Toll样受体4抑制过氧化物酶体增殖物激活受体γ加重血脂蓄积的分子机制

doi:10.6040/j.issn.1671-7554.0.2020.0102

摘要/Abstract

摘要： 目的选取高脂饮食模型大鼠和氧化低密度脂蛋白(oxLDL)暴露下巨噬细胞模型,验证在血脂蓄积过程中Toll样受体4(TLR4)和过氧化物酶体增殖物激活受体γ(PPARγ)的具体干预机制。方法健康雄性Wistar大鼠20只,随机分为对照组和高脂组,每组10只。测定各组大鼠总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白水平,采用苏木精-伊红染色检测颈动脉血管内膜中膜厚度比,采用Western blotting法检测TLR4、PPARγ蛋白表达水平。体外培养小鼠巨噬细胞RAW264.7,以oxLDL(50 mg/L)刺激巨噬细胞制备模型,且应用siRNA-TLR4沉默巨噬细胞内的TLR4因子制备TLR4沉默模型,将细胞分为空白组(A组)、oxLDL组(B组)、oxLDL+siRNA组(C组)、oxLDL+siRNA-TLR4组(D组)、oxLDL+siRNA-TLR4+PPARγ激动剂组(E组)、oxLDL+siRNA-TLR4+PPARγ抑制剂组(F组)。采用油红O染色法观察巨噬细胞内血脂蓄积情况,定量检测巨噬细胞内胆固醇含量,采用Western blotting法检测 TLR4、PPARγ蛋白表达水平。结果在动物模型实验中,与对照组相比,高脂组总胆固醇、甘油三酯、低密度脂蛋白水平、颈动脉内膜中膜厚度比、TLR4蛋白相对表达含量明显增高(P<0.01),血清高密度脂蛋白水平、PPARγ蛋白相对表达含量明显降低(P<0.01),且TLR4与PPARγ呈负相关性(r=-0.928 1,P<0.001)。在oxLDL暴露巨噬细胞实验中,与A组比较,B、C组巨噬细胞内胆固醇含量、油红O颗粒、光密度值及TLR4蛋白相对表达含量明显增多(P<0.01),PPARγ蛋白相对表达含量明显减少(P<0.05),且B组TLR4与PPARγ呈负相关性(r=-0.986 7,P<0.001)。与B组相比,C组巨噬细胞内胆固醇含量、油红O颗粒、光密度值及TLR4、PPARγ蛋白相对表达含量未见明显改变(P>0.05)。与B组相比,D组巨噬细胞内胆固醇含量、油红O颗粒及光密度值及TLR4蛋白相对表达含量明显减少(P<0.01),PPARγ蛋白相对表达含量明显增多(P<0.05)。与D组相比,E组巨噬细胞内胆固醇含量、油红O颗粒及光密度值明显减少(P<0.01),PPARγ蛋白相对表达含量明显增多(P<0.05),TLR4蛋白相对表达含量未见明显改变(P>0.05)。与D组相比,F组巨噬细胞内胆固醇含量、油红O颗粒及光密度值明显增多(P<0.01),PPARγ蛋白相对表达含量明显减少(P<0.05),TLR4蛋白相对表达含量未见明显改变(P>0.05)。结论细胞内的血脂蓄积是动脉粥样硬化形成的机制之一,PPARγ可以抑制巨噬细胞血脂蓄积进而参与动脉粥样硬化调节,是巨噬细胞血脂蓄积过程的保护性因子。而TLR4作为PPARγ上游调控位点,通过抑制PPARγ的表达,加重血脂蓄积的过程,进而干预动脉粥样硬化进程。

