Effects Of Circadian Rhythm Gene Bmal1 On The Function Of RAW264.7 Cells

Objective A plasmid vector of Bmal1 gene was constructed to transfect macrophages in vitro.ox-LDL was used to stimulate macrophages and to observe and evaluate the changes in the degree of foam,migration,inflammation and apoptosis of macrophages.Methods(1)We selected mouse macrophage RAW264.7 as the research object,divided into four groups: control group(blank control group),ox-LDL group(50 μg/mL ox-LDL intervention group),Ad-GFP + ox-LDL group(Ad-Scramble transfection for 3 days plus 50 μg/mL ox-LDL intervention group)and Ad-Bmal1 + ox-LDL group(Bmal1 adenovirus transfection for 3 days plus 50 μg/mL ox-LDL intervention group).(2)Fluorescence microscopy was used to observe the transfection efficiency of adenovirus in RAW264.7 cells.(3)Oil red O staining was used to detect the cell foam degree of RAW264.7 at different circadian rhythm time points(0 h,6 h,12 h,24 h).(4)Transwell chamber method and scratch test were used to detect the migration of RAW264.7 cells.(5)The apoptosis of RAW264.7 cells was detected by flow cytometry.(6)The expressions of Bmal1、IL6、IL10、Bax、Mcl1 were measured by q RT-PCR.(7)The expressions of Caspase3、Caspase9 were measured by western blot.Results(1)The green fluorescent protein(GFP)was expressed in Ad-GFP+ox-LDL group and Ad-Bmal1+ox-LDL group under fluorescence microscope,which indicated that the transfection efficiency of recombinant adenovirus in vitro was high;After 72 hours of Ad-Bmal1 transfection,the expression of Bmal1 m RNA increased significantly,suggesting that Ad-Bmal1 can effectively induce overexpression of Bmal1.(2)Oil red O staining showed that obvious lipid droplets were observed in the cells after stimulation of ox-LDL for 6,12 and 24 hours,the area of lipid droplets was maximal at 24 h.Compared with 0 h and 6 h groups,the area of intracellular lipid droplets in 12 h and 24 h groups increased(P<0.05).(3)Transwell chamber method and scratch test showed that 50 μg/mL ox-LDL could stimulate normal cells to migrate obviously,but Ad-GFP and Ad-Bmal1 had no obvious inhibition on migration.(4)Flow cytometry showed that the percentage of apoptotic cells in ox-LDL group was higher than that in Control group(P<0.05).However,compared with the Ad-GFP+ ox-LDL group,the apoptosis rate of cells were decreased in the Ad-Bmal1+ ox-LDL group(P<0.05).(5)Stimulation at ox-LDL for 12 hours,compared with the Ad-GFP+ ox-LDL group,the m RNA expression level of IL6 were decreased in the Ad-Bmal1+ ox-LDL group,while the m RNA expression level of IL10 were increased(P<0.05).(6)Stimulation at ox-LDL for 24 hours,compared with the Ad-GFP+ ox-LDL group,the m RNA expression level of Bax were decreased in the Ad-Bmal1+ ox-LDL group,while the m RNA expression level of Mcl1 were increased(P<0.05).(7)Stimulation at ox-LDL for 6,12 hours,,compared with the Ad-GFP+ ox-LDL group,the protein expression levels of pre-caspase3 and pre-caspase9 were decreased in the Ad-Bmal1+ ox-LDL group(P<0.05).(8)Stimulation at ox-LDL for 6,12,24 hours,compared with the Ad-GFP+ ox-LDL group,the protein expression level of cleaved caspase-3 were decreased in cells of the Ad-Bmal1+ ox-LDL group(P<0.05).Conclusion The circadian rhythm gene Bmal1 can inhibit the inflammatory response and apoptotic effect of macrophages stimulated by ox-LDL,and provide experimental basis for exploring the role of circadian rhythm in atherosclerosis.