Effect of <i>NLRP3</i> gene silencing on expression of proinflammatory agents-induced inflammatory factors in rat brain microvascular endothelial cells_West China Medical Journal

Authors：

ZHANG Jing ^1,2 ,  PAN Xiaohua ² , LIU Guorong ³ , GUO Lizhi ² , NI Zhaowen ² , WANG Yue ² , YU Yingying ¹

1. Baotou Clinical College, Inner Mongolia Medical University, Baotou, Inner Mongolia 014040, P. R. China;
2. Department of Neurology, Baotou Eighth Hospital, Baotou, Inner Mongolia 014040, P. R. China;
3. Department of Neurology, Baotou Central Hospital, Baotou, Inner Mongolia 014040, P. R. China;

Corresponding author：

PAN Xiaohua, Email: 88088647@qq.com

Keywords：

NOD-like receptor family, pyrin domain containing protein 3; silence gene expression; brain microvascular endothelial cell; cerebral small vessel disease

DOI：

10.7507/1002-0179.202205008

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Objective To study the effect of silencing the NOD-like receptor family, pyrin domain containing protein 3 (NLRP3) gene on the production of inflammatory factors induced by lipopolysaccharide (LPS) and adenosine triphosphate (ATP) in rat brain microvascular endothelial cells (BMECs), and whether NLRP3 inflammasome signaling pathway plays a role in the BMEC model of cerebral small vessel disease induced by proinflammatory agents. Methods BMECs from male Wistar rats were extracted in vitro and the morphology and purity of endothelial cells were identified. BMECs in normal culture were divided into blank control group and LPS+ATP group. The expression levels of NLRP3 inflammasome and downstream inflammatory factor Caspase-1 were detected by Western blot and real-time polymerase chain reaction, and compared by student’s t test between the two groups. Small interfering RNA (siRNA) was used to silence the specific gene NLRP3 in BMECs. After transfection of siRNA NLRP3 and siRNA plasmid negative control into BMECs, the transfected cells were divided into four groups, namely, siNC group (non silenced target gene), siNLRP3 group (silenced target gene), siNC+LPS+ATP group (non silenced target gene and added proinflammatory agents) and siNLRP3+LPS+ATP group (silenced target gene and added proinflammatory agents). The expression levels of NLRP3 and Caspase-1 were detected by Western blot and real-time polymerase chain reaction, and analyzed by analysis of variance for 2-factor factorial design. Results The microvascular segments of rat BMECs were “beaded” after 24 h of isolation and culture; after 48 h, “island” cell clusters were formed; after 72 h, “paving stone” like monolayer cells adhered to the wall and grew. After that, the cells gradually became dense and reached the convergence degree of 80%. The positive rate of BMECs detected by immunofluorescence staining was 96%. In the normally cultured cells, the protein and mRNA expression levels of NLRP3 and Caspase-1 in the LPS+ATP group were higher than those in the blank control group (P<0.05). In the RNA interference cultured cells, the protein and mRNA expression levels of NLRP3 and Caspase-1 in the siNLRP3 group were lower than those in the siNC group, and those expression levels in the siNLRP3+LPS+ATP group were lower than those in the siNC+LPS+ATP group (P<0.05); the protein and mRNA expression levels of NLRP3 and Caspase-1 in the siNC+LPS+ATP group were higher than those in the siNC group, and those expression levels in the siNLRP3+LPS+ATP group were higher than those in the siNLRP3 group (P<0.05). Plasmid transfection and proinflammatory agents intervention had statistically significant interaction effect on the mRNA expression of NLRP3 and Caspase-1 (P<0.05). Conclusions LPS and ATP can promote the release of NLRP3 and Caspase-1 in BMECs. Silencing NLRP3 gene expression can reduce the induction of proinflammatory agents. NLRP3 inflammasome signaling pathway may play a role in the cerebral small vessel disease cell model of rat BMECs induced by proinflammatory agents.

