| Rheumatoid arthritis (RA) is a chronic, systemic autoimmune diseases, that joint synovial inflammation as the main pathogenesis symptoms. Synovial joints are affected first. Then under the stimulation of a variety of inflammatory factors, Fibroblast-like Synoviocytes(FLS) dramaticlly proliferated, formed new blood vessels, appeared joint capsule, joint effusion and synovial bursa effusion. Pannus appeared on the synovial membrane surface, gradually migrate to the joint surface and erode of the articular cartilage, and ultimately destruct the articular cartilage and loss of function. The main reason that destruction of RA cartilage and joint is pannus’migration and erosion. FLS and vascular endothelial cells(EC) were the most important components in the pannus tissue.Angiotensin Ⅱ (Ang Ⅱ) is one of components in the Renin-Angiotensin system. There are two type of receptor including type 1 receptor (AT1R) and type 2 receptor (AT2R), which are both of G protein coupled receptors. Ang Ⅱ binds with AT1R can stimulate angiogenesis, inhibit FLS apoptosis, promote collagen protein synthesis and the growth factors. Research shows that high levels of Angiotensin converting enzyme (ACE), Ang Ⅱ and AT1R protein, were found in synovial tissue of RA patients. That prompt Ang Ⅱ and its receptors were involved in the process of RA disease.Tumor necrosis factor-α (TNF-α) is a key inflammatory factor which is implicated in RA. TNF-α exerts its effects by interacting with two different receptors:TNF receptor 1 (TNFR1) and TNFR2. TNFRl express on most cells and tissues and triggers cell death processes. In contrast to TNFR1, TNFR2 only express on immune cells and by regulating TNF receptor-associated factor 2 (TRAF2) triggering of cell death processes. Previous studies by our group identified the TNF-a raised the expression of TRAF2, separated with G protein coupled receptor kinase 2 (GRK2), and promoted the level of GRK2 on membrane to cause EP4 receptors desensitization and endocytosis. Then decreased the levels of cAMP, promoted human FLS proliferation. Further research shows that Paeoniflorin (Pae) by inhibiting the activity of GRK2 can reduce the highly expressed of GRK2 in the synovial tissues and synovial cells of collagen arthritis rats(CIA), inhibit the synovial cell proliferation therefore has therapeutic effect on CIA rats.TNF-a and Ang Ⅱ were both existed in synovial tissue of RA, however, there has no reports about this two kinds of inflammatory factors combination affects cell function and the mechanism. Paeoniflorin-6-oxygen-benzene sulfonic code CP-25, which was the structural modifications of Pae. But the functions of CP-25 affect on cells is unclear. Therefore, this experiment by cultivating Human umbilical vein endothelial cells (HUVEC) and FLS or the co-culture system, stimulated with Ang Ⅱ, TNF-α and their combination to observe the effects on cells and the effects of CP-25. Reveal the mechanisms of TNF-a, Ang Ⅱ promoted cell abnormal activation and the mechanism of CP-25, to further understand the pathological mechanism of RA, looking for new targets for its clinical treatment.Objective:These works were carried out to investigate effects of TNF-α、AngⅡand their combination on HUVEC, FLS and the effects of CP-25. Reveal the molecular mechanisms that TNF-a receptor signaling pathway and AngⅡ receptor signaling pathways involved in RA synovial inflammation in HUVEC, FLS and the mechanism of CP-25, to provide new experimental basis for RA clinical treatment.Methods:Different concentrations of Ang Ⅱ and TNF-a treated with HUVEC. Proliferation of HUVEC was detected by CCK8 assay. scratch assay and Transwell chambers were used to test the migration of HUVEC. Tube formation assay to test the angiogenesis of HUVEC. The levels of AT1R, AT2R were quantified by flow cytometry. The expression of total AT1R、 total AT2R of cytoplasm or membrane proteins、 p-ERK1/2、 TRAF2、 p-Ikpβα GRK2 were measured by immunofluorescence, laser confocal or Western blotting. The ability of TRAF2 interacted biochemically with GRK2 