Effect Of Thalidomide On The Expression Of VEGF And TNF-α And Erythropoiesis In Rats With Collagen Induced Arthritis

Rheumatoid arthritis (RA) is a kind of autoimmune disease characterized by chronic and symmetrical multijoint inflammation, which result in joint malformation even lead to sever disability. The major damaged-target is synovial membrane. Neovascularization is the major cause of pannus causing synovitis and destroying bone. Therefore, the key point for the treatment of RA is to inhibit synovial neovascularization. Several studies showed that many highly expressed factors contributed to the forming of blood vessels in RA patients, among which vascular endothelial growth factor (VEGF) plays important function in synovitis and neovascularization. The expression of VEGF is regulated by tumor necrosis factor-α(TNF-α), interleukiu-1(IL-1), transforming growth factor-β(TGF-β), CD40-CD40L, hypoxia and other factors. As a key factor in the cytokines net, TNF-αplays vital role and interacts with VEGF, accelerating the progression of RA.Anemia in RA patients is the most common extra-joint symptom and the degree of anemia is often consistent with the activity of joint inflammation. This kind of anemia is commonly considered as anemia of chronic disease (ACD), and is related with abnormity in iron absorption, obstruction of iron releasing from macrophage and deficient expression of erythropoietin (EPO). Cytokines such as TNF-αand IL-6 are shown to suppress the proliferation of erythroid progenitor cells and the expression of erythropoietin EPO. TNF-αand IL-6 are also shown to interfere the response of bone marrow cells to EPO and iron metabolism.In the past decade, Thalidomide regains the research hotspot for its immune regulation, anti-inflammation and anti-angiogenesis effects. Studies showed that Thalidomide is effective in the treatment of several diseases such as cancer, rheumatoid disease and refractory anemia. However, there are few reports about the clinical and experimental research on the efficiency and mechanism of thalidomide in the treatment of RA, especially for RA with anemia. We, therefore, established the modified collagen-induced arthritis (CIA ) rats model with anemia by subcutaneous injecting collagen and adjuvant, repetitiously. Through these CIA rats, we made a further study on the effect of thalidomide on joint inflammation, angiogenesis, expression of cytokines, apoptosis and erythropoiesis, in order to select an effective drug in the treatment of RA, without severe side-effects. This experiments consist the following four parts.Part one: The effect of thalidomide on expression of VEGF and TNF-αin CIA rats.Objective:To explore the dynamic expression of VEGF and TNF-αmRNA and the protein in the blood and synovial membrane, to analyze the relationship between VEGF, TNF-αand the joint activity, microvessel density in synovial membrane, to study the effect of thalidomide on the expression of VEGF ,TNF-αand the joint activity, microvessel density in CIA rats.Methods: 128 male Wistar rats were divided into five groups,Ⅰ:normal group (n=24),Ⅱ: Collagen-induced arthritis rats (CIA) model group (n=26),Ⅲ: model group with high dosage of thalidomide group(200mg/kg/d, n=26),Ⅳ: model group with low dosage of thalidomide group(100mg/kg/d, n=26),Ⅴ: model group with Methotrexate ( MTX) group,(2.7mg/kg/w, n=26). CIA rats were made by subcutaneous injecting collagen and complete freud's adjuvant (CFA). Each group was divided into 7 subgroups according to the time point (7d, 14d, 21d, 28d, 35d, 42d, 60d) after the first injection. Radionuclide imaging,χ-ray, electron microscope, pathological technic were used to evaluate the arthritis index (AI), paw thickness and other parameters of CIA rats. Reverse transcription-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and immunohis-chemistry were used to detect the expression of VEGF and TNF-αlevel at different time point; microvessel density (MVD) was determined immunohischemistryically by CD31. The effect of Thalidomide and MTX were compared.Results:①AI, paw thickness, and concentration of radionuclide of CIA rats reached peak value on the 21st day, among which the change of radionuclide in the joints was more sensitive and objective than other index , and had high value in diagnosing early joint disease.