| Objective:Rheumatoid arthritis(RA)is a chronic autoimmune rhematic disease,mainly manifested as recurrent synovitis,that leads to multiple joints swelling,pain,deformation,loss of normal function,which is often evolved into disability.As a lifelong disease,RA has a highest disability rate among joint diseases,seriously affects life quality of people.The specific cause of RA is not clear.This topic is based on the wet stickiness and turbidity,long-term formation of toxic,long-term heat-induced phlegm,retardation of meridians and veins,combined with the western etiology of“body fluid theory”,proposed that“toxic dampness obstruction”may lead to the etiology of RA.Into the network of wet toxicity,the flow or migration of body fluids may stimulate the abnormal expression of aquaporins(AQPs).Inflammatory exudation,synovial congestion and edema during RA activity may also be associated with abnormal metabolism of water.It has been reported that AQPs play a role in the development of RA and can mediate cell migration during its pathological changes.In addition,there is no cure for RA in clinical practice.Most of them stay in symptomatic treatment and lack of treatment.This topic aims at the cause“toxic dampness obstruction”of RA and uses empirical prescription sanhuang yilong decoction(SYD)in combination with methotrexate(MTX)to treat RA with damp-heat-obstruction symptom pattern.The pathogenesis of RA and the mechanism of action of Sanhuang Yilong Decoction or combined with MTX have been studied in clinical and animal experiments.Methods:The first part of the paper focused on peripheral blood and synovial fluid from RA patients with damp-heat-obstruction symptom pattern and conducted preliminary studies on the expression and regulation of cytokines,AQPs and JAK-STAT pathway signaling factors.ELISA methods were applied in the detection of relevant indicators.The second part replicated the animal model of toxic dampness obstruction ones.Taking rat peripheral blood,bones,joints,lungs,spleen,and kidneys as research objects,this paper had a deep study on inflammatory factors,AQPs,Na~+,K~+-ATP enzyme,and JAK-STAT pathway signaling factors and the changes and regulation of joint histopathology.ELISA methods,RT-PCR and histopathology were applied in the detection.Main results:First.Changes of inflammatory factors,AQPs and JAK-STAT pathway signaling factors in RA patients with damp-heat-obstruction symptom pattern,and the regulation and mechanism of SYD combined with MTX.1.Effect of SYD combined with MTX on inflammatory factors in RA patients with damp-heat-obstruction symptom pattern(1)The swelling index,tenderness index,ESR,CRP,DAS28 were significantly increased in the active stage of RA patients with damp-heat-obstruction symptom pattern,and could be improve quickly by SYD combined MTX.(2)The expression levels of TNF-αand IFN-γof RA patients with damp-heat-obstruction symptom pattern were significantly higher than those in healthy control group,and TNF-αand IFN-γin synovial fluid were significantly higher than that in peripheral blood.(3)The expression levels of TNF-αand IFN-γwere decreased significantly after treatment by SYD plus MTX.2.Changes of AQPs in RA patients with damp-heat-obstruction symptom pattern and effects of SYD combined with MTX on them.(1)The expression levels of AQP1,AQP2 and AQP3 in peripheral blood and synovial fluid of RA patient with damp-heat-obstruction symptom pattern were significantly lower than those in healthy subjects.(2)The expression of AQP1,AQP2 and AQP3 in peripheral blood were slightly higher than those in synovial fluid,of which AQP1 had the highest expression,followed by AQP3 and AQP2.(3)After treatment by SYD plus MTX,the expression of AQP1,AQP2,and AQP3 were significantly increased.3.Changes of JAK-STAT pathway signaling factors in RA patients with damp-heat-obstruction symptom pattern and effects of SYD combined with MTX on them.(1)The expression levels of STAT1,STAT3,PIAS1,PIAS3,SOCS-1 and SOCS-3 in RA patient with damp-heat-obstruction symptom pattern were lower than those in healthy subjects,among which the expression of STAT1 was higher than STAT3,PIAS1 higher than PIAS3,SOCS-1 and SOCS-3 almost the same.(2)The expression of STAT1,PIAS1,PIAS3and SOCS-1 in peripheral blood of RA were higher than those in synovial fluid,while the levels of STAT3 and SOCS-3 in peripheral blood were basically equivalent to those in synovial fluid.(3)After the treatment of SYD plus MTX,STAT1,STAT3,PIAS1,PIAS3,and SOCS-1 in peripheral blood of RA patients with damp-heat-obstruction symptom pattern were all down-regulated,but there was no significant change in SOCS-3.Second.Changes of inflammatory factors,AQPs,Na~+,K~+-ATPase,JAK-STAT pathway signaling factors,and joint histopathological changes in RA rat models with toxic dampness obstruction and the regulation and mechanism of SYD Combined with MTX1.Changes of inflammatory factors in RA rat models with toxic dampness obstruction and the effects of SYD or Combined MTX.(1)The count of platelet and ratio of neutrophils and mononuclear cells in the blood of RA model rats with toxic dampness obstruction were significantly higher than those in the normal group,and the lymphocyte ratio was significantly lower than that in the normal group.SYD or SYD combined with MTX can better reduce the ratio of neutrophils and monocytes and upregulate the ratio of lymphocytes.