Study On The Mechanism Of Duhuo Jisheng Decoction And Senkyunolide Ⅰ Regulating Lymphatic Reflux In The Treatment Of Rheumatoid Arthritis

Object:To explore the effects of Duhuo Jisheng Decoction on the lymphatic system and immune response of mice with rheumatoid arthritis.To explore the effect of Senkyunolide I on lymphatic return function and inflammatory response of mice with rheumatoid arthritis.To investigate the effect of Senkyunolide I on the inflammatory response of lymphatic endothelial cells and the Notch and NF-κB signaling pathways,as well as the effect on the functional genes of lymphatic smooth muscle cells.Methods:1.Study of DHJST on lymphatic system and immune response of TNF-Tg mice15 mice were used in this experiment,of which 10 3-month-old TNF-α transgenic(TNFTg)mice were randomly divided into model group and DHJST group,and another 5 wildtype littermates were taken as negative controls.DHJST group mice were treated with Duhuo Jisheng Decoction.In the control and model group,the same volume of normal saline was intragastrically administered.All mice were dosed once a day for 12 weeks.ABOG(Alcian Blue-Hematoxylin)staining is used to observe bone mass and synovial inflammation.And through the IF(immunofluorescence staining)method,explore the distribution of M1 macrophages that promote inflammation and the situation of lymphangiogenesis.2.Effects of Senkyunolide I on lymphatic reflux and inflammatory response in TNF-Tg mice18 mice were used in this experiment,of which 18 2-month-old TNF-α transgenic(TNFTg)mice were randomly divided into model group and SEI group,and another 6 wildtype littermates were taken as negative controls.SEI group mice were treated with Senkyunolide I.In the control and model group,the same volume of normal saline was intragastrically administered.All mice were dosed once a day for 10 weeks.HE(Hematoxylin-Eosin)staining is used to observe liver and kidney toxicity and synovial inflammation.ABOG staining is used to observe bone mass and cartilage.Trap(Tartrateresistant acid phosphatase)staining is used to observe the number of osteoclasts.IF staining to observe lymphangiogenesis and distribution.ICG-NIR(Indocyanine GreenNear Infrared)lymphatic imaging system for detection of lymphatic reflux function.PLN three-dimensional imaging system detects lymph nodes.The threshold of plantar pain in mice was measured by heat pain instrument.3.Study on the mechanism of Senkyunolide I on lymphatic endothelial and smooth muscle cellsTo investigate the effect of Senkyunolide I on inflammatory phenotype of lymphatic endothelial cells in TNF-α-induced inflammatory environment.Lymphatic endothelial cells and lymphatic smooth muscle cells were co-cultured to detect the effects of Senkyunolide I on the functional genes of lymphatic smooth muscle cells in the inflammatory environment.Q-PCR was used to detect the influence of Notch signaling pathway;Western blot was used to detect the influence of NF-κB signaling pathway;cell immunofluorescence staining was used to observe the nuclear entry of P65Results:1.DHJST promotes lymphangiogenesis and reduces joint inflammation in TNF-Tg mice.After 12 weeks of DHJST,the results of histomorphological staining of ankle joint showed that cartilage and bone erosion and synovial inflammation were significantly improved,the number of pro-inflammatory M1 macrophages was decreased,lymphangiogenesis was increased,and collecting lymphatic vessels were widely distributed.2.Senkyunolide I promotes lymphatic return in TNF-Tg mice and reduces joint inflammationAfter 10 weeks of Senkyunolide I treatment,ICG-NIR test results showed that Senkyunolide I could improve lymphatic pulsation frequency and lymphatic clearance rate.Three-dimensional PLN imaging results show that SEI can reduce the swelling degree of lymph nodes.The results showed that Senkyunolide I could increase the threshold of heat pain in the foot base of mice.The histomorphological staining results of ankle joint showed that Senkyunolide I could reduce synovial inflammation,reduce the number of osteoclasts,improve cartilage and bone erosion and promote lymphatic angiogenesis,and had no obvious toxic and side effects on liver and kidney.3.Senkyunolide reduces the level of inflammation in lymphatic endothelial cells and promotes gene expression in lymphatic smooth muscle cells.Senkyunolide I inhibited TNFα-induced endothelial inflammatory cytokines,Notch and NF-κB signaling pathways.Senkyunolide I promotes the recovery of functional gene expression levels in lymphatic smooth muscle cells after TNFα induction.Conclusion1.DHJST can reduce the progression of rheumatoid arthritis by regulating the lymphatic system and improving joint inflammation in mice2.Senkyunolide I improves the symptoms of rheumatoid arthritis by promoting lymphatic system reflux and reducing inflammatory articular cartilage and bone erosion3.Senkyunolide I can reduce TNFα-induced levels of inflammatory factors in lymphatic endothelial cells,restore functional gene expression of lymphatic smooth muscle cells,and improve lymphatic return function4.Senkyunolide I can inhibit the expression of Notch and NF-κB signaling pathways,which may be related to the mechanism of anti-inflammatory action,but the tandem relationship between Notch and NF-κB signaling pathways needs further study Figure 43 Table 0 Reference 132...