apo E^-/-fed with a high-fat diet and intra-peritoneally injected with hydrogen-rich saline daily.WT mice were fed with a regular ch-ow diet and normal saline was injected as a control group.Apo E^-/-mice fed with a high-fat diet and injected with normal saline was used as a model group.After 8 weeks of experi-mentation,frozen section were prepared using the heart tissues of the above mice,HE stai-ning and oil red O staining were conducted to analyze the aortic root plaque changes.Indu-cible nitric oxide synthase（i NOS）was used as a M1 type macrophage marker and Arg-inase 1（Arg-1）was used as a M2 type marker to analyze macrophage polarization.Cell experiments:Wild type mice of 8-12 week-old were selected to isolate bone marrow-derived macrophage（BMDM）.The BMDM of model group was induced by lipo-polysaccharide（LPS）at a final concentration of 100 ng/ml in DMEM for 12 hours.The B-MDM of hydrogen-rich medium group was pre-protected with hydrogen-rich DMEM for 3hours,and then 100 ng/ml of LPS was added and incubate for 12 hours.The control group was added with the same volume of phosphate buffer solution（PBS）as the LPS in the mo-del group in DMEM.Inflammatory factors including Toll-like receptor 4（TLR4）,signal tr-ansducer and activator of transcription 3（STAT3）,phosphorylated signal transducer and a-ctivator of transcription 3（P-STAT3）,suppressor of cytokine signaling 3（SOCS3）,nuclear factor kappa B（NF-κB）,nuclear factor kappa B inhibitory protein alpha（Inhibitor of NF-κB-α,IκB-α）,phosphorylated inhibitor of NF-κB-α（P-IκB-α）and cysteinyl aspartate spe-cific proteinase-1（Caspase-1）were detected by western blot（WB）.The expression of in-terleukin-1β（IL-1β）in the medium after incubation of three groups of cells were detected by enzyme-linked immunosorbent assay（ELISA）.Statistical analysis:Statistical analysis was performed using Graph Pad Prism 5 so-ftware after 3 to 4 replicates of each experiment.Data were usually expressed as mean±s-tandard error,and data between groups were analyzed by t test.The difference was stati-stically significant when P<0.05.The results（1）Animal experiment:Compared with the control group,the model group accu-mulated a large number of foam cells in the aortic root and protruded into the lumen.Co-mpared with the model group,the hydrogen-rich saline group contains reduced foam cells and lipid accumulation.Compared with the model group,the number of M1 macrophages decreased,and the number of M2 macrophages increased in the hydrogen-rich saline group.（2）Cell experiments:After stimulation of BMDM into M1 macrophages after LPS in-cubation,total protein,membrane protein and nuclear protein were extracted respectively,and TLR4,STAT3,SOCS3,p-IκB-α,caspase-1,NF-κB,and p-STAT3 were detected.The WB results showed that the expression levels of above proteins in model group increased,and the those of the hydrogen-rich medium group decreased.After LPS induction,IκB-αwas detected by total protein extraction.The results of WB showed that the expression of IκB-αdecreased in the model group,and the expression of the hydrogen-rich medium gro-up increased.Cell medium after intervention of all groups were collected,and the supe-rnatant was centrifuged for ELISA.After analyzing the data,we found that IL-1βsecretion of model group was significantly up-regulated compared with the control group,and the h-ydrogen-rich medium group declined compared with the model group.Conclusion（1）Daily hydrogen-rich saline injection can reduce plaque area and lipid deposition in the aortic root of Apo E^-/-mice,which may results from hydrogen’s anti-inflammatory cap-acity to down-regulate M1 macrophages and up-regulate M2 macrophages of hydrogen molecules;（2）Hydrogen-rich medium reduced the expression of LPS-induced TLR4,downrgu-lated the expression of downstream signaling molecules such as p-STAT3,SOCS3 and ca-spase-1,inhibited the nuclear translocation of NF-κB,and further inhibited the polarization of LPS-induced M1 macrophages.