Abstract
Brain microvascular endothelial cells are a key part of the blood-brain barrier. This experiment was set up to investigate the effect and molecular mechanism of Dendrobium polysaccharide on oxidized low-density lipoprotein (ox-LDL)-induced damage to the human brain microvascular endothelial cells. For this purpose, human brain microvascular endothelial cells HBMEC were divided into control group (without any treatment), ox-LDL group (50 μg/mL ox-LDL), Dendrobium polysaccharide low, medium and high concentration group (0.1 μg/L, 0.2 μg/L, 0.4 μg/L Dendrobium polysaccharide+50 μg/mL ox-LDL), ox-LDL+miR-NC group (transfection miR-378 mimic negative control+50 μg/mL ox-LDL), ox-LDL+miR-378 group (transfected miR-378 mimics+50 μg/mL ox-LDL), ox-LDL+DP+anti-miR-NC group (transfected miR-378 inhibitor negative control +0.4 μg/L Dendrobium polysaccharide+50 μg/mL ox-LDL), ox-LDL+DP+anti-miR-378 group (transfected miR-378 inhibitor+0.4 μg/L Dendrobium polysaccharide+50 μg/mL ox-LDL ). The kit was used to detect the levels of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) and catalase (CAT); flow cytometry to detect apoptosis; and Western blot to detect B-cell lymph tumor/leukemia-2 (Bcl-2) and Bcl-2 related X (Bax) protein expression; real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of miR-378. Results showed that after treatment with different concentrations of Dendrobium polysaccharides, MDA levels were decreased in ox-LDL-induced human brain microvascular endothelial cells, SOD and CAT activities were increased, apoptosis rate was decreased, Bcl-2 expression was increased, Bax expression was decreased, miR-378 expression was increased, in a dose-dependent manner (P<0.05). Overexpression of miR-378 inhibits ox-LDL-induced oxidative stress and apoptosis in human brain microvascular endothelial cells. Inhibition of miR-378 expression reversed the effect of Dendrobium polysaccharide on ox-LDL-induced damage to human brain microvascular endothelial cells. Then dendrobium polysaccharide may inhibit ox-LDL-induced oxidative stress and apoptosis in human brain microvascular endothelial cells by up-regulating the expression of miR-378.
Highlights
The blood-brain barrier (BBB) is the area between the extracellular fluid of the brain in the central nervous system and the circulatory system in the body so that if colored substances are injected into the bloodstream, it can be seen that there is no trace of this substance in the brain
MEC were purchased from Shanghai Kanglang Biotechnology Co., Ltd.; fetal bovine serum and RPMI1640 medium were purchased from Shenzhen Ziker Biotechnology Co., Ltd.; oxidized low-density lipoprotein, Dendrobium polysaccharides were purchased from Beijing Chreagen Biological Technology Co., Ltd.; superoxide dismutase (SOD) kit, malonaldehyde (MDA) kit, catalase (CAT) were purchased from Nanjing Jiancheng Bioengineering Institute; Annexin V-FITC/PI apoptosis detection kit was purchased from Beijing Solarbio Science & Technology Co., Ltd.; real-time fluorescence quantitative PCR detection kit, protein extraction kit, and bicinchoninic acid (BCA) kit were purchased from Beijing Biao Laibo Technology Co., Ltd
Logarithmic growth phase HBMEC cells were taken and cultured in 50 μg/mL oxidized low-density lipoprotein (ox-LDL) to establish a cell injury model which was recorded as an ox-LDL group; the control group (Con) was added with an equal amount of medium; 0.1 μg/L, 0.2 μg/L. 0.4 μg/L Dendrobium polysaccharide was cultured for 2 h and added with 50 μg/mL ox-LDL for continued cultivation, which was recorded as low, medium and high-concentration Dendrobium polysaccharide groups
Summary
The blood-brain barrier (BBB) is the area between the extracellular fluid of the brain in the central nervous system and the circulatory system in the body so that if colored substances are injected into the bloodstream, it can be seen that there is no trace of this substance in the brain. This curtain or barrier consists of special capillaries that, unlike the normal structure of capillaries, do not have the usual pores and the intercellular connection in them is of the strong connection type, and as a result, many molecules and micro-molecules as well as bacteria are able to pass through them (via diffusion) and reaching the cerebrospinal fluid is not in the brain.
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