摘要
目的探讨姜黄素对系统性红斑狼疮(SLE)患者外泌体刺激后巨噬细胞吞噬能力的影响。方法选取2016年2月至2017年11月广州市皮肤病防治所和中山大学孙逸仙纪念医院诊治的活动期SLE患者18例,健康对照18例。提取受试者血清外泌体,分为健康对照者外泌体(CON-exo)、SLE患者外泌体(SLE-exo)、CON-exo +姜黄素.SLE-exo +姜黄素用于刺激THP-1源性巨噬细胞,pHrodo-Red指示剂与巨噬细胞共孵育,流式细胞仪检测平均荧光强度,免疫荧光染色计算pHrodo-Red阳性细胞比例,评价巨噬细胞吞噬功能。Western印迹法检测CON-exo组、SLE-exo组、SLE-exo +姜黄素(浓度分别为1、5、20μmol/L)组CD14蛋白水平。两组比较使用Student-Z检验,多组间对比采用单因素方差分析,两两比较采用LSD-t检验。结果流式细胞仪检测显示,CON-exo组、CON-exo +姜黄素组>SLE-exo组、SLE-exo +姜黄素组相对荧光强度分别为101.3%± 14.05%,94.27 ± 14.13%、41.02%± 9.54%.87.33%± 15.01%,差异有统计学意义(F= 10.81,P < 0.01);免疫荧光染色pHrodo检测显示,阳性细胞比例分别为82.16%± 5,20%.81.33%± 4.51%.54.20%± 9.31%.71.23%± 5.43%,差异有统计学意义(F=12.42, P< 0.01), SLE-exo组巨噬细胞荧光强度、阳性细胞比例均低于CON-exo组Q值分别为5.26.5.35,均P <0.01)和SLE-exo +姜黄素组Q值分别为3.97.3.26,均P <0.05)。Western印迹法显示,CON-exo组、SLE-exo组SLE-exo +姜黄素(浓度分别为1、5、20μmol/L)组细胞CD14蛋白水平分别为96.33%± 13.65%.30.67%± 5.86%,45.24%±&89%,72.81 %± 6.62%.90.67%±12.66%,差异有统计学意义(F = 24.57,P < 0.01), SLE-exo 组低于 Con-exo组(t= 8.06, P< 0.001)和SLE-exo +姜黄素(5、20μmol/L)(t值分别为5.08.7.38,均P<0.001),随着姜黄素剂量增加,CD14水平有升高的趋势。结论SLE患者外泌体明显抑制巨噬细胞吞噬功能,而姜黄素可逆转SLE患者外泌体的作用。
Objective To evaluate the effect of curcumin on phagocytic function of macrophages after the stimulation with exosomes from patients with systemic lupus erythematosus (SLE). Methods From February 2016 to November 2017, 18 patients with active SLE and 18 healthy controls were enrolled from Guangzhou Institute of Dermatology and Sun Yat-sen Memorial Hospital, Sun Yat-sen University, and serum exosomes were extracted from these subjects. The exosomes from the healthy controls (CON-exo) and SLE patients (SLE-exo) as well as different concentrations (1, 5, 20 μmol/L) of curcumin were used alone or in combination to stimulate the acute monocytic leukemia cell line THP-1 - derived macrophages. After coincubation of the stimulated macrophages with pHrodo TM Red indicator, flow cytometry was perfonned to determine the average fluorescence intensity, immunofluorescence staining to calculate the proportion of pHrodo TM Red - positive macrophages, and then the phagocytic activity of macrophages was assessed. Western blot analysis was conducted to determine the protein expression of CD14 in the stimulated macrophages. Statistical analysis was carried out by Student-i test for comparison between two groups, oneway analysis of variance for comparison among several groups, and least significant difference (LSD)-t test for multiple comparisons. Results Flow cytometry showed that there were significant differences in the relative fluorescence intensity of macrophages among the CON-exo group, CON-exo + 20 pimol/L curcumin group, SLE-exo group and SLE-exo + 20 |xmol/L curcumin group (101.3%± 14.05%, 94.27%± 14.13%, 41.02%± 9.54% and 87.33%± 15.01%, respectively;F = 10.81, P < 0.01), and immunofluorescence staining revealed that the proportion of pHrodo Red-positive macrophages significantly differed among the above 4 groups (82.16%± 5.20%, 81.33%± 4.51%, 54.20%± 9.31% and 71.23%± 5.43% respectively;F = 12.42, P < 0.01). The fluorescence intensity of macrophages and proportion of pHrodo - positive macrophages were both significantly lower in the SLE-exo group than in the CON-exo group \t = 5.26, 5.35 respectively, both P < 0.01) and SLE-exo + 20 pimol/L curcumin group (t= 3.97, 3.26 respectively, both P < 0.05). Western blot analysis showed that there were significant differences in the protein expression of CD14 among the CON-exo group, SLE-exo group, SLE-exo + 1μmol/L curcumin group, SLE-exo + 5 pumol/L curcumin group and SLE-exo + 20μmol/L curcumin group (96.33%± 13.65%, 30.67%± 5.86%, 45.24%± 8.89%, 72.81%± 6.62% and 90.67%± 12.66% respectively;F = 24.57, P < 0.01). The protein expression of CD14 was significantly lower in the SLE-exo group than in the CON-exo group(Z = 8.06, P < 0.001), SLEexo + 5 pcmol/L curcumin group and SLE-exo + 20 μmol/L curcumin group(t = 5.08, 7.38, both P < 0.001), and the CD14 level showed an increasing trend along with the increase in the concentration of curcumin. Conclusion Exosomes from SLE patients markedly inhibit the phagocytosis of macrophages, while curcumin can reverse this inhibitory effect.
作者
张淑娟
唐亚萍
梁景耀
刘玉梅
Zhang Shujuan;Tang Yaping;Liang Jingyao;Liu Yumei(Department of Dermatology,Guangzhou Institute of Dermatology,Guangzhou 510095,China)
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2019年第6期378-382,共5页
Chinese Journal of Dermatology
基金
广东省医学科学技术研究基金(A2016554、B201808)
广州市医药卫生科技项目(西医类)(20161A010075).
