The Effect And Possible Mechanism Of Triptolide On Rheumatoid Arthritis Based On Treg/Th17 Imbalance

ObjectiveWe aim to investigate the relationship between T regulatory(Treg)cells/T-helper(Th)17 cells and disease stage as well as disease activity in patients with rheumatoid arthritis(RA),and whether the aberrant DNA methylation is implicated in the imbalance of Treg/Th17 cells.MethodsPeripheral blood mononuclear cells were isolated from RA and healthy control(HC)populations.Circulating Treg/Th17 ratio was determined by flow cytometry.Treg/Th17 related cytokines were detected by BCA and ELISA.Levels of DNA methylation related enzymes were measured by q RT-PCR.Treg-specific demethylated region(TSDR)/retinoic acid?related orphan receptor(ROR)C methylation was measured by bisulfite sequencing PCR.ResultsBy recruiting 65 patients with multi-staging RA and 20 HC,we found that patients with active RA exhibited relative lymphopenia and higher white blood cells,neutrophils,and platelets.Circulating Treg/Th17 in patients with early active RA was significantly decreased.The expression of IL-6 and IL-17 A was significantly increased in early active RA,whereas that of IL-10 and TGF-? was on the contrary.Furthermore,the frequency of Treg cells and Treg/Th17 were negatively correlated with DAS28,and the frequency of Th17 cells was on the contrary.Levels of DNA methylation related enzymes had significant difference between early active RA and HC.Relative hypermethylation was observed at the gene level for TSDR and hypomethylation for RORC in early active RA.ConclusionThe ratio of Treg/Th17 cells was unbalanced in patients with active RA,and it was negatively correlated with disease activity DAS28.Abnormal DNA methylation might be related to Treg/Th17 imbalance.ObjectiveThis study aims to investigate the therapeutic effect of triptolide(TP)on collagen-induced arthritis(CIA)mice and its effect on Treg/Th1/Th17 cells,and to explore whether its mechanism is related to the activation of JAK/PTEN-STAT3 signaling pathway.MethodsC57 mice were injected with chicken collagen type II emulsion at the base of the tail to establish a CIA model.They were administrated with saline or TP(32?g/kg/d)for 35 days.Body weights and arthritis scores were measured twice a week.Micro-CT,safranin O staining,tartrate-resistant acid phosphatase(TRAP)and hematoxylin-eosin(HE)staining were used to evaluate the pathological changes in the joints.The levels of anti-collagen type II(CII)immunoglobulin(Ig)G1,anti-CII Ig G2 a,anti-tumor necrosis factor-?(TNF-?),and interleukin-6(IL-6)in the serum were determined by ELISA.The ratios of Treg,Th1 and Th17 cells in mouse spleen were measured by flow cytometry.The m RNA expression of transcription factors Foxp3 and ROR-?t were measured by q RT-PCR.The expression of JAK/PTEN-STAT3 pathway in mouse joints was analyzed by western blot,and the expression of IL-17 A and p STAT3 in mouse spleen was measured by immunofluorescence.ResultsThe dose of TP was within a safe range,and it could significantly alleviate joint swelling,reduce bone destruction,and downregulate serum anti-CII Ig G1,TNF-?,and IL-6 levels in CIA mice.TP improved the imbalance of Treg/Th17 cells in the spleen of CIA mice.Meanwhile,TP significantly inhibited the activation of JAK/PTEN-STAT3 signaling pathway and reduced the expression of p STAT3 and IL-17.ConclusionTP can inhibit local and systemic inflammation and alleviate bone destruction in CIA mice.The underlying mechanism are related to the regulation of the imbalance of Treg/Th17 cells through the JAK/PTEN-STAT3 pathway.ObjectiveThis study aims to explore the possible binding pose,binding energy and binding stability of triptolide(TP)against targeted proteins and the effects of TP on the JAK/PTEN-STAT3 signaling pathway in mouse spleen lymphocytes.MethodsReverse docking was used to characterize the binding pose,binding energy and binding stability of TP against targeted proteins Foxp3,ROR-?t,JAK1,JAK2,PTEN,and STAT3.Mouse spleen lymphocytes were obtained by the lysis of red blood cells,and CCK-8 assay was used to measure the effect of TP on the survival rate of lymphocytes.After IL-6 stimulation,the expression of JAK/PTEN-STAT3 pathway proteins were analyzed by western blot.ResultsResults of TP against Foxp3,ROR-?t,JAK1,JAK2,PTEN and STAT3 showed high binding affinity.The inhibitory effect of TP on lymphocytes was concentration-dependent.Next,the concentrations of 20 n M and 40 n M were used as further experimental doses.After IL-6 stimulation,TP significantly inhibited the activation of the JAK-STAT3 signaling pathway.ConclusionTP against Foxp3,ROR-?t,JAK1,JAK2,PTEN,and STAT3 can form tight bindings.In an inflammatory environment,TP can regulate the activation of STAT3 signaling pathway in mouse spleen lymphocytes.