目的:本研究旨在探讨NLRP3炎性小体在乙型肝炎病毒相关性肾炎(Hepatitis B virus associat-ed-Glomerulonephritis, HBV-GN)足细胞焦亡中的作用。方法:通过将携带HBx基因的载体及NLRP3 siRNA载体转染至人肾足细胞中,使足细胞株分为空白对照组、空转染组、HBx转染组和HBx+NLRP3 siRNA转染组后,完成以下实验。通过流式细胞术及Hoechst 33342染色,分别检测各组足细胞焦亡率及细胞核的数量及形态学变化。通过实时荧光定量逆转录聚合酶链式反应(Quantitative real-time reverse transcription polymerase chain reaction, qRT-RCR)、蛋白质免疫印迹(Western blot, WB)以及酶联免疫吸附实验(Enzyme linked immunosorbent assay, ELISA)检测4组足细胞的NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speek-like protein containing CARD, ASC)、caspase-1、白细胞介素(Interleukin, IL)-1β、IL-18的表达。通过ELISA试剂盒检测乳酸脱氢酶(lactate dehydrogenase, LDH)水平。通过免疫荧光染色检测各组足细胞中Nephrin蛋白、Desmin蛋白的表达。结果:HBx过表达使NLRP3炎性小体、Caspase-1、IL-1β、IL-18、LDH、Desmin表达均升高,同时导致Nephrin表达下降,最终引起了足细胞焦亡(P < 0.05)。然而,抑制NLRP3炎症小体的生成可以减轻足细胞焦亡,减少了焦亡相关蛋白的表达(P < 0.05)。结论:NLRP3炎性小体介导的炎症信号通路在HBx过表达引起的足细胞焦亡中发挥了重要作用,NLRP3炎性小体有望成为HBV-GN的潜在治疗靶点之一。
Objective: This study was designed to investigate the role of NLRP3 inflammasome in podocyte py-roptosis in Hepatitis B virus associated-Glomerulonephritis (HBV-GN). Methods: The vectors carry-ing HBx gene and NLRP3 siRNA were transfected into human renal podocytes. The podocyte lines were divided into blank control group, empty plasmid group, HBx group and HBx+NLRP3 siRNA group. The podocyte pyroptosis rate and the numerical and morphological changes of nuclei were detected by flow cytometry and Hoechst 33342 staining, respectively. NLRP3, apoptosis associated speek-like protein containing CARD (ASC), caspase-1, IL-1β and IL-18 were detected by RT-PCR, Western blot and ELISA. The level of lactate dehydrogenase (LDH) was detected by ELISA kit. The expressions of nephrin protein and desmin protein in podocytes of each group were detected by immunofluorescence staining. Results: The overexpression of HBx increased the expression of NLRP3, caspase-1, IL-1β, IL-18, LDH and desmin, which led to the decrease of nephrin and finally mediated podocyte pyroptosis (P < 0.05). Moreover, the addition of NLRP3 siRNA decreased podo-cyte pyroptosis as well as the expression of pyrosis-related proteins compared with the HBx group (P < 0.05). Conclusion: NLRP3 inflammasome plays an important role in HBx-induced podocyte py-roptosis, suggesting that NLRP3 inflammasome is a potential therapeutic target for alleviating HBV-GN inflammation.
Objective: This study was designed to investigate the role of NLRP3 inflammasome in podocyte pyroptosis in Hepatitis B virus associated-Glomerulonephritis (HBV-GN). Methods: The vectors carrying HBx gene and NLRP3 siRNA were transfected into human renal podocytes. The podocyte lines were divided into blank control group, empty plasmid group, HBx group and HBx+NLRP3 siRNA group. The podocyte pyroptosis rate and the numerical and morphological changes of nuclei were detected by flow cytometry and Hoechst 33342 staining, respectively. NLRP3, apoptosis associated speek-like protein containing CARD (ASC), caspase-1, IL-1β and IL-18 were detected by RT-PCR, Western blot and ELISA. The level of lactate dehydrogenase (LDH) was detected by ELISA kit. The expressions of nephrin protein and desmin protein in podocytes of each group were detected by immunofluorescence staining. Results: The overexpression of HBx increased the expression of NLRP3, caspase-1, IL-1β, IL-18, LDH and desmin, which led to the decrease of nephrin and finally mediated podocyte pyroptosis (P < 0.05). Moreover, the addition of NLRP3 siRNA decreased podocyte pyroptosis as well as the expression of pyrosis-related proteins compared with the HBx group (P < 0.05). Conclusion: NLRP3 inflammasome plays an important role in HBx-induced podocyte pyroptosis, suggesting that NLRP3 inflammasome is a potential therapeutic target for alleviating HBV-GN inflammation.
将TRIzol试剂(DP405,天根生化科技有限公司,北京)用于从冰冻足细胞中提取总RNA。使用高容量cDNA反转录试剂盒(美国加利福尼亚州福斯特市应用生物系统公司)将提取的RNA反转录成cDNA。在ABI 7500快速实时PCR系统(美国应用生物系统公司)上,使用SYBR Green I扩增第一链cDNA,以量化mRNA的相对表达。扩增后,确定阈值周期(Ct),并根据2−ΔΔCt法计算。GAPDH被用作数据规范化的内部控制。
孙静宜,蒋 伟. NLRP3炎性小体在乙型肝炎病毒相关性肾炎足细胞焦亡中的作用The Role of NLRP3 Inflammasome Activation in Podocyte Pyroptosis in Hepatitis B Virus Associated-Glomerulonephritis[J]. 临床医学进展, 2022, 12(08): 7012-7025. https://doi.org/10.12677/ACM.2022.1281010
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