目的 通過探究左西孟旦對巨噬細(xì)胞極化的影響，觀察其抗動脈粥樣硬化（AS）的作用機(jī)制。方法 將45只健康清潔級ApoE-/-小鼠按照數(shù)字表法隨機(jī)分為模型組、左西孟旦（2 mg/kg）組、左西孟旦（2 mg/kg）+脂多糖［LPS，2 mg/kg，Toll樣受體4（TLR4）激動劑］組，每組15只，以高脂飼料喂養(yǎng)；取15只健康清潔級C57BL/6小鼠作為對照組，以普通飼料喂養(yǎng)。喂養(yǎng)8周后，ip給藥，模型組和對照組ip等體積生理鹽水，每天1次，持續(xù)4周。全自動生化儀檢測血清總膽固醇（TC）、三酰甘油（TG）、低密度脂蛋白-膽固醇（LDL-C）及高密度脂蛋白膽固醇（HDL-C）水平；HE染色與Masson染色觀察主動脈組織形態(tài)變化與纖維化水平，油紅O染色檢測全主動脈及主動脈根部動脈斑塊形成情況；免疫組織化學(xué)染色檢測主動脈M1型標(biāo)記蛋白誘導(dǎo)型一氧化氮合酶（iNOS）與M2型標(biāo)記蛋白分化群206 （CD206）的表達(dá)；實時熒光定量 PCR（qRT-PCR）法檢測主動脈M1型巨噬細(xì)胞相關(guān)基因腫瘤壞死因子-α（TNF-α）、白細(xì)胞介素（IL-6）與M2型巨噬細(xì)胞相關(guān)基因轉(zhuǎn)化生長因子-β（TGF-β）、精氨酸-1（Arg-1）的表達(dá)；Western blotting檢測主動脈TLR4與磷酸化的核轉(zhuǎn)錄因子 kB p65（p-NF-κB p65）蛋白表達(dá)。結(jié)果 與對照組比較，模型組小鼠血清TG、TC和LDL-C水平顯著升高，HDL-C水平顯著降低（P<0.05）；主動脈內(nèi)膜增厚，斑塊面積顯著增加，伴隨大量纖維化（P<0.05）；iNOS陽性細(xì)胞比例顯著升高，CD206陽性細(xì)胞比例顯著下降（P<0.05）；TNF-α、IL-6 mRNA水平顯著上調(diào)，TGF-β、Arg-1 mRNA水平顯著下調(diào)（P<0.05）；同時TLR4、p-NF-κB p65蛋白表達(dá)顯著上調(diào)（P<0.05）。與模型組比較，左西孟旦組小鼠血清TG、TC和LDL-C水平顯著降低，HDL-C水平顯著升高（P<0.05）；主動脈病理現(xiàn)象減輕，斑塊面積顯著減小，纖維化程度顯著變小（P<0.05）；iNOS陽性細(xì)胞比例顯著下降而CD206陽性細(xì)胞比例顯著升高（P<0.05）；TNF-α、IL-6 mRNA水平顯著下調(diào)，TGF-β、Arg-1 mRNA水平顯著上調(diào)（P<0.05）；TLR4、p-NF-κB p65蛋白表達(dá)顯著下調(diào)（P<0.05）。與LPS聯(lián)用基本抵消左西孟旦對上述病理現(xiàn)象的改善作用。結(jié)論 左西孟旦能夠抑制TLR4的表達(dá)，阻止下游NF-κB信號途徑的激活，調(diào)控巨噬細(xì)胞極化狀態(tài)，從而發(fā)揮抗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 C57BL/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.

R965

新疆維吾爾自治區(qū)自然科學(xué)基金資助項目（2019D01C005）