Inhibition of P2X7R modulates ECM deposition in HSCs and crosstalk with macrophages in liver fibrosis

OBJECTIVE Modulation of immune response and reduction of extracellular matrix(ECM)deposition are both essential in the therapy of liver fibrosis.Regulating P2 X7 R might be a potential therapeutic strategy to treat liver fibrosis, we investigated whether the blockade of P2 X7 R could reverse liver fibrosis and how P2 X7 R is involved in fibrogenesis during hepatic stellate cells(HSCs) and macrophages crosstalk. METHODS In vivo,liver fibrosis model was established by thioacetamide(TAA) intraperitoneal administration in male C57 BL/6 mice. In vitro, LX-2 cells were treated with TGF-β and LPS/ATP respectively. Supernatant from LPS/ATP stimulated THP-1 macrophages were supplemented to LX-2 cells to mimic cellular crosstalk between HSCs and macrophages. RESULTS Blockade of P2 X7 R with its selective antagonist A438079, not only decreased liver injury and ECM deposition but also ameliorated inflammation by inhibiting NLRP3 inflammasome, NF-κB activation and IL-1β production in TAA-induced liver fibrosis. And the recruitment of macrophages, monocytes and granulocytes were also inhibited. In TGF-β-stimulated LX-2 cells,ECM deposition was reduced by the inhibiting of P2 X7 RTLR4-NLRP3 axis. Protein synthesis and cleavage of IL-1β and its m RNA level was dramatically increased by LPS 4 h combined with ATP 30 min than those in HSC treated with LPS or ATP alone. Additionally, LX-2 cells primed with LPS/ATP greatly increased m RNA and protein expression of caspase-1, NLRP3 and P2 x7 R, as well as liver fibrosis markers, α-SMA and typeⅠcollagen.These events were remarkably suppressed by A438079 pretreatment. si RNA against P2 x7 R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type Ⅰ collagen induced by LPS/ATP. Ectopic overexpression of P2 X7 R reduced the threshold of ECM deposition in HSCs induced by TGF-β.Inhibiting upstream receptors of NLRP3 inflammasome,P2 X7 receptor-selective antagonist(A438079), TLR4 inhibitor(CLI-095) reduced the fibrotic markers in both models of TGF-β and LPS/ATP-activated HSCs. The cultured medium of the THP-1 macrophages by LPS/ATP aggravated ECM deposition in LX-2 cells. The decreased IL-1β by the pharmacological inhibitors of P2 X7 R,caspase-1 and TLR4 treated to THP-1 macrophages attenuates ECM deposition in LX-2 cells. CONCLUSION Both ECM producing in HSCs and inflammatory cytokines secreting from macrophages were regulated by P2 X7 R, suggesting a therapeutic utility of P2 x7 R blockade in liver fibrosis treatment.