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