IL-18 And Cox2 Mediate The ET-1 On The Regulation Of ANP Secretion In Hypoxic Atria

Background:Hypoxia is a common pathophysiological process in many diseases.During hypoxia,the hypoxia-inducible factor-1α(HIF-1α)can promotes mammalian cells to adapt to hypoxic environment by regulating the hypoxia response elements and its target genes.As a target gene of HIF-1α,endothelin-1(ET-1)is widely existed in the cardiovascular system and plays various biological effects,such as constricting blood vessels,promoting cell mitogenesis,stimulating the secretion of neuropeptides and atrial natriuretic peptide(ANP).ANP is a member of the family of cardiac natriuretic peptides that it is mainly synthesized and secreted by atrial myocytes.ANP is primarily involved in the regulation of body fluid volume and blood pressure homeostasis.In addition,ANP possesses resisting of ET-1,anti-inflammation,antihypertrophy,anti-fibrosis and preventing of myocardium to against ischemia/reperfusion injury.It has been demonstrated that hypoxia potently stimulates the secretion of ANP leading to cellular adaptation to hypoxia and protection of the ischemic heart,palliation of heart failure development and prevention of pathological remodeling in dilated cardiomyopathy.Therefore,the elucidating mechanism of hypoxia-induced ANP secretion and its intervention is expected to be an effective strategy for the prevention and treatment of related diseases.In previous study,it has been demonstrated that endogenous ET-1 upregulates the expression of nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4)through activation of its type A and B receptors(ETRA&ETRB)leading to activation of the non-receptor protein tyrosine kinase Src,thereby promotes the secretion of ANP in isolated beating rat hypoxic atria.Moreover,ET-1 mediates inflammatory processes by increasing interleukin-18(IL-18)release through oxidative stress.As a pro-inflammatory cytokine,IL-18 upregulates the expression of GATA binding protein 4(GATA4)through activation of phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)pathway,thereby increases the mRNA levels and expression of ANP.ET-1 also promotes the metabolism of arachidonic acid through activation of cyclooxygenase 2(COX2),resulting generation of prostaglandin(PG)H2.Lipocalin-type prostaglandin D synthase(L-PGDS)is a downstream biosynthase of COX2 that converts the PGH2 to PGD2,which is clarified a protective role in the ischemic/hypoxic heart and play an important role in anti-inflammation.However,the effects of endogenous ET-1 on IL-18 and COX2 as well as its downstream L-PGDS,and its role on the regulation of atrial ANP secretion during hypoxia are not clear.Objective:This study is to investigate the effects of endogenous ET-1 on IL-18 and COX2 as well as L-PGDS,and its role on the regulation of ANP expression or secretion in atria in vitro,in vivo and in HL-1 atrial myocytes during hypoxia.Methods:1.Preparation of hypoxic models:(1)The isolated perfused beating left atrial hypoxic model of SD rats were established by the N2 replaced O2 method.(2)The hypoxic model of HL-1 atrial myocytes was prepared with an anoxic cell incubator(1%O2,94%N2,5%CO2).(3)The in vivo hypoxic atrial model of SD rats was established by a normal pressure of hypoxic chamber(10%O2,90%N2).2.Atrial pulse pressure was recorded by a physiograph via a pressure transducer.3.The mRNA levels of ANP,ET-1,IL-18,activating transcription factor 3(ATF3),lymphoid enhancer factor 1(LEF1)and COX2 were detected by quantitative real-time PCR(q-PCR).4.The concentrations of ANP and ET-1 in perfusates and tissues were measured using radioimmunoassay.5.The expression of IL-18 and its receptor subunits,NOX4,NOX2,Src,guanine nucleotide exchange factors selective for the Rho family of G-proteins(RhoGEF;p115RhoGEF),RhoA,ATF3,T cell factor(TCF)3,TCF4,LEF1,HIF-1α,COX2,LPGDS,nuclear factor erythroid-2-related factor 2(Nrf2)and peroxisome proliferatoractivate receptor γ(PPARγ)were evaluated by western blot(WB).6.The hydrogen peroxide(H2O2)assay kit was used to measure the content of H2O2.7.The levels of IL-18 and PGD2 were quantified using the enzyme linked immunosorbent assay(ELISA)kit.8.HL-1 cell activity was detected by CCK-8 kit.9.Reactive oxygen species(ROS)levels in HL-1 cells were measured by a ROS fluorescent probe(DCFH-DA).10.IL-18-siRNA,ATF3-siRNA,LEF1-siRNA and COX2-siRNA were transfected by lipofectamine 2000 to interfere the IL-18,ATF3,LEF1 and COX2 in HL-1 cells.11.Immunofluorescence(IF)was used to observe the expression of IL-18 and COX2.12.The changes of oxygen pressure(pO2)were measured with blood gas analyzer.13.Hematoxylin-eosin(HE)and Masson staining were used to observe atrial pathological changes.Results:Part 1:Effects of ET-1-IL-18 signaling on ANP secretion in hypoxic atria.1.IL-18 was involved in hypoxia-induced ANP secretion in isolated beating rat atria:(1)Hypoxia significantly increased the ANP mRNA level,expression and secretion concomitantly with strongly inhibition of pulse pressure.