目的 基于NLRP3炎癥小體探討黃芪赤風(fēng)湯（HQCF）通過改善動脈粥樣硬化（AS）內(nèi)皮損傷的分子機制。方法 氧化低密度脂蛋白（ox-LDL）誘導(dǎo)bEnd.3細胞構(gòu)建內(nèi)皮細胞損傷模型，結(jié)合si-激活NOD樣受體家族含pyrin結(jié)構(gòu)域蛋白3（NLRP3）基因敲低技術(shù)及HQCF (50、100、200μg·mL^-1)干預(yù)。乳酸脫氫酶（LDH）試劑盒檢測bEnd.3細胞受損傷程度；試劑盒法檢測bEnd.3細胞內(nèi)總膽固醇（TC）、游離膽固醇（FC）、膽固醇酯（CE）水平和CE/TC；實時熒光定量PCR（qRTPCR）檢測bEnd.3細胞炎性因子白細胞介素-1β（IL-1β）、IL-6、IL-18和腫瘤壞死因子-α（TNF-α）和黏附分子細胞間黏附分子-1（ICAM-1）、血管細胞黏附分子-1（VCAM-1）基因表達水平；DCFH-DA探針通過流式細胞儀檢測bEnd.3細胞內(nèi)活性氧（ROS）含量；Western blotting檢測bEnd.3細胞中Toll樣受體4（TLR4）、p-核因子κB（NF-κB）/NF-κB、核苷酸結(jié)合寡聚化結(jié)構(gòu)域樣受體蛋白3（NLRP3）、含半胱氨酸的天冬氨酸蛋白水解酶-1（Caspase-1）和凋亡相關(guān)斑點樣蛋白（ASC）蛋白表達水平；qRT-PCR評估si-NLRP3序列的轉(zhuǎn)染效率；油紅O染色評估b End.3細胞內(nèi)脂質(zhì)聚積情況。結(jié)果 與模型組相比，HQCF組LDH釋放量顯著減少（P<0.05、0.01）,TC、FC、CE水平及CE/TC顯著降低（P<0.05、0.01）,ROS水平顯著下調(diào)（P<0.01）,IL-1β、IL-6、IL-18、TNF-α m RNA表達水平顯著下調(diào)（P<0.01）,TLR4、p-NF-κB/NF-κB、NLRP3、Caspase-1和ASC蛋白表達水平顯著下調(diào)（P<0.01）。與ox-LDL+si-NC組相比，ox-LDL+si-NLRP3組和ox-LDL+si-NLRP3+HQCF組細胞脂質(zhì)蓄積情況明顯改善，且以ox-LDL+si-NLRP3+HQCF組改善效果最佳；ox-LDL+si-NLRP3組細胞TC和CE水平顯著下調(diào)（P<0.01）,FC和CE/TC有下調(diào)趨勢但無統(tǒng)計學(xué)意義，ox-LDL+si-NLRP3+HQCF組細胞TC、FC、CE、CE/TC的水平顯著下調(diào)（P<0.01）;ox-LDL+si-NLRP3組與ox-LDL+si-NLRP3+HQCF組bEnd.3細胞ICAM-1和VCAM-1 mRNA表達水平顯著下調(diào)（P<0.01）。結(jié)論 HQCF通過抑制NLRP3炎癥小體通路激活，減輕ox-LDL誘導(dǎo)的內(nèi)皮細胞脂質(zhì)蓄積及炎癥，從而發(fā)揮抗AS內(nèi)皮細胞損傷的作用。

Objective To explore the molecular mechanism of Huangqi Chifeng Tang（HQCF） in improving endothelial damage of atherosclerosis（AS） based on NLRP3 inflammatory inflammasome. Methods ox-LDL-induced bEnd.3 cells were used to construct an endothelial cell injury model, combined with si-NLRP3 gene knockdown technology and HQCF(50, 100, 200 μg·mL^-1) intervention. Lactate dehydrogenase（LDH） assay kit was used to detect bEnd.3 cell viability; The assay kit was used to detect the levels of total cholesterol（TC）, free cholesterol（FC）, cholesterol esters（CE）, and CE/TC in bEnd.3 cells; Real-time fluorescence quantitative PCR（qRT-PCR） was used to detect the gene expression levels of inflammatory factors interleukin-1β（IL-1β）, IL-6, IL-18, tumor necrosis factor-α（TNF-α）, intercellular adhesion molecule-1（ICAM-1）, and vascular cell adhesion molecule-1（VCAM-1） in bEnd.3 cells; DCFH-DA probe was used to detect the intracellular reactive oxygen species（ROS） content in bEnd.3 cells by flow cytometry; Western blotting was used to detect the protein expression levels of Toll like receptor 4（TLR4）, p-nuclear factor kappa B（p-NF-κB）/NF-κB, NOD-, LRR-and pyrin domain-containing protein 3（NLRP3）, Cysteinyl aspartate specific proteinase-1（Caspase-1）, and Apoptosis-associated speck-like protein containing a caspase recruitment domain（ASC） in bEnd.3 cells; transfection efficiency of the si-NLRP3 sequence was evaluated by qRT-PCR; Oil red O staining was used to evaluate the intracellular lipid accumulation in bEnd.3 cells. Results Compared with model group, the LDH release in the HQCF group was significantly reduced（P＜0.05, 0.01）, the levels of TC, FC, CE and CE/TC were significantly decreased（P＜0.05, 0.01）, the ROS level was significantly down-regulated（P＜0.01）, the mRNA expression levels of IL-1β, IL-6, IL-18 and TNF-α were significantly down-regulated（P＜0.01）, and the protein expression levels of TLR4, p-NF-κB/NF-κB, NLRP3, Caspase-1 and ASC were significantly down-regulated（P＜0.01）. Compared with the ox-LDL + si-NC group, the lipid accumulation in cells of the ox-LDL + si-NLRP3 group and the ox-LDL + si-NLRP3 + HQCF group was significantly improved, and the improvement effect was the best in the ox-LDL + si-NLRP3 + HQCF group; the levels of TC and CE in cells of the ox-LDL + si-NLRP3 group were significantly down-regulated（P＜0.01）, FC and CE/TC showed a downward trend but without statistical significance, and the levels of TC, FC, CE and CE/TC in cells of the ox-LDL + si-NLRP3 + HQCF group were significantly down-regulated（P＜0.01）; the mRNA expression levels of ICAM-1 and VCAM-1 in bEnd.3 cells of the ox-LDL + si-NLRP3 group and the ox-LDL + si-NLRP3 + HQCF group were significantly down-regulated（P＜0.01）. Conclusion HQCF exerts an anti-AS endothelial cell injury effect by inhibiting the activation of NLRP3 inflammasome pathway, reducing ox-LDL induced lipid accumulation and inflammation in endothelial cells.

R285.5

黑龍江省自然科學(xué)基金項目(PL2024H239)