title: Potentiation of hepatic stellate cell activation by extracellular ATP is dependent on P2X7R-mediated NLRP3 inflammasome activation.
abstract: Purinergic receptor P2x7 (P2x7R) is a key modulator of liver inflammation and fibrosis. The present study aimed to investigate the role of P2x7R in hepatic stellate cells activation. Lipopolysaccharide (LPS) or the conditioned medium (CM) from LPS-stimulated RAW 264.7 mouse macrophages was supplemented to human hepatic stellate cells, LX-2 for 24h and P2x7R selective antagonist A438079 (10μM) was supplemented to LX-2 cells 1h before LPS or CM stimulation. In addition LX-2 cells were primed with LPS for 4h and subsequently stimulated for 30min with 3mM of adenosine 5'-triphosphate (ATP). A438079 was supplemented to LX-2 cells 10min prior to ATP. Directly treated with LPS on LX-2 cells, mRNA expressions of interleukin (IL)-1β, IL-18 and IL-6 were increased, as well as mRNA expressions of P2x7R, caspase-1, apoptosis-associated speck-like protein containing CARD (ASC) and NOD-like receptor family, pyrin domain containing 3 (NLRP3) mRNA. LPS also increased α-smooth muscle actin (α-SMA) and type I collagen mRNA expressions, as well as collagen deposition. Interestingly treatment of LX-2 cells with LPS-activated CM exhibited the greater increase of above factors than those in LX-2 cells directly treated with LPS. Pretreatment of A438079 on LX-2 cells stimulated by LPS or LPS-activated CM both suppressed IL-1β mRNA expression. LPS combined with ATP dramatically increased protein synthesis and cleavage of IL-1β and its mRNA level than those in HSC treated with LPS or ATP alone. Additionally LX-2 cells primed with LPS and subsequently stimulated for 30min with ATP greatly increased mRNA and protein expression of caspase-1, NLRP3 and P2x7R, as well as liver fibrosis markers, α-SMA and type I collagen. These events were remarkably suppressed by A438079 pretreatment. siRNA against P2x7R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type I collagen. The involvement of P2X7R-mediated NLRP3 inflammasome activation in IL-1β production of HSC might contribute to ECM deposition and suggests that blockade of the P2x7R-NLRP3 inflammasome axis represents a potential therapeutic target to liver fibrosis.
authors.affiliation: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China.
authors.forename: Jin-Hua
authors.initials: JH
authors.lastname: Zheng
authors.affiliation: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Li
authors.affiliation: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China.
authors.forename: You-Li
authors.initials: YL
authors.lastname: Yao
authors.affiliation: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China.
authors.forename: Yan-Ling
authors.initials: YL
authors.lastname: Wu
authors.affiliation: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China.
authors.forename: Shun-Zong
authors.initials: SZ
authors.lastname: Song
authors.affiliation: Clinical Research Center, Yanbian University Hospital, Yanji, Jilin Province 133002, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Sun
authors.affiliation: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China; Clinical Research Center, Yanbian University Hospital, Yanji, Jilin Province 133002, China. Electronic address: jxnan@ybu.edu.cn.
authors.forename: Ji-Xing
authors.initials: JX
authors.lastname: Nan
authors.affiliation: Key Laboratory for Natural Resource of Changbai Mountain & Functional Molecules, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province 133002, China. Electronic address: lhlian@ybu.edu.cn.
authors.forename: Li-Hua
authors.initials: LH
authors.lastname: Lian
mesh_terms: D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D020170:Caspase 1 / Q000378:metabolism; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein / Q000378:metabolism*; D000067996:RAW 264.7 Cells; D012333:RNA, Messenger / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D000199:Actins; D024042:Collagen Type I; D016207:Cytokines; D058847:Inflammasomes; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D012333:RNA, Messenger; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate; D020170:Caspase 1
keywords: A438079 (PubChem CID: 11673921); Hepatic stellate cells; Inflammasome; Liver fibrosis; NLRP3; P2X7 receptor