Effect of Orally-administration Niclosamide on serum level of VCAM-1 and E-selectin in Collagen induced arthritis: Experimental study
1Department of Pharmacy, Al-Esraa University, Baghdad, Iraq.
2Department of Pharmacology, College of Medicine, AL-Mustansiriya University, Baghdad, Iraq.
3Department of Medicine, College of Medicine, University of Baghdad, Baghdad, Iraq.
*Corresponding Author E-mail: ahmedsalimmahmood@gmail.com
Soluble adhesion molecules (mainly VCAM-1 and E-selectin) have a vital role in the pathogenesis of the rheumatoid arthritis (RA) and consider as angiogenic mediators for this disease. The main goal for this research is to evaluate the efficacy of orally administer niclosamide (NCS) in prevention the angiogenic mediators (VCAM-1 and E-selectin) using collagen induced arthritis model in rats (CIA). Fifty male Spraque-Dawley rats underwent collagen induced arthritis (CIA) model. When arthritis was fully developed, the rats were either treated orally with low-dose (50mg/kg) NCS or high-dose (100mg/kg) NCS or treated intrapertonially (IP) with 30mg/kg NCS or leave without treatment for 4 weeks. Body weight measurement and arthritis index were monitor before and after treatment in all groups. At the end of the treatment period serum level of vascular cell adhesion molecule-1 (VCAM-1), E-selectin and TNFα were measured together with collection of articular synovial tissue to evaluate the pathological changes. The experiment showed that NCS significantly reduce the arthritis index, foot pad thickness and ankle swelling (p value < 0.05) when given orally in a high dose and IP to the experimental animal. Comparing to the CIA model group, significant reduction in the serum level of VCAM1 and E-selectin has been observed in those rats treated with high dose of oral NCS or IP injection of NCS (p value < 0.05). Niclosamide can effectively decrease, in dose dependent manner, the clinical scores, joint swelling, VCAM1, E-selectin and pathological changes in arthritic rats induced by collagen type II.
KEYWORDS: Niclosamide, Rheumatoid arthritis, Soluble adhesion molecules, Angiogenic mediators.
Rheumatoid arthritis (RA) is one type of autoimmune diseases, where the immune system of the body attacks its own articular cartilage and synovial membrane of the joint1,2. The disease is prevalence in many countries, Approximately 1.3 million people have RA in the USA3. This disease characterized by synovial inflammation and hyperplasia (“swelling”), autoantibody production (RF and anti–ACPA), cartilage and bone destruction (“deformity”), and systemic features, including cardiovascular, pulmonary, psychological, and skeletal disorders4. Inflammation of synovial membrane in RA starts by migration and infiltration of inflammatory cells to the synovium.
The migration of T-lymphocytes from blood circulation to the synovial compartment occurs due to activation of endothelial membrane in the joints which increases the expression of adhesion molecules including integrins, selectins and chemokines. These events together with hypoxic condition, cytokines release and insufficient lymphangiogenseis stimulate the angiogenesis. These micro environmental changes, combined with profound synovial architectural reorganization and local fibroblast activation, permit the buildup of synovial inflammatory tissue in rheumatoid arthritis5,6. Many research demonstrated that the rheumatoid synovial fluid has chemotactic activity for endothelial cells and angiogenic activity and the main mediators for angiogenesis in rheumatoid joints are E-selectin and VCAM-1 molecules7. In addition to that, early RA associated with high serum concentration of soluble adhesion molecules: ICAM-1, VCAM-1 and E-selectin8. Another clinical study revealed that there is positive correlation between the serum level of VCAM-1 and the disease prognosis in patients with RA, consequently, measurement the serum level of this mediators can reflect the disease condition and efficacy of drugs used in treatment of RA9. The first line therapy used for treatment of RA are leflunomide, methotrexate, sulphasalazine and infliximabe which are known as disease- modifiying anti rheumatic drugs (DEARDs). However, the used of DEMARDS frequently associated with serious adverse effect like liver injury and hepatotoxicity10,11. A new research shows that niclosamide (an old anthementhic drug) effectively inhibits inflammatory and angiogenesis by blocking the adhesion of mononuclear leukocytes to endothelial cells and suppresses the expression of ICAM-1 and VCAM-1 in vitro12. In addition to that, the severity of arthritis in mice (induced by collagen type II) reduced when treated with once-daily intraperitoneal (IP) injection of NCS13. As NCS available as oral tablet only, this animal study has been designed to evaluate the efficacy of NCS in reducing the serum level of E- selectin and VCAM-1 when given orally in different doses.
