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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).
CONCLUSIONS: 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.
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).
CONCLUSIONS: 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.
摘要：
目的：观察脑络心通汤(NLXTD)对氧糖剥夺/再灌注(OGD/R)大鼠脑微血管内皮细胞(BMECs)功能及血管生成的影响,并探讨其可能的作用机制。
方法：将有或没有caspase-1siRNA转染的大鼠BMECs在10%含药血清（或空白血清）存在下培养，并暴露于OGD/R。CCK-8测定，Transwell腔室分析，和试管形成试验用于评估增殖，迁移,和细胞的管形成能力。使用商业测定试剂盒测定培养上清液中乳酸脱氢酶（LDH）的活性，ELISA法检测炎症因子IL-1β和IL-18水平。caspase-1,caspase-1,NLRP3,GasderminD,和血管生成相关蛋白VEGF和VEGFR2使用蛋白质印迹法检测。
结果：BMEC在OGD/R暴露后表现出明显的损伤。与空白血清相比，含药血清显著提高了细胞活力，迁移能力，和管腔形成能力（P<0.01），并降低了OGD/R暴露后细胞的IL-1β和IL-18水平以及LDH释放（P<0.01）。蛋白质印迹显示，在暴露于OGD/R的BMECs中，含药血清强烈上调VEGF和VEGFR2蛋白的表达（P<0.01），降低pro-caspase-1，caspase-1，NLRP3和GasderminD的蛋白表达（P<0.01）。转染caspase-1siRNA可促进VEGFR2蛋白在细胞中的表达(P<0.01)。
结论：NLXTD可以促进细胞增殖，迁移,OGD/R损伤的BMECs可能通过抑制细胞凋亡中的caspase-1/GasderminD通路来促进血管生成，减轻细胞损伤，并上调VEGF和VEGFR2的表达。
方法：将有或没有caspase-1siRNA转染的大鼠BMECs在10%含药血清（或空白血清）存在下培养，并暴露于OGD/R。CCK-8测定，Transwell腔室分析，和试管形成试验用于评估增殖，迁移,和细胞的管形成能力。使用商业测定试剂盒测定培养上清液中乳酸脱氢酶（LDH）的活性，ELISA法检测炎症因子IL-1β和IL-18水平。caspase-1,caspase-1,NLRP3,GasderminD,和血管生成相关蛋白VEGF和VEGFR2使用蛋白质印迹法检测。
结果：BMEC在OGD/R暴露后表现出明显的损伤。与空白血清相比，含药血清显著提高了细胞活力，迁移能力，和管腔形成能力（P<0.01），并降低了OGD/R暴露后细胞的IL-1β和IL-18水平以及LDH释放（P<0.01）。蛋白质印迹显示，在暴露于OGD/R的BMECs中，含药血清强烈上调VEGF和VEGFR2蛋白的表达（P<0.01），降低pro-caspase-1，caspase-1，NLRP3和GasderminD的蛋白表达（P<0.01）。转染caspase-1siRNA可促进VEGFR2蛋白在细胞中的表达(P<0.01)。
结论：NLXTD可以促进细胞增殖，迁移,OGD/R损伤的BMECs可能通过抑制细胞凋亡中的caspase-1/GasderminD通路来促进血管生成，减轻细胞损伤，并上调VEGF和VEGFR2的表达。
