摘要
以转化生长因子-β(TGF-β)/SMAD家族成员2/3(Smad2/3)信号通路为切入点,探讨痰瘀同治方治疗小鼠动脉粥样硬化(AS)的可能作用机制及组方配伍意义。8只C57BL/6J小鼠作为正常组,给予普通饲料喂养,35只同品系ApoE^(-/-)小鼠通过高脂饲料喂养8周制备AS模型。造模成功后随机分为模型组、痰瘀同治组(18.2 mg·kg^(-1))、化痰组(10.4mg·kg^(-1))、祛瘀组(7.8 mg·kg^(-1))各8只。除正常组外,其余各组均采用高脂饲料连续8周喂养制备AS模型,之后各组小鼠均灌胃给药8周。采用酶联免疫吸附测定(ELISA)检测总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)以及白细胞介素1β(IL-1β)、白细胞介素18(IL-18)水平;苏木素-伊红(HE)染色、油红O染色及Russell-Movat五色染观察小鼠主动脉组织的病理学形态变化,并计算主动脉斑块面积占比、红染脂质面积占比、胶原纤维及弹力纤维占比;免疫荧光法检测基质金属蛋白酶2(MMP2)及组织金属蛋白酶抑制物2(TIMP2)蛋白表达水平;Western blot检测主动脉TGF-β1、TGF-β2、Smad2/3、Smad7的蛋白表达;实时荧光定量PCR法检测小鼠主动脉组织转化生长因子-β受体(TGF-βR)、TGF-β1、Smad2/3、Smad7、细胞黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)mRNA表达量。结果显示,与正常组比较,模型组血浆TC、LDL-C升高,HDL-C显著降低;血浆IL^(-1)β、IL-18显著升高;主动脉斑块面积占比、红染脂质面积占比及胶原纤维占比增加;主动脉弓中MMP2表达显著上调,TIMP2表达显著下调;主动脉组织TGF-βR、TGF-β1、p-Smad2/3蛋白及mRNA表达水平显著升高,Smad7表达水平降低;与模型组比较,痰瘀同治组血浆TC、LDL-C显著降低,HDL-C显著升高;血浆IL-1β、IL-18显著降低;主动脉斑块明显缩小,主动脉斑块病变程度显著减小;主动脉弓中MMP2表达明显下调;ICAM-1、VCAM-1 mRNA表达水平显著降低;主动脉组织TGF-β1、p-Smad2/3蛋白及mRNA表达水平显著降低,Smad7蛋白表达水平不同程度升高;与痰瘀同治组比较,祛瘀组LDL-C显著升高,血浆IL-1β、IL-18升高;化痰组主动脉弓中MMP2表达上调,TIMP2表达下调。综上,痰瘀同治方基于痰毒、瘀血致病组方配伍而成,化痰组主要发挥调节血脂代谢、调控炎症因子等作用,而祛瘀组主要发挥维持血管壁通透性、抑制斑块发展、稳定斑块的作用,全方维持血管稳态,其作用机制可能涉及抑制主动脉中TGF-β1的表达,减少Smad2/3的磷酸化,同时提高Smad7的表达水平。
This study aimed to investigate the potential mechanism and the compatibility significance of Tanyu Tongzhi Formula in treating atherosclerosis(AS)in mice based on the transforming growth factor-β(TGF-β)/Smad2/3 signaling pathway.Eight C57BL/6J mice were as assigned to a normal control group and fed a regular diet,while 35 ApoE^(‒/‒) mice of the same strain were fed a high-fat diet for 8 weeks to establish an AS model.The model mice were randomly divided into a model group,a Tanyu Tongzhi group(18.2 mg·kg^(-1)),a Huatan(phlegm-resolving)group(10.4 mg·kg^(-1)),and a Quyu(blood stasis-resolving)group(7.8 mg·kg^(-1)),with 8 mice in each group.Except for the normal group,all other groups continued to be fed a high-fat diet for 8 weeks to maintain the AS model,and then the mice were treated by gavage for 8 weeks.Plasma levels of total cholesterol(TC),triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),interleukin-1β(IL-1β),and interleukin-18(IL-18)were measured using enzyme-linked immunosorbent assay(ELISA).).Hematoxylin and eosin(HE)staining,oil red O staining,and Russell-Movat pentachrome staining were performed to observe the pathological changes in the aortic tissue.The proportions of aortic plaque area,lipid-stained area,collagen fibers,and elastic fibers were calculated.Immunofluorescence was used to detect the protein expression levels of matrix metalloproteinase-2(MMP2)and tissue inhibitor of metalloproteinases-2(TIMP2).Western blot was used to detect the protein expression levels of TGF-β1,TGF-β2,Smad2/3,and Smad7 in aortic tissue.Real-time