Immunization grade bovine type II collagen and Incomplete Freund's adjuvant were purchased from Chondrex (USA). Niclosamide was obtained from Alexandria Co. Industries (Egypt). The following animal Elisa kits for rat were purchased from Shanghai (China): TNF-α, VCAM-1 and E-selectin. Ketamine produced by Hikma Pharmaceuticals Plc, Jordan.
Fifty male rats of Spraque-Dawley species weighting approximately (100-110g) were obtained from Animal breeding center The Center of Quality Control and Drug Research, Baghdad and transported to the animal house of the Iraqi Center for Cancer and Medical Genetics Research, Baghdad. The animals were kept in a temperature (22–25°C) with a 12 h light/dark cycle and humidity of 25-35% with access to water and standard rat chow. The animal care protocols were approved by the Ethics Committee of the college of medicine/Al- Mustansiriyia University (No. 1369 in 9/03/2017). After seven days of acclimatization the animals were evenly randomized into 2 groups, CIA model group (40 rats) and negative control group (10 rats).
The arthritis induced by immunization the rat with 0.2ml of collagen type II dissolved in 0.05M acetic acid in incomplete Freund's adjuvant emulsion injected intradermal at the base of the tail. Second booster dose was used after one week to increase the incidence of arthritis. Normal control group injected only with 0.05M acetic acid14.
Niclosamide oral solution was freshly prepared daily to be used orally by oral gavage. Fist, NCS tablet (500mg) was triturated by mortar and pestle the transfer into plan tube to prepare stock solution by 10ml of normal saline.
After 2 weeks of starting the experiment, 28 rats of successfully developed RA in CIA group were randomly divided into four groups (7 rats for each) (B-E) while group A also include 7 rats isolated from normal control group. All these groups treated for 28 days with following regimes: Group A received no treatment, Group B: treated with oral normal saline, Group C: treated with NCS orally (50mg/kg), Group D: treated with NCS orally (100mg/kg) and group E: treated with IP injection of NCS in a dose 30mg/kg. The body weight was measured each week. The evaluation of RA in rats was started on day 0 before induction of arthritis and once weekly over 6 weeks by calculating the arthritis index (AI).
Blood specimens were collected after anthesized the rats by ketamine. Then, about 3-4ml of the blood were drawn from the heart before and after the experiment and centrifuged at 3000rpm for 20 minutes to separate serum and then transferred into Eppendorf tube and kept freezing at (-20°C) to be used for estimation the inflammatory markers. At the end of the experiment the rats were sacrificed by cervical dislocation. The ankles of the hind paws were dissected and preserved in 10 % buffered formalin solution. The preserved ankles were then dehydrated and then processed and embedded in paraffin. Specimens were cut longitudinally to the midline, and 5µm sections mounted for staining with hematoxylin and eosin (H &E).
Arthritis severity was determined at the baseline and after treatment period (28 days) according to the following interpretation: 0, normal; 1, mild swelling and redness limited to single joint; 2, moderate swelling and redness limited to ankle and wrist; 3, severe redness and swelling of wrist or ankle; 4, sever inflammation and swelling of the entire limb affecting multiple joints15.
Measurement the inflammatory markers: Determination the rat serum level for TNF- α, E-selectin and VCAM-1 was done using ready-made enzyme- linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocols and instructions.
Evaluation the pathological changes in the joints were done at the Department of Pathology Al-Mustansiriya University, college of Medicine. The ankle joints of each rat were fixed in 10% formalin for 14 days, decalcified with CalEx decalcification solution for 35 days, and processed in paraffin. The fixed tissues of the ankle joints were longitudinally cut into 5μm sections, and stained with hematoxylin and eosin. Grading of cellular infiltration, synovial hyperplasia, pannus formation, joint space narrowing, and cartilage and bone erosion of the ankle joints was blindly investigated by pathologist.
Data were analyzed using SPSS v20. Data were presented as mean±SD. Paired student’s t-test and the one-way ANOVA followed by Dunnett’s post hoc test were used. P-values of 0.05 or less were considered significant.
Initial comparison between normal control group and CIA was done after two weeks which demonstrates high elevation in AI score in CIA group (P <0.001) with no incidence of any sign of redness and/or swelling in normal control group as shown in table (1). Only rats that showed 4-5 score of AI where include in CIA model group (totally 28 rats). Severe sings of arthritis have been observed clearly in CIA model including swelling, redness, deformity and ankyloses in the hind paws, forelimbs and ankle joints (Fig.1 B). The hind paw of normal rats showed significant difference from the hind paw of arthritic control rats with normal thickness and color (Fig. 1 A).