②The expression level of VEGF and TNF-αmRNA of CIA rats on the 21st day were 0.75±0.06, 2.01±0.13, which was significantly higher than that of normal group, P<0.01. Only high dose thalidomide could down regulate VEGF mRNA, while both thalidomide and MTX could down regulate TNF-αmRNA.③The results of immunohischemistry test showed that the expression of VEGF and TNF-αin synovial membrane was strongest on the 21st day (15683.15±319.23, 9037.71±403.03), higher than that of normal group, P<0.01. Thalidomide and MTX could reduce the expression of VEGF and TNF-α, and the high dose thalidomide showed the effect the most rapidly.④ELISA results showed the same change of VEGF and TNF-αin blood (2147.3±37.56 and 2685.0±112.09, respectively) as that in synovial membrane of CIA rats, which were higher compared with that in normal group, P<0.01. All the drugs could reduce VEGF and TNF-αlevel, and the effect of high dose thalidomide was almost the same as that of MTX.⑤The MVD of CIA rats was highest on the 28th day (22.45±2.43), higher than that of normal group, P<0.01. All the drugs could decrease MVD of the synovial membrane, and the effect of thalidomide was more rapidly.⑥The level of VEGF and TNF-αin blood and synovial membrane of CIA rats was positively related with the change of paw thickness, P<0.05; the value of VEGF was also positively related with that of MVD, P<0.01.Conclusion: The expression level of VEGF and TNF-αwas higher in CIA rats, the change of which was correlated with the joint disease. There were increased newly developed microvessels in synovial membrane. Thalidomide could down regulate the expression level of VEGF and TNF-αmRNA and the protein, decrease the MVD, improve the joint disease. The effect of thalidomide was better than or equal to MTX. High dose thalidomide had more effect.Part two: The effect of thalidomide on the expression of VEGF and TNF-αand the apoptosis of the FLS in CIA rats.Objective:To explore the effect of thalidomide on the expression of VEGF and TNF-αand the apoptosis on Fibroblast-like synoviocytes (FLS) of CIA rats stimulated by lipopolysaccharide (LPS).Methods: Axenic synovial membranes from knees of CIA rats were obtained, and synovial cells were separated and cultured in vitro. The cultured cells were divided into six groups as following: normal group, LPS group,LPS+ high dose thalidomide (50mg/L) group, LPS+ middle dose thalidomide (5mg/L) group, LPS+ low dose thalidomide (0.5mg/L) group, LPS+MTX group. The shape of FLS was observed by convert microscope and transmission electron microscope; ELISA was used to determine the expression level of VEGF and TNF-αin the culture medium; RT-PCR and Western Blot method were used to determine VEGF, TNF-αmRNA and protein; Apoptosis of FLS was determine by FCM .Results:①The original synovial cells were to be confluent after 10～14 day and form a new generation by separated culture. FLS held predominance after 3 generation, which presented shuttle or rhombic shape.②ELISA results showed the expression level of VEGF and TNF-αin the culture medium of LPS group was higher compared with normal group(859.00±22.74pg/mL, 1315.20±19.38pg/mL), P<0.01. Thalidomide could reduce the concentration of VEGF and TNF-α, and the higher the dosage, the better the effect. The effect of MTX was better than thalidomide.③RT-PCR,Western Blot results showed that thalidomide could down regulate the expression level of VEGF and TNF-αmRNA and the protein of FLS, and the effect was partly in dose-dependent manners.④Thalidomide could induce the FLS apoptosis, also in a dose-dependent manner in the definite dosage. The apoptotic rates at 48hr were 8.07%±1.45%, 5.72%±1.42%, and 0.40%±0.04%,among which the former two were higher than that of LPS group (0.24%±0.05%), P<0.01;The nerotic rates at 48hr were 20.34%±4.43%,12.96%±4.32% and 1.94% ±1.16%, higher than that of LPS group(0.13%±0.09%), P<0.01.There was no difference between thalidomide (50mg/L) group and MTX group, P>0.05.