南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (7): 1093-1101.doi: 10.12122/j.issn.1673-4254.2023.07.05

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脑络欣通促进氧糖剥夺/再灌注损伤大鼠的脑微血管内皮细胞血管新生：基于激活caspase-1/Gasdermin D通路

李佩佩，胡音琦，刘 佳，王丽娜，吴元洁，胡建鹏   

  1. 安徽中医药大学新安医学教育部重点实验室，安徽 合肥 230038；安徽中医药大学中医学院，安徽 合肥 230012
  • 出版日期:2023-07-20 发布日期:2023-07-20

Naoluo Xintong Decoction activates caspase-1/Gasdermin D pathway to promote angiogenesis of rat brain microvascular endothelial cells after oxygen glucose deprivation/reperfusion injury

LI Peipei, HU Yinqi, LIU Jia, WANG Lina, WU Yuanjie, HU Jianpeng   

  1. Ministry of Education Key Laboratory of Xin'an Medicine, Anhui University of Chinese Medicine, Hefei 230038, China; School of Traditional Chinese Medicine, Anhui University of Chinese Medicine, Hefei 230012, China
  • Online:2023-07-20 Published:2023-07-20

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摘要： 目的 探讨脑络欣通（NLXTD）含药血清对氧糖剥夺/再灌注（OGD/R）大鼠脑微血管内皮细胞（BMECs）焦亡及血管新生的影响和可能作用机制。方法 BMECs体外培养，分别经OGD/R诱导、NLXTD含药血清处理及siRNA转染caspase-1，设置Control组：BMECs+10%空白血清；OGD/R组：BMECs+OGD/R+10%空白血清；NLXTD组：BMECs+OGD/R+10% NLXTD含药血清；siRNA-NC组：BMECs转染siRNA-NC+OGD/R+10%空白血清；si-caspase-1组：BMECs转染si-caspase-1+OGD/R+10%空白血清；si-caspase-1+NLXTD组：BMECs转染si-caspase-1+OGD/R+10%NLXTD含药血清。采用CCK-8法、Transwell小室实验、管腔形成实验分别检测细胞增殖、迁移、成管能力；试剂盒测定上清液中乳酸脱氢酶（LDH）活性；ELISA法检测培养液中炎性因子IL-1β、IL-18的含量；Western blot检测细胞焦亡相关蛋白pro-caspase-1、caspase-1、NLRP3、Gasdermin D及血管生成关键蛋白VEGF，VEGFR2的表达。结果 BMECs经OGD/R诱导后出现明显的损伤；与OGD/R组比较，10%的NLXTD含药血清能显著提高OGD/R损伤BMECs的存活率、迁移能力及成管能力（P<0.01），降低IL-1β、IL-18的含量以及LDH的释放（P<0.01）；Western blot实验结果显示，NLXTD含药血清可上调OGD/R损伤BMECs的VEGF和VEGFR2蛋白表达（P<0.01），并降低pro-caspase-1、caspase-1、NLRP3、Gasdermin D的蛋白表达（P<0.01），加入caspase-1 siRNA后，能进一步促进VEGFR2蛋白表达（P<0.01）。结论 NLXTD可以提高OGD/R损伤后的BMECs增殖、迁移、成管能力，促进血管新生，其机制可能与抑制细胞焦亡caspase-1/Gasdermin D通路，减轻BMECs损伤，上调VEGF、VEGFR2水平有关。

关键词: 脑络欣通；脑微血管内皮细胞；氧糖剥夺/再灌注；血管新生；细胞焦亡；血管内皮生长因子

Abstract: Objective To investigate the effects of Naoluo Xintong Decoction (NLXTD) on pyroptosis and angiogenesis of brain microvascular endothelial cells (BMECs) and explore the possible mechanisms in rats with oxygen-glucose deprivation/reperfusion (OGD/R). Methods Rat BMECs with or without caspase-1 siRNA transfection were cultured in the presence of 10% medicated serum from NLXTD-treated rats (or blank serum) and exposed to OGD/R. CCK-8 assay, Transwell chamber assay, and tube formation assay were used to assess proliferation, migration, and tube- forming abilities of the cells. The activity of lactate dehydrogenase (LDH) in the culture supernatant was determined using a commercial assay kit, and the levels of inflammatory factors IL-1β and IL-18 were detected with ELISA. The cellular expressions of pro-caspase-1, caspase-1, NLRP3, Gasdermin D, and angiogenesis-related proteins VEGF and VEGFR2 were detected using Western blotting. Results The BMECs showed obvious injuries after OGD/R exposure. Compared with the blank serum, the medicated serum significantly improved the cell viability, migration ability, and lumen-forming ability (P<0.01) and lowered the levels of IL-1β and IL-18 and the LDH release (P<0.01) of the cells with OGD/R exposure. Western blotting showed that in the BMECs exposed to OGD/R, the medicated serum strongly upregulated the expression of VEGF and VEGFR2 proteins (P<0.01) and reduced the protein expressions of pro-caspase-1, caspase-1, NLRP3, and Gasdermin D (P<0.01), and transfection of the cells with caspase-1 siRNA further promoted the expressions of VEGFR2 protein in the cells (P<0.01). Conclusion NLXTD can improve the proliferation, migration, and tube- forming ability and promote angiogenesis of BMECs with OGD/R injury probably by inhibiting the caspase-1/Gasdermin D pathway in pyroptosis, alleviating cell injury, and upregulating the expressions of VEGF and VEGFR2.

Key words: Naoluo Xintong Decoction; brain microvascular endothelial cells; oxygen-glucose deprivation/reperfusion; angiogenesis; pyroptosis; vascular endothelial growth factor