Role And Mechanism Study Of Xanthine Oxidoreductase(XOR) Mediated Alveolar Macrophages Polarization In Paraquat-Induced Lung Injury

Paraquat(PQ)is a potent herbicide widely used in agriculture and forestry production.However,the extremely high mortality rate of oral suicide by paraquat has also caused serious social problems.The metabolism of paraquat produces a large amount of reactive oxygen radicals,causing severe oxidative stress damage.Upon absorption into the body,paraquat is enriched in the lungs and induces acute lung injury(ALI)or even acute respiratory distress syndrome(ARDS),with irreversible pulmonary fibrosis and multi organ failure at advanced stage,leading to a fatal outcome.Xanthine oxidoreductase(XOR)is mainly characterized by two interconvertible enzymatic activities,xanthine dehydrogenase(XDH)and xanthine oxidase(XOD),both of which catalyze the elimination of purines to uric acid.The electrons generated by XDH reduce NAD~+to NADH.However,the electrons generated by XOD activity are accepted by oxygen to produce hydrogen peroxide and superoxide anion(O2˙-),causing oxidative stress damage by the accumulation of reactive oxygen species(ROS)in many pathological processes.Some researchers found that serum XOD activity was increased in paraquat-poisoned patients.XOD activity was also increased in lung tissue of paraquat-poisoned rats,and XOR inhibitors such as allopurinol could reduce mortality.Current studies have shown that lungs of paraquat-poisoned patients and animals have significant macrophage infiltration.Paraquat induces the migration and activation of macrophages,and the expression and release of pro-inflammatory cytokines.Alveolar macrophages(AMs)are innate immune cells directly exposed to the airway and are one of the most important regulators of the local inflammatory microenvironment and inflammatory response in the lung.The activation of AMs is considered to be an important initiating factor of ALI/ARDS.Therefore,we propose that the induction of AM polarization by paraquat through the XOR/ROS pathway is an important mechanism of lung injury in paraquat poisoning.Objectives:First,our aim is to observe the phenotype,XOR expression and activity of AMs stimulated with paraquat.Then,to investigate whether XOR/ROS could mediate paraquat-induced changes in the function and phenotype of AMs.Finally,to investigate whether intervention of XOR/ROS can ameliorate paraquat-induced lung injury.Methods:1.MH-S cells,a mouse AM cell line,were stimulated using gradient concentrations of paraquat.The cell viability of MH-S cells was measured using CCK-8(cell counting kit-8)to determine the appropriate paraquat concentration.XOR expression and XOD activity,tumor necrosis factor alpha(TNF-α),interleukin 6(IL-6),interleukin 10(IL-10),inducible nitric oxide synthase(i NOS)and Arginase-1(Arg-1)were measured by q RT-PCR.The relative expression of markers of macrophage polarization CD86 and CD206,was measured by flow cytometry.2.In vitro paraquat-stimulation experiment.MH-S cells were stimulated with paraquat to observe changes of XOR expression and activity,and phenotypes.Cells were divided into control(NC)group and a paraquat(PQ)group.3.In vitro XOR-inhibition experiment.Two agents were used to inhibit XOR in MH-S cells,RNA interference(XOR-si)to inhibit XOR expression and allopurinol to inhibit XOD activity,to verify that XOR is a mediator in the M1 polarization,inflammatory response and oxidative stress damage in MH-S cells caused by paraquat.Cells were divided into control(NC)group,RNA interference(XOR-si)group,allopurinol(Allo)group,paraquat(PQ)group,paraquat plus RNA interference(PQ+XOR-si)group,and paraquat plus allopurinol(PQ+Allo)group.4.In vitro ROS-inhibition experiment.Without the stimulation of paraquat,the role of ROS as a downstream molecule of XOR in MH-S cells M1 polarization and inflammatory damage was verified by overexpression of XOR gene and inhibition of ROS using N-Acetylcysteine(NAC).Cells were divided into control(NC)group,XOR overexpression(XOR-OE)group,NAC group,and XOR overexpression plus NAC(XOR-OE+NAC)group.5.In the in vitro paraquat-stimulation experiment,XOR-inhibition experiment and ROS-inhibition experiment,the following indicators were measured:XOR protein expression by Western blot and XOD activity;ROS,malonaldehyde(MDA)and superoxide dismutase(SOD)activity to evaluate the extent of oxidative