目的 观察生地黄水煎总提物对动脉粥样硬化(AS)ApoE-/-小鼠脂质斑块的改善作用以及对“IL-34”-Rho/Rock通路的影响。方法 ①动物实验:将40只ApoE-/-小鼠分成空白对照组(CTL组)、模型对照组(Model组)、5 μg·g-1生地黄干预组(Reh-L组)和10 μg·g-1生地黄干预组(Reh-H组)。空白对照组小鼠给予普通饲料,其余组小鼠腹腔注射人血清低密度脂蛋白(LDL)2周,并喂以高脂饲料。连续灌胃4周,检测小鼠血脂指标,并计算AS指数(AI)。②细胞实验:将RAW264.7细胞分为空白对照组(con组)、ox-LDL组、IL-34组和Reh组;油红O染色观察泡沫细胞形成,检测细胞内胆固醇酯含量和胆固醇流出率。免疫荧光染色法、qRT-PCR法和Western blot法检测RhoA和Rock1表达量。结果 ①动物实验:Reh-H组小鼠外周血TC、TG、LDL-C、内皮素-1(ET-1)、白介素-34(IL-34)、单核细胞趋化蛋白1(MCP-1)、巨噬细胞炎性蛋白2(MIP-2)水平、AI值以及AS斑块面积比低于Model组和Reh-L组,而HDL-C水平高于Model组和Reh-L组(P<0.05)。经qRT-PCR法和Western blot法检测,Reh-H组小鼠主动脉组织RhoA和Rock1表达量较Model组和Reh-L组降低(P<0.05)。②细胞实验:经ox-LDL和IL-34共诱导,RAW264.7细胞内胆固醇酯含量、胆固醇流出率以及RhoA和Rock1蛋白表达量较con组和ox-LDL组细胞增加(P<0.05)。而加入Reh干预后泡沫细胞数量、胆固醇酯含量、胆固醇流出率以及RAW264.7细胞RhoA和Rock1蛋白表达量较IL-34组细胞明显减少(P<0.05)。结论 生地黄可通过抑制“IL-34”-Rho/Rock信号通路抑制巨噬细胞吞噬脂质形成泡沫细胞,从而有效地预防AS的形成和进展。
Abstract
OBJECTIVE To investigate the protective effects and mechanism of extracts of Rehmanniae against atherosclerosis by “IL-34”-Rho/Rock pathway in ApoE-/-mice. METHODS ①Animal experiment 40 ApoE-/- mice were randomly divided into CTL group, Model group, Reh-L group (5 μg·g-1) and Ren-H group (10 μg·g-1), which were induced by low-density lipoprotein (LDL) for AS models. The serum levels of interleukin-34 (IL-34), LDL, mouse monocyte chemotactic protein 1 (MCP-1), mouse macrophage inflammatory protein 2(MIP-2) and endothelin-1 (ET-1) were detected by ELISA. AS index (AI) was calculated according to the serum lipids levels. ②Macrophage experiment RAW264.7 macrophages were cultured in vitro and divided into con group, ox-LDL group, IL-34 group and Reh group. IL-34-indeced foam cells formation by oil red O staining. The cholesterol ester levels and cholesterol efflux of RAW264.7 macrophages were detected. The immunofluorescence, qRT-PCR, Western blot analysis were used to detect RhoA and Rock1 expression. RESULTS ①Animal experiment the serum levels of TC, TG, LDL-C, ET-1, IL-34, MCP-1, MIP-2 and AI, atherosclerotic lesion areas of the thoracic aorta of mice in Reh-H group were lower than Model group and Reh-L group, but with higher HDL-C level (P<0.05). Compared with the CTL group, the RhoA and Rock1 levels of aorta tissues in Model group were significantly increased by qRT-PCR and Western blot (P<0.05). But the RhoA and Rock1 levels of aorta tissues in Reh-H group were lower than Model group or Reh-L group (P<0.05). ②Macrophage experiment the cholesterol ester level and cholesterol efflux, RhoA and Rock1 expressions of RAW264.7 macrophages induced by ox-LDL and IL-34 were more than con group and ox-LDL group (P<0.05). Compared with IL-34 group, the cholesterol ester level and cholesterol efflux, RhoA and Rock1 expressions of RAW264.7 macrophages in Reh group were significantly decreased (P<0.05). CONCLUSION The extracts of Rehmanniae can protectagainst atherosclerosis in ApoE-/- AS mice models induced by LDL through inhibiting “IL-34”-Rho/Rock pathway.
关键词
动脉粥样硬化 /
生地黄 /
IL-34 /
Rho/Rock信号通路 /
RAW264.7巨噬细胞
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Key words
atherosclerosis /
rehmanniae /
IL-34 /
Rho/Rock pathway /
RAW264.7 macrophages
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中图分类号：
R965
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脚注
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基金
国家自然科学基金项目资助(81500362)
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