Citation： ZHANG Jing, PAN Xiaohua, LIU Guorong, GUO Lizhi, NI Zhaowen, WANG Yue, YU Yingying. Effect of NLRP3 gene silencing on expression of proinflammatory agents-induced inflammatory factors in rat brain microvascular endothelial cells. West China Medical Journal, 2022, 37(6): 855-862. doi: 10.7507/1002-0179.202205008 Copy

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9.	王丽, 刘国荣, 潘晓华. NLRP3 炎症小体相关炎症因子 IL-1β 和 IL-18 在原代脑微血管内皮细胞中的激活及作用. 包头: 内蒙古科技大学包头医学院, 2019.
10.	刘蓓桦, 候梁, 罗伟珏, 等. LPS 与 ATP 共同诱导巨噬细胞中 NLRP3 炎症小体的激活. 北京农学院学报, 2018, 33(1): 74-78.
11.	Wang X, Xu B, Xiang M, et al. Advances on fluid shear stress regulating blood-brain barrier. Microvasc Res, 2020, 128: 103930.
12.	Tanaka KA, Kurihara S, Shibakusa T, et al. Cystine improves survival rates in a LPS-induced sepsis mouse model. Clin Nutr, 2015, 34(6): 1159-1165.
13.	Feng S, Zou L, Wang H, et al. RhoA/ROCK-2 pathway inhibition and tight junction protein upregulation by catalpol suppresses lipopolysaccaride-induced disruption of blood-brain barrier permeability. Molecules, 2018, 23(9): 2371.
14.	Sakurai E. Elucidation of new function in endothelial cells for efficient delivery strategy of drug to tissues. Yakugaku Zasshi, 2020, 140(1): 51-62.
15.	王义宝, 刘云会, 刘丽波, 等. 大鼠脑微血管内皮细胞的原代培养及其生物学行为初步探讨. 神经解剖学杂志, 2006(2): 224-228.
16.	鹿文葆, 秦伟伟, 刘淑英, 等. 大鼠脑微血管内皮细胞的纯化培养与鉴定. 中国脑血管病杂志, 2011, 8(10): 535-538.
17.	徐平湘, 齐特, 陆莉, 等. 原代大鼠脑微血管内皮细胞培养方法的改进. 首都医科大学学报, 2016, 37(5): 693-698.
18.	赵琳, 郭虹, 康立源, 等. 脑微血管内皮细胞培养技术的研究进展. 中国药理学通报, 2008, 24(8): 992-995.
19.	卢晓星, 张小强, 苗歆雨, 等. 原花青素对 LPS/ATP 诱导的 NLRP3 炎症小体激活及 NF-κBp65 磷酸化的抑制作用. 南京医科大学学报(自然科学版), 2020, 40(8): 1111-1118.
20.	李钰婷, 蔡大可, 郑柳怡, 等. NLRP3 基因敲除增强芹菜素对 triton-WR1339 所致高脂血症的降血脂及抗炎作用研究. 中国药理学通报, 2020, 36(4): 543-549.
21.	薛鸿, 林昌建, 谢宝松, 等. 沉默 NLRP3 炎性小体表达降低香烟烟雾提取物导致的人肺动脉内皮细胞凋亡. 基础医学与临床, 2020, 40(4): 490-495.

