| Background:Rheumatoid Arthritis is a chronic systemic autoimmune disease,with joint synovitis as the main pathological feature.The specific pathogenesis is not yet clear.Neutrophils play an important role in the pathological process of RA:Neutrophils secrete a lot of inflammatory cytokines,which leads to the continuous activation of inflammatory response;Cells secrete a variety of cytotoxic proteases,which mediate host joint tissue damage through oxidative damage;Delayed apoptosis leads to a continuous inflammatory response at the joint;Neutrophil extracellular traps are an important source of self-antigens,promoting the production of anti-citrullinated protein antibodies and aggravating the progress of RA.Andrographolide is the main active ingredient from Andrographis paniculata,which is a diterpene lactone compound.Multiple studies from basic and clinical studies have confirmed that andrographolide has anti-inflammatory,antibacterial,anti-tumor,liver protection and immunomodulatory activities.Previous basic studies have reported that andrographolide inhibits neutrophil ROS production,NF-?B activity,migration and adhesion.A prospective,randomized,placebo-controlled clinical study involving 60 RA patients found that andrographolide was effective in relieving symptoms and had good safety.However,whether andrographolide can treat RA by regulating the activity of neutrophils is still lack of clear evidence.Objectives:We took Freund's complete adjuvant-induced RA mouse model and purified mouse peritoneal neutrophils as research objects to explore the effect of andrographolide on RA mice and the mechanism mediated by neutrophils,with a view to providing experimental evidence for clinical medication.Methods:(1)Observe the effect of andrographolide on RA through AA mouse model:the measurement of ankle joint diameter and joint pathological score were used to evaluate ankle joint swelling;ankle joint pathological status was evaluated by using H&E staining and safranine green staining;The CB A reagent kit detects the secretion of cytokines in the serum of mice to assess the systemic inflammatory state.(2)The effect of andrographolide on the migration of neutrophils:the expression of MPO and NE in the ankle joints of mice in each group was evaluated by immunohistochemistry experiment,and the migration of neutrophils in the tissues was evaluated;The migration and changes of neutrophils was evaluated by the mice back airbag experiment induced by LPS and transwell experiment.(3)The effect of andrographolide on neutrophil apoptosis:the changes of neutrophil apoptosis were detected by Annexin V/PI double staining method and flow cytometry.The differential expression of Bax,Bcl-2 and Cleaved-caspase3 were detected by Western blotting.(4)The effect of andrographolide on the formation of NETs:the difference in the formation of NETs and the release of MPO,NE and CitH3 was evaluated by cellular immunofluorescence experiments and confocal microscopy;the differential expression of PAD4 and CitH3 in the ankle joints of each group were detected by immunohistochemistry experiments.(5)The effect of andrographolide on the formation of autophagy:the differential expression of LC3 was detected by cellular immunofluorescence experiments and confocal microscopy.The related proteins including LC3,Beclinl and p62 were detected by Western blotting.(6)Andrographolide regulates the activity of neutrophils in the inflammatory microenvironment mainly through the TNF signaling pathway.TNF is the key regulator molecule:Using mRNA sequencing technology to study the type and number of mRNA transcribed from neutrophils by andrographolide in the presence or absence of andrographolide,we also searched for the important gene groups closely related to changes in functional status through biological statistical analysis.Results:(1)Andrographolide ameliorated Adjuvant-Induced Arthritis(AA)in Mice:After 27 days administration,the ankle diameter and joint pathology score of mice in the AA model group were significantly higher than the normal control group;The degree of joint swelling,ankle joint diameter and joint pathology score were significantly relieved after andrographolide treatment.In the high-dose treatment group,the diameter of the ankle joint was reduced to 3.6±0.1 mm and the joint pathology score was reduced to around 2 points.H&E staining showed that compared with the normal control group,the RA group showed obvious inflammatory pathological changes,such as inflammatory cell infiltration,bone destruction and synovial hyperplasia;inflammatory lesions at the joints were significantly relieved after andrographolide treatment Safranin O-fast green staining showed that the bone damage of the RA group was significantly higher than the normal control,and the bone damage was significantly reduced after andrographolide treatment.CBA test showed that the levels of TNF-?,IFN-?,IL-6,IL-17 and IL-2 in the serum of mice in AA group were significantly higher than those in Control group.After treatment with andrographolide,the content of TNF-?,IFN-?,IL-6 and IL-17 decreased significantly.(2)Andrographolide inhibited the migration of neutrophils:The immunohistochemical experiment of mouse ankle showed that the expression of MPO and NE in the AA group was significantly increased compared with the normal control group,and the expression of MPO and NE was significantly reduced after andrographolide treatment.Andrographolide significantly inhibited the number of neutrophil migration induced by LPS in the dorsal airbag of mice.In addition,Transwell experiments showed that andrographolide significantly inhibited fMLP-induced neutrophil migration.(3)Andrographolide accelerates neutrophil apoptosis in the Presence of LPS:The results of Annexin V/PI double staining showed that the apoptosis of neutrophils in the LPS stimulated group was significantly delayed compared to the normal control group,and andrographolide can reverse this phenomenon;Immunoblotting experiments showed that compared with the LPS-stimulated group,the andrographolide intervention group could significantly increase the expression of Bax and Cleared caspase-3 and decrease the expression of Bcl-2.(4)Andrographolide inhibited the formation of NETs:The immunohistochemical experiment of mouse ankle showed that the expression levels of PAD4 and CitH3 in RA mice were significantly increased compared with the normal control group,and the expression levels of PAD4 and CitH3 were significantly decreased after treatment with andrographolide.Cellular immunofluorescence experiments showed that andrographolide can significantly inhibit the formation of NETs and the release of MPO,NE and CitH3.(5)Andrographolide inhibited neutrophil autophagy:Cell immunofluorescence experiments showed that andrographolide can significantly inhibit the expression of LC3 compared to the PMA stimulation group.Western blot experiments showed that andrographolide significantly inhibited the expression of LC3-? and Beclinl and up-regulated the expression of p62.(6)mRNA sequencing analysis of the effect of andrographolide on neutrophil activity stimulated by LPS:RNA-seq results showed that there were 284 differentially expressed genes between LPS stimulation group vs normal control group and andrographolide intervention group vs LPS stimulation group.The KEGG pathway enrichment analysis and the STRING database for protein interaction network analysis of common differentially expressed genes suggest that the TNF signal transduction pathway is the most significant enrichment pathway,and TNF is the key regulatory molecule.Conclusions:(1)Andrographolide relieved RA symptoms effectively in mice.(2)Andrographolide inhibited the migration and recruitment of neutrophils and the secretion of TNF-?,IFN-?,IL-6 and IL-17.(3)Andrographolide induced apoptosis of activated neutrophils.(4)Andrographolide inhibited the formation of NETs.(5)Andrographolide inhibited neutrophils autophagy.(6)Andrographolide regulates the activity of neutrophils in the inflammatory microenvironment mainly through the TNF signaling pathway.TNF is the key regulator molecule. |
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