Mechanism Of Histone Methylation Imbalance Affecting Macrophage Migration Via NF-κB Pathway

ObjectiveTo observe the expression of histone H3 lysine trimethylation at position 27（H3K27me3）in an oxidized low density lipoprotein（oxLDL）exposed macrophage（Raw264.7）model,and to evaluate the expression by cellular function Gain and deletion experiments to explore the specific mechanism of H3K27me3 affecting macrophage migration through the nuclear factor kappa-B（NF-κB）pathway at the level of gene epigenetics,and to clarify gene epigenetics—Histone A The regulation mechanism of basalization imbalance on atherosclerosis（AS）provides a new experimental basis and data support for the early detection and targeted therapy of AS.MethodThe passaged Raw264.7 macrophages were grouped and cultured in a cell incubator at 37 degrees Celsius（degrees celsius,℃）and 5%carbon dioxide（CO2）.The final concentration of oxLDL exposed to macrophages was 30 mg/L,and a macrophage inflammatory migration model was constructed.Experimental grouping and drug intervention:group A,control group:macrophages were cultured for 40 hours without any treatment after passage（hour,h）;group B,macrophages+oxLDL group:macrophages were cultured for 16 hours,and the medium was replaced,and then added 30 mg/L oxLDL for 24 hours;group C,macrophages+histone demethylase inhibitor（GSKJ4）+oxLDL group:macrophages were pretreated with 10μmol/L GSKJ4 for 16 hours,and then the medium was changed,and then added 30 mg/L oxLDL for 24 hours;group D,macrophages+histone methylase inhibitor（GSK126）+oxLDL group:macrophages were pretreated with 10μmol/L GSK126 for 16 hours,the medium was changed,and then Add 30mg/L oxLDL and continue to culture for 24 h;group E,macrophages+phorbol ester（NF-κB stimulant）+oxLDL group:macrophages were pretreated with 5μmol/L phorbol ester for 16 h,and the medium was fresher,and then added 30 mg/L oxLDL for 24 hours;group F:macrophages+BAY 11-7082（NF-κB inhibitor）+oxLDL group:macrophages were pretreated with 5μmol/L BAY 11-7082 for 16 hours,and the culture was replaced basal,and then added 30 mg/L oxLDL to continue the culture for 24 h.Transwell assay was used to detect the migration ability of macrophages.The protein expressions of H3K27me3,NF-κB and interleukin-6（IL-6）in macrophages were detected by western blot（WB）.Result1.Growth of macrophagesThe macrophages were subcultured at 1:2-1:4,and the subcultured cells were grouped for intervention.Under the light microscope,the macrophages adhered to the wall and grew at a uniform density,indicating that the growth was in good condition and the macrophage model was successfully prepared.2.Expression of related proteins and migration of macrophages in cells of each group（1）Compared with group A,the migration of macrophages in group B was increased,and the relative expressions of NF-κB and IL-6 proteins were increased in group B,and the difference was statistically significant（P<0.01）.（2）Compared Compared with group A,the ratio of H3K27me3/H3 in group B was decreased,and the difference was statistically significant（P<0.01）,and the relative expression of H3K27me3 and NF-κB protein in group B was negatively correlated,Pearson r~2=0.8017,the difference was significant Statistical significance（P<0.01）;the relative expression of H3K27me3 and IL-6 protein was negatively correlated,Pearson r~2=0.8393,the difference was statistically significant（P<0.01）.（3）Compared with group A,the ratio of H3K27me3/H3 in group B decreased,the relative expression of NF-κB and IL-6 proteins increased,and the migration of macrophages increased,and the differences were statistically significant（P<0.01）.,the ratio of H3K27me3/H3 in group C increased,the relative expression of NF-κB and IL-6 proteins decreased,and the migration of macrophages decreased,and the difference was statistically significant（P<0.01）.The H3 ratio decreased,the protein expressions of NF-κB and IL-6 increased,and the migration of macrophages increased,and the differences were statistically significant（P<0.01）.（4）Compared with group A,the ratio of H3K27me3/H3 in group B decreased,the relative expression of NF-κB and IL-6 proteins increased,and the migration of macrophages increased,and the differences were statistically significant（P<0.01）.,the ratio of H3K27me3/H3 in group E did not change significantly,and the difference was not statistically significant（P>0.05）.Compared with group B,the ratio of H3K27me3/H3 in group F did not change significantly,and the difference was not statistically significant（P>0.05）.The difference was statistically significant（P<0.01）.Conclusion1.AS is a chronic inflammatory disease,and macrophages are the most important inflammatory cells involved in the inflammatory response.Ox LDL promotes the migration of macrophages and increases the expression of inflammatory factors NF-κB and IL-6,which are important mechanisms for the occurrence and development of AS.2.In the oxLDL-exposed macrophage inflammatory migration model,the expression of H3K27me3 protein was decreased,and the protein expression of NF-κB and IL-6 was increased,and H3K27me3 was negatively correlated with the protein expression of NF-κB and IL-6.It is suggested that epigenetics represented by histone methylation is involved in the regulation of AS,and the reduction of H3K27me3expression may be a key factor in triggering the inflammation and migration of macrophages.3.Up-regulation of H3K27me3 protein expression can lead to decreased NF-κB and IL-6 protein expression,and reduced macrophage migration;conversely,down-regulation of H3K27me3 protein expression level can cause increased NF-κB and IL-6 protein expression and macrophage migration.Increase.It was confirmed that H3K27me3 has a protective effect on AS,which may inhibit the transcriptional expression of downstream inflammatory factor genes at the gene transcription level through gene epigenetics,reduce macrophage migration,and thus delay the progression of AS.4.Up-or down-regulation of NF-κB protein expression did not alter H3K27me3protein expression,but enhanced or attenuated inflammatory factor expression and macrophage migration.It is suggested that H3K27me3 improves inflammation and affects the migration of macrophages through NF-κB pathway.5.This study provides new experimental basis and data support for the early detection and targeted therapy of AS.Histone methylation modification may become a potential target for early detection and prevention of diseases.