Table No. 1: Effect of Collagen type II on AI scoring
|
Groups |
N |
AI scoring (mean ± SD) |
|
|
Baseline |
After 2 Week |
||
|
Normal control group |
10 |
0.0 ± 0.0 |
0.0 ± 0.0 |
|
CIA |
35 |
0.0 ± 0.0 |
3.63 ± 0.49 * |
|
P. value between groups |
Not calculated |
< 0.001 |
|
n= number of the animal for each group, * p. value comparing to normal control group.
Fig. 1: Incidence of RA in rats after 2 weeks of immunization by collagen type II. A: normal control rat, B: arthritic control rat showing redness, swelling, deformity and ankyloses with severe arthritis (symptoms maximum in this group)
Significant changes in the body weight were observed between Group A and other groups in the second week before starting the treatment (p value < 0.001). Furthermore, insignificant increase in the body weight was observed in the CIA rats (group B), low oral dose NCS treated rats (group C) and high oral dose NCS treated rats (group D) during the course of the treatment. On the other hand, an absolute increment in the weight of groups A and E was found with no significant differences between them (P value > 0.05) (figure 2). Those rats in treated with low dose of NCS and high dose of NCS (group C and D respectively) shows significant differences in the weight when compare with normal group (group A) counterpart (p value < 0.001).
A significant elevation in the mean of arthritic score was observed in CIA control rats (group B) from the second week to 6th week during development of RA. Group C rats, treated with NCS orally (50mg/kg) showed insignificant decline in AI during the course of the therapy with no significant changes at the end of the experiment when compare their value with CIA (group B). In contrast, group D and E rats observed a significant lowering in the AI during the development of the arthritis (table 2).
Fig. 2: Body weight of rats from week 2 to the week 6 post collagen immunization. * P < 0.001, vs group A for each group
Table No. 2: Mean AI score of rats from week 2 to the 6th week post collagen immunization.
|
Group |
n |
Arthritis index (mean ± SE) |
||||
|
Week 2 |
Week 3 |
Week 4 |
Week 5 |
Week 6 |
||
|
Group A |
7 |
00.00 ± 00.00 |
00.00 ± 00.00 |
00.00 ± 00.00 |
00.00 ± 00.0 |
00.00 ± 00.0 |
|
Group B |
7 |
3.71 ± 0.48 * |
3.71 ± 0.48 * |
3.00 ± 0.57 * |
3.29 ± 0.48 |
3.43± 0.53 |
|
Group C |
7 |
3.29 ± 0.48 * |
3.29 ± 0.48 * |
2.71 ± 0.48 * |
3.00 ± 0.57 |
2.86 ± 0.69 |
|
Group D |
7 |
3.71 ± 0.48 * |
3.71 ± 0.48 * |
3.43 ± 0.53* |
2.43± 0.53 ** |
2.57 ± 1.13 ** |
|
Group E |
7 |
3.86 ± 0.37 * |
3.86 ± 0.37 * |
2.43 ± 0.43 * |
2.00 ± 0.00** |
1.14 ± 0.9 ** |
n= no. of the animal, * p value < 0.05 compare to group A, ** p value > 0.05 compare to group B
Effect of Niclosamide on the serum level of TNF-α: Intraperitoneal administration of NCS has shown to reduce the serum level of TNF-α significantly (p. value
>0.05) after 4 weeks of treatment when compare their levels before treatment. On the other hand, oral therapy of NCS in a dose (100mg/kg or 50mg/kg) did not show a significant reduction in the above inflammatory biomarkers (p value > 0.05). However, one way Anova shows a significant difference in the mean of TNF- α (p value < 0.05) in group of rats treated with high dose of NCS (group D) when compare with positive control group (group B), whereas, low dose of NCS shows no significant changes in TNF-α when compare with group B (Fig. 3).
Fig. 3: Changes in the level of TNF-α in experimental group. Data are expressed as the mean ± standard deviation of the mean (n=7).
*P<0.05, vs. baseline level; ** P<0.01, vs. positive control group (B).
