Quercetin Ameliorates Chronic Arthritis In Mice By Modulating Neutrophil Activities

Background:Rheumatoid arthritis(RA)is a systemic inflammatory autoimmune disorder characterized by the inflammation of synovial joints,leading to the destruction of articular cartilage and erosion of the bones at joints.Many studies have shown that neutrophils are believed to be responsible for RA pathogenesis.Therefore,neutrophils may serve as important therapeutic targets in RA.Quercetin is a flavonoid widely distributed in nature which is claimed to exert anti-oxidant and anti-inflammatory activities.Recently,a number of studies have demonstrated that quercetin has anti-inflammatory effect through down-regulating NF-kappa B pathway,regulating NO release,and alleviating oxidative damage in the tissue.Quercetin inhibits LPS-induced delay in spontaneous apoptosis and activation of neutrophils.Quercetin induces human FLS apoptosis by enhancing the accumulation of cleaved-caspase-3 and caspase-9 and promoting their activities in RA.However,whether quercetin can ameliorate chronic inflammation in RA remains largely unknown.Objective:We used the adjuvant-induced arthritis murine model to investigate the impact of quercetin on RA,and then to investigate the activation of neutrophils through in vivo and in vitro.Methods:(1)Freund’s complete adjuvant-induced arthritis was used as the murine model of rheumatoid arthritis(RA).Quercetin was intraperitoneally administrated daily after onset of the disease.The joint diameter and inflammatory score were evaluated daily during the treatment period.(2)The expressiom of cytokines was measured by CBA kit of plasma of mice.(3)MPO and NE activities were evaluated by immunohistochemical analysis.(4)Neutrophils infiltration was evaluated by air pouch andtranswell assays.(5)Expression of pro-inflammatory cytokine IL-6 was determined by quantitative real-time PCR.(6)The expression of cleaved caspase-3 was detected by Western blot analysis.The neutrophil apoptosis was examined by Annexin V/PI staining by flow cytometry.(7)NETs formation,release of MPO and NE and autophagy were assessed by immunostaining and confocal microscopy.(8)ROS was detected by DCF-DA by flow cytometry.Results:(1)Quercetin alleviated RA in mice:We used a murine model challenged with Freund’s complete adjuvant(FCA)to examine whether quercetin could inhibit joint inflammation.Compared with the control group,the RA group manifested characterized symptoms including the swollen paw and ankle joint.Treatment with quercetin significantly decrease the diameter of ankle joints and arthritis score.(2)Histological analysis of ankle joint in mice:FCA challenge resulted in typical inflammation that wascharacterized by cartilage erosion,synovial hyperplasia and multiple inflammatory cells infiltration.Treatment with quercetin significantly alleviated the arthritis in mice.(3)Quercetin reduced the secretion of pro-inflammatory cytokines in the plasma of RA mouse:CBA kit analysis showed that quercetin treatment can significantly reduce the secretion of TNF-α,IL-6 and IFN-γMeanwhile,anti-inflammatory cytokine IL-10increased remarkably.(4)Effect of quercetin on MPO and NE expression in ankle joint of RA mice:Immunohistochemistry analysis showed that the expression of MPO and NE were significantly upregulated in RA mice compared with that of the control group.And quercetin treatment significantly suppressed the MPO and NE induced by FCA.(5)Quercetin inhibited LPS-induced neutrophil influx in vivo and fMLP-induced migration in vitro:Air pouch experiments were used to investigate the impact of quercetin on LPS-induced neutrophil infiltration.The treatment of quercetin significantly reduced the total leukocytes as well as neutrophils in the air pouch.Quercetin treatment can also inhibit fMLP-induced migration of neutrophil.(6)Effect of quercetin on IL-6 expression in LPS-activated neutrophils.The qRT-PCR analysis showed that quercetin significantly down-regulated the mRNA level of IL-6.(7)Quercetin induced the apoptosis of LPS-treated neutrophils:We detected the expression of cleaved caspase-3 by Western blot analysis.Quercetin significantly increased the expression of cleaved caspase-3.Besides,flow cytometry assay indicated that quercetin raised the percentage of dead neutrophils after LPS induction.Thus,quercetin treatment promoted neutrophil apoptosis.(8)Quercetin inhibited NETs formation,release of MPO and NE and autophagy in vitro:PMA-induced NETs formation and autophagy was visualized by staining with anti-MPO or anti-NE and anti-LC3-B,respectively.Which were observed by immunofluorescence confocal microscopy.Quercetin inhibited NETs and autophagy formation,as well as NETs-associated MPO and NE release.(9)Quercetin reducd intracellular ROS production of neutrophil.Conclusion:Our findings showed that quercetin significantly alleviated murine arthritis by modulating the inflammatory activities of neutrophils.These results indicated that quercetin could serve as an alternative or adjunct modality for the treatment of RA.