(2)Hypoxia also significantly increased the expression and production of IL-18,and upregulated the expression of its receptor α and β subunits(IL-18Rα and IL18Rβ);a selective blocker of IL-18,IL-18 binding protein(IL-18BP),obviously attenuated the hypoxia-induced increase of mRNA level,expression and secretion of atrial ANP.2.The signal pathway of endogenous ET-1 on the regulation of IL-18 expression in beating rat hypoxic atria:(1)Hypoxia significantly increased the atrial release of ET-1 and upregulated expression of ETRA and ETRB;selective antagonists of ETRA and ETRB(BQ123 and BQ788)blocked the hypoxia-induced upregulation of IL-18 and its receptor.(2)The expression of NOX4,generation of H2O2 and expression of Src were significantly increased by hypoxia;BQ123 and BQ788 completely blocked the hypoxia-induced increase of NOX4 and Src expression.(3)GLX351322(GLX),a blocker of NOX4,also eliminated the production of H2O2 and Src expression induced by hypoxia.(4)A ROS scavenger nacetyl cysteine(NAC)was mimicked the inhibitory effect of GLX on Src expression during hypoxia.(5)Src inhibitor 1(SrcI),an inhibitor of Src,completely blocked the effects of hypoxia on IL-18 and its receptor subunits by inhibiting RhoGEF and RhoA,resulting a dramatic attenuation of hypoxia-induced ANP secretion.3.Downstream signaling of ET-1-IL-18 on the regulation of hypoxia-induced ANP secretion in beating rat atria:Hypoxia upregulated the expression of ATF3,TCF3/and TCF4/LEF1,these effects were completely abolished by BQ123,BQ788 and IL-18BP resulting a dramatic attenuation of hypoxia-induced ANP secretion.4.The effects of ET-1-IL-18 on the regulation of ANP expression in hypoxic HL-1 atrial myocytes:(1)Hypoxia also significantly increased mRNA level and expression of ET-1,and upregulated expression of NOX4,IL-18,ATF3 and LEF1;the hypoxia-induced upregulation of NOX4,IL-18 and LEF1 expression were blocked by BQ123 and BQ788.(2)The intervention of IL-18 with IL-18-siRNA markedly inhibited the effects of hypoxia on ATF3 and LEF1,resulting an attenuation of hypoxia-induced increase of ANP expression.(3)The effect of hypoxia on LEF1 also completely blocked by ATF3-siRNA,while LEF1-siRNA significantly inhibited the hypoxia-induced increase of mRNA level and expression of ANP.5.Effect of ET-1-IL-18 on the regulation of atrial ANP expression in hypoxic rat in vivo:Endogenous ET-1 also regulated the atrial ANP mRNA level and expression in hypoxic rats through IL-18 signaling,which was consistent with the results of isolated beating atria and HL-1 atrial myocytes during hypoxia.Part 2:Effects of ET-1-COX2 signaling on ANP secretion in hypoxic atria1.ET-1-COX2 regulates ANP secretion in isolate beating hypoxic atrial:(1)Hypoxia eminently upregulated the expression of COX2 and L-PGDS,and increased PGD2 production and PPARy phosphorylation(p-PPARy),these effects were abrogated by BQ123,BQ788.(2)Celecoxib(Cele),an inhibitor of COX2,also completely eliminated the effects of hypoxia on L-PGDS,PGD2 and p-PPARγ;the PPARy inhibitor GW9662 also attenuated the hypoxia-induced ANP secretion,which were similarly to the ET receptor,COX2 and PGD2 and PGF2α receptor antagonists(AH6809 and AL8810).(3)PGD2 also positively feedback regulate the expression of L-PGDS by activation of Nrf2.2.Cross-talking between IL-18 and COX2 signaling pathways regulated by ET-1:The expression of IL-18 and its receptor induced by hypoxia were facilitated by antagonists of COX2,L-PGDS and PPARy as well as ANP receptor(A71915)in isolated beating rat atria.In contrast,the hypoxia increased expression of COX2,LPGDS and PPARy were recovered by IL-18BP.3.Validation of the effects of ET-1-COX2 on hypoxia-induced ANP expression in HL-1 cells:In hypoxic HL-1 cells cultured in vitro,the interference of COX2 by COX2siRNA significantly reduced the upregulation of ANP expression induced by hypoxia through inhibition of L-PGDS and its downstream PPARy,which was extremely similar to the role of COX2 on regulation of hypoxia-induced ANP expression and secretion in isolated beating atria.4.The effect of ET-1-COX2 on the regulation of atrial ANP expression in rat in vivo:(1)In the rat atrium in vivo,COX2 also regulates the mRNA and expression of ANP through L-PGDS and its downstream PPARy,these were similar to the results in isolated beating rat atria and HL-1 cells during hypoxia.(2)Hypoxia leads to disordered arrangement of atrial myocytes,interstitial inflammatory cell infiltration and collagen fiber deposition,which were significantly reduced by IL-18BP but facilitated by Cele and A71915 pretreatment.Conclusions:1.Endogenous ET-1 regulates IL-18 production through activation of NOX4 in hypoxic atria.2.NOX4-IL-18 controlled by endogenous ET-1 promotes ANP secretion via ATF3-TCF3/TCF4-LEF1 signaling under hypoxic condition.3.COX2 controlled by endogenous ET-1 regulates the ANP expression and secretion in hypoxic atria by L-PGDS-PPARy signaling during hypoxia.4.L-PGDS-derived PGD2 regulates L-PGDS expression through Nrf2 positive feedback during hypoxia,which is also one of the mechanism of hypoxia-induced ANP secretion by endogenous ET-1.5.The cross-talking between the IL-18 and COX2-L-PGDS-PPARy signaling in atria also affect the process of endogenous ET-1 on the regulation of ANP secretion during hypoxia.6.ANP plays an important resistant role in IL-18-induced atrial fibrosis during hypoxia.