参莲提取物对ox-LDL诱导的巨噬细胞泡沫化和凋亡的药效学研究及提取部位活性比较
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摘要
目的:研究参莲(SL)提取物对氧化型低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)诱导的腹腔巨噬细胞泡沫化和凋亡的药效及SL不同提取部位的活性比较。方法:从C57BL/6J雌性小鼠腹腔中收集腹腔巨噬细胞,体外培养72 h后,分为7组,阴性对照组(NC),模型组(Model,ox-LDL,20 mg·L~(-1)),丹参水溶性提取物组(DS,4.32 mg·L~(-1)+20 mg·L~(-1) ox-LDL),丹参脂溶性提取物组(DZ,2.5 mg·L~(-1)+20 mg·L~(-1) ox-LDL),穿心莲脂溶性提取物组(C,3.18 mg·L~(-1)+20 mg·L~(-1) ox-LDL),SL提取物组(10 mg·L~(-1)+20 mg·L~(-1) ox-LDL),内质网应激诱导剂衣霉素组(TM,1 mg·L~(-1)),药物处理24 h。油红O染色观察脂质积累的情况;钙离子荧光染色联合流式细胞仪检测细胞内钙离子浓度的变化;Annexin V-FITC/PI染色观察细胞的凋亡情况;比色法检测Caspase-3的酶活性;Western blot检测Caspase-12的表达。结果:与NC组相比,Model组和TM组腹腔巨噬细胞内脂质蓄积,凋亡率增加,Caspase-3的酶活性增加,钙离子浓度增加,Caspase-12的活化水平上调。与Model组相比,SL组和C组显著改善细胞内脂质积累的情况;SL组和DZ组显著降低细胞的凋亡率,降低Caspase-3的酶活性,有效降低细胞内钙离子的浓度,显著降低Caspase-12的活化水平。结论:综上所述,以巨噬细胞为靶点,SL提取物可能通过缓解ox-LDL诱导的内质网应激,抑制巨噬细胞源泡沫细胞的凋亡。
Objective:To study the effects of Shenlian on ox-LDL-induced macrophage-derived foam cell formation and its apoptosis and comparison of its fractions.Methods:Peritoneal macrophages were collected from the C57 BL/6 J mice.After culturing for 72 hours,the peritoneal macrophages were randomly divided into 7 groups:negative control group(NC),model group(model,20 mg·L~(-1) ox-LDL),water-soluble extract of Salvia miltiorrhiza group(DS,4.32 mg·L~(-1)+20 mg·L~(-1) ox-LDL),liposoluble extract of S.miltiorrhiza group(DZ,2.5 mg·L~(-1)+20 mg·L~(-1) ox-LDL),liposoluble extract of Andrographis paniculata group(C,3.18 mg·L~(-1)+20 mg·L~(-1) ox-LDL),and the Shenlian extract group(SL,10 mg·L~(-1)+20 mg·L~(-1) ox-LDL),endoplasmic reticulum stress inducer group(TM,1 g·L~(-1)).After treatment for 24 hours,the accumulation of lipids were stained of oil red oxygen.The intracellular calcium concentration was detected by flow cytometry and fluorescent probes.The cell apoptosis was detected by Annexin V-FITC double staining.The Caspase-3 activity was measured by chromatometry and the activation of casepase-12 was detected by Western blot.Results:Compared with the negative control group,both the modeling group and the endoplasmic reticulum stress inducer group significantly increased the accumulation of lipids,the rates of apoptosis,the activity of Caspase-3,the concentration of intracellular calcium and the activation of Caspase-12.Compared with the ox-LDL modeling group,the Shenlian extract and liposoluble extract of A.paniculata decreased the accumulation of lipids.The Shenlian extract and liposoluble extract of S.miltiorrhiza significantly inhibited peritoneal macrophages apoptosis and decreased the activity of Caspase-3,and effectively reducesed the concentration of intracellular calcium and the activation of Caspase-12.Conclusion:By targeting macrophages,SL extract may inhibit the apoptosis of peritoneal macrophage-derived foam cells by alleviating ox-LDL-induced endoplasmic reticulum stress.