(2)The expression levels of IL-6 and TNF-αin the peripheral blood of RA model rats with toxic dampness obstruction were significantly lower than those in the normal group.The expression of IL-6 and TNF-αin the bone and joints were significantly higher than those in the normal group.SYD or SYD together with MTX can upregulate the expression of IL-6 and TNF-αin peripheral blood,and down-regulate IL-6 and TNF-αin bone and joints.(3)The expression of VEGF in the peripheral blood of model rats with toxic dampness obstruction was no different from that in the normal group,but the expression of VEGF in the bone and joints was significantly higher than that in the normal group.SYD or SYD combined with MTX can reduce the expression of VEGF in bone and joints.(4)The content of TGF-β1mRNA in lung tissue of model rats with toxic dampness obstruction was no different from that in the normal group,but the content of TGF-β1mRNA in the soft tissue surrounding inflammatory joint was significantly lower than that in the normal group.SYD combined with MTX can promote the expression of TGF-β1mRNA in the soft tissues around inflammatory joints.2.Changes of aquaporins,Na~+,K~+-ATPase in model rats with toxic dampness obstruction and the effect of SYD combined with MTX.(1)The expression levels of AQP1,AQP2,and AQP3 in peripheral blood and bone and joint tissues of model rats with toxic dampness obstruction were significantly lower than those in normal rats.(2)The expression of AQP2 was highest in peripheral blood of model rats,and was followed by AQP3.The expression of AQP3 in bone and joints was the highest,followed by AQP2.(3)The content of AQP1mRNA in the lung tissue of model rats was significantly lower than that in the normal group,and the content of AQP1mRNA in kidney tissue was not different from that in the normal group.((4)The activity of Na~+,K~+-ATPase in the spleen tissue of model rats was significantly decreased compared with the normal group.(5)Both SYD and SYD plus MTX can promote the expression of AQP1,AQP2 and AQP3 in the peripheral blood and bone and joints of model rats.SYD plus MTX can also significantly enhance the expression of AQP1mRNA in lung tissue.(6)SYD and SYD plus MTX can significantly increase the activity of Na~+,K~+-ATPase in the spleen tissue of model rats.3.Changes of signaling factors of JAK-STAT signaling pathway in model rats with toxic dampness obstruction and the effect of SYD combined with MTX.(1)The expression levels of JAK1,JAK2 and JAK3 in peripheral blood of model rats with toxic dampness obstruction were significantly lower than those in normal rats.JAK1 and JAK3 in the bone and joints of model rats were significantly higher than those of the normal group,and JAK2 was significantly lower than that of the normal group.(2)The content of JAK3mRNA in lung tissue of model rats was no different from that in the normal group,and the content of JAK3mRNA in kidney tissue was significantly higher than that in the normal group.(3)The expression of STAT1 in peripheral blood and bone and joints of model rats has no difference compared with the normal group.(4)The expression of STAT3 in the peripheral blood of model rats was significantly lower than that in the normal group,and STAT3 in the bone and joints was not different from that in the normal group.(5)SYD and SYD plus MTX can increase the expression of JAK1,JAK2,JAK3 in the peripheral blood and JAK2 in bone and joints of model rats.(6)SYD combined with MTX could down-regulate the expression of JAK1 and JAK3 in the bone and joints of model rats.(7)SYD and SYD plus MTX can upregulate the expression of STAT3 in peripheral blood.(8)SYD and SYD plus MTX can inhibit the expression of JAK3mRNA in renal tissue.4.Changes of joint histopathology in model rats with toxic dampness obstruction and the effect of SYD combined with MTX.(1)The histopathology of joints in model rats with toxic dampness obstruction showed macrophage infiltration with fibrous tissue hyperplasia.(2)SYD combined with MTX can inhibit the proliferation of fibrous tissue and infiltration of lymphocytes in themodel rats with toxic dampness obstruction.Conclusion:This study confirms that toxic dampness obstruction is the basic pathogenesis of RA.RA induced by toxic dampness obstruction is closely related to AQPs,Na~+,K~+-ATPase,JAK,etc..It showed that the expression of AQP1,AQP2,AQP3 and AQP1 mRNA and the activity of Na~+,K~+-ATPase were decreased in peripheral blood,bone and joint,lung,spleen and kidney etc.,and the production of proinflammatory cytokines IL-6,TNF-αand vascular endothelial growth factor(VEGF)increased,TGF-β1 mRNA in soft tissue decreased,signal factor JAK1,JAK3 and JAK3 mRNA abnormal expression,and Macrophages infiltrate with fibrous tissue in the joints.It is confirmed that the pulmonary main waterway and spleen transport function are involved in the occurrence and development of RA with toxic dampness obstruction.It was proved that SYD and SYD combined with MTX can reduce joint swelling,tenderness index,reduce ESR,CRP,DAS28 and other inflammatory markers,inhibit the production of proinflammatory cytokine IL-6,TNF-α,IFN-γand VEGF,promote the secretion of TGF-β1 mRNA,increase the expression of AQP1,AQP2,AQP3 and AQP1mRNA in the peripheral blood and bone joints,and the activity of Na~+,K~+-ATPase in the spleen,and regulate the expression of signal factors such as JAK1,JAK2,JAK3 and STAT3.They can act on multiple tissues and organs,and regulate through multiple targets,improve the metabolism of tissue cells,relieve inflammation of synovial membrane,and inhibit angiogenesis of synovial tissuee.This provides a theoretical basis for clinical application and promotion. |
PDF Full Text Request