| Objective Chemerin is a newly discovered adipokine. The preliminary study of this project showed,. the expression of chemerin m RNA and protein was significantly higher in epicardial adipose tissue from patients with CAD, and was positively correlated with the severity of coronary atherosclerosis. Chemerin was found to promote monocyte adhesion and formation of foam cells, and these two pathological processes plays an important role in the occurrence and development of As. Macrophage apoptosis occurres in all stages of atherosclerosis. In the advanced atherosclerotic lesions, macrophage apoptosis causes secondary cell necrosis and inflammatory reaction, promote the formation and enlargement of plaque lipid core, and the protease which release after the macrophages apoptosis may damage the fibrous cap, resulting in the formation of unstable plaque(i.e. vulnerable plaque). The aim of this study is to observe whether recombinant human chemerin plays a role in FC-induced macrophage apoptosis and to clear the effect of JNK and P38 MAPK signaling pathway on this apoptosis process, so as to provide new intervention target for prevention and treatment of atherosclerosis and CAD.Methods(I)Culture of human monocyte and differentiation of monocyte to macrophage RPMI 1640 medium with 10% fetal bovine serum was used to culture THP-1 monocytes and CD14-FITC was used to identify, incubated 72 h after adding 100nmol/l phorbol myristate acetate(PMA),THP-1 human monocytes were induced to differentiate into macrophages and THP-1 macrophages were obtained.(II)Preparation of free cholesterol loading(FC-loading) model According to reference, DMEM medium with 1% fetal bovine serum which was added 100μg/ml acetylated low density lipoprotein and 10μg/ml cholesterol acyltransferase inhibitor-58035 was used to promote FC accumulation, induced macrophage apoptosis.(III)Cell apoptosis analysis Cell apoptosis was assessed by Annexin-FITC/PI double-staining assay. The cell suspension was made from the macrophages washed by PBS solution, adding 5μl Annexin-V and 5μl PI, incubated 15 min at room temperature without light and then add 200μl 1x Buffer buffer before the assessment of flow cytometry. Each sample was counted for 5000 cells, with an excitation wavelength of 488 nm, emission wavelength of 515 nm, 560 nm standard. Analysed from flow cytometry cell scatter plot, Annexin-V+ /PI- cells were apoptotic cells(green fluorescence); Annexin-V+/PI+ cells were necrotic cells(red-orange fluorescence); Annex-in-V-/PIcells were live cells. The percentage of apoptotic macrophages accounted for total macrophages was apoptotic rate.(IV)Effect of different concentrations of recombinant human chemerin on FC-loading induced macrophage apoptosis Added recombinant human chemerin according to the concentration gradient of 0.1μg/l, 10μg/l, 500μg/l,established the blank control group and anti-chemerin group, made chemerin and chemerin antibody response 30 min in advance in anti-chemerin group, incubated for 8h in 5%CO2 incubator at 37℃; These groups were added to macrophage medium, incubated for 18 h, then incubated for another 12 h in the state of FC-loading. Macrophage apoptosis rate was analysed according to the method above.(V)Clear the effect of JNK and P38 MAPK signaling pathway Experimental concentrations of recombinant human chemerin is determined according to the experiments above, establish recombinant chemerin group and anti-chemerin group; 10μmol/l JNK inhibitor(sp600125) and 10μmol/l P38 MAPK inhibitor(SB203580) were used to intervene recombinant chemerin group and anti-chemerin group respectively, then repeat the experiment above.Results(I) Effect of different concentrations of recombinant human chemerin on FC-loading induced macrophage apoptosis Recombinant human chemerin can promote the FC-loading induced macrophage apoptosis, and with the concentration of chemerin increased, macrophage apoptosis enhanced: control group 19.04%±0.39%, recombinant chemerin 0.1μg/l 22.77%±0.86%, 10μg/l 26.37%±1.06%, 500μg/l 33.44%±0.87%. Each macrophage apoptosis rate of chemerin intervene group were compared with that of control group, the differences were statistically significant(P(27)0.05). After the intervention of chemerin antibody, the differences between macrophage apoptosis rate of each group and the control group had no statistical significance( P>0.05).(II) Role of JNK and P38 MAPK signaling pathway in the process of macrophage apoptosis promoted by chemerin Macrophage apoptosis rate of recombinant chemerin group 23.81%±0.83%, anti-chemerin group 17.75%±1.23%, recombinant chemerin+JNK inhibitor group 13.96%±0.68%, anti-chemerin+JNK inhibitor group 11.62%±0.36%, recombinant chemerin+P38MAPK inhibitor group 11.99%±0.23%, and anti-chemerin+P38MAPK inhibitor group 10.10%±0.43%. The difference of macrophage apoptosis percentages before and after the intervention of JNK inhibitor: recombinant chemerin group 9.85%±0.34%, anti-chemerin group 6.13%±1.09%. The former was obviously higherthan the latter, the difference was statistically significant(P(27)0.05). Therefore, blocking the JNK signaling pathway can significantly weaken the role of chemerin in promoting macrophage apoptosis. But there was still difference between recombinant chemerin group and anti-chemerin group after the intervention of JNK inhibitor, the difference was statistically significant(P(27)0.05). It suggested that there were other pathways involved in the process of macrophage apoptosis promoted by chemerin. Then P38 MAPK pathway was blocked, the difference of macrophage apoptosis percentages before and after the intervention of P38 MAPK inhibitor: recombinant chemerin group 11.82%±0.60%, anti-chemerin group 7.65%±0.83%. The former was obviously higher than the latter, the difference was statistically significant(P(27)0.05). Therefore, P38 MAPK signaling pathway was also involved in the process of macrophage apoptosis promoted by chemerin.Conclusion Recombinant human chemerin can promote the FC-induced macrophage apoptosis. Moreover, JNK and P38 MAPK signaling pathway participate in this apoptosis progress. |
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