Objective:To study the effects of Shenlian on ox-LDL-induced macrophage-derived foam cell formation and its apoptosis and comparison of its fractions.Methods:Peritoneal macrophages were collected from the C57 BL/6 J mice.After culturing for 72 hours,the peritoneal macrophages were randomly divided into 7 groups:negative control group(NC),model group(model,20 mg·L~(-1) ox-LDL),water-soluble extract of Salvia miltiorrhiza group(DS,4.32 mg·L~(-1)+20 mg·L~(-1) ox-LDL),liposoluble extract of S.miltiorrhiza group(DZ,2.5 mg·L~(-1)+20 mg·L~(-1) ox-LDL),liposoluble extract of Andrographis paniculata group(C,3.18 mg·L~(-1)+20 mg·L~(-1) ox-LDL),and the Shenlian extract group(SL,10 mg·L~(-1)+20 mg·L~(-1) ox-LDL),endoplasmic reticulum stress inducer group(TM,1 g·L~(-1)).After treatment for 24 hours,the accumulation of lipids were stained of oil red oxygen.The intracellular calcium concentration was detected by flow cytometry and fluorescent probes.The cell apoptosis was detected by Annexin V-FITC double staining.The Caspase-3 activity was measured by chromatometry and the activation of casepase-12 was detected by Western blot.Results:Compared with the negative control group,both the modeling group and the endoplasmic reticulum stress inducer group significantly increased the accumulation of lipids,the rates of apoptosis,the activity of Caspase-3,the concentration of intracellular calcium and the activation of Caspase-12.Compared with the ox-LDL modeling group,the Shenlian extract and liposoluble extract of A.paniculata decreased the accumulation of lipids.The Shenlian extract and liposoluble extract of S.miltiorrhiza significantly inhibited peritoneal macrophages apoptosis and decreased the activity of Caspase-3,and effectively reducesed the concentration of intracellular calcium and the activation of Caspase-12.Conclusion:By targeting macrophages,SL extract may inhibit the apoptosis of peritoneal macrophage-derived foam cells by alleviating ox-LDL-induced endoplasmic reticulum stress.
引文
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[22] CAI B,THORP E B,DORAN A C,et al.MerTK receptor cleavage promotes plaque necrosis and defective resolution in atherosclerosis[J].J Clin Invest,2017,127(2):564-568.
[23] HEO K S,CUSHMAN H J,AKAIKE M,et al.ERK5 activation in macrophages promotes efferocytosis and inhibits atherosclerosis[J].Circulation,2014,130(2):180-191.
[2] LIBBY P,BORNFELDT K E,TALL A R.Atherosclerosis:Successes,Surprises,and Future Challenges[J].Circ Res,2016,118(4):531-534.
[3] PATEL K M,STRONG A,TOHYAMA J,et al.Macrophage sortilin promotes LDL uptake,foam cell formation,and atherosclerosis[J].Circ Res,2015,116(5):789-796.
[4] FEBBRAIO M,PODREZ E A,SMITH J D,et al.Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice[J].J Clin Invest,2000,105(8):1049-1056.
[5] GONZALEZ L,TRIGATTI B L.Macrophage Apoptosis and Necrotic Core Development in Atherosclerosis:A Rapidly Advancing Field with Clinical Relevance to Imaging and Therapy[J].Can J Cardiol,2017,33(3):303-312.
[6] SEIMON T A,NADOLSKI M J,LIAO X,et al.Atherogenic lipids and lipoproteins trigger CD36-TLR2-dependent apoptosis in macrophages undergoing endoplasmic reticulum stress[J].Cell Metab,2010,12(5):467-482.
