Research On The Mechanism Of Tetrandrine In The Treatment Of Rheumatoid Arthritis By Regulating The Activity Of Neutrophils

Background:Rheumatoid arthritis(RA)is a chronic autoimmune joint disease with joint synovial inflammation as the main pathological manifestation.Its pathogenesis has not yet been fully elucidated.Neutrophil,as an important part of innate immunity,plays an important role in the initiation and development of RA.Neutrophil exudates from blood vessels and migrates to articular sites.Neutrophil also releases cytokines,chemokines and protein enzymes that delay the remission of the inflammatory response,and forms neutrophil extracellular traps(NETs),eventually leading to tissue damage.The release of NETs and their components may cause damage to surrounding tissue and promote the production of citrullinated autoantigens,thereby generating self-antigen antibodies,which further breaks immune tolerance.Therefore,neutrophils are important targets for RA treatment.At present,clinically promoted RA treatment drugs are mainly traditional disease-modifying anti-rheumatic drugs(cDMARDs)and nonsteroidal anti-inflammatory drugs(NSAIDs),which mainly relieve the symptoms.The application of expensive biologics is relatively fewer.Traditional Chinese medicine has been applied in many researches.Tetrandrine(TET)is an important alkaloid in the traditional Chinese medicine Stephania tetrandra S.Moore.It has been widely studied in malignant tumors and organ fibrosis.Clinical trials and experiment assays have proved that the efficacy of Tetrandrine in RA patients and RA animal models,respectively.But the study focused on the effect of tetrandrine on neutrophil in rheumatoid arthritis animal models is still lacking..Objective:The aim of this study is to explore the protective effect of tetrandrine on adjuvant induced arthritis(AA)mouse model and furthermore assess its regulatory effect on neutrophil activity both in vivo and in vitro,thus laying the foundation of further clinical application as novel therapeutic targets.Methods:(1)To explore the effect of tetrandrine on arthritis in mice through Freund’s complete adjuvant-induced arthritis model,mouse ankle paraffin sections were prepared to perform H&E staining and safranin O solid green staining to evaluate joint protective effect of tetrandrine;CBA kit was used to detect the expression of major inflammatory cytokines in mice.(2)The expression of myeloperoxidase(MPO)and peptidylarginine deiminase 4(PAD4)in ankle joints of AA mice was detected by immunohistochemistry in ankle paraffin sections.(3)Real-time qPCR and enzyme-linked immunosorbent assay were used to detect the expression levels of neutrophil cytokines TNF-α,IL-1β and IL-6 after LPS stimulation with or without tetrandrine.(4)The effect of tetrandrine on neutrophil migration in vitro and in vivo was evaluated by the transwell assay and the mouse air punch assay,respectively.(5)The effect of tetrandrine on apoptosis was detected by Annexin V/PI double staining.Western blot was used to detect the expression levels of apoptosis-related proteins PARP,Bcl-2,Bax and cleaved caspase-3.(6)Neutrophil extracellular trap formation and neutrophil elastase(NE)release was evaluated using indirect immunofluorescence and laser confocal microscopy.(7)The transcriptome sequencing was used to detect the regulatory effect of LPS on neutrophil after two-hour stimulation on genes and pathways,and to compare the regulatory differences of LPS on neutrophil and macrophage.(8)The concentration of proinflammatory cytokines TNF-α,IFN-γ,IL-1β IL-2,IL-4,IL-6,IL 10 and IL-17A after neutrophil culture in vitro was detected by enzyme-linked immunosorbent assay(ELISA)and flow cytometry analysis.(9)The transcriptome sequencing was applied to explore the transcriptional regulation of neutrophil induced by tetrandrine upon LPS stimulation.Western Blot was performed to verify the sequencing results.Results:(1)Alleviated symptoms of AA mice treated with tetrandrine:Freund’s complete adjuvant(FCA)was applied to induce mouse arthritis model to assess whether tetrandrine can inhibit arthritis.The ankle diameter of model group had significantly increased compared to that of normal control mice.The arthritis score and ankle diameter of the mice treated with tetrandrine intraperitoneally aggressively decreased(p<0.01).(2)The histological analysis of ankle joints in AA mice:typical inflammation manifestations were observed in the ankle joint paraffin sections of AA model mice compared with the normal control group,such as cartilage damage and synovial hyperplasia.The pathological changes in the joints of the tetrandrine treatment group mice were significantly improved.Cartilage damage and synovial hyperplasia were alleviated.(3)The effect of tetrandrine on the secretion of TNF-α,IFN-γ and IL-6 in the sera of AA mice:the concentration of TNF-α,IFN-γ and IL-6 in the sera of mice was detected by the CBA kit.The concentration of TNF-α and IL-6 in AA mice sera was significantly higher than that of normal control group(p<0.01).After the treatment with tetrandrine,the concentration of IL-6 decreased significantly(p<0.05).(4)The effect of tetrandrine on the expression of MPO and PAD4 in the ankle joints of mice:after immunohistochemical analysis,the expression of MPO and PAD4 in the ankle joints of AA mice was aggressively increased compared with that of normal mice.However,the expression of MPO and PAD4 in the tetrandrine treatment group was significantly reduced compared to the AA model group mice.