[汤剂激活半胱天冬酶-1/Gasdermin D通路促进氧糖剥夺/再灌注损伤后大鼠脑微血管内皮细胞血管生成]
[ Decoction activates caspase-1/Gasdermin D pathway to promote angiogenesis of rat brain microvascular endothelial cells after oxygen glucose deprivation/reperfusion injury].
作者信息
Li P, Hu Y, Liu J, Wang L, Wu Y, Hu J
机构信息
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.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2023 Jul 20;43(7):1093-1101. doi: 10.12122/j.issn.1673-4254.2023.07.05.
OBJECTIVE
To investigate the effects of 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 ( < 0.01) and lowered the levels of IL-1β and IL-18 and the LDH release ( < 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 ( < 0.01) and reduced the protein expressions of pro-caspase-1, caspase-1, NLRP3, and Gasdermin D ( < 0.01), and transfection of the cells with caspase-1 siRNA further promoted the expressions of VEGFR2 protein in the cells ( < 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
探讨脑络欣通方（NLXTD）对氧糖剥夺/复灌注（OGD/R）大鼠脑微血管内皮细胞（BMECs）焦亡及血管生成的影响，并探究其可能机制。
方法
将转染或未转染caspase-1小干扰RNA（siRNA）的大鼠BMECs，置于含10%脑络欣通方处理大鼠的含药血清（或空白血清）中培养，然后进行OGD/R处理。采用CCK-8法、Transwell小室法和管腔形成实验分别评估细胞的增殖、迁移及管腔形成能力。使用商用试剂盒测定培养上清液中乳酸脱氢酶（LDH）的活性，采用酶联免疫吸附测定（ELISA）法检测炎症因子白细胞介素-1β（IL-1β）和白细胞介素-18（IL-18）的水平。采用蛋白质免疫印迹法检测前半胱天冬酶-1（pro-caspase-1）、半胱天冬酶-1（caspase-1）、NLR家族含pyrin结构域蛋白3（NLRP3）、Gasdermin D及血管生成相关蛋白血管内皮生长因子（VEGF）和血管内皮生长因子受体2（VEGFR2）的细胞表达。
结果
OGD/R处理后BMECs出现明显损伤。与空白血清相比，含药血清显著提高了OGD/R处理细胞的活力、迁移能力和管腔形成能力（P<0.01），降低了细胞的IL-1β和IL-18水平及LDH释放（P<0.01）。蛋白质免疫印迹法显示，在OGD/R处理的BMECs中，含药血清显著上调了VEGF和VEGFR2蛋白的表达（P<0.01），降低了pro-caspase-1、caspase-1、NLRP3和Gasdermin D的蛋白表达（P<0.01），用caspase-1 siRNA转染细胞进一步促进了细胞中VEGFR2蛋白的表达（P<0.01）。
结论
脑络欣通方可能通过抑制焦亡中的caspase-1/Gasdermin D途径、减轻细胞损伤并上调VEGF和VEGFR2的表达，改善OGD/R损伤的BMECs的增殖、迁移和管腔形成能力，促进血管生成。
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