关键词: 参红补血颗粒, 血管内皮功能障碍, 氧化应激, Toll样受体4, 人脐静脉内皮细胞

Abstract:

Objective To investigate the protective effect of Shenhong Buxue Granules （SBG） on vascular endothelium of the mice with vascular endothelial dysfunction （VED） of Qi stagnation and blood stasis type and its effect on the expression levels of Toll-like receptor 4 （TLR4），myeloid differentiation factor （MyD88），nuclear factor κB p65 （NF-κB p65），intercellular adhesion molecule-1 （ICAM-1） proteins in the human umbilical vein endothelial cells （HUVECs），and to elucidate its relevant action mechanism. Methods A total of 60 Kunming mice were randomly divided into normal control group， VED group， positive control group （0.62 g·kg^－1·d^－1 Xuefuzhuyu Capsule）， low dose of SBG （3 g·kg^－1·d^－1） group， medium dose of SBG （6 g·kg^－1·d^－1） group and high dose of SBG （9 g·kg^－1·d^－1） group，and there were 10 mice in each group. Except for normal control group， the mice in other groups were used to construct the VED models of Qi stagnation and blood stasis type with ice water bath method，and the mice were continuously administered for 21 d. The behavior of the mice in various groups were observed，the whole blood viscosities of the mice in various groups were measured by blood rheometer，HE staining was used to observe the pathomorphology of lung and thoracic aorta tissues of the mice in various groups，the serum levels of von Willebrand factor （vWF）， thrombomodulin （TM），ICAM-1， and tumor necrosis factor-α （TNF-α），and interleukin-6 （IL-6） of the mice in various groups were detected by ELISA method， and the kits were used to measure the levels of nitric oxide （NO） in serum and the activities of glutathione peroxidase （GSH-Px） and superoxide dismutase （SOD） in thoracic aorta tissue of the mice in various groups.The human umbilical vein endothelial cells （HUVECs） were cultured in vitro and divided into normal control group， tert-butyl hydroperoxide （TBHP） group and low，middle and high doses of SBG groups.Except for normal control group， the HUVECs in the other 4 groups were induced by TBHP to establish the oxidative damage model of HUVECs. The survival rates of HUVECs in various groups were detected by MTT assay， Western blotting method was used to detect the expression levels of TLR4， MyD88， NF-κB p65， and ICAM-1 proteins in the HUVECs in various groups. Results Compared with normal control group， the grasping force value and the number of autonomous activities of the mice in VED group were significantly decreased （P<0.05），and the whole blood viscosity was increased significantly （P<0.05）； compared with VED group， the grasping force values of the mice in positive control group and the low， middle and high doses of SBG groups were significantly increased （P<0.05）， the whole blood viscosities were significantly decreased （P<0.05），and the numbers of autonomous activities of the mice in positive control group and high dose of SBG group were significantly increased （P<0.05）.The HE staining results showed that compared with normal control group， the lung tissue of the mice in VED group showed obvious telangiectasia and congestion， and the tissue was accompanied by a large number of inflammatory cell infiltration； compared with VED group， the degrees of telangiectasia and congestion of the alveolar wall of the mice in positive control group and different doses of SBG groups were decreased， and the number of inflammatory cell infiltration was significantly decreased in a dose-dependent manner； compared with normal control group， the inner wall of thoracic aorta of the mice in VED group was disordered， with defects and falling off； compared with VED group， the falling off lesion of vascular intima of the mice in positive control group and different doses of SBG groups was improved and the integrity of the intima was maintained. Compared with normal control group， the serum levels of vWF，TM，ICAM-1， TNF-α ，and IL-6 of the mice in VED group were significantly increased （P<0.05）， and the serum level of NO and serum activities of GSH-Px and SOD in aorta tissue were significantly decreased （P<0.05）； compared with VED group， the serum levels of vWF， TM， ICAM-1， TNF-α and IL-6 of the mice in positive control group and the high dose of SBG group were significantly decreased （P<0.05），and the serum level of NO and activities of GSH-Px and SOD in aorta tissue were significantly increased （P<0.05）. Compared with normal control group， the survival rate of HUVECs in TBHP group was significantly decreased （P<0.05），and the expression levels of TLR4， MyD88， NF-κB p65， and ICAM-1 proteins in the HUVECs in TBHP group were significantly increased （P<0.05）； compared with TBHP group， the survival rate of the HUVECs in high dose of SBG group was significantly increased （P<0.05），and the expression levels of TLR4， MyD88， NF-κB p65， and ICAM-1 proteins in the HUVECs in high dose of SBG group were significantly decreased （P<0.05）. Conclusion SBG can protect the vascular endothelium of the mice with VED of Qi stagnation and blood stasis type by inhibiting the oxidative stress and alleviating the inflammatory response.

Key words: Shenhong Buxue Granules, Vascular endothelial dysfunction, Oxidative stress, Toll-like receptor 4, Human umbilical vein endothelial cells