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

Efficacies Identification of Shenlian Extracts on Ox-LDL-induced Macrophage-derived Foam Cell Formation and Its Apoptosis and Comparison of Its Fractions

目的:研究参莲(SL)提取物对氧化型低密度脂蛋白(oxidizedlowdensitylipoprotein,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72h后,分为7组,阴性对照组(NC),模型组(Model,ox-LDL,20mg·L^-1),丹参水溶性提取物组(DS,4.32mg·L^-1+20mg·L^-1ox-LDL),丹参脂溶性提取物组(DZ,2.5mg·L^-1+20mg·L^-1ox-LDL),穿心莲脂溶性提取物组(C,3.18mg·L^-1+20mg·L^-1ox-LDL),SL提取物组(10mg·L^-1+20mg·L^-1ox-LDL),内质网应激诱导剂衣霉素组(TM,1mg·L^-1),药物处理24h。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;AnnexinV-FITC/PI染色观察细胞的凋亡情况;比色法检测Caspase-3的酶活性;Westernblot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 C57BL/6J 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.