基于TLR4/NF-κB途径探究左西孟旦调控巨噬细胞极化减轻动脉粥样硬化的作用

Exploring effect of levosimendan in regulating macrophage polarization and reducing atherosclerosis based on TLR4/NF-κB pathway

目的通过探究左西孟旦对巨噬细胞极化的影响,观察其抗动脉粥样硬化(AS)的作用机制。方法将45只健康清洁级ApoE-/-小鼠按照数字表法随机分为模型组、左西孟旦(2 mg/kg)组、左西孟旦(2 mg/kg)+脂多糖[LPS,2 mg/kg,Toll样受体4(TLR4)激动剂]组,每组15只,以高脂饲料喂养;取15只健康清洁级C57BL/6小鼠作为对照组,以普通饲料喂养。喂养8周后,ip给药,模型组和对照组ip等体积生理盐水,每天1次,持续4周。全自动生化仪检测血清总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白-胆固醇(LDL-C)及高密度脂蛋白胆固醇(HDL-C)水平;HE染色与Masson染色观察主动脉组织形态变化与纤维化水平,油红O染色检测全主动脉及主动脉根部动脉斑块形成情况;免疫组织化学染色检测主动脉M1型标记蛋白诱导型一氧化氮合酶(iNOS)与M2型标记蛋白分化群206 (CD206)的表达;实时荧光定量PCR(qRT-PCR)法检测主动脉M1型巨噬细胞相关基因肿瘤坏死因子-α(TNF-α)、白细胞介素(IL-6)与M2型巨噬细胞相关基因转化生长因子-β(TGF-β)、精氨酸-1(Arg-1)的表达;Western blotting检测主动脉TLR4与磷酸化的核转录因子kB p65(p-NF-κB p65)蛋白表达。结果与对照组比较,模型组小鼠血清TG、TC和LDL-C水平显著升高,HDL-C水平显著降低(P<0.05);主动脉内膜增厚,斑块面积显著增加,伴随大量纤维化(P<0.05);iNOS阳性细胞比例显著升高,CD206阳性细胞比例显著下降(P<0.05);TNF-α、IL-6 mRNA水平显著上调,TGF-β、Arg-1 mRNA水平显著下调(P<0.05);同时TLR4、p-NF-κB p65蛋白表达显著上调(P<0.05)。与模型组比较,左西孟旦组小鼠血清TG、TC和LDL-C水平显著降低,HDL-C水平显著升高(P<0.05);主动脉病理现象减轻,斑块面积显著减小,纤维化程度显著变小(P<0.05);iNOS阳性细胞比例显著下降而CD206阳性细胞比例显著升高(P<0.05);TNF-α、IL-6 mRNA水平显著下调,TGF-β、Arg-1 mRNA水平显著上调(P<0.05);TLR4、p-NF-κB p65蛋白表达显著下调(P<0.05)。与LPS联用基本抵消左西孟旦对上述病理现象的改善作用。结论左西孟旦能够抑制TLR4的表达,阻止下游NF-κB信号途径的激活,调控巨噬细胞极化状态,从而发挥抗AS的作用。

Objective By exploring the effect of levosimendan on the polarization of macrophages, observe its anti-atherosclerosis(AS) mechanism. Methods Forty-five healthy and clean ApoE-/-mice were randomly divided into model group, levosimendan(2 mg/kg) group, levosimendan(2 mg/kg)+LPS [2 mg/kg, Toll-like receptor 4(TLR4)] group with 15 mice in each group according to the number table method, and fed with high-fat diet. Fifteen healthy and clean C57 BL/6 mice were taken as control group and fed with ordinary diet. After eight weeks of feeding, the model group and the control group were ip given equal volume of normal saline once a day for four weeks. Automatic biochemical analyzer was used to detect serum total cholesterol(TC), hyperlipidemia(TG), lowdensity lipoprotein-cholesterol(LDL-C) and high-density lipoprotein cholesterol(HDL-C) levels. HE staining and Masson staining were used to observe the changes of aortic tissue morphology and fibrosis level. Oil red O staining was used to detect the formation of plaque in the whole aorta and the aortic root. immunohistochemical staining was used to detect the expression of aortic M1 marker protein inducible nitric oxide synthase(iNOS) and M2 marker protein differentiation group 206(CD206). Real-time fluorescent quantitative PCR(qRT-PCR) was used to detect tumor necrosis factor-α(TNF-α) and interleukin(IL-6) in M1 macrophage-related gene and stransforming growth factor-β(TGF-β) and arginine-1(Arg-1) in M2 macrophage-related gene.Western blotting was used to detect TLR4 and nuclear transcription factor κB p65(p-NF-κB p65) protein expression. Results Compared with control group, the serum TG, TC and LDL-C levels of model group were significantly increased, and HDL-C levels was significantly reduced(P < 0.05). The aortic intima was thickened, and the plaque area was significantly increased(P < 0.05).The proportion of iNOS positive cells was significantly increased, and the proportion of CD206 positive cells was significantly decreased(P < 0.05). The mRNA levels of TNF-α and IL-6 were significantly increased, and the mRNA levels of TGF-β and Arg-1 were significantly down-regulated(P < 0.05). At the same time, the expression protein of TLR4 and p-NF-κB p65 were significantly up-regulated(P < 0.05). Compared with model group, the serum TG, TC and LDL-C levels of the levosimendan group mice were significantly reduced, and the HDL-C level was significantly increased(P < 0.05). The pathological phenomenon of the aorta was reduced, and the plaque area was significantly reduced, the degree of fibrosis became smaller(P < 0.05). The proportion of iNOS positive cells was significantly decreased while the proportion of CD206 positive cells was significantly increased(P < 0.05). The mRNA levels of TNF-α and IL-6 were significantly down-regulated, the mRNA levels of TGF-β and Arg-1 were significantly increased(P < 0.05). The expression protein of TLR4 and p-NF-κB p65 were also suppressed(P < 0.05). Compared with levosimendan group, the mice that were treated with levosimendan after the TLR4 agonist effect showed no significant improvement in the above-mentioned pathological phenomena. Conclusion Levosimendan can inhibit the expression of TLR4, prevent the activation of the downstream NF-κB signaling pathway, and regulate the polarization state of macrophages, thereby exerting an antiatherosclerotic effect in mice.