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基于TLR4/NF-κB/ICAM-1信号通路探讨铁皮石斛茎水提取物对高黏血症大鼠的影响

[Effect of Dendrobii Officinalis Caulis water extract on hyperviscosity rats based on TLR4/NF-κB/ICAM-1 signaling pathway].

作者信息

Chen Zi-Yan, Yan Mei-Qiu, Dong Ying-Jie, Su Jie, Yu Jing-Jing, Lin Qi-Wei, Hu Xue-Ling, Fu Meng, Chen Su-Hong, Lyu Gui-Yuan, Chen Jian-Zhen

机构信息

School of Pharmaceutical Sciences, Zhejiang Chinese Medical University Hangzhou 310053, China.

Zhejiang University of Technology Hangzhou 310014, China.

出版信息

Zhongguo Zhong Yao Za Zhi. 2024 Oct;49(19):5261-5272. doi: 10.19540/j.cnki.cjcmm.20240604.401.

Abstract

This study aims to reveal the effect and mechanism of Dendrobii Officinalis Caulis water extract on the rat model of hyperviscosity induced by a high-sugar, high-salt, and high-fat diet. Thirty-six male SD rats were randomized into normal, model, Compound Danshen Tablets(0.5 g·kg(-1)), and low-, medium-, and high-dose(0.25, 0.5, and 1.0 g·kg(-1), respectively) Dendrobii Officinalis Caulis water extract groups. Except the normal group, the remaining groups were fed with a high-salt and high-fat diet and received regular gavage of sucrose solution(20 g·kg(-1)) for 12 consecutive weeks. The modeling was accompanied by gavage of corresponding drugs. During the experimental period, general signs(such as grip strength, automatic activity, vertigo time, and pain threshold), blood rheology, and blood flow in the tail microcirculation were measured. Flow cytometry was employed to detect the positive cells of intercellular cell adhesion molecule 1(ICAM-1/CD54) and vascular cell adhesion molecule 1(VCAM-1/CD106) in the peripheral blood and the Ca(2+) fluorescence intensity. Enzyme-linked immunosorbent assay was employed to measure the levels of ICAM-1, VCAM-1, platelet endothelial cell adhesion molecule 1(PECAM-1/CD31), endothelin 1(ET-1), thromboxane A2(TXA2), prostaglandin I2(PGI2), nitric oxide(NO), interleukin-1β(IL-1β), C-reactive protein(CRP), interleukin-6(IL-6), tumor necrosis factor-α(TNF-α), and lipopolysaccharide(LPS). Hematoxylin-eosin, Masson, and Gomori's aldehyde-fuchsin staining were used to observe the pathological and morphological changes in rat colon and aorta. Fluorescence quantitative PCR was conducted to determine the mRNA levels of Toll-like receptor 4(TLR4), myeloid differentiation factor 88(MyD88), nuclear factor-kappaB(NF-κB), ICAM-1, and VCAM-1 in the aortic tissue. Western blot was employed to determine the protein levels of occludin and claudin-1 in the colon tissue and ICAM-1, VCAM-1, and IL-6 in the aortic tissue. Immunohistochemistry and immunofluorescence were employed to detect the expression of TLR4 and NF-κB in the aortic tissue. The results showed that Dendrobii Officinalis Caulis water extract significantly improved the general signs, reduced the whole blood viscosity, whole blood reducing viscosity, and whole blood flow resistance, increased the blood flow of tail microcirculation, reduced the rates of CD54, CD106-positive cells in peripheral blood, and attenuated the blood flow obstruction in the rat model of hyperviscosity. The water extract significantly lowered the serum levels of ICAM-1, VCAM-1, PECAM-1, and ET-1, elevated the PGI2 and NO level, and alleviated vascular endothelial injury. In addition, the water extract decreased the content of IL-1β, TNF-α, IL-6, and CRP in the serum and reduced the vascular inflammatory injury. Moreover, the Dendrobii Officinalis Caulis water extract improved the morphology of colon and aortic tissues, promoted the expression of occludin and claudin-1 in the colon, and down-regulated the mRNA levels of TLR4, MyD88, NF-κB, ICAM-1, and VCAM-1 as well as the protein levels of TLR4, NF-κB, and IL-6 in the aortic tissue. In conclusion, the results suggest that the Dendrobii Officinalis Caulis water extract may regulate the endothelial inflammatory injury via the TLR4/NF-κB/ICAM-1 pathway, thereby ameliorating hyperviscosity.

摘要

本研究旨在揭示铁皮石斛水提取物对高糖、高盐、高脂饮食诱导的大鼠高粘滞血症模型的作用及机制。将36只雄性SD大鼠随机分为正常组、模型组、复方丹参片组(0.5 g·kg⁻¹)以及低、中、高剂量铁皮石斛水提取物组(分别为0.25、0.5、1.0 g·kg⁻¹)。除正常组外,其余各组给予高盐高脂饮食,并连续12周每日定时灌胃蔗糖溶液(20 g·kg⁻¹)。造模同时给予相应药物灌胃。实验期间,测定一般体征(如握力、自主活动、眩晕时间和痛阈)、血液流变学指标以及尾微循环血流量。采用流式细胞术检测外周血中细胞间黏附分子1(ICAM - 1/CD54)和血管细胞黏附分子1(VCAM - 1/CD106)阳性细胞及Ca²⁺荧光强度。采用酶联免疫吸附测定法检测ICAM - 1、VCAM - 1、血小板内皮细胞黏附分子1(PECAM - 1/CD31)、内皮素1(ET - 1)、血栓素A2(TXA2)、前列腺素I2(PGI2)、一氧化氮(NO)、白细胞介素 - 1β(IL - 1β)、C反应蛋白(CRP)、白细胞介素 - 6(IL - 6)、肿瘤坏死因子 - α(TNF - α)和脂多糖(LPS)水平。采用苏木精 - 伊红染色、Masson染色和Gomori醛复红染色观察大鼠结肠和主动脉的病理及形态学变化。采用荧光定量PCR法检测主动脉组织中Toll样受体4(TLR4)、髓样分化因子88(MyD88)、核因子 - κB(NF - κB)、ICAM - 1和VCAM - 1的mRNA水平。采用蛋白质免疫印迹法检测结肠组织中闭合蛋白和紧密连接蛋白 - 1以及主动脉组织中ICAM - 1、VCAM - 1和IL - 6的蛋白水平。采用免疫组织化学法和免疫荧光法检测主动脉组织中TLR4和NF - κB的表达。结果表明,铁皮石斛水提取物显著改善一般体征,降低全血黏度、全血还原黏度和全血流动阻力,增加尾微循环血流量,降低外周血中CD54及CD106阳性细胞率,减轻高粘滞血症大鼠模型的血流障碍。该水提取物显著降低血清中ICAM - 1、VCAM - 1、PECAM - 1和ET - 1水平,升高PGI2和NO水平,减轻血管内皮损伤。此外,该水提取物降低血清中IL - 1β、TNF - α、IL - 6和CRP含量,减轻血管炎症损伤。而且,铁皮石斛水提取物改善结肠和主动脉组织形态,促进结肠中闭合蛋白和紧密连接蛋白 - 1的表达,下调主动脉组织中TLR4、MyD88、NF - κB、ICAM - 1和VCAM - 1的mRNA水平以及TLR4、NF - κB和IL - 6的蛋白水平。综上所述,结果提示铁皮石斛水提取物可能通过TLR4/NF - κB/ICAM - 1通路调节内皮炎症损伤,从而改善高粘滞血症。

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