[關(guān)鍵詞]
[摘要]
目的 基于代謝組學(xué)探討黃芪赤風(fēng)湯對(duì)內(nèi)皮細(xì)胞損傷的保護(hù)作用及其機(jī)制。方法 通過(guò)氧化低密度脂蛋白(ox-LDL)誘導(dǎo)小鼠腦微血管內(nèi)皮bEnd.3細(xì)胞建立體外內(nèi)皮細(xì)胞損傷模型。bEnd.3細(xì)胞隨機(jī)分為對(duì)照組、模型組和黃芪赤風(fēng)湯低、中、高劑量組。CCK-8法分別檢測(cè)不同濃度ox-LDL和黃芪赤風(fēng)湯對(duì)bEnd.3細(xì)胞活力的影響;油紅O染色評(píng)估bEnd.3細(xì)胞內(nèi)脂質(zhì)聚積情況;酶聯(lián)免疫吸附試驗(yàn)(ELISA)法檢測(cè)bEnd.3細(xì)胞總膽固醇(TC)、游離膽固醇(FC)、膽固醇酯(CE)、白細(xì)胞介素-1β(IL-1β)、IL-6、腫瘤壞死因子-α(TNF-α)、細(xì)胞間黏附分子-1(ICAM-1)和血管細(xì)胞黏附分子-1(VCAM-1)水平和CE/TC;非靶向代謝組學(xué)分析bEnd.3細(xì)胞的代謝物譜,以鑒定篩選出與內(nèi)皮損傷相關(guān)的差異代謝物,從而探究黃芪赤風(fēng)湯保護(hù)內(nèi)皮損傷的代謝通路及潛在作用機(jī)制。結(jié)果 CCK-8結(jié)果確定50μg/mL為ox-LDL的造模濃度,50、100和200μg/mL為黃芪赤風(fēng)湯干預(yù)bEnd.3細(xì)胞的低、中、高劑量。與模型組比較,黃芪赤風(fēng)湯能有效改善bEnd.3細(xì)胞的泡沫化程度,脂滴蓄積明顯減少,黃芪赤風(fēng)湯各劑量組TC、CE和TNF-α水平顯著下調(diào)(P<0.05、0.01),黃芪赤風(fēng)湯中、高劑量組FC、IL-6、VCAM-1水平和CE/TC顯著下調(diào)(P<0.05、0.01),黃芪赤風(fēng)湯高劑量組IL-1β和ICAM-1水平顯著下調(diào)(P<0.05、0.01)。代謝組學(xué)鑒定出15種差異代謝產(chǎn)物,包括5-羥基吲哚乙醛、同型半胱氨酸、花生四烯酸、鞘氨醇-1-磷酸、苯丙酮酸、谷胱甘肽、氧化谷胱甘肽和L-磷酸精氨酸等,主要富集于谷胱甘肽代謝、煙酸和煙酰胺代謝、苯丙氨酸代謝、半胱氨酸和蛋氨酸代謝等代謝途徑。結(jié)論 黃芪赤風(fēng)湯可有效改善內(nèi)皮細(xì)胞損傷,抑制ox-LDL誘導(dǎo)的bEnd.3細(xì)胞內(nèi)脂質(zhì)蓄積、炎性因子和黏附分子表達(dá),其機(jī)制可能與調(diào)節(jié)受損內(nèi)皮細(xì)胞內(nèi)的代謝功能,調(diào)節(jié)細(xì)胞內(nèi)氨基酸等物質(zhì)代謝相關(guān)。
[Key word]
[Abstract]
Objective To investigate the protective effect of Huangqi Chifeng Tang on endothelial cell injury and its mechanism based on metabolomics. Methods An in vitro endothelial cell injury model was established by ox-LDL induced mouse brain endothelial bEnd.3 cells. bEnd.3 cells were randomly divided into control group, model group and Huangqi Chifeng Tang low, medium, and high dose groups. Cell counting Kit-8(CCK-8) was used to detect the effects of different concentrations of ox LDL and Huangqi Chifeng Tang on bEnd.3 cell viability. Oil red O staining was used to evaluate the intracellular lipid accumulation in bEnd.3 cells. Enzymelinked immunosorbent assay(ELISA) was used to detect the levels of total cholesterol(TC), free cholesterol(FC), cholesterol esters(CE), interleukin-1β(IL-1β), IL-6, tumor necrosis factor-α(TNF-α), intercellular adhesion molecule-1(ICAM-1), and vascular cell adhesion molecule-1(VCAM-1) in bEnd.3 cells, as well as CE/TC. Non-targeted metabolomics analysis of the metabolite profile of bEnd.3 cells to identify and screen differential metabolites related to endothelial injury, in order to explore the metabolic pathways and potential mechanisms of Huangqi Chifeng Tang in protecting endothelial injury. Results CCK-8 determined 50 μg/mL as the modeling concentration for ox-LDL, and 50, 100, and 200 μg/mL were the low, medium, and high dose of Huangqi Chifeng Tang intervention in bEnd.3 cells. Compared with the modal group, Huangqi Chifeng Tang can effectively improve the foam degree of bEnd.3 cells, and significantly reduced the accumulation of lipid droplets. Compared with the modal group, the levels of TC, CE, and TNF-α were significantly decreased in each dose group of Huangqi Chifeng Tang(P<0.05, 0.01), the levels of FC, IL-6, VCAM-1, and CE/TC were significantly decreased in the middle and high dose groups of Huangqi Chifeng Tang(P<0.05, 0.01), and the levels of IL-1β and ICAM-1 were significantly decreased in the high dose group of Huangqi Chifeng Tang(P<0.05, 0.01). Metabolomics results showed that a total of 15 differential metabolites were identified, including 5-hydroxyindolealdehyde, homocysteine, arachidonic acid, sphingosine-1-phosphate, phenylpyruvic acid, glutathione, oxidized glutathione and L-Phosphoarginine, mainly enriched in metabolic pathways such as glutathione metabolism, niacin and nicotinamide metabolism, phenylalanine metabolism, cysteine and methionine metabolism. Conclusion Huangqi Chifeng Tang can effectively improve endothelial cell damage, inhibit ox-LDL induced lipid accumulation, inflammatory factors, and adhesion molecule expression in bEnd. 3 cells. Its mechanism may be related to regulating the metabolic function of damaged endothelial cells and regulating the metabolism of intracellular amino acids and other substances.
[中圖分類號(hào)]
R285.5
[基金項(xiàng)目]
黑龍江省自然科學(xué)基金資助項(xiàng)目(PL2024H239)