⑤Observed with transmission electron microscope, the apoptotic phenomena were obvious: the chromatins condensed and shrunk and aggregated along inside of nuclear membrane to exist on the form of fall, petal and crescent. apoptotic bodies were, sometimes, formed.Conclusion: FLS cells could be harvested and purified by culturing and passaging. Thalidomide could induce apoptosis of FLS and inhibit the expression of VEGF and TNF-αmRNA and the protein of FLS, partly in dose-dependent manners.Part three: The effect of thalidomide on the anemia of chronic disease in CIA rats.Objective:To investigate the effect of thalidomide on the anemia of chronic disease in CIA ratsMethods: The rat model of arthritis accompanied with anemia was established though the modified CIA model, referring to the part one, and each group was divided into 6 subgroups according the time point (14d, 21d, 28d, 35d, 42d, 60d) after the first injection. The hemoglobin concentration and the degree of anemia were determined; Serum iron, total iron binding capacity, ferritin, EPO, morphology of bone marrow, iron inside and outside RBC of BM were determined or observed; Serum TNF-αconcentration and the paw thickness of model rats were measured by the methods mentioned above, and the relationship between these parameters and hemoglobin were analyzed; The relationship between EPO and hemoglobin was analyzed also.Results:①On the 28th day, anemia appeared in model rats, the hemoglobin was 121.75 g/L±9.18g/L, lower than that of normal group(147.25 g/L±5.74 g/L), P<0.01.②The characteristics of anemia include lower serum iron and total iron binding capacity, higher ferritin and EPO, erythroid cells in BM were in hyperplasia, lower iron inside RBC of BM, unchanged iron outside RBC of BM and the normal shape of RBC.③High dose thalidomide could increase hemoglobin concentration on the 60th day compared with model group on the same time point, P<0.05.④Serum TNF-αconcentration was positively related with the change of paw thickness (P<0.01) , and negatively related with hemoglobin (P<0.05);There was no correlation between hemoglobin and paw thickness (P>0.05).⑤There was no correlation between hemoglobin and EPO concentration (P>0.05).Conclusion:Model rats with arthritis and anemia were successfully made by the meliorative method. The more serious of arthritis, the higher concentration of TNF-αand the more serious of anemia. Thalidomide could abate joint swelling and anemia, reducing TNF-αconcentration. It is hopeful to apply thalidomide in the treatment of RA with ACD patients.Part four: The effect of thalidomide on erythroid progenitor cells in CIA rats accompanied with anemia.Objective : To investigate the effect of thalidomide on erythroid progenitor cells of bone marrow in experimental arthritis and anemia rats. Methods: 40 male Wistar rats were divided into 5 groups. The methods of making model CIA rats grouping were the same as that in part one. Rats were killed on the third and eighth week (the 60th day), and the BM mononuclear cells (BMMNCs) were harvested and cultured in semisolid methylcellulose culture medium. The numbers of BFU-E and CFU-E were counted; The number of CD34+/CD71+cells in BM and the percentage of apoptotic cells were determined with FCM; TNF-αconcentration was determined by ELISA.Result:①The numbers of BFU-E and CFU-E on the third and eighth week were 13.33±4.32BFU-E/2.5×105 BMMCs,16.33±5.44 BFU-E/2.5×10~5 BMMCs and 62.27±4.98 CFU-E/2.5×10~5 BMMCs,73.67±7.20 CFU-E/2.5×10~5 BMMCs,lower than that of the normal group on the same time point, P<0.05. Thalidomide could increase the formation of BFU-E and CFU-E, which was better than MTX.②The lower number of CD34+/CD71+ cells was in model rats than that of the normal group (P<0.05) and the numbers were 0.30%±0.10% and 0.47%±0.06%, respectively, on the third and eighth week. Thalidomide could also increase the number of CD34~+/CD71~+ cells of model rats. High dosage of thalidomide could increase the number of CD34~+/CD71~+ cells the number is 0.52%±0.02%和0.57%±0.11%,P<0.05;low dosage of thalidomide had the effect just on the 3rd week(0.44%±0.03%).③The percentage of CD34~+/CD71~+ apoptotic cells in model rats (31.30%±2.64%) was higher than that of normal group (21.96%±2.51% ) (P<0.05) and thalidomide could decrease the percentage of CD34+/CD71+ apoptotic cells. Both high and low dosage of thalidomide had the function, the percentage of CD34+/CD71+ was 20.57%±2.43%, 14.77%±4.52% and 23.14%±3.78%, 16.04%±2.28%.④Serum TNF-αconcentration of model rats was significantly negative correlated with the number of BFU-E (P<0.05) , and positive correlated with the percentage of CD34+/CD71+ apoptotic cell (P<0.05), while no correlation was found between the number of CD34+/CD71+ cells and the number of CFU-E (P>0.05).Conclusion: The ability of forming BFU-E and CFU-E was decreased, the number of CD34~+/CD71~+ cells was lower, and the percentage of CD34~+/CD71~+ apoptotic cell was higher in the BM of rats with arthritis and ACD. Thalidomide could improve these parameters.