1. Seoane PI, Lee B, Hoyle C, et al. The NLRP3-inflammasome as a sensor of organelle dysfunction. J Cell Biol, 2020, 219(12): e202006194.
2. McKernan DP. Pattern recognition receptors as potential drug targets in inflammatory disorders. Adv Protein Chem Str, 2020, 119: 65-109.
3. Khoshnam SE, Winlow W, Farzaneh M. The interplay of microRNAs in the inflammatory mechanisms following ischemic stroke. J Neuropathol Exp Neurol, 2017, 76(7): 548-561.
4. Periyasamy P, Thangaraj A, Guo ML, et al. Epigenetic promoter DNA methylation of miR-124 promotes HIV-1 tat-mediated microglial activation via MECP2-STAT3 axis. J Neurosci, 2018, 38(23): 5367-5383.
5. 刘思佚, 曾燕, 刘疏柯, 等. 嗜铬粒蛋白 A 衍生多肽 CHR 抑制 LPS 诱导的永生化脑微血管内皮细胞高通透性. 第三军医大学学报, 2018, 40(15): 1364-1369.
6. Wardlaw JM, Smith C, Dichgans M. Small vessel disease: mechanisms and clinical implications. Lancet, 2019, 18(7): 684-696.
7. 中国研究型医院学会脑小血管病专业委员会《中国脑小血管病诊治专家共识 2021》编写组. 中国脑小血管病诊治专家共识 2021. 中国卒中杂志, 2021, 16(7): 716-726.
8. 刘国荣, 潘晓华, 刘秀珍, 等. 炎症小体 NLRP3 相关炎症因子 IL-1β、IL-18 与脑小血管病相关性研究. 脑与神经疾病杂志, 2019, 27(3): 153-157.
9. 王丽, 刘国荣, 潘晓华. NLRP3 炎症小体相关炎症因子 IL-1β 和 IL-18 在原代脑微血管内皮细胞中的激活及作用. 包头: 内蒙古科技大学包头医学院, 2019.
10. 刘蓓桦, 候梁, 罗伟珏, 等. LPS 与 ATP 共同诱导巨噬细胞中 NLRP3 炎症小体的激活. 北京农学院学报, 2018, 33(1): 74-78.
11. Wang X, Xu B, Xiang M, et al. Advances on fluid shear stress regulating blood-brain barrier. Microvasc Res, 2020, 128: 103930.
12. Tanaka KA, Kurihara S, Shibakusa T, et al. Cystine improves survival rates in a LPS-induced sepsis mouse model. Clin Nutr, 2015, 34(6): 1159-1165.
13. Feng S, Zou L, Wang H, et al. RhoA/ROCK-2 pathway inhibition and tight junction protein upregulation by catalpol suppresses lipopolysaccaride-induced disruption of blood-brain barrier permeability. Molecules, 2018, 23(9): 2371.
14. Sakurai E. Elucidation of new function in endothelial cells for efficient delivery strategy of drug to tissues. Yakugaku Zasshi, 2020, 140(1): 51-62.
15. 王义宝, 刘云会, 刘丽波, 等. 大鼠脑微血管内皮细胞的原代培养及其生物学行为初步探讨. 神经解剖学杂志, 2006(2): 224-228.
16. 鹿文葆, 秦伟伟, 刘淑英, 等. 大鼠脑微血管内皮细胞的纯化培养与鉴定. 中国脑血管病杂志, 2011, 8(10): 535-538.
17. 徐平湘, 齐特, 陆莉, 等. 原代大鼠脑微血管内皮细胞培养方法的改进. 首都医科大学学报, 2016, 37(5): 693-698.
18. 赵琳, 郭虹, 康立源, 等. 脑微血管内皮细胞培养技术的研究进展. 中国药理学通报, 2008, 24(8): 992-995.
19. 卢晓星, 张小强, 苗歆雨, 等. 原花青素对 LPS/ATP 诱导的 NLRP3 炎症小体激活及 NF-κBp65 磷酸化的抑制作用. 南京医科大学学报(自然科学版), 2020, 40(8): 1111-1118.
20. 李钰婷, 蔡大可, 郑柳怡, 等. NLRP3 基因敲除增强芹菜素对 triton-WR1339 所致高脂血症的降血脂及抗炎作用研究. 中国药理学通报, 2020, 36(4): 543-549.
21. 薛鸿, 林昌建, 谢宝松, 等. 沉默 NLRP3 炎性小体表达降低香烟烟雾提取物导致的人肺动脉内皮细胞凋亡. 基础医学与临床, 2020, 40(4): 490-495.

West China Medical Journal

Effect of NLRP3 gene silencing on expression of proinflammatory agents-induced inflammatory factors in rat brain microvascular endothelial cells

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