| Objective:It is now well-accepted that macrophage are essential components of atherosclerosis progress.Monocytes are released into the peripheral blood from the bone marrow,and then recruited to the lesion where they differentiate into macrophages subsequently.Macrophage has many subtypes,different subtypes play different role in the process of atherosclerosis.Yang xin shi is a compound preparation composed of many kinds of traditional Chinese medicine,which may interfere with the process of atherosclerosis from multi-targets,and is used to treat coronary heart disease.To provide experimental evidence for revealing the mechanism of Yang xin shi for prevention and treatment in coronary heart disease,we cultured human THP-1 macrophage with treated by Yang xin shi.We suspected that its anti-inflammation effect in atherosclerosis maybe through affecting the inflammatory activation and phenotype of macrophage.Methods:Cultured cells The THP-1 cells were cultured in RPMI 1640 medium suppl emented with 10%heat-inactivated fetal bovine serum and incubated at 37? and 5%C02.These human THP-1 cells stimulated by 160 nmol/L phorbol ester(P MA)for 24 hours to transform into macrophages.We treated human THP-1 ma crophages with different concentrations of ox-LDL(0mg/L,10mg/L,25 mg/L,50 mg/L)and hours(0h,8h,16h,24h),then detected MIF and MCP-1 in ce ll supernatant by ELISA,and the content of CD16,CD68 were tested in the cel 1 fluid by flow cytometry.We treated human THP-1 macrophages with different concentrations of Yang xin shi,then we stimulated these cells by ox-LDL and d etected MIF,MCP-1,CD 16 and CD68 using the method described above.Results:1.Effect of inflammatory activation and phenotype of human THP-1 macrophage treated with ox-LDL(1)Stimulated human THP-1 macrophages cells with different concentrations of ox-LDL(0mg/L,10mg/L,25 mg/L,50mg/L)for 24h,we found that ox-LDL can induce the expression of MIF and MCP-1 of human THP-1 macrophages in a dose-dependent manner.Control group(MIF:4.61±0.40ng/L,MCP-1:5.71±1.94pg/L),10mg/L group(MIF:5.74±0.38ng/L,MCP-1:10.66±3.13pg/L),25mg/L group(MIF:7.09±0.41 ng/L,MCP-1:20.08±1.57pg/L),50mg/L group(MIF:18.67±0.15ng/L,MCP-1:29.30±1.48pg/L).MIF:There were significant differences between every two group(p<0.01).MCP-1:There were significant differences between every two group(p<0.01).(2)Stimulating cells for different time(0h,8h,16h,24h)with 50mg/L ox-LDL showed that ox-LDL can induce the expression of MIF and MCP-1 of human THP-1 macrophages in a time-dependent manner.Control group(MIF:4.36±0.29ng/L,MCP-1:4.27±1.03pg/L),8h group(MIF:6.83±0.22ng/L,MCP-1:9.13±2.04pg/L),16h group(MIF:7.02±0.45ng/L,MCP-1:14.37±4.08pg/L),24 group(MIF:18.72±0.09ng/L,MCP:1:24.47±3.28pg/L).MIF:There was no significant difference between 8h group and 16 group(p>0.05),all other groups have obvious difference(p<0.01).MCP-1:There was no significant difference between control group and 8h group(p>0.05),no significant difference between 8h group and 16h group(p>0.05),all other groups have obvious difference(p<0.01).(3)Stimulated cells with different concentrations of ox-LDL for 24h,we found that low concentrations of ox-LDL increased the expression of CD68 and CD 16 slightly based on the result of the mean fluorescence intensity:Omg/L group(CD16:29.8,CD68:25.2),10mg/l group(CD 16:30.9,CD68:28.3).As ox-LDL concentration increases,the expression of CD 16 and CD68 were down-regulated,25mg/l group(CD 16:29.9,CD68:23.7),50mg/L group(CD16:26.9,CD68:22).2.Effect of inflammatory activation and phenotype of human THP-1 macrophage treated with Yang xin shi(1)The human THP-1 macrophages were pretreated by different concentrations of Yang xin shi(0?g/ml?10?g/ml?50?g/ml?100?g/ml?200?g/ml)for 12h and stimulated with 50mg/L ox-LDL 24h subsequently,the results indicated that Yang xin shi could inhibit MIF and MCP-1 that secreted from the activated human THP-1 macrophage stimulated by ox-LDL in a dose-dependent manner.Control group(MIF:18.65±0.15ng/L,MCP-1:33.30±2.37pg/L),10?g/ml group(MIF:1 5.50±0.27ng/L,MCP-1:26.78±1.48pg/L),50?g/ml group(MIF:9.07 ±0.26ng/L,MCP-1:26.78±1.48pg/L),10O?g/ml group(MIF:4.85±0.12ng/L,MCP-1:9.95±2.09pg/L),200?g/ml group(MIF:4.58±0.36ng/L,MCP-1:8.53±1.37pg/L).MIF:There was no significant difference between 100?g/ml and 200?g/ml group(p>0.05),all other groups have obvious difference(p<0.01).MCP-1:There was no significant difference between 10?g/ml and 50?g/ml(p>0.05),no significant difference between 100?g/ml and 200?g/ml(p>0.05),all other groups have obvious di fference(p<0.01).(2)The human THP-1 macrophages were pretreated by different concentrations of Yang xin shi for 12h and then stimulated with 50mg/L ox-LDL 24h.The results indicated that Yang xin shi could up-regulate the expression of CD 16 and CD68 in a dose-dependent manner.The mean fluorescence intensity of 10?g/ml group(CD16:71.2,CD68:54.7),50?g/ml group(CD16:92.7,CD68:60.7),100?g/ml(CD16:96,CD68:91.6),the effect of 100?g/ml group is more significant than other groups.As the concentration increases,the expression of CD 16 and CD68 were down-regulated in 200?g/ml group(CD16:80.6,CD68:72.1).Conclusions:Yang xin shi may execute its anti-atherosclerotic effect by inhibitin g inflammatory activation and affect macrophages polarization. |
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