| Background:Rheumatoid arthritis(RA)is a chronic inflammatory disease characterized by synovial hyperplasia,joint destruction and extra-articular manifestations with a significant impact on both morbidity and mortality.Although the etiology and pathogenesis of this disease have not been fully elucidated,it is known that joint inflammation and damage in RA is mediated by the influx of innate and adaptive immune cells into the synovial joint space,such as neutrophils,macrophages,synovial fibroblasts,T cells and B cells.Neutrophils are the most abundant immune cells present in synovial fluid from the joints of RA patients and are also abundant at the pannus/cartilage interface,the site of active tissue damage.Neutrophils,terminally differentiated cells with a short lifespan in circulation,are the most abundant leukocytes in the human body,with homeostasis maintained by their continuous release from the bone marrow.Complex interactions between receptors on neutrophils and vascular endothelial cells regulate their attachment and subsequent migration from the circulation into tissues during inflammation.Various functional abnormalities of neutrophils have been reported in RA.Many clinical studies have revealed the pivotal contribution of cytokines in the pathogenesis of RA.The critical role of TNF and IL-6 therein has been unequivocally demonstrated by successful clinical targeting of these cytokines in RA using biological therapies.Neutrophil granule proteins such as myeloperoxidase(MPO),matrix metalloproteinases(MMPs),neutrophil elastase(NE),cathepsin G and proteinase 3 are found in high concentrations in RA synovial fluid and may be responsible for damage to cartilage and tissue,activation of soluble cytokines and receptors,inhibition of chondrocyte proliferation,and activation of synoviocyte proliferation and invasion.Circulating and synovial fluid neutrophils in RA patients are more prone to form neutrophil extracellular traps(NETs)compared with neutrophils from healthy controls and from patients with osteoarthritis.Levels of NETosis correlate with the presence and levels of anti-citrullinated peptide antibodies(ACPAs)and with markers of systemic inflammation.Further,in RA,dysregulation of neutrophil apoptosis can lead to their extended survival in inflamed tissues,which prolongs the release of neutrophil-derived immune regulatory cytokines,chemokines and cytotoxic products,which can lead to persistent inflammation.Therefore,neutrophils serves as the important target for RA therapy.Tanshinone ⅡA(TⅡA),a phenanthrenequinone derivative extracted from Salvia miltiorrhiza Bunge,has been used clinically to manage many diseases,such as angina pectoris,myocardial infarction and stroke.Previous studies have demonstrated that TⅡA may exert anti-inflammatory effects.TⅡA inhibits the production of pro-inflammatory mediators such as NO,TNFα,IL-1β,and IL-6 via inhibiting NF-κB activation in RAW 264.7 cells stimulated with LPS.In brain microvascular endothelial cells,TⅡA inhibits the expression of VCAM-1 and ICAM-1 a concentration dependent fashion through the inhibition of NF-κB activation and ROS generation induced by TNFa.TⅡA also induces inflammation resolution in vivo both by inducing neutrophil apoptosis and promoting reverse migration of neutrophils.However,whether TⅡA can ameliorate chronic inflammation in RA remains largely unknown.We hypothesized that TⅡA reduced neutrophil infiltration and activation,which may eventually attenuates the inflammatory response in RA.We investigated whether the treatment of TⅡA could ameliorate adjuvant-induced arthritis in murine model of RA.Objective:Rheumatoid arthritis(RA)is a chronic inflammatory disease mediated by the influx of immune cells into the synovial joint space.As Tanshinone ⅡA(TⅡA)has potent antioxidant and anti-inflammatory activities,we used the adjuvant-induced arthritis(AA)murine model of RA to investigate the impact of TⅡA on RA and the activation of immune cells.Methods:(1)The anti-arthritic activity of TⅡA was investigated in adjuvant-induced arthritis model of RA in mice.