Table No. 3: Effect of Niclosamide on the serum level of soluble adhesion molecules:
|
Groups |
E-selectin (mean ± SD) |
VCAM-1 (mean ± SD) |
||
|
Before |
After |
Before |
After |
|
|
Group A |
0.58 ± 0.47 |
0.31 ± 0.39 a |
0.12 ± 0.01 |
0.08 ± 0.03 |
|
P value |
0.48 |
0.07b |
||
|
Group B |
3.98 ± 1.74 |
4.19 ± 2.48 |
1.05 ± 0.2 |
0.59 ± 0.62 |
|
P value |
0.84 |
0.13 |
||
|
Group C |
2.51 ± 1.19 |
2.72 ±2.62 |
0.92 ± 0.2 |
0.31 ± 0.52 |
|
P value |
0.9 |
0.08 b |
||
|
Group D |
2.92 ± 1.73 |
2.43 ± 2.08 |
0.99 ± 0.17 |
0.18 ± 0.32 |
|
P value |
0.77 |
0.01 b |
||
|
Group E |
2.86 ± 1.38 |
0.22 ± 0.24 a |
0.86 ± 0.21 |
0.17 ± 0.21 |
|
P value |
0.016 b |
0.009 b |
||
Values express as mean ± SD, a p value < 0.05 comparing to group B. b p value < 0.05 comparing to baseline before starting treatment. n= is number of the animals for each groups.
Those groups of rats treated with high dose oral NCS (group D) or IP injection of NCS (group E) exert a significant reduction in E-selectin and VCAM-1 after 4 weeks of therapy with significant differences comparing to mean value in the untreated arthritic rats (group B). On the other hand, oral administration of NCS in a low dose (group C) lowers significantly the serum level of VCAM-1 with no significant effect on E-selectin concentration (Table 3)
Histopathological examination:
At the end of the experiment histopathological examination of ankle joints from each group was investigated by pathologist. Slide from normal rats demonstrated normal cartilage thickness, smooth and continuous lining of the joint surfaces without any bone or cartilage destruction. While light microscope demonstrates the accumulation of large number of inflammatory cells, fragment of eroded cartilage and pannus formation in Group B who are subjected to collagen type II only. Tissue sample from rat joints in group C shows absence of pannus formation but still there is destruction in the cartilage with infiltration of inflammatory cells. In contrast , the microscopically examination of the joint of rats from group D observes normal cartilage with inflammatory cell infiltration. Group E does not show any high pathological changes in the joint with only little infiltration of inflammatory cell as shown in the figure (4).
Fig. 4: Right hind paws of experimental groups’ rats demonstrate good bone architecture. H&E stained hitopatholical images of the
hinpaws from normal control group (image A), CIA group without given treatment (image B), Low oral NCS treated rat 50mg/kg (image C), High oral NCS treated rat 100mg/kg (image D), and IP injection of NCS treated group 30mg/kg (image E).
The pathophysiology of RA is hetrogenous in nature, starting with presence of autoantibodies that formed immune complexes with citrulline containing antigens and subsequent binding of rheumatoid factor causing activation of many complement. Activated fibroblasts, together with the accumulated activated T cells and B cells, and monocytes and macrophages which stimulate the release of cytokines like TNF, IL-1, IL-6, and IL1716. Adhesion molecules alos play a role in the pathogenesis of the RA diseases. Activation of endothelial layer of synovial micro vessels lead to increase in the expression of soluble adhesion molecules (including E-selectin) which facilitate the angiogenesis that induced by local hypoxia, cytokines and insufficient lymphangiogenesis. These changes permit the development of synovial inflammatory tissue in the RA17. They are involved in the regulation of mononuclear cells activation, circulation, migration and infiltration to rheumatoid synovium. In addition to that, VCAM-1 and E-selectin have seen to play a part in initiation and progression of RA18. VCAM-1 is a major regulator of leukocyte adhesion and trans endothelial migration through interaction with α4β1 integrin. α4β1 integrin expressed on leukocytes adheres to VCAM-1 on the surface of endothelial cells, and activates signaling pathways within the activated endothelial cells that allow the trans endothelial migration of leukocytes19. On the other hand, E-selectin mediates endothelial cell chemotaxis and potentially increasing the ingress of mononuclear cells into inflamed RA synovial tissue7. The migration of inflammatory cell is increased due to the formation of neovascularization in the synovial tissue which mainly stimulated by endothelial growth factors. Other study demonstrated that soluble forms of VCAM-1 (sVCAM- 1) and of E-selectin might act as angiogenic mediators which progress the inflammation of synovial membrane20. Many anti-rheumatoid arthritis drugs already in use can block soluble adhesion molecules, however, their effect is for short-term and many research are requiring to evaluate their tolerability upon long term use. For example, etanercept an anti-TNF drug has the ability to modulate indirectly different biological responses that are stimulated or regulated by TNF, such as the expression of adhesion molecules E-selectin and to a lesser extent ICAM-121. In the last decade new research has been done exploring the anti-inflammatory and anti-angiogenesis of NCS both in vitro and in vivo when given by IP route to the animals12. For this reason, this study