الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 195 |  |  |
| | | from | 195 | | |
Abstract
Methotrexate is a folic acid antagonist developed mainly for the treatment of malignant tumors. It competitively inhibits several main enzymes that are required for DNA synthesis and cell division. Therefore, MTX exerts its primary toxic effects against highly proliferating cells such as the gut mucosa, and mucositis is a common side effect of MTX therapy. Despite its toxicity, MTX is widely used by most rheumatologists as the first-line disease-modifying anti-rheumatic drug (DMARD) for rheumatoid arthritis (RA) patients due to its anti-inflammatory and immunosuppressive effects. Intriguingly, during MTX-induced mucositis the mucosal immune responses remain resilient and are still able to respond to bacterial stimuli. Thus, it is believed that gut microbiota can be targeted to improve efficacy or alleviate the toxicities of MTX. This entails probiotic-based therapies. In addition, a variety of natural compounds such as plant-derived substances have been used to treat MTX-induced intestinal toxicity due to their anti-apoptotic, anti-inflammatory, and antioxidant effects.
Several systems such as pharmaceutical formulations and food-based products have been developed for the delivery of probiotics to the gastrointestinal tract. Lactéol® fort sachets are an example of a pharmaceutical form currently used to deliver probiotics. Such sachets are characterized by the high shelf-life of the probiotic strains used in this form and have the advantage to avoid the possible effects of milk allergy.
Along with the probiotic-based therapy, it has been also inferred that consumption of pomegranate fruit or its juice protects against and may even improve the course of various prevalent disorders and exerts physiological effects on the intestines.
Therefore, the aim of the present study was to explore the ability of either probiotic Lactobacillus acidophilus LB (Lactéol® fort) or pomegranate juice to modulate intestinal immunity and architecture, apoptosis, and inflammatory mediators in an animal model of human rheumatoid arthritis, following methotrexate therapy.
Eighty-one healthy adult albino rats were used in this study. Animals were divided into nine groups (n = 9 each). group I (control group) was provided with a normal diet ad libitum and received 1 ml distilled water by oral intubation. group II (LB group) received daily dose of Lactobacillus acidophilus LB (1 x 109 CFU; sachet/rat) by oral intubation for 4 weeks. group III (PJ group) was administered pomegranate juice by oral intubation at a daily dose of 1 ml/day for 4 weeks. The remaining animals were injected with CFA (0.1 ml) in a single dose into the subplantar region of the right hind paw for the induction of arthritis, then divided into 6 groups. group IV (AA group) did not receive any treatment after the induction of arthritis. group V (MTX group): treated with MTX in an oral dose of 0.3 mg/kg b.w. twice a week for 4 weeks initiated from day 10 after the induction of arthritis and continued up to day 38. group VI (Prophylactic Lactobacillus LB group; Pro. LB): received a daily dose of L. acidophilus LB (109 CFU; sachet) suspended in 1 ml distilled water by oral intubation from the first day of CFA injection (day 0) and continued until the rats were sacrificed. MTX was later administered to these animals in an oral dose of 0.3 mg/kg bw twice a week on day 10 post the onset of arthritis for 4 weeks. group VII (Concurrent Lactobacillus LB group; Con. LB): administered a daily dose of MTX concurrent with L. acidophilus LB in the same doses as group VI, for 4 weeks starting from day 10 after the induction of arthritis and continued up to the end of the experiment. group VIII (Prophylactic pomegranate juice group; Pro. PJ): received a daily dose of pomegranate juice (1 ml) by oral intubation prior to MTX in the same protocol as group VI. group IX (Concurrent pomegranate juice group; Con. PJ): administered a daily dose of MTX concomitant with 1 ml of pomegranate juice by oral intubation in the same protocol as group VII.
Blood samples were collected to measure various immunological parameters such as Pro-inflammatory cytokines and chemokines (TNF-α, IFN-γ, and IL-8), in addition to anti-inflammatory cytokines (IL-4) and immunoglobulin-A (IgA). Furthermore, Ileum tissue was removed to perform qualitative and quantitative histological studies and to asses caspase-3 dependent apoptotic pathway semiquantitavely.
Arthritis evaluation
The results of the present study revealed that subplantar injection of Complete Freund’s Adjuvant (CFA) into the right paw of rats induced polyarthritis signs. Oral administration of methotrexate to arthritic rats alleviated the inflammatory response of CFA injection as evidenced by a discernibly reduced arthritis score. Prophylactic or concurrent administration of L. acidophilus LB with MTX resulted in a noticeable reduction in the visible arthritic signs, with a significant improvement in the severity of arthritic score. This reduction was more pronounced when L. acidophilus LB was given in concurrent regimen. Moreover, both prophylactic and concomitant administration of PJ with MTX significantly reduced the arthritis score compared with the MTX-treated group. However, this reduction was more noticeable in the prophylactic group.
Immunological studies
The data of the current study displayed that serum levels the pro-inflammatory cytokines (TNF-α and IFN-γ) and chemokines (IL-8) were significantly elevated in healthy control rats after L. acidophilus LB administration. Moreover, CFA-injection resulted in a marked increase in serum levels of TNF-α, IFN-γ, and IL-8. Treatment of arthritic animals with MTX alone or concurrently combined with L. acidophilus LB significantly lowered the elevated TNF-α, IFN-γ, and IL-8 serum levels. In some way, the prophylactic supplementation of L. acidophilus LB prior to MTX treatment of arthritic rats produced a different response on the selected pro-inflammatory cytokines; given that, they significantly decreased the concentrations of these cytokines in arthritic rats, but it did not reach the normal values or the reduced levels attained by the administration of L. acidophilus LB concurrently-combined with MTX. Oral administration of pomegranate juice caused a slight increase of the serum levels of TNF-α, IFN-γ, and IL-8. Both prophylactic and concurrent administration of PJ with MTX to the arthritic rats significantly reduced the elevated serum levels of the proinflammatory cytokines and chemokines.