(2)Myeloperoxidase and neutrophil elastase expression in ankle joints were assessed by immunohistochemistry analysis.(3)Neutrophils infiltration was evaluated in air pouch experiments.(4)Expression of pro-inflammatory cytokines such as TNF-α、IL-6、iNOS was determined by quantitative real-time PCR,enzyme-linked immunosorbent assay and Western blot analysis.(5)The expression of Bcl-2,Bax and cleaved caspase-3 was detected by Western blot analysis.The neutrophil apoptosis was also examined by Annexin V/propidium iodide(PI)staining assay by using flow cytometry.(6)NETs formation and MPO and NE release were assessed by immunostaining and confocal microscopy.Results:(1)TⅡA alleviated RA in mice:We used a murine model challenged with Freund’s complete adjuvant(FCA)to examine whether TⅡA could inhibit joint inflammation.Compared with the control mice,the adjuvant induced paw edema can be significantly reduced by intraperitoneal injection of TⅡA,as well as ankle joint diameter and arthritis scores.(2)Histological analysis of ankle joint in mice:FCA challenge resulted in typical inflammation,characterized by cartilage erosion,synovial hyperplasia,and multiple inflammatory cells infiltration,compared with the normal mice.Treatment with TⅡA significantly alleviated the arthritis in mice based on histological analysis.(3)Effect of TⅡA on TNF-a and IL-6 in the plasma of RA mouse.ELISA analysis showed that TⅡA treatment can significantly reduce the secretion of TNF-a and IL-6 proteins in the plasma of RA mouse.(4)Effect of TⅡA on MPO and NE expression in ankle joint of RA mice.Immunohistochemistry analysis showed that the expression of MPO and NE was significantly upregulated in RA mice compared with the control group.However,TⅡA treatment significantly suppressed the up-regulation of MPO and NE induced by FCA.(5)TⅡA inhibited LPS-induced neutrophil influx in vivo:Air pouch experiments were used to investigate the impact of TⅡA on LPS-induced neutrophil infiltration.The treatment of TⅡA significantly reduced the total leukocytes as well as neutrophils in the air pouch.(6)Effect of TⅡA on TNF-α and IL-6 expression in LPS-activated neutrophils.The qRT-PCR analysis showed that TⅡA significantly down-regulated the mRNA level of TNF-αand IL-6 in LPS-activated neutrophils.Consistently,ELISA analysis showed that TⅡA treatment also significantly reduced the secretion of TNF-α and IL-6 proteins in a dose-dependent manner.(7)Effect of TⅡA on iNOS expression in LPS-activated neutrophils:The qRT-PCR and Western blot were used to analyze the expressions of iNOS mRNA and protein in neutrophils.TⅡA significantly reduced the expression of iNOS mRNA and protein expressions.(8)TⅡA induced the apoptosis of LPS-treated neutrophils:We detected the expression of Bcl-2,Bax and cleaved caspase-3 by Western blot analysis.TⅡA significantly increased the expression of Bax and cleaved caspase-3,and reduced the expression of Bcl-2.Thus,TⅡA treatment promoted neutrophil apoptosis.TⅡA also significantly increased the percentage of cells labeled as Annexin V(+)PI(-)and Annexin V(+)PI(+)by flow cytometry.(9)TⅡA inhibited NETs formation and MPO and NE release by neutrophils in vitro:PMA-induced NETs formation was visualized by staining with DAPI and anti-MPO or anti-NE,and observed by immunofluorescence confocal microscopy.TⅡA inhibited NETs formation,as well as NETs-associated MPO and NE release.Conclusion:(1)TⅡA can inhibit the migration and infiltration of neutrophils into the inflammatory site.(2)TⅡA reduced the expression and secretion of TNFα,IL-6 and iNOS,thus inhibited the activation of neutrophils.(3)TⅡA promoted the apoptosis of neutrophils,thus decreased the activated neutrophils in the inflamed joints.(4)TⅡA inhibited NETs formation and NETs-associated MPO and NE release,thus reduced tissue damage and inflammation.Our findings showed that TⅡA can effectively ameliorate RA by targeting neutrophils,indicating that TⅡA may act as a potential therapeutics for RA. |
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