[7] YAO S,SANG H,SONG G,et al.Quercetin protects macrophages from oxidized low-density lipoprotein-induced apoptosis by inhibiting the endoplasmic reticulum stress-C/EBP homologous protein pathway[J].Exp Biol Med(Maywood),2012,237(7):822-831.
[8] 田华,李严严,丁明德,等.载脂蛋白A-I模拟肽D4F通过抑制caspase-12减轻氧化低密度脂蛋白诱导的巨噬细胞凋亡[J].中国病理生理杂志,2015,31(10):1750-1755.
[9] HUANG A,PATEL S,MCALPINE C S,et al.The Role of Endoplasmic Reticulum Stress-Glycogen Synthase Kinase-3 Signaling in Atherogenesis[J].Int J Mol Sci,2018,19(6):1607.
[10] LIN Y,ZHUANG J,LI H,et al.Vaspin attenuates the progression of atherosclerosis by inhibiting ER stress-induced macrophage apoptosis in apoE-/- mice[J].Mol Med Rep,2016,13(2):1509-1516.
[11] LIU M Q,CHEN Z,CHEN L X.Endoplasmic reticulum stress:a novel mechanism and therapeutic target for cardiovascular diseases[J].Acta Pharmacol Sin,2016,37(4):425-443.
[12] 李玉洁,朱晓新,杨庆,等.参莲提取物对动脉粥样硬化家兔血管病理形态和脂质代谢的影响[J].中草药,2011,42(4):760-764.
[13] GUO Y,LIU X C,WANG Y J,et al.Effects of Shenlian extract on experimental atherosclerosis in ApoE-deficient mice based on ultrasound biomicroscopy[J].BMC Complement Altern Med,2016,16(1):469.
[14] 郭琰.基于炎症消散的参莲提取物抗动脉粥样硬化作用的机理研究[D].北京:中国中医科学院中药研究所,2015.
[15] 刘思思,李琦,孙立东,等.参莲提取物对LPS诱导的巨噬细胞炎症反应的影响[J].中国实验方剂学杂志,2017,23(10):85-91.
[16] TABAS I.Macrophage death and defective inflammation resolution in atherosclerosis[J].Nat Rev Immunol,2010,10(1):36-46.
[17] TAO H,YANCEY P G,BABAEV V R,et al.Macrophage SR-BI mediates efferocytosis via Src/PI3K/Rac1 signaling and reduces atherosclerotic lesion necrosis[J].J Lipid Res,2015,56(8):1449-1460.
[18] ELLIOTT M R,KOSTER K M,MURPHY P S.Efferocytosis Signaling in the Regulation of Macrophage Inflammatory Responses[J].J Immunol,2017,198(4):1387-1394.
[19] YANCEY P G,BLAKEMORE J,DING L,et al.Macrophage LRP-1 controls plaque cellularity by regulating efferocytosis and Akt activation[J].Arterioscler Thromb Vasc Biol,2010,30(4):787-795.
[20] SENEVIRATNE A N,EDSFELDT A,COLE J E,et al.Interferon Regulatory Factor 5 Controls Necrotic Core Formation in Atherosclerotic Lesions by Impairing Efferocytosis[J].Circulation,2017,136(12):1140-1154.
[21] YURDAGUL A,DORAN A C,CAI B,et al.Mechanisms and Consequences of Defective Efferocytosis in Atherosclerosis[J].Front Cardiovasc Med,2018.doi:10.3389/fcvm.2017.00086.
[22] CAI B,THORP E B,DORAN A C,et al.MerTK receptor cleavage promotes plaque necrosis and defective resolution in atherosclerosis[J].J Clin Invest,2017,127(2):564-568.
[23] HEO K S,CUSHMAN H J,AKAIKE M,et al.ERK5 activation in macrophages promotes efferocytosis and inhibits atherosclerosis[J].Circulation,2014,130(2):180-191.