and GRK2 with AT 1R were tested by co-immunoprecipitation.AngⅡ(1×10-9, 1×10-8,1×10-7, 1×10-6, 1×10-5 mol/L) combination with or without TNF-a (20 ng/mL) stimulated on FLS for 48 h, and then CCK8 assay and Transwell chamber were used to test the proliferation, migration and invasion of FLS. The expression of Ang Ⅱ receptors (AT1R and AT2R) and GRK2 were measured by immunofluorescence, laser confocal or Western blotting. Transwell Chambers were used to establish FLS and HUVEC co-culture system; The optimum concentrations of AngⅡand TNF-α were used alone or combined respectively on these two kinds of cells to observe another cell migration, invasion, or tube formation.Results:1 The effects of TNF-α、Ang Ⅱ and their combination on HUVEC proliferation, migration and tube formation and effects of CP-25.1.1 The effects of TNF-α and Ang Ⅱ on HUVEC proliferation. The results of CCK suggested that the suboptimal concentration of TNF-α was 5 ng/ml and optimum time point was 24 h for HUVEC proliferation. The optimum concentration of Ang Ⅱ was 10-7mol/L and optimum time point was 24 h though AngⅡ(1×10-9, 1×10-8, 1×10-7, 1×10-6mol/L) also have effects on HUVEC proliferation. And results showed that under TNF-α plus Ang Ⅱ stimulated, cells has a sthenia proliferation. ATIR inhibitor Losartan (1×10-6 mol/L) can inhibit it, But not AT2R inhibitor PD123319 (1×10-6mol/L)1.2 The effects of TNF-α and Ang Ⅱ on HUVEC migration and tube formation. The migration and tube formation of HUVEC were significantly induced by Ang Ⅱ, TNF-α or their combination compared with control group; And the influence on HUVEC migration and tube formation, Ang Ⅱ was stronger than TNF-α. The combined group’s influence on cell migration and tube formation were stronger than Ang Ⅱ or TNF-α alone. Losartan (1×10-6 mol/L) can significantly inhibit Ang Ⅱ plus TNF-α induced HUVEC migration and tube formation, PD123319(1 ×10-6mol/L) had no effect on these.1.3 CP-25 inhibit the migration and tube formation of HUVEC. CP-25(1×10-7, 1×10-6, 1×10-5mol/L) inhibited TNF-a plus AngⅡ induced HUVEC migration and tube formation, But have no effect on HUVEC proliferation. GRK2 inhibitor (50 μmol/L) can significantly inhibit TNF-α plus Ang Ⅱ induced HUVEC migration and tube formation.2 The effects of TNF-α、 Ang Ⅱ and their combination on FLS proliferation, migration and invasion and effects of CP-25.2.1 The effects of TNF-a and Ang II on FLS proliferation AngⅡ(1×10-7, 1×10-6, 1×10-5 mol/L) can promote the proliferation of FLS at 48 h, 1×10-7mol/L is the optimum concentration; TNF-a (20 ng/mL) significantly enhance the proliferation of FLS; Ang Ⅱ (1 × 10-7,1× 10-6,1 × 10-5 mol/L) combination with TNF-α (20 ng/mL) had a further promoting on FLS proliferation than used TNF-α alone.2.2 The effects of TNF-α and Ang Ⅱ on FLS migration and invasion. The migration and invasion of FLS were significantly induced by Ang Ⅱ, TNF-α or their combination compared with control group; There have a significant differences between Ang Ⅱ and TNF-α on the migration and invasion of FLS, TNF-α is stronger than AngⅡon the influence of FLS function. Combined group’s influence on cell migration and invasion were stronger than Ang Ⅱ or TNF-α alone. GRK2 inhibitor (50 μmol/L) can significantly inhibit Ang Ⅱ plus TNF-α induced FLS migration and invasion.2.3 CP-25 inhibit FLS proliferation, migration and invasion. CP-25 (1×10-7, 1×10-6, 1×10-5 mol/L) inhibit TNF-α plus Ang Ⅱ induced FLS proliferation, migration and invasion which were increased abnormally by TNF-α plus Ang Ⅱ.3 The effects of TNF-α、 Ang Ⅱ and their combination on HUVEC and FLS co-culture system and the effects of CP-25.3.1 HUVEC affect FLS migration.TNF-α、Ang Ⅱ and their combination treated with HUVEC for 24 h then co-cultured with FLS for 48 h, the results were shown as follow, TNF-α and the combination with Ang Ⅱ could promote the FLS migration after treated with HUVEC. Ang Ⅱ had no significant influence on FLS migration of this system; CP-25 (1×10-7, 1×10-6, 1×10-5 mol/L) had no effect on TNF-α plus Ang Ⅱ induced FLS migration