| 何志迪,金笑平,朱星蓉,王恩,王凤,吴刚,徐逸雯.连翘苷通过非Calpain途径升高ABCA1抑制动脉粥样硬化斑块形成[J].浙江中西医结合杂志,2024,34(3): |
| 连翘苷通过非Calpain途径升高ABCA1抑制动脉粥样硬化斑块形成 |
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| 投稿时间：2023-08-17 修订日期：2024-01-26 |
| DOI： |
| 中文关键词: 连翘苷 动脉粥样硬化 ABCA1 |
| 英文关键词:Phillyrin Atherosclerosis ABCA1 |
| 基金项目:浙江省公益技术研究基金（LGF18H090019） |
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| 中文摘要: |
| 摘要 目的：验证连翘苷是否通过抑制钙激活中性蛋白酶(Calpain)途径调节ATP结合盒转运蛋白A1（ABCA1）起到抗动脉粥样硬化作用。方法：利用雄性 APOE基因敲除小鼠建立动脉粥样硬化模型，随机分为空白对照组、模型对照组、连翘苷低剂量组、连翘苷高剂量组、阿托伐他汀组，每组5只。试剂盒检测小鼠血清高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇(LDL-C)、甘油三酯（TG）、总胆固醇(TC)、葡萄糖、纤维蛋白原及超敏C反应蛋白水平；组织化学染色分析连翘苷对小鼠主动脉斑块的作用；免疫组织化学检测ABCA1、肝脏X受体α(LXR-α)、过氧化物酶体增殖物激活受体γ（PPAR-γ）、钙蛋白酶抑制蛋白(Calpastatin)、Calpain的表达。使用佛波酯（PMA）和氧化低密度脂蛋白建立THP-1源性泡沫细胞模型，分为THP-1细胞组、泡沫细胞组、低浓度连翘苷组、高浓度连翘苷组，进行油红O染色观察脂质沉积，使用Western blot检测ABCA1蛋白表达。结果：与模型对照组比较，连翘苷高剂量组小鼠血清LDL-C、TC、TG水平显著降低，HDL-C水平升高 [LDL-C :(7.10±1.10)mmol/L比（16.09±2.08）mmol/L;TC（44.10±3.38）mmol/L比（65.57±7.47）mmol/L;TG:(6.03±0.66)mmol/L比（15.03±1.20）mmol/L; HDL-C:(3.15±0.27)mmol/L比(1.33±0.14)mmol/L;P＜0.05]。连翘苷高剂量组血清葡萄糖水平[（4.38±0.08）mmol/L比（5.74±0.22）mmol/L，P<0.05]水平、纤维蛋白原[（19.67±2.27）ug/mL比（56.11±2.28）ug/mL，P＜0.05]、超敏C反应蛋白[（6.65±0.87）ng/mL比（17.51±4.39）ng/mL，P＜0.05]水平较模型对照组显著下降。病理结果显示，连翘苷可以抑制血管内皮动脉粥样硬化斑块的形成，抑制胶原分解。免疫组化染色（AOD）：与模型对照组比较，连翘苷高剂量组ABCA1、LXR-α、PPAR-γ表达显著上调[ABCA1:(0.163±0.004)比(0.143±0.011);LXR-α:(0.215±0.031)比(0.170±0.008);PPAR-γ:(0.201±0.022)比(0.150±0.007)；P<0.05],而 Calpastatin和Calpain蛋白表达水平在两组间无统计学差别（P＞0.05）。 油红O染色提示连翘苷可以抑制泡沫细胞内脂质沉积。Western blot结果提示连翘苷的干预能增加泡沫细胞中ABCA1蛋白的表达。结论：连翘苷可能通过非Calpain途径升高ABCA1抑制动脉粥样硬化。 |
| 英文摘要: |
| Abstract Purpose: To validate whether Phillyrin inhibits ATP-binding cassette transporter A1 (ABCA1) and exerts anti-atherosclerotic effects through the inhibition of calpain, a calcium-activated neutral protease. Methods: Atherosclerosis models were established in male APOE gene knockout mice. They were randomly divided into blank control group, model control group, low-dose Phillyrin group, high-dose Phillyrin group, and atorvastatin group, with 5 mice in each group. Serum levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), total cholesterol (TC), glucose, fibrinogen, and high-sensitivity C-reactive protein were measured using assay kits. The effects of Phillyrin on aortic plaque in mice were analyzed using histological staining. Immunohistochemistry was used to detect the expression of ABCA1, liver X receptor alpha (LXR-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), calpastatin, and calpain. A THP-1-derived foam cell model was established using phorbol 12-myristate 13-acetate (PMA) and oxidized low-density lipoprotein. The cells were divided into THP-1 cell group, foam cell group, low-concentration Phillyrin group, and high-concentration Phillyrin group. Lipid deposition was observed using Oil Red O staining, and ABCA1 protein expression was detected using Western blot. Results: The high-dose Phillyrin group demonstrated notable decreases in serum levels of TC, TG, and LDL-C relative to the model control group, along with a rise in HDL-C [LDL-C :(7.10±1.10)mmol/L vs（16.09±2.08）mmol/L;TC（44.10±3.38）mmol/L vs（65.57±7.47）mmol/L;TG:(6.03±0.66)mmol/L vs（15.03±1.20）mmol/L; HDL-C:(3.15±0.27)mmol/L vs (1.33±0.14)mmol/L;P＜0.05]. In addition, the high-dose Phillyrin group demonstrated significant reductions in serum glucose levels [(4.38±0.08) mmol/L vs. (5.74±0.22) mmol/L; P<0.05] ，fibrinogen [(19.67±2.27) ug/mL vs. (56.11±2.28) ug/mL] and high-sensitivity C-reactive protein [(6.65±0.87) ng/mL vs. (17.51±4.39) ng/mL; P＜0.05] , when compared to the model control group. Pathological findings shown that Phillyrin could prevent collagen breakdown and the development of atherosclerotic plaques in vascular endothelial arteries. The high-dose Phillyrin group exhibited significantly higher expression levels of ABCA1, LXR-α, and PPAR-γ than the model group [ABCA1: (0.163±0.004) vs. (0.143±0.011); LXR-α: (0.215±0.031) vs. (0.170±0.008); PPAR-γ: (0.201±0.022) vs. (0.150±0.007); P<0.05]. In contrast, there was no statistically significant difference between the two groups for the protein expression levels of Calpastatin and Calpain (P>0.05). The inhibition of lipid deposition in foam cells by Phillyrin was suggested by the use of oil red O staining. According to Western blot data, foam cells' expression of the ABCA1 protein might be elevated by Phillyrin intervention. Conclusion: Phillyrin may elevate ABCA1 and inhibit atherosclerosis through a non-calpain pathway. |
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