| BackgroundRheumatoid arthritis(RA)is a chronic autoimmune disease of the whole body.The main pathological features of RA are synovial invasive hyperplasia and pannus formation,resulting in the destruction of articular cartilage and bone.Pannus is composed of new capillaries,hypertrophic synoviocytes,inflammatory cells and organized cellulose.Pannus is similar to tumor tissue and is the main pathological basis of joint lesion and cartilage destruction.Synovial angiogenesis plays an important role in the formation and maintenance of Pannus,and endothelial cell activation is an important material basis for angiogenesis,its ability of proliferation,migration,adhesion and tube formation plays a central role in the process of promoting angiogenesis.The treatment of RA with traditional Chinese medicine has the advantage of multi-component and multi-target,and the bone destruction in RA patients is closely related to the formation of progressive synovitis and Pannus,our group has demonstrated in previous studies that DTYM can significantly improve the clinical symptoms and laboratory parameters of patients with rheumatoid arthritis,and that DTYM can effectively inhibit the inflammatory proliferation of synovium at animal and cellular levels,however,the role of DTYM in the inhibition of pannus formation in RA has not been well studied.Therefore,we hypothesized that DTYM might play a role in pannus endothelial cell activation.In this study,Collagen-Induced arthritis(CIA)in mice and Human umbilical vein Endothelium(HUVEC)in vitro were used to investigate the effects of DTYM on synovial Pannus and HUVEC activation,to explore the mechanism of DTYM inhibiting pannus formation through miR-337-3p mediated VEGF pathway.This study will provide scientific basis for clinical application of DTYM and lay a foundation for drug research of RA.Part 1 DTYM inhibits VEGF pathway to inhibit pannus formation in CIA mice ObjectivesTo study the inhibitory effect of DTYM on pannus formation in CIA mice and its mechanism.MethodsTwenty-four PF grade DBA/1 male mice aged 7-8 weeks were randomly divided into four groups: Blank Group(NC Group),Model Group(CIA group),Methotrexate Group(MTX group)and DTYM Group(DTYM Group).Except the normal group,all the mice in other groups were given collagen and adjuvant emulsifier to make model.After the second immunization,the drug and menstruum were given by Gavage for 35 days.The general condition and arthritis index of mice were observed.35 days after the second immunization,the knee and ankle were taken for Micro CT scanning,and the knee and ankle were stained with HE and safranin-fixing green,the expression of CD31,VEGF-?,VEGFR2 and p-VEGFR2 were detected by immunohistochemistry.To detect the expression of miR-337-3p in the plasma of CIA mice.ResultsAfter the second immunization,a small number of mice ankle appeared red and swollen changes 3 days later,to 7-14 days after the second immunization,except the normal group,all other groups of mice ankle red and swollen,most of the mice have limited movement,ankylosed ankle joints appeared in some of the CIA mice 21 days after the second immunization.Compared with CIA group,DTYM Group and MTX group significantly reduced the degree of ankle swelling.Compared with CIA group,the arthritis index scores of DTYM Group and MTX group decreased significantly after three weeks(P<0.05 or P<0.01).The results of HE staining showed that compared with CIA group,DTYM Group and MTX group had relatively intact knee and ankle joint surface,no obvious thickening of synovium and a few inflammatory cells infiltration.Compared with CIA group,DTYM Group and MTX group showed relatively intact articular surface of knee and ankle joints,some cartilage surface was rough and cartilage thickness increased obviously.Compared with CIA group,MTX Group and DTYM Group also suffered significantly less bone destruction,and compared with CIA group,MTX Group and DTYM group significantly reduced bone destruction scores of knee and ankle joints(P<0.05 or P<0.01).Compared with CIA group,MTX group had normal articular surface,no obvious synovial invasion,and only a small amount of synovial invasion was found at the adhesion of synovial membrane and bone Compared with CIA group,DTYM group had intact articular surface with some signs of synovial invasion,but no brown areas were found at the site of invasion.The optical density of CD31 in the DTYM group was significantly lower than that in the CIA