血府逐瘀胶囊对动脉粥样硬化小鼠巨噬细胞极化的影响

Effect of Xuefu Zhuyu Capsules on Polarization of M acrophages in M ice with Atherosclerosis

目的:以果蝇双翅边缘缺刻同源基因1(Notch1)/锯齿典型Notch配体1(Jagged1)/Hes家族BHLH转录因子1(Hes1)信号通路为切入点,探讨血府逐瘀胶囊调控巨噬细胞极化抗动脉粥样硬化的作用机制。方法:载脂蛋白E敲除(ApoE-/-)小鼠高脂饮食4周制备动脉粥样硬化小鼠模型,随机分为模型组、血府逐瘀胶囊组、阿托伐他汀组,以C57BL/6小鼠常规饲养作为正常组。血府逐瘀胶囊组给予血府逐瘀胶囊(0.728 g·kg^(-1)·d^(-1))灌胃,阿托伐他汀组给予阿托伐他汀片(6.07 mg·kg^(-1)·d^(-1))灌胃,正常组、模型组给予等体积去离子水灌胃,连续干预12周。采用苏木素-伊红(HE)染色观察主动脉斑块形态,油红O染色观察主动脉斑块面积及脂质沉积情况,免疫组化法检测主动脉组织CD86、CD206阳性表达,酶联免疫吸附测定法(ELISA)检测小鼠血清肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β、转化生长因子(TGF)-β1、IL-10表达,实时荧光定量聚合酶链式反应(Real-time PCR)检测主动脉组织诱导型一氧化氮合酶(iNOS)、精氨酸酶-1(Arg^(-1))、Notch1、Jagged1、Hes1mRNA相对表达,蛋白免疫印迹法(Western blot)检测主动脉组织iNOS、Arg^(-1)、Notch1、Jagged1、Hes1蛋白相对表达。结果:与正常组比较,模型组有明显的主动脉斑块和脂质沉积,促炎因子TNF-α、IL-1β表达显著升高(P<0.01),抗炎因子TGF-β1表达有下降趋势,但差异无统计学意义;巨噬细胞标志物CD86、CD206出现阳性表达;iNOS mRNA及蛋白表达显著升高(P<0.01),Arg^(-1)蛋白表达显著下降(P<0.01);相关通路分子Jagged1 mRNA及Notch1、Jagged1、Hes1蛋白表达明显升高(P<0.05,P<0.01)。与模型组比较,血府逐瘀胶囊组斑块面积与脂质沉积有下降趋势,TNF-α、IL-1β表达有下降趋势;TGF-β1表达明显升高(P<0.05),巨噬细胞标志物CD86表达下降,CD206表达显著升高(P<0.01);iNOS mRNA及蛋白表达显著下降(P<0.01)Arg^(-1)mRNA及蛋白表达明显升高(P<0.05,P<0.01);相关通路分子Notch1、Jagged1、Hes1 mRNA及蛋白表达显著下降(P<0.01)。结论:血府逐瘀胶囊可减少动脉粥样硬化小鼠主动脉斑块面积和脂质沉积,减轻炎症,抑制M1型巨噬细胞、促进M2型巨噬细胞的表达,其机制可能与调控Notch1/Jagged1/Hes1信号通路有关。

Objective::To investigate the mechanism of Xuefu Zhuyu capsules against atherosclerosis via regulating polarization of macrophages based on Notch1/jagged canonical Notch ligand 1(Jagged1)/Hes family BHLH transcription factor 1(Hes1)signaling pathway.M ethod::The mouse models with atherosclerosis were prepared by feeding the mice with an ApoE-/-high-fat diet for four weeks,and they were randomly divided into the model group,Xuefu Zhuyu capsule group,and atorvastatin group.C57BL/6 mice were fed as a normal group.The Xuefu Zhuyu capsule group was intragastrically given Xuefu Zhuyu capsules(0.728 g·kg^(−1)·d^(-1)),and the atorvastatin group was intragastrically given atorvastatin tablet(6.07 mg·kg^(−1)·d^(-1)).The normal group and the model group were given equal volume of the deionized water by intragastric administration,and the intervention lasted for 12 weeks.Aortic plaque morphology was observed by hematoxylin-eosin(HE)staining,and aortic plaque area and lipid deposition were observed by oil red O staining.The positive expression levels of CD86 and CD206 in aortic tissue were detected by immunohistochemistry,and serum levels of tumor necrosis factor(TNF)-α,interleukin(IL)-1β,transforming growth factor(TGF)-β1,and IL-10 were detected by enzyme-linked immunosorbent assay(ELISA).The relative mRNA expressions of inducible nitric oxide synthase(iNOS),arginase-1(Arg-1),Notch1,Jagged1,and Hes1 in aortic tissue were detected by real-time fluorescence quantitative polymerase chain reaction(Real-time PCR).The relative protein expression of iNOS,Arg-1,Notch1,Jagged1,and Hes1 in aortic tissue was detected by Western blot.Result::Compared with the normal group,the model group had significant aortic plaque and lipid deposition,and the expression levels of pro-inflammatory cytokines TNF-αand IL-1βwere increased(P<0.01).The expression level of anti-inflammatory cytokine TGF-β1 showed a downward trend,but the difference was not statistically significant.The mRNA and protein expressions of iNOS were increased(P<0.01).The protein expression of Arg-1 was decreased(P<0.01),and the mRNA expression of related pathway molecule Jagged1,as well as the protein expressions of Notch1,Jagged1,and Hes1 were increased in the model group(P<0.05,P<0.01).Compared with those in the model group,the plaque area and lipid deposition had a decreasing trend in the Xuefu Zhuyu capsule group,and the expressions of TNF-αand IL-1βshowed a downward trend.The expression of TGF-β1was increased(P<0.05),and the expression of macrophage marker CD86 was decreased.The mRNA and protein expressions of iNOS were decreased(P<0.01).The mRNA and protein expressions of Arg-1 were increased(P<0.05,P<0.01).Furthermore,the mRNA and protein expressions of Notch1,Jagged1,and Hes1were decreased(P<0.01).Conclusion::Xuefu Zhuyu capsules can reduce aortic plaque area and lipid deposition in mice with atherosclerosis,alleviate inflammation,inhibit M1 macrophages,and promote the expression of M2 macrophages,and the mechanism may be related to the regulation of Notch1/Jagged1/Hes1 signaling pathway.