fluorescence quantitative PCR(RT-qPCR)was used to measure the mRNA expression levels of TGF-βreceptor(TGF-βR),TGF-β1,Smad2/3,Smad7,intercellular adhesion molecule-1(ICAM-1),and vascular cell adhesion molecule-1(VCAM-1)in aortic tissue.The results showed that compared with the normal control group,the model group had increased plasma TC and LDL-C,significantly decreased HDL-C,and significantly elevated plasma IL-1βand IL-18 levels.The model group also exhibited an increased proportion of aortic plaque area,lipid-stained area,and collagen fiber area,along with significantly upregulated MMP2 and downregulated TIMP2 expression in the aortic arch.Additionally,the expression levels of TGF-βR,TGF-β1,and p-Smad2/3 proteins and mRNA in the aortic tissue were significantly elevated,while Smad7 expression was decreased.Compared with the model group,the Tanyu Tongzhi group showed significantly reduced plasma TC and LDL-C levels,significantly increased HDL-C levels,and significantly decreased plasma IL-1βand IL-18 levels.The Tanyu Tongzhi group also exhibited a significant reduction in aortic plaque size and severity,a significant downregulation of MMP2 expression in the aortic arch,and significantly decreased ICAM-1 and VCAM-1 mRNA expression levels.Moreover,the Tanyu Tongzhi group demonstrated significantly reduced expression levels of TGF-β1 and p-Smad2/3 proteins and mRNA in the aortic tissue,and an increased expression level of Smad7 protein to varying degrees.Compared with the Tanyu Tongzhi group,the Quyu group had significantly higher LDL-C levels and elevated plasma IL-1βand IL-18 levels.The Huatan group showed upregulated MMP2 expression and downregulated TIMP2 expression in the aortic arch.In conclusion,Tanyu Tongzhi Formula,which is composed based on the pathogenesis of phlegm and blood stasis,maintains vascular homeostasis by primarily regulating lipid metabolism and controlling inflammatory factors through the Huatan group,and maintaining vascular wall permeability,inhibiting plaque development,and stabilizing plaques through the Quyu group.The mechanism of action may involve inhibiting TGF-β1 expression in the aorta,reducing Smad2/3 phosphorylation,and simultaneously increasing Smad7 expression.
作者
崔晓珊
张会雨
陈原原
李亮
高佳明
郝伟
谢成志
刘建勋
付建华
郭浩
CUI Xiao-shan;ZHANG Hui-yu;CHEN Yuan-yuan;LI Liang;GAO Jia-ming;HAO Wei;XIE Cheng-zhi;LIU Jian-xun;FU Jian-hua;GUO Hao(Institute of Basic Medicine,Xiyuan Hospital,China Academy of Chinese Medical Sciences,Beijing 100091,China;National Clinical Research Center for Traditional Chinese Medicine of Cardiovascular Diseases,Beijing 100091,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2024年第23期6429-6438,共10页
China Journal of Chinese Materia Medica
基金
国家自然科学基金面上项目(82074060)
中国中医科学院科技创新工程项目(CI2021A04602)
中国中医科学院西苑医院能力提升项目(XYZX0304-02)。