group(P<0.01),according to the Quantitative analysis.The expression of VEGF-,VEGFR2 and p-VEGFR2 was observed in the Pannus invasion site in the CIA group.Compared with the CIA group,the expression of VEGF-,VEGFR2 and p-VEGFR2 in the MTX group was normal,but no synovial invasion was observed,compared with the CIA group,the DTYM group had intact articular surface,some of them had pannus invasion,a few brown areas and a few synovial hyperplasia,there was erosion of synovial and osseous attachments,but no significant brown areas were seen.VEGF-?,VEGFR2 and P-VEGFR2 optical density Quantitative analysis were significantly lower in DTYM group than those in CIA group(P<0.01).The results showed that the level of miR-337-3p was significantly higher in DTYM group than that in CIA group(P< 0.01).ConclusionsDTYM may inhibit pannus formation by regulating the VEGF pathway in CIA model mice,and DTYM may up-regulate the expression of miR-337-3p in CIA model mice.Part 2 Inhibitory effect of DTYM on Cell Activation of HUVEC Model in Vitro ObjectivesTo verify the inhibitory effect and mechanism of the DTYM on the activation of HUVEC model cells in vitro.MethodsThe effect of DTYM on the viability of HUVEC cells was detected by CCK-8method.EDU cell proliferation assay kit was used to detect the changes of HUVEC cell proliferation after intervention with DTYM.Then the in vitro migration of Transwell and the expression of actin in cells were detected by ghost pen cyclic peptide fluorescence staining.The effect of DTYM on the tube formation ability of HUVEC cells was detected by matrix gel method.RT-q PCR was used to detect the m RNA expression of HUVEC adhesion molecules E-selectin,ICAM1 and VCAM1.Western blotting was used to detect the expression of HUVEC cell-related activating proteins(VWF,CD31,ANG-1,CYR61).The effect of DTYM on the expression of CD31 protein was detected by cellular immunofluorescence.Western blotting was used to detect the expression of VEGF signal pathway protein.ResultsThe results of CCK-8 experiment showed that compared with the blank control group,the cell activity of HUVEC in DTYM group(2000?g/ml)decreased significantly(P<0.01).The proliferation of HUVEC was detected by EDU method,the results showed that DTYM could significantly reduce the proliferation rate of HUVEC induced by VEGF(P<0.01).The experimental results of HUVEC migration induced by VEGF in Transwell chamber culture showed that the number of HUVEC migrated to the lower chamber decreased significantly after intervention with DTYM as compared with that of VEGF group.The effects of two dose groups of DTYM on the number of cell migration were statistically significant(P<0.01),in a dose-dependent manner.The results of ghost pen cyclic peptide fluorescence staining showed that after the intervention of DTYM,compared with TNF-? decoction group,DTYM could significantly reduce the expression of HUVEC-shaped fibrous actin(P<0.01).The results of tube formation experiment showed that after the intervention of DTYM,compared with VEGF group,DTYM could significantly reduce the number of lumen nodes and the number of intersecting points of lumen connection formed by HUVEC(P<0.01).The results of RT-q PCR method showed that compared with TNF-? group,the expression of E-selectin,ICAM1 and VCAM1 m RNA in HUVEC decreased significantly after intervention with DTYM,and the difference was statistically significant in a dose-dependent manner.The results of Western blot showed that DTYM could significantly down-regulate the increased protein expression levels of VWF,CD31,ANG-1 and CYR61 induced by TNF-? in a dose-dependent manner.Cellular immunofluorescence showed that the immunofluorescence intensity of CD31 cells in TNF-groups was significantly higher than that in NC group,while that in DTYM groups was significantly lower than that in TNF-? groups(P<0.01).The results of Western blot showed that the relative expression level of VEGF-? protein and P-VEGFR2 protein in HUVEC decreased significantly after DTYM intervention,while the relative expression level of VEGFR2 protein had no significant change compared with TNF-? group.ConlusionsDTYM can inhibit the proliferation,migration,adhesion and tube formation of HUVEC,thus inhibiting the activation of endothelial cells.The mechanism may be related to DTYM inhibiting the expression of HUVEC-related activating proteins(CD31,VWF,CYR61,Ang-1)and regulating VEGF signal pathway.Part 3 Study on the Mechanism of DTYM regulating Cell Activation