参红补血颗粒对气滞血瘀型血管内皮功能障碍小鼠血管内皮的保护作用及其机制

doi:10.13481/j.1671-587X.20220609

Abstract

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

CLC Number:

Junxiu LIU,Jia ZHOU,Guangfu LYU,Yuchen WANG,Xuefeng ZHUANG,Jiarui ZHAO,Xiaowei HUANG,Ruili LI. Protective effect of Shenhong Buxue Granule on vascular endothelium of mice with vascular endothelial dysfunction of Qi stagnation and blood stasis type and its mechanism[J].Journal of Jilin University(Medicine Edition), 2022, 48(6): 1437-1447.

Figures/Tables 11

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References 29

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Group	Grasping forcevalue(m/g)	Number of autonomous activity
Normal control	263.22±76.51	53.17±5.21
VED	158.67±37.34^*	35.00±7.96^**
Positive control	262.15±43.91^△△	50.83±6.57^△△
SBG
Low dose	210.75±32.47^△	41.83±5.43
Middle dose	229.82±23.01^△△	44.00±9.63
High dose	255.18±20.17^△△	48.67±9.74^△

Group	Whole blood viscosity
Group	10·s^－1	60·s^－1	150·s^－1
Normal control	8.45±1.24	5.84±0.82	4.44±0.69
VED	16.23±4.42^**	9.63±2.81^*	7.37±2.62^*
Positive control	8.55±2.27^△△	6.59±0.74^△	4.57±0.49^△
SBG
Low dose	10.49±1.67^△	6.55±0.66^△	4.60±0.61^△
Middle dose	9.85±1.65^△	6.26±1.28^△	4.59±0.50^△
High dose	9.60±0.60^△△	6.03±1.26^△	4.55±0.63^△

Group	vWF [λ_B/(U·L^－1)]	TM [ρ_B/(pg·L^－1)]	ICAM-1 [ρ_B/(ng·L^－1)]	NO [c_B/(μmol·L^－1)]
Normal control	255.53±44.10	18.11±2.37	184.01±8.32	26.40±2.09
VED	486.13±82.92^*	25.83±2.73^*	240.10±1.36^**	16.08±2.09^**
Positive control	262.53±45.90^△	19.63±1.49^△	197.79±5.50^△△	24.93±2.09^△
SBG
Low dose	353.53±47.39	23.11±1.24	230.48±8.49	16.08±4.17
Middle dose	334.87±94.10	22.69±1.51	212.53±1.63^△△	19.03±3.61
High dose	290.87±47.23^△	18.58±1.92^△	211.25±7.85^△△	21.98±2.09^△

Group	TNF-α [ρ_B/(ng·L^－1)]	IL-6 [ρ_B/(pg·L^－1)]	GSH-Px [λ_B/(U·mg^－1)]	SOD [λ_B/(U·mg^－1)]
Normal control	626.88±127.88	54.62±11.83	260.81±12.79	52.09±2.43
VED	1 006.04±96.97^*	100.40±15.85^*	150.35±6.68^**	32.96±2.97^**
Positive control	652.92±99.49^△	57.68±13.95^△	232.50±10.40^△△	42.74±2.02^△
SBG
Low dose	718.54±112.45	72.30±8.53	182.22±7.84	34.87±2.54
Middle dose	688.33±128.50^△	70.79±6.66	187.06±19.76	38.51±1.91
High dose	656.46±121.80^△	57.55±13.75^△	229.52±19.73^△△	43.54±1.33^△

Group	TNF-α [ρ_B/(ng·L^－1)]	IL-6 [ρ_B/(pg·L^－1)]	NO [c_B/(μmol·L^－1)]	LDH [λ_B/(U·L^－1)]	MDA [m_B/(μmol·g^－1)]	GSH-PX [λ_B/(U·mg^－1)]	SOD [λ_B/(U·mg^－1)]
Normal control	66.97±4.35	39.65±5.33	27.88±6.26	202.78±60.48	2.13±0.21	203.67±11.62	40.12±1.88
TBHP	135.27±9.87^**	98.60±6.80^**	14.44±0.23^*	495.37±65.21^*	5.15±0.44^**	115.35±4.44^**	18.62±1.14^**
SBG
Low dose	122.72±6.97	82.07±5.38	19.03±3.61	400.93±68.61	3.92±0.22^*	139.93±12.44	21.58±1.98
Middle dose	102.14±6.33^△	72.64±8.17^△	21.98±2.09^△△	309.26±47.39^△	3.39±0.25^△△	152.74±10.18^△△	30.52±2.27^△△
High dose	78.52±4.90^△△	53.61±10.78^△△	23.45±3.61^△	243.06±80.64^△	2.87±0.25^△△	185.77±8.04^△△	36.04±3.18^△△

Protective effect of Shenhong Buxue Granule on vascular endothelium of mice with vascular endothelial dysfunction of Qi stagnation and blood stasis type and its mechanism

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Group	Survival rate
Normal control
5% blank serum	100.00±0.00
10% blank serum	100.00±0.00
20% blank serum	100.00±0.00
Low dose of SBG
5% drug serum	95.75±5.55
10% drug serum	98.08±1.15
20% drug serum	93.63±3.32
Middle dose of SBG
5% drug serum	99.04±3.86
10% drug serum	95.68±2.56^*
20% drug serum	86.39±5.78^*
High dose of SBG
5% drug serum	99.84±3.93
10% drug serum	85.17±0.40^*
20% drug serum	70.54±7.36^*

Group	Survival rate
Group	(t/h) 24	48
Normal control	100.00±0.00	100.00±0.00
TBHP	73.27±6.09^*	49.63±7.79^*
SBG
Low dose	83.56±4.27	51.24±4.56
Middle dose	92.67±5.04^△	62.95±5.53
High dose	96.92±1.78^△	76.81±3.88^△

Group	TLR4	MyD88	NF-κB p65	ICAM-1
Normal control	1.00±0.00	1.00±0.00	1.00±0.00	1.00±0.00
TBHP	1.72±0.21^**	1.88±0.31^*	1.97±0.24^**	2.18±0.39^*
SBG
Low dose	1.63±0.13	1.74±0.29	1.81±0.23	1.86±0.17
Middle dose	1.55±0.24^△	1.79±0.37	1.99±0.36	1.76±0.27
High dose	1.16±0.04^△	1.24±0.11^△	1.34±0.08^△	1.22±0.10^△

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