The Mechanism Of Triptolide And Tripterine In The Treatment Of Rheumatoid Arthritis By Regulating Neutrophil Activity

Rheumatoid arthritis(RA)is an autoimmune disease characterized by chronic synovitis.Neutrophils are recognized to play a crucial role in the pathogenesis of RA,especially in the early phase RA.Neutrophils can produce a repertoire of inflammatory mediators in early RA.Neutrophil extracellular traps(NETs)externalize various autoantigens and granule enzymes,which may perpetuate a vicious cycle leading to generation of specific autoantibodies and inflammatory responses and are closely associated with autoantibody formation in early RA patients.Thus,neutrophils are now viewed as major orchestrators of inflammation in RA and emerging as an important target for RA therapy.Tripterygium wilfordii Hook F(TwHF)is an effective drug for the treatment of RA.However,there is a lack of clear instruction for the use of TwHF.Abusing of TwHF often leads to irreversible damage of RA patients.Considering the important role of neutrophils in the pathogenesis of early RA,we planned to analyze the function of triptolide and celastrol on neutrophils activation.We demonstrated that triptolide and celastrol could ameliorate arthritis by inhibiting neutrophil-mediated inflammation.The results are helpful to elucidate the molecular mechanism of TwHF in the treatment of RA.The results also interpret the phenomena that the onset of TwHF treatment would be earlier than other drugs.It would provide theoretical direction for the clinical application of TwHF.Part ?.Triptolide alleviates RA by inhibiting the inflammatory activities of neutrophilsObjective:to explore the influence of triptolide on neutrophil inflammatory activities in arthritic joints of AA mice and examined the mechanisms in the inhibition of neutrophil-mediated inflammation.Methods:we analyzed the efficacy of triptolide on neutrophil-mediated inflammation in vivo and in vitro.Freund's complete adjuvant-induced arthritis was used as the murine model of AA.Triptolide(70 ?g/kg)was injected intraperitoneally(i.p.)starting from the onset of AA and then continued uninterrupted until the day before tissue harvesting.Joint swelling was assessed by measuring ankle joint diameter using a pocket thickness gauge.The inflammatory joints were examined by H-E(hematoxylin-eosin)staining.MPO(myeloperoxidase)and NE(neutrophil elastase)in the inflammatory joints were analyzed by immunohistochemical analysis.The plasma of mice was harvested and the expression of cytokines were measured by ELISA.To address the mechanisms underlying the observed effects of triptolide on inflammatory mediators,we investigated the effect of triptolide on LPS-activated neutrophils in vitro.Neutrophils were stimulated with LPS for 2 h and treated in the presence of 0,10,50 and 200 nM triptolide.The expression of TNFa,IL-6,GM-CSF,CCL-5 mRNA was measure by quantitative Q-PCR in neutrophils.The cytokine proteins levels in the culture supernatants were measured by ELISA.We evaluated the expression of the protein Bcl-2,Bax,Caspase-3 and p-ERK after the treatment of tritpolide in LPS-induced neutrophil in vitro by western blot.We tested the effect of triptolide on PMA-induced NETs formation.The released NETs were confirmed by immunofluorescence staining of neutrophil granule MPO(red)and NE(green).Results:triptolide ameliorated adjuvant induced arthritis in AA mice.Statistically significant effects of triptolide on both hind paw swelling and clinical scores were seen(P<0.01).Histologic analysis with H-E staining revealed that triptolide reduced inflammation and reduced cartilage erosion.Triptolide treatment suppressed the MPO and NE enzyme expression in AA mice.Treatment with triptolide displayed a significant decrease in serum of TNFa and IL-6 in comparison to the RA mice(P<0.05).We studied the effects of the triptolide on LPS-induced neutrophil inflammatory mediators.We found that triptolide decreased the LPS-induced mRNA expression of TNFa,IL-6,CCL-5 and GM-CSF in a concentration-dependent manner by Q-PCR,respectively.We also found that triptolide suppressed LPS-induced TNFa and IL-6 production in a concentration-dependent manner by ELISA.Triptolide induced apoptosis of neutrophils.Western blot analysis revealed that triptolide increased the levels of Bax in LPS-induced neutrophils.On the contrary,triptolide declined the expression of Bcl-2 and p-ERK in LPS-induced neutrophils.Triptolide treatment to LPS-induced neutrophils resulted in the expected caspase-3 cleavage.The levels of cleaved caspase-3 increased with the treatment of triptolide while the levels of pro-caspase-3 decreased.Tritpolide inhibited NETs formation in vitro.By confocal microscopy analysis,we found that the robust NET formation induced by PMA was effectively inhibited by triptolide treatment.Conclusion:triptolide could suppresse autoimmune arthritis by inhibiting the inflammatory activities of neutrophils.Part ?.Celastrol alleviates RA by inhibiting the inflammatory activities of neutrophilsObjective:Neutrophils play an important role in the pathogenesis of rheumatoid arthritis(RA).In this study,we used the adjuvant-induced arthritis murine model to evaluate the efficacy of celastrol neutrophil-mediated inflammation in RA.Methods:Freund's complete adjuvant-induced arthritis was used as the murine model of RA.Celastrol was intraperitoneally administrated daily after onset of the disease.The joint diameter and inflammatory score were evaluated daily during the treatment period.MPO and NE activities were evaluated by immunohistochemical analyses.Quantitative Q-PCR and ELISA were used to quantify the expression of cytokines.NETs were visualized byfluorescence microscopy.The expression of apoptosis related proteins Bcl-2,Bax and caspase-3 were evaluated by Western blot.Results:Celastrol suppressed inflammation in joints of arthritic mice and diminished the expression of MPO and NE in the joint tissue.Celastrol significantly inhibited the expression of TNFa and IL-6 induced by LPS in neutrophils in a dose-dependent manner in vitro.Celastrol inhibited the formation of NETs.Moreover,Celastrol induced apoptosis of LPS-stimulated neutrophils by increasing the expression of Bax and cleaved caspase-3 while decreasing Bcl-2.Conclusion:Our findings showed that celastrol significantly alleviated murine arthritis by modulating the inflammatory activities of neutrophils.These results indicate that celastrol could serve as an alternative or adjunct modality for the treatment of RA.