[Mechanism of herbal cake-separated moxibustion combined with atorvastatin in treating ApoE(-/-) atherosclerotic mice based on the integrin/YAP/JNK signaling pathway].

摘要

OBJECTIVES: To investigate the effects of herbal cake-separated moxibustion combined with atorvastatin on lipid metabolism, aortic pathological structure, plaque deposition, and integrin/ yes-associated protein (YAP)/ c-Jun N-terminal kinase (JNK) signaling pathway in ApoE-/- atherosclerosis (AS) mice, and to explore its preventive and therapeutic mechanisms for AS. METHODS: Male C57BL/6J mice were used as the blank group, and male ApoE-/- mice were randomly divided into the model group, medicine group, herbal cake-separated moxibustion group, and combined treatment group, with 9 mice per group. High-fat diet was used to establish the AS model. The herbal cake-separated moxibustion group received moxibustion at "Danzhong" (CV17) and "Shenque" (CV8) acupoints, once every other day, 3 times a week. The medicine group received atorvastatin calcium tablet via gavage, 3 mg·kg-1·d-1, once daily. The combined treatment group received an integrated approach that combined the interventions of the medicine group and herbal cake-separated moxibustion group. All groups were treated for 8 weeks. Body weight were observed before and after treatment. HE staining was used to observe aortic pathological morphology, and Oil Red O staining to observe aortic lipid plaque area. Biochemical analysis was used to detect serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) levels. Serum vascular endothelial growth factor (VEGF), endothelin-1 (ET-1), interleukin (IL)-6, IL-8, and tumor necrosis factor-α (TNF-α) levels were detected by ELISA, and serum nitric oxide (NO) level was detected by colorimetric assay. Western blot was used to detect protein expression levels of integrin αVβ3, YAP, phosphorylated (p)-YAP, p-JNK1/2, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in the aorta. RESULTS: Compared to the blank group, the model group exhibited increased body weight (P<0.01)；thickened aortic intima, foam cells and lipid deposition, hypertrophic edema of smooth muscle cells, thinning of elastic fibers, widened endothelial cell gaps, and rupture of elastic membranes；significantly increased arterial lipid plaque area (P<0.01)；elevated serum TC, TG, LDL-C, IL-6, IL-8, TNF-α, ET-1, and VEGF levels (P<0.01), while HDL-C and NO levels decreased (P<0.01)；increased protein expression of integrin αVβ3, YAP, p-JNK1/2, ICAM-1, and VCAM-1 (P<0.01), and reduced p-YAP protein expression (P<0.01) in the aorta. Compared to the model group, the medicine group, herbal cake-separated moxibustion group, and combined treatment group showed reduced body weight (P<0.01), with a significantly lower weight change difference than that of the model group (P<0.01)；more regular aortic lumen structure, thinner intima, reduced foam cells and lipid deposition, alleviated smooth muscle cell