stress injury;the relative expression of CD86 and CD206 using flow cytometry,and expression of i NOS,Arg-1,peroxisome proliferator-activated receptor gamma(PPAR-γ),and the phosphorylation of signal transducer and activator of transcription(STAT-1)using Western blot,to collectively assess the polarization of MH-S cells;assay of TNF-α,IL-6 and IL-10 in the cell culture medium supernatant by ELISA to evaluate the inflammation.6.The effects of XOR and ROS inhibition on pulmonary pathological injury,alveolar macrophage polarization,inflammatory injury and oxidative stress injury were investigated in paraquat-poisoned mice.The mice were divided into control(NC)group,paraquat(PQ)group,paraquat plus allopurinol(PQ+Allo)group,paraquat plus NAC(PQ+NAC)group,and paraquat with allopurinol and NAC combined treatment(PQ+Allo+NAC)group.The following tests were performed in lungs:XOR protein expression and XOD activity;assessment of HE pathological damage using four indicators:pulmonary hemorrhage,infiltration of granulocytes,pulmonary edema,and interstitial thickening;assay of MDA and SOD to assess oxidative stress damage of lung tissue;detection of Siglec-F~+CD86~+cells by immunofluorescence,and determination of i NOS,Arg-1 and PPAR-γexpression,and STAT-1 phosphorylation to jointly evaluate the level of alveolar macrophage M1 polarization;TNF-α,IL-6,IL-10to assess the inflammatory damage.Results:1.The expression levels of XOR,i NOS,TNF-αand IL-6 m RNA in MH-S cells showed a dose-dependent effect with paraquat concentration.m RNA expression levels of Arg-1 and IL-10 decreased with increasing paraquat concentration.Expression of M1 polarization molecule marker CD86 was upregulated with increasing paraquat concentration in MH-S cells,and expression of M2 polarization marker CD206decreased with increasing paraquat concentration.2.In the in vitro paraquat-stimulation experiment,the PQ group showed upregulated XOR expression,increased XOD activity,elevated oxidative stress damage,upregulated i NOS expression,increased secretion of pro-inflammatory cytokines TNF-αand IL-6,decreased Arg-1 expression and secretion of anti-inflammatory cytokine IL-10 compared to the NC group.3.In the in vitro XOR-inhibition assay,RNA interference significantly inhibited XOR protein expression,allopurinol significantly inhibited XOD activity;compared with the PQ group,the PQ+XOR-si and PQ+Allo groups significantly decreased cellular oxidative stress injury,M1 polarization and pro-inflammatory cytokine secretion,and significantly elevated anti-inflammatory cytokine secretion and M2polarization;compared with the NC group,the XOR-si and Allo groups significantly decreased oxidative stress,M1 polarization and secretion of pro-inflammatory cytokines,and significantly elevated M2 polarization and secretion of anti-inflammatory cytokine.4.In the in vitro ROS-inhibition experiment,compared with the NC group,the XOR-OE group significantly increased XOR expression,XOD activity,oxidative stress injury,M1 polarization and secretion of pro-inflammatory cytokines in MH-S cells,and significantly decreased M2 polarization and anti-inflammatory cytokine secretion;compared with the XOR-OE group,the XOR-OE+NAC group significantly decreased XOD activity,oxidative stress level,M1 polarization and secretion of pro-inflammatory cytokines,and significantly increased M2 polarization and anti-inflammatory cytokine secretion.5.In the paraquat-poisoned mouse model,compared with the NC group,the XOR protein and XOD activity in the lungs of the PQ group were significantly increased,the proportion of M1 AMs was significantly increased,and the pathological injury,oxidative stress injury and inflammatory injury were significantly aggravated;compared with the PQ group,the PQ+Allo group,PQ+NAC group or PQ+Allo+NAC group could significantly reduce the XOR expression,the proportion of M1 AMs,and the pathological injury,oxidative stress injury and inflammatory injury.Conclusion:Paraquat induces upregulation of XOR expression and XOD activity,M1polarization,inflammatory respoonse and oxidative stress damage in MH-S cells.Paraquat induces M1 polarization,inflammatory response and oxidative stress damage in MH-S cells via XOR/ROS pathway.Inhibition of XOR and ROS ameliorates pathological damage,inflammatory damage and oxidative stress damage,and decreased M1 polarization of AM in lung tissue of paraquat-poisoned mice.