This study showed that supplementation of pomegranate juice or L. acidophilus LB induced a significant increase in the serum concentrations of the anti-inflammatory cytokine Interleukin-4 (IL-4) compared to their corresponding controls. Likewise, the serum levels of this cytokine exhibited significant elevations in untreated arthritis rats. On the other hand, MTX administration alone or in combination with L. acidophilus LB in both prophylactic and concurrent regimens significantly inhibited the production of IL-4 in arthritic animals. Although the administration of pomegranate juice combined with MTX in both concurrent and prophylactic protocols significantly decreased the elevated levels of IL-4, these values were still high than the normal values.
In the present study, oral administration of pomegranate and L. acidophilus LB each alone to healthy animals resulted in a marked increase in the serum levels of IgA. Also, Adjuvant-induced arthritis was associated with a pronounced increase in IgA serum levels. Treatment of arthritic rats with methotrexate alone ameliorated the CFA-induced elevated levels of IgA, reaching nearly the normal values. Likewise, administration of L. acidophilus LB or pomegranate juice along with methotrexate either in prophylactic or concurrent treatment strategy significantly lowered the serum levels of IgA. However, these levels were markedly higher than those of the control or MTX groups.
Histopathological studies
The present data indicated that the induction of arthritis was accompanied by noticeable alterations in the histological structure of the ileum, including disturbance in the normal villous architecture, distorted crypts with signs of cryptitis, intensified inflammatory cellular infiltration in the lamina propria, pyknosis, as well as changes in the composition of PPs with absence of distinct germinal centers. Examination of ileal sections from MTX-treated arthritic rats revealed the presence of pronounced destructive changes in the histological architecture compared to arthritic non-treated animals. These include severe villous shortening and fusion, atrophy of villi, total loss of villi in some areas as well as goblet cells, ulceration, development of crypts abscess and crypt loss, intense inflammatory cell infiltration in the lamina propria, and many pyknotic lymphocytes. The GALT area also showed severe alterations including atrophid lymphatic follicles with non-distinguished sub-areas. Consequently, arthritic rats treated with MTX manifested the highest score of ulceration, goblet cell depletion, inflammation, and intestinal injury, which may be attributed to the direct irritant, cytotoxic and oxidative effect of the MTX.
Daily administration of L. acidophilus LB with MTX, either in the prophylactic or concurrent protocol, resulted in pronounced improvement in the mucosal architecture and decreased the histological damage score of ileal tissue induced by MTX. Such effect of L. acidophilus LB is thought to be due to the ability of probiotic bacteria to enhance crypt cell proliferation and growth that attributed to the protective effect of probiotics on the crypt’s stem cells. There is not yet a known mechanism(s) that underlies probiotic effects on intestinal health, but the results of the current study may indicate that this probiotic works in more than one way to modulate inflammation and prevent intestinal damage.
In the same context, administration of pomegranate prior to or concomitantly with MTX protected the intestinal architecture and maintained the structural and functional integrity of ileal mucosa, and reduced the severity of the intestinal damage score. The intestinal protective effects exhibited by pomegranate herein are probably associated with its anti-inflammatory properties and its ability to regulate vital cellular functions, such as cell proliferation and differentiation, as well as its potent antioxidant activity.
The presence of an increased population of apoptotic cells in the ileum tissue induced by methotrexate was confirmed by a marked elevation of caspase-3 positive immunostaining. Again, administration of Lactobacillus acidophilus LB and pomegranate each alone in combination with methotrexate in prophylactic and concurrent treatment stratigies counteracted the effects of methotrexate on caspase-3 expression. This could be due to the inhibition of specific apoptotic pathways that converge to trigger the activity of caspase-3, ending in overall inhibition of caspase-3.
Conclusion & recommendations
Conclusively, the findings of this study reveal that methotrexate alleviates the severity of arthritis through its anti-inflammatory effect. However, it induces severe histological changes and increases apoptosis in the small intestine due to its cytotoxic and oxidative effect. Also, the present data indicate that prophylactic or concomitant administration of probiotic Lactobacillus acidophilus LB (Lactéol® fort) or pomegranate juice in combination with methotrexate is able to modulate and ameliorate all the alterations induced by methotrexate.
Accordingly, the present study provides new insights on the use of the pharmaceutical form of probiotics (Lactobacillus acidophilus LB; Lactéol® fort) or pomegranate juice for alleviating inflammation and minimizing the deleterious effects of methotrexate in rheumatoid arthritis. This study shows, for the first time, the protective effects of probiotic Lactobacillus acidophilus LB (Lactéol® fort) and pomegranate juice as an adjunctive therapy on methotrexate-induced intestinal toxicity in the arthritis rat model. In general, it seems that both of them can be effective, inexpensive, and safe remedies. These data may be of clinical importance for considering the use of Lactobacillus acidophilus LB probiotics or pomegranate juice as adjunctive therapy with methotrexate in RA patients, with imparted protective effects on the intestine. Further investigations are required to determine the molecular mechanism(s) by which Lactobacillus acidophilus LB and pomegranate juice exert their beneficial properties, and to examine their potential effects on human subjects with active rheumatoid