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

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.
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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.	劉思佚, 曾燕, 劉疏柯, 等. 嗜鉻粒蛋白 A 衍生多肽 CHR 抑制 LPS 誘導的永生化腦微血管內皮細胞高通透性. 第三軍醫大學學報, 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.	中國研究型醫院學會腦小血管病專業委員會《中國腦小血管病診治專家共識 2021》編寫組. 中國腦小血管病診治專家共識 2021. 中國卒中雜志, 2021, 16(7): 716-726.
8.	劉國榮, 潘曉華, 劉秀珍, 等. 炎癥小體 NLRP3 相關炎癥因子 IL-1β、IL-18 與腦小血管病相關性研究. 腦與神經疾病雜志, 2019, 27(3): 153-157.
9.	王麗, 劉國榮, 潘曉華. NLRP3 炎癥小體相關炎癥因子 IL-1β 和 IL-18 在原代腦微血管內皮細胞中的激活及作用. 包頭: 內蒙古科技大學包頭醫學院, 2019.
10.	劉蓓樺, 候梁, 羅偉玨, 等. LPS 與 ATP 共同誘導巨噬細胞中 NLRP3 炎癥小體的激活. 北京農學院學報, 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.	王義寶, 劉云會, 劉麗波, 等. 大鼠腦微血管內皮細胞的原代培養及其生物學行為初步探討. 神經解剖學雜志, 2006(2): 224-228.
16.	鹿文葆, 秦偉偉, 劉淑英, 等. 大鼠腦微血管內皮細胞的純化培養與鑒定. 中國腦血管病雜志, 2011, 8(10): 535-538.
17.	徐平湘, 齊特, 陸莉, 等. 原代大鼠腦微血管內皮細胞培養方法的改進. 首都醫科大學學報, 2016, 37(5): 693-698.
18.	趙琳, 郭虹, 康立源, 等. 腦微血管內皮細胞培養技術的研究進展. 中國藥理學通報, 2008, 24(8): 992-995.
19.	盧曉星, 張小強, 苗歆雨, 等. 原花青素對 LPS/ATP 誘導的 NLRP3 炎癥小體激活及 NF-κBp65 磷酸化的抑制作用. 南京醫科大學學報(自然科學版), 2020, 40(8): 1111-1118.
20.	李鈺婷, 蔡大可, 鄭柳怡, 等. NLRP3 基因敲除增強芹菜素對 triton-WR1339 所致高脂血癥的降血脂及抗炎作用研究. 中國藥理學通報, 2020, 36(4): 543-549.
21.	薛鴻, 林昌建, 謝寶松, 等. 沉默 NLRP3 炎性小體表達降低香煙煙霧提取物導致的人肺動脈內皮細胞凋亡. 基礎醫學與臨床, 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. 劉思佚, 曾燕, 劉疏柯, 等. 嗜鉻粒蛋白 A 衍生多肽 CHR 抑制 LPS 誘導的永生化腦微血管內皮細胞高通透性. 第三軍醫大學學報, 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. 中國研究型醫院學會腦小血管病專業委員會《中國腦小血管病診治專家共識 2021》編寫組. 中國腦小血管病診治專家共識 2021. 中國卒中雜志, 2021, 16(7): 716-726.
8. 劉國榮, 潘曉華, 劉秀珍, 等. 炎癥小體 NLRP3 相關炎癥因子 IL-1β、IL-18 與腦小血管病相關性研究. 腦與神經疾病雜志, 2019, 27(3): 153-157.
9. 王麗, 劉國榮, 潘曉華. NLRP3 炎癥小體相關炎癥因子 IL-1β 和 IL-18 在原代腦微血管內皮細胞中的激活及作用. 包頭: 內蒙古科技大學包頭醫學院, 2019.
10. 劉蓓樺, 候梁, 羅偉玨, 等. LPS 與 ATP 共同誘導巨噬細胞中 NLRP3 炎癥小體的激活. 北京農學院學報, 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. 王義寶, 劉云會, 劉麗波, 等. 大鼠腦微血管內皮細胞的原代培養及其生物學行為初步探討. 神經解剖學雜志, 2006(2): 224-228.
16. 鹿文葆, 秦偉偉, 劉淑英, 等. 大鼠腦微血管內皮細胞的純化培養與鑒定. 中國腦血管病雜志, 2011, 8(10): 535-538.
17. 徐平湘, 齊特, 陸莉, 等. 原代大鼠腦微血管內皮細胞培養方法的改進. 首都醫科大學學報, 2016, 37(5): 693-698.
18. 趙琳, 郭虹, 康立源, 等. 腦微血管內皮細胞培養技術的研究進展. 中國藥理學通報, 2008, 24(8): 992-995.
19. 盧曉星, 張小強, 苗歆雨, 等. 原花青素對 LPS/ATP 誘導的 NLRP3 炎癥小體激活及 NF-κBp65 磷酸化的抑制作用. 南京醫科大學學報(自然科學版), 2020, 40(8): 1111-1118.
20. 李鈺婷, 蔡大可, 鄭柳怡, 等. NLRP3 基因敲除增強芹菜素對 triton-WR1339 所致高脂血癥的降血脂及抗炎作用研究. 中國藥理學通報, 2020, 36(4): 543-549.
21. 薛鴻, 林昌建, 謝寶松, 等. 沉默 NLRP3 炎性小體表達降低香煙煙霧提取物導致的人肺動脈內皮細胞凋亡. 基礎醫學與臨床, 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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