edema, hypertrophy, and inflammatory infiltration；all other indicators also showed significant improvement compared to the model group (P<0.01, P<0.05). Compared to the herbal cake-separated moxibustion group, the medicine group and combined treatment group exhibited reduced plaque area (P<0.05, P<0.01), increased serum NO level (P<0.01), decreased LDL-C, ET-1, and VEGF levels (P<0.01), and reduced protein expressions of p-JNK1/2, ICAM-1, and VCAM-1 in the aorta (P<0.05, P<0.01)；the medicine group also showed decreased serum TC, TG, IL-6, IL-8, and TNF-α levels (P<0.01), increased HDL-C level (P<0.01), reduced integrin αVβ3 and YAP protein expressions in the aorta (P<0.01, P<0.05), and increased p-YAP protein expression (P<0.05). Compared to the medicine group and the herbal cake-separated moxibustion group, the combined treatment group exhibited a greater reduction in body weight (P<0.05, P<0.01), decreased serum levels of TC, TG, IL-6, IL-8, and TNF-α, as well as reduced expressions of integrin αVβ3 and YAP proteins in the aorta (P<0.01, P<0.05), elevated serum HDL-C level (P<0.01), and increased p-YAP protein expression (P<0.01). CONCLUSIONS: Herbal cake-separated moxibustion combined with atorvastatin exerts synergistic effects, ameliorates serum lipid profiles, mitigates endothelial dysfunction of aorta, and attenuates inflammatory responses in AS mice, potentially via modulation of the integrin/YAP/JNK signaling pathway in the aorta. 目的: 观察隔药饼灸联合阿托伐他汀对ApoE⁻/⁻动脉粥样硬化（AS）小鼠血脂代谢、主动脉病理结构、斑块沉积及整合素（integrin）/Yes相关蛋白（YAP）/c-Jun N末端激酶（JNK）信号通路的影响，探讨其防治AS的作用机制。方法: 雄性C57BL/6J小鼠作为空白组，雄性ApoE⁻/⁻小鼠随机分为模型组、西药组、隔药饼灸组和联合治疗组，每组9只。高脂饲料喂养复制AS模型。隔药饼灸组于“膻中”和“神阙”施以隔药饼灸，隔日1次，每周3次;西药组予阿托伐他汀钙片3 mg·kg-1·d-1，每日1次;联合治疗组联合运用西药组和隔药饼灸组的干预方法。各组均连续干预8周。治疗前后观察各组小鼠体质量，HE染色观察主动脉病理形态，油红O染色观察主动脉脂质斑块面积，生化分析仪检测血清甘油三酯（TG）、总胆固醇（TC）、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）含量，ELISA法检测血清血管内皮生长因子（VEGF）、内皮素-1（ET-1）、白细胞介素（IL）-6、IL-8 和肿瘤坏死因子-α（TNF-α）含量，比色法检测血清一氧化氮（NO）含量，Western blot法检测主动脉integrin αVβ3、YAP、磷酸化（p）-YAP、p-JNK1/2、细胞间黏附分子-1（ICAM-1）、血管细胞间黏附分子-1（VCAM-1）的蛋白表达水平。结果: 与空白组相比，模型组体质量增加（P<0.01）;主动脉内膜增厚，泡沫细胞和脂质堆积，平滑肌细胞增生水肿，弹性纤维变薄，内皮细胞间隙增宽，弹性膜发生断裂;动脉脂质斑块面积显著增加（P<0.01）;血清TC、TG、LDL-C、IL-6、IL-8、TNF-α、ET-1、VEGF含量升高（P<0.01），HDL-C和NO水平降低（P<0.01）;主动脉integrin αVβ3、YAP、pJNK1/2、ICAM-1、VCAM-1蛋白表达增加（P<0.01），p-YAP蛋白表达减少（P<0.01）。与模型组相比，西药组、隔药饼灸组和联合治疗组小鼠体质量减轻（P<0.01），且体质量变化的差值明显低于模型组（P<0.01）;主动脉管腔结构较规则，内膜变薄，泡沫细胞和脂质沉积减少，平滑肌细胞水肿、肥大和炎性反应浸润减轻;其余各项指标也均较模型组有明显改善（P<0.01，P<0.05）。与隔药饼灸组相比，西药组和联合治疗组斑块面积减小（P<0.05，P<0.01），血清NO含量上升（P<0.01），LDL-C、ET-1和VEGF含量降低（P<0.01），主动脉p-JNK1/2、ICAM-1和VCAM-1蛋白表达减少（P<0.05，P<0.01）;西药组血清TC、TG、IL-6、IL-8、TNF-α水平降低（P<0.01），HDL-C水平升高（P<0.01），主动脉integrin αVβ3、YAP蛋白表达减少（P<0.01，P<0.05），p-YAP蛋白表达增加（P<0.05）。与西药组和隔药饼灸组相比，联合治疗组小鼠体质量减轻幅度更大（P<0.05，P<0.01），血清TC、TG、IL-6、IL-8、TNF-α含量和主动脉integrin αVβ3、YAP蛋白表达降低（P<0.01，P<0.05），血清HDL-C水平升高（P<0.01），p-YAP蛋白表达增加（P<0.01）。结论: 隔药饼灸联合阿托伐他汀具有协同增效作用，可调节AS小鼠血脂水平，减轻主动脉内皮损伤，降低炎性反应，其作用可能与调控主动脉integrin/YAP/JNK信号通路相关。.