关键词: 氧化低密度脂蛋白, Toll样受体4, 过氧化物酶体增殖物激活受体γ, 动脉粥样硬化, 血脂, 胆固醇, 巨噬细胞, 基因沉默

Abstract: Objective To investigate the molecular mechanism of Toll-like receptor 4(TLR4)and peroxisome proliferator-activated receptor γ(PPARγ)in the process of lipid accumulation in rat models of high-fat diet and oxidized low-density lipoprotein(oxLDL). Methods A total of 20 healthy male Wistar rats were randomly divided into control group(n=10)and high fat group(n=10). Total cholesterol(TG), triglycerides(TC), high-density lipoprotein(HDL)and low-density lipoprotein(LDL)levels were measured in both groups. The carotid intima-media thickness ratio was detected with HE staining, and the protein expressions of TLR4 and PPARγ were determined with Western blotting. Rat macrophages RAW264.7 were cultured in vitro and stimulated by oxLDL(50 mg/L)to prepare the macrophage models, and TLR4 factors in the macrophages were silenced with siRNA-TLR4 to prepare the TLR4 silencing models. The cells were divided into blank group(group A), oxLDL group(group B), oxLDL+siRNA group(group C), oxLDL+siRNA-TLR4 group(group D), oxLDL+siRNA-TLR4+PPARγ agonist group(group E)and oxLDL +siRNA-TLR4+PPARγ inhibitor group(group F). The lipid accumulation in macrophages was observed with oil red O staining, cholesterol level in macrophages was quantitatively detected, and protein expressions of TLR4 and PPARγ were tested with Western blotting. Results In the animal model experiments, compared with control group, high fat group had significantly increased levels of TG, TC, LDL, ratio of carotid intima-media thickness, and relative expression of TLR4(P<0.01), but decreased levels of serum HDL and PPARγ(P<0.01), and there was a negative correlation between TLR4 and PPARγ (r=-0.928 1, P<0.001). In the oxLDL exposure to macrophages experiments, compared with group A, groups B and C had significantly increased cholesterol content(P<0.01), oil red O particles, optical density(OD)value and relative expression of TLR4, but decreased relative expression of PPARγ (P<0.05). Besides, there was a negative correlation between TLR4 and PPARγ(r=-0.986 7, P<0.001)in group B. Compared with group B, group C had no significant changes in cholesterol content, oil red O particles, OD value and relative expressions of TLR4 and PPARγ(P>0.05). Compared with group B, group D had significantly decreased cholesterol content, oil red O particles, OD value and relative expression of TLR4(P<0.01), but significantly increased relative expression of PPARγ(P<0.05). Compared with group D, group E had significantly decreased cholesterol content, oil red O particles and OD value(P<0.01), but significantly increased relative expression of PPARγ(P<0.05), and the expression of TLR4 remained unchanged(P>0.05). Compared with group D, group F had significantly increased cholesterol content(P<0.01), oil red O particles and OD value, but significantly decreased relative expression of PPARγ(P<0.05), and the relative expression of TLR4 protein did not significantly change(P>0.05). Conclusion The accumulation of blood lipids in cells is involved in the pathogenesis of atherosclerosis(AS). As a protective factor, PPARγ can inhibit the accumulation of blood lipids in macrophages and then participate in AS regulation. As an upstream regulatory site of PPARγ, TLR4 aggravates lipid accumulation by inhibiting the expression of PPARγ, and then interferes with the progression of AS.

Key words: Oxidized low-density lipoprotein, Toll-like receptor 4, Peroxisome proliferator-activated receptor γ, Atherosclerosis, Blood lipids, Cholesterol, Macrophages, Gene silencing

中图分类号:

R543.5

付洁琦,张曼,张晓璐,李卉,陈红. Toll样受体4抑制过氧化物酶体增殖物激活受体γ加重血脂蓄积的分子机制[J]. 山东大学学报 (医学版), 2020, 1(7): 24-31.

FU Jieqi, ZHANG Man, ZHANG Xiaolu, LI Hui, CHEN Hong. Molecular mechanism of Toll-like receptor 4 in the aggravation of blood lipid accumulation by inhibiting the peroxisome proliferator-activate receptor γ[J]. Journal of Shandong University (Health Sciences), 2020, 1(7): 24-31.

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