基于NLRP3介导的细胞焦亡探讨当归黄芪超滤物对冠状动脉微血管疾病的影响

Discussion on the Effects of Angelicae Sinensis Radix and Astragali Radix Extract on Coronary Microvascular Disease Based on NLRP3-mediated Pyroptosis

目的观察当归黄芪超滤物对人脐静脉内皮细胞(HUVEC)焦亡的影响,探讨其治疗冠状动脉微血管疾病的作用机制。方法将HUVEC分为空白组、模型组、MCC950组和当归黄芪低、中、高剂量组,造模及含药血清处理后,CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,鬼笔环肽染色检测细胞骨架形态,免疫荧光染色检测细胞血管内皮生长因子(VEGF)、内皮型一氧化氮合成酶(eNOS)、血管生成素-2(Ang-2)、活性氧(ROS)、内皮素-1(ET-1)、血栓素A2(TXA2)表达,ELISA测定细胞上清液白细胞介素(IL)-1β、IL-18含量,Weasten blot检测细胞NOD样受体蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、胱天蛋白酶-1(Caspase-1)、GSDMD蛋白表达。结果与空白组比较,模型组HUVEC细胞活力降低(P<0.01),细胞凋亡率升高(P<0.01),细胞微丝结构紊乱、打结,VEGF、eNOS表达降低,Ang-2、ROS、ET-1、TXA2表达升高,细胞上清液IL-1β、IL-18含量增加(P<0.01),细胞NLRP3、ASC、Caspase-1、GSDMD蛋白表达升高(P<0.01);与模型组比较,低、中、高剂量当归黄芪含药血清可提高细胞活力(P<0.05),降低细胞凋亡率(P<0.05),改善细胞微丝结构,升高VEGF、eNOS表达,降低Ang-2、ROS、ET-1、TXA2表达,减少细胞上清液IL-1β、IL-18含量(P<0.05),降低NLRP3、ASC、Caspase-1、GSDMD蛋白表达(P<0.05),且当归黄芪中剂量组作用更明显(P<0.05)。结论当归黄芪超滤物可抑制血管紧张素Ⅱ诱导的HUVEC焦亡,减轻内皮细胞功能障碍,进而治疗冠状动脉微血管疾病,其机制可能与抑制NLRP3炎症小体组装相关。

Objective To observe the effects of Angelica Sinensis Radix and Astragali Radix extract(ASR-AR)on HUVEC pyroptosis;To explore its mechanism of treating coronary microvascular disease. Methods HUVECwere divided into blank group, model group, MCC950 group, ASR-AR low-, medium- and high-dosage groups. Aftermodeling and treatment with drug containing serum, cell viability was detected by CCK-8 method, and cell apoptosiswas detected by flow cytometry, phalloidin staining was used to detect cytoskeletal morphology, immunofluorescencestaining was used to detect the expressions of VEGF, eNOS, Ang-2, ROS, ET-1 and TXA2, ELISA was used to detectthe contents of IL-1β and IL-18 in cell supernatant, Western blot was used to detect the expressions of NLRP3, ASC,Caspase-1 and GSDMD protein in cells. Results Compared with the blank group, the model group showed adecrease in HUVEC cell viability (P<0.01) and an increase in cell apoptosis rate (P<0.01), cellular microfilamentstructure was in disorder and knotting, the expressions of VEGF and eNOS decreased, and expressions of Ang-2,ROS, ET-1 and TXA2 increased, the contents of IL-1β and IL-18 in cell supernatant increased (P<0.01), and theexpressions of NLRP3, ASC, Caspase-1 and GSDMD protein in cells increased (P<0.01). Compared with the modelgroup, ASR-AR low- , medium- and high-dosage containing serum could increase cell viability (P<0.05), decreasecell apoptosis rate (P<0.05), improve cell microfilament structure, elevate VEGF and eNOS expressions, decreaseAng-2, ROS, ET-1, TXA2 expressions, reduce IL-1β and IL-18 contents in cell supernatant (P<0.05), and decreaseNLRP3, ASC, Caspase-1 and GSDMD protein expressions (P<0.05). ASR-AR medium-dosage group was moreobvious (P<0.05). Conclusion ASR-AR can inhibit pyroptosis of HUVEC induced by AngⅡ , attenuate endothelialcell dysfunction, thus treating coronary microvascular disease, and its mechanism may be related to the inhibition ofthe assembly of NLRP3 inflammasome.