(5)The effect of tetrandrine on TNF-α,IL-1β and IL-6 secreted by neutrophil:the differentially expressed gene analysis was accomplished by real-time quantitative PCR.Based on former results,TNF-α,IL-1β and IL-6 expression was tested.Compared with the LPS-stimulated group,the tetrandrine treatment group showed a certain degree of inhibition of the expression of IL-1β and IL-6(p<0.01).ELISA was used to detect cell supernatants recovered at 2 h and 4 h,and found that tetrandrine inhibited IL-6 at both 2h and 4h(p<0.01),while IL-1β was merely reduced at 2 h(p<0.05).(6)The effect of tetrandrine on neutrophil iNOS expression:Western Blot was used to detect the effect of tetrandrine on iNOS protein expression level in neutrophil activated by LPS.Compared with the LPS-stimulated group,iNOS expression was inhibited in the tetrandrine-treated group in the dose dependent manner.(7)The effect of tetrandrine on neutrophil recruitment in mice:Transwell assays showed that tetrandrine significantly inhibited fMLP-induced neutrophil migration in vitro(p<0.01).In the mouse air punch assay,the total number of leukocyte and neutrophil in the punch of LPS-stimulated group significantly rose up compared with that of the PBS group(p<0.01).The number of neutrophil from the punch of the tetrandrine treatment was significantly reduced(p<0.01)compared with the LPS group.(8)The effect of tetrandrine on neutrophil apoptosis:flow cytometry analyses showed that tetrandrine could reverse the apoptosis delay caused by LPS stimulation(p<0.01).Western Blot was used to detect the expression of PARP,Bcl-2,Bax and cleaved caspase-3 proteins.Compared with the LPS stimulation group,the tetrandrine treatment group could significantly increase the expression of Bax,cleaved caspase-3 and cleaved PARP expression.At the same time,tetrandrine could significantly reduce the expression of Bcl-2.(9)The effect of tetrandrine on neutrophil NETs formation and NE release:the indirect immunofluorescence staining showed that tetrandrine could significantly inhibit the formation of NETs and NE release upon PM A stimulation.(10)The sequencing analysis of neutrophil stimulated by LPS:after LPS stimulation,1158 genes were up-regulated and 1021 genes were down-regulated,in which "fold change>2" and "adjusted p value<0.05" was set as the cutoff.The results of cluster analysis of differentially expressed genes indicated that the expression pattern of the LPS-stimulated group and the blank control group could be completely separated.Differential genes could be enriched based on the KEGG database,such as TNF signaling pathway,NOD-like receptor signaling pathway,cytokine signaling pathway and so on.Then the differential genes acquired from neutrophil were compared with those obtained from bone marrow-derived macrophages stimulated by LPS,where difference criteria were the same.With genes intersected,neutrophil and macrophage were found to share the same up-regulated 386 genes that enriched the tumor necrosis factor pathway,cancer pathway,metabolic pathway and so on.They also had the same down-regulated 303 genes that enriched to metabolic pathways,cancer pathways and herpes virus infection pathways.(11)The effect of LPS on cytokines secreted by neutrophil in cell culture supernatants:the supernatants test was accomplished using CBA kits and ELISA.A two-hour stimulation by LPS significantly increased TNF-α,IL-1β,IL-10 and IL-6 concentration(p<0.01)while LPS had no significant effect on IL-2,IL-4,IFN-y and IL-17A.LPS stimulation for 4 h can significantly increase the concentration of TNF-α,IL-6 and IL-10(p<0.01).(12)The limited regulatory effect of tetrandrine on transcriptome of neutrophil stimulated by LPS:tetrandrine could up-regulate 268 genes and down-regulate 426 genes,where "fold change>1.5" and "adjusted p value<0.05" was set as the cutoff.(13)The modulatory effect of tetrandrine on JNK expression and phosphorylation:Western blot showed that LPS induced the protein level of p-JNK,which was lessened by tetrandrine.Conclusion:(1)Tetrandrine can relieve the arthritis symptoms of AA mouse model by inhibiting the migration of neutrophil to the inflammatory sites,reducing not only the release of proinflammatory cytokines(IL-1β and IL-6)but also the expression of related proteins(NE and PAD4).(2)Tetrandrine can reduce the expression of IL-1β,IL-6 and iNOS,thereby inhibiting neutrophil activation.(3)Tetrandrine can promote timely apoptosis of the activated neutrophil to promote the resolution of RA inflammation.(4)Tetrandrine can inhibit the formation of NETs and the release of NE during the formation of NETs,thus reducing the inflammatory response and tissue damage of RA.(5)LPS can cause differential gene expression in neutrophil,which is mainly involved in biological process.(6)Tetrandrine can regulate the neutrophil transcriptome induced by LPS in a complex and limited way.In summary,tetrandrine can relieve joint symptoms in rheumatoid arthritis animal models,improve pathological changes and regulate local neutrophil infiltration as well as activity.Furthermore,tetrandrine can regulate neutrophil activity in vitro.Therefore,the fact that tetrandrine can regulate the activity of neutrophil may be one of the therapeutic targets of tetrandrine so as to alleviate rheumatoid arthritis.