of this co-culture system.3.2 FLS affect HUVEC migration and tube formationTNF-α、 Ang Ⅱ and their combination treated with FLS for 48 h then co-cultured with HUVEC for 24 h, the results were shown as follow, TNF-α、 Ang Ⅱ and their combination could promote the HUVEC migration and tube formation after HUVEC treated for 48 h then co-cultured with FLS. Compared with Ang Ⅱ group, the combination group had a stronger influence on HUVEC migration and tube formation. CP-25 (1×10-7, 1×10-6, 1×10-5 mol/L) could inhibit TNF-α plus Ang Ⅱ induced HUVEC migration and tube formation of this co-culture system.4 The molecular mechanism of TNF-α、 Ang Ⅱ and their combination effected the functions of HUVEC、 FLS.4.1 The expression of AT1R on HUVEC after cells treated by TNF-α、 Ang Ⅱ and theircombination.The expression of AT1R were increased obviously by TNF-α、 AngⅡand their combination treated on HUVEC, and combined group performed significantly better promotion in protein expression than Ang Ⅱ or TNF-α group.4.2 The effect on ERK signaling pathway of HUVEC induced by TNF-α、 Ang Ⅱ and their combination.Because ERK signaling pathway is known to induce cells proliferation, migration and invasion by activated mediator NF-κB, we tested the expression of p-ERK and p-Ikβα after cells stimulated by TNF-α、 Ang Ⅱ and their combination. Results showed that the expression of p-ERK and p-Ikβαa were significantly increased by Ang Ⅱ、TNF-α and their combination; And the combination group could significantly increase the expression of p-ERK and p-Ikβα compared with Ang Ⅱ、 TNF-α group.4.3 The effect of TNF-α on the level of TRAF2-GRK2 complex in HUVEC.The expression of GRK2 was significantly increased by Ang Ⅱ、 TNF-α and their combination. And the combination group could significantly increase the expression of GRK2 compared with Ang Ⅱ、TNF-α group. The Immune coprecipitation showed that an interaction occurs between TRAF2 and GRK2 or GRK2 and AT1R, and the TRAF2-GRK2 or GRK2-AT1R interactions were weaken after cells stimulated by TNF-α.4.4 The effects of CP-25 on the expressions of AT1R, GRK2 and ERK pathway in HUVEC.CP-25 (1×10-7, 1×10-6, 1×10-5mol/L) could inhibit the expression of AT1R, GRK2, p-ERK and p-Ikβα induced by TNF-α and Ang Ⅱ combination.4.5 The expression of ATR on FLS after cells treated by TNF-α Ang Ⅱ and theircombination.The expression of AT1R was increased by Ang Ⅱ、 TNF-α or their combination and the combination group could significantly increase the expression of AT1R compared with Ang II group. The expression of AT2R had a rising trend but had no significant difference when cells treated by Ang Ⅱ or TNF-α. But TNF-α plus Ang Ⅱ could significantly increased AT2R expression. GRK2 inhibitor (50 μmol/L) could significantly down-regulate the expression of AT1R and AT2R on FLS.Conclusion:1. TNF-α as well as Ang Ⅱ could promote HUVEC proliferation, migration and tube formation and promote FLS proliferation, migration and invasion; But TNF-α promoted FLS migration and invasion was stronger than Ang II, Ang II promoted HUVEC migration and tube ability was stronger than TNF-α.2. The HUVEC and FLS co-culture system, TNF-α Ang Ⅱ and their combination or CP-25 stimulated FLS could significantly change migration and tube formation of HUVEC, but HUVEC stimulated by Ang Ⅱ or CP-25 had no effect with FLS migration and invasion. The changes of FLS functions could effect the functions of HUVEC.3. The expression of GRK2 protein was increased after HUVEC treated by TNF-α, and the interactions between TRAF2-GRK2 or GRK2-AT1R were decreased, then the level of uncombined GRK2 was raised, ERK-NFκB pathway were abnormally activated. It is may be the important mechanism that influence the function of HUVEC.4. CP-25 could inhibit the proliferation, migration, invasion of FLS and the proliferation, migration, tube formation of HUVEC induced by TNF-α、 Ang Ⅱ and their combination, its mechanism may be related to inhibit the expression of AT1R and GRK2, and inhibit the activity of ERK pathway. |
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