was proposed to evaluate the efficacy of orally administered NCS in reducing the serum level of VCAM-1 and E-selectin when use for long term in experimental animals using CIA model. Orally administered NCS especially when used in high dose or IP can effectively lower the serum level of VCAM-1 and E-selectin after 4 weeks’ treatment course. The mechanism underlying this reduction of these two molecules are not yet known, but it may be due to the reduction in TNF-α that seen in v arthritic rats when treated with NCS orally (in high dose) or IP. Tumor necrosis factor alpha TNF-α is a pro-inflammatory cytokine that triggers the expression of inflammatory molecules, including other cytokines and cell adhesion molecules. TNFα induces the expression of VCAM-1 which was originally identified as a cell adhesion molecule that helps regulate inflammation-associated vascular adhesion and the Trans endothelial migration of leukocytes, such as macrophages and T cells22. It has been demonstrated that niclosamide suppresses TNF-α induced production of IL-1b, IL-6, IL-8, IL-17A and IFN-c in cultured RA fibroblast like synoviocyte13. Interestingly, similar finding was also obtained by pervious in vitro study which showed the ability of NCS to inhibit adhesion of mononuclear leukocytes to endothelial cells and suppresses the expression of ICAM-1 and VCAM-123. The present study shows for the first time the ability of NCS in decreasing the serum level of E-selectin in arthritic rats when used orally for 4 weeks suggesting the possibility of NCS in reducing the complication of RA like bone erosion, cartilage destruction as well as cardiovascular complication especially when used as adjuvant in treatment of RA. There is a definite evidence that E-selectin is increased in synovial fluid in RA patient and play apart in the migration of inflammatory leukocytes into synovial fluid and synovial tissues24. Tak PP and his colleagues demonstrated the ability of anti-TNF-α in decreasing the immunohistochemical “expression scores” for VCAM-1 and E-selectin of synovial biopsy tissue obtained from RA patients25. Several studies have been done about many compounds that have anti-adhesion properties or blocking the E-selectin. For example, the anti- inflammatory effect of Fucoidin (selectin-binding carbohydrates) has been tested experimentally; the drug inhibits the leukocyte rolling and adhesion in a rat ischemia/ reperfusion model26. Another study demonstrated that VCAM-1 IgG (human-human) fusion protein which binds to an a4pl activation-dependent determinant also has anti-inflammatory effect and may have potential promise if tested in rheumatoid disease27. The histopathological study proves the ability of orally administered NCS in reducing the inflammatory changes in the joints exerts by collagen type II by decreasing the infiltration of leukocyte, hyperplasia and bone erosion together with reduction in the arthritis index that seen in the arthritic joints before starting treatment. In addition to that, long term use of NCS orally caused significant reduction of body weight and this considers an advantage especially in those obese RA patients. This finding is agree with the early study by Al-Gareeb IA et al (2017) that showed oral administration of NCS significantly reduced the body weight and food intake in obese mice fed on high fat diet for 2.5 months28. Recently, many drugs are used in treatment of RA, each one has different mechanism of actions. However, some of these medicines show thereapeutic failure mainly when used as monotherapy29. On the other hand, combination thereapy of anti-rheumatoid drugs show improvement in the signs and symptoms of the RA than when used each one alone. Accordingly, the addition of NCS to the treatment regimen of RA may increase the effecacy of other anti-rheumatoid drugs. Furthermore NCS has antioxidant effect which can reverse the oxidant/antioxidant balance in RA patients that causes activation of antigen presenting cells usch as dentric cells that trigger the immun reaction30,31.
This study demonstrated that NCS has anti-inflammatory effect depending on the dose given partially due to reduction in the TNF-α together with reducing in the VCAM-1 and E-selectin which also has a role in initiation and progression of rheumatoid arthritis. These data give suggestions to use NCS safely in treatment of RA as adjuvant or alone for long-term period.
AI Conceived the presented idea. AI, FI and AS designed the research. AS carried out the experiment. FI and AS analyzed data. AS wrote the manuscript. AI and FI supervised the project. All authors read and approved the manuscript.
The authors are grateful to all staff in the Iraqi Center for Genetics and Cancer Research in Al-Mustansiriya University for technical assistance. Also, truthful appreciation and respect go to all staff in the department of pharmacology/college of medicine/ AL-Mustansiriya University.
The authors declare that they have no competing interests.
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Received on 24.10.2020 Modified on 17.12.2020
Accepted on 15.01.2021 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(10):5150-5156.
DOI: 10.52711/0974-360X.2021.00896