of HUVEC Model in Vitro by up-regulating the expression of miR-337-3p ObjectivesTo verify the mechanism of DTYM inhibiting cell activation of HUVEC model in vitro by up-regulating the expression of miR-337-3p.MethodsThe transfection efficiency of miRNA in HUVEC cells was detected by fluorescence staining.The relative expression of miR-337-3p in HUVEC was detected after intervention of DTYM and liposome transfection.The effect of miR-337-3p on the proliferation of HUVEC cells was detected by EDU cell proliferation assay kit.Transwell migration in vitro and cyclopeptide fluorescence staining were used to label the expression of actin in vitro to detect the migration function of cells in vitro.The effect of miR-337-3p on tube formation of HUVEC cells was detected by matrix gel method.Western blotting was used to detect the expression of miR-337-3p on HUVEC cell-related activating proteins(VWF,CD31,ANG-1,CYR61)and VEGF signal pathway.Western blotting was used to detect the expression of HUVEC cell-related activating proteins(VWF,CD31,ANG-1,CYR61)and VEGF signal pathway induced by DTYM and miR-337-3p.ResultsThe results of transfection fluorescence experiment showed that the transfection efficiency was good under inverted microscope.The level of miR-337-3p in HUVEC transfected with miR-337-3p mimic was significantly higher than that in NC-mimic(NCM)group,and the level of miR-337-3p in HUVEC transfected with miR-337-3p inhibitor was significantly lower than that in NC-inhibitor(NCI)group(P<0.01).Compared with NC group,the level of miR-337-3p in HUVEC increased significantly after intervention with two doses of DTYM in a dose-dependent manner.The results of cell proliferation by EDU assay showed that the proliferation rate of HUVECs in miR-337-3p mimic(337M)group was significantly lower than that in NCM group,while that in miR-337-3p inhibitor(337I)group was significantly higher than that in NCI group(P<0.01).The results of Transwell chamber showed that compared with NCM group,the number of cells moving out of Transwell chamber was significantly decreased after miR-337-3p mimic intervention under VEGF stimulation,and increased after miR-337-3p inhibitor intervention compared with NCI group(P<0.01).The results of ghost pen cyclic peptide fluorescence staining showed that compared with NCM group,the expression of HUVEC fibrillar actin in TNF-? treated with miR-337-3p mimic decreased significantly,and compared with NCI,the expression of HUVEC fibrillar actin increased after miR-337-3p inhibitor intervention,but the difference was not statistically significant(P>0.05).The results of tube formation experiment showed that compared with NCM group,the number of lumen nodes and lumen connection intersections in HUVEC were significantly decreased after VEGF stimulation with miR-337-3p mimic,but there was no significant difference compared with NCI group after miR-337-3p inhibitor intervention,but the difference was not statistically significant(P>0.05).The results of tube formation experiment showed that after miR-337-3p inhibitor intervention,the number of lumen nodes and lumen connection intersections in HUVEC group were significantly lower than those in HUVEC group(P<0.05),but there was no significant difference between HUVEC group and HUVEC group(P > 0.05).The results of immunoblotting showed that the relative expression levels of VWF,CD31,ANG-1,CYR61,VEGF-? protein and P-VEGFR2 in 337 M group were significantly lower than those in NCM group.Compared with NCI group,the relative expression levels of CD31 and CYR61 protein in HUVEC of 337 I group were significantly increased under TNF-?stimulation,while VWF and ANG-1 had no significant change.Compared with the NCI group,the relative expression level of VEGF-? protein in HUVEC of 337 I group was significantly increased,but there was no significant change in P-VEGFR2.Compared with the group treated with DTYM or miR-337-3p mimic alone,the relative expression levels of VWF,CD31,ANG-1,CYR61,VEGF-? protein and P-VEGFR2 in HUVEC treated with DTYM and miR-337-3p mimic were significantly lower than those treated with DTYM and miR-337-3p mimic alone under the stimulation of DTYM.ConclusionsDTYM may partly up-regulate the expression of miR-337-3p to inhibit the expression of VWF,CD31,CYR61 and Ang-1,and inhibit the activation of VEGF pathway to regulate the activation of endothelial cells,so as to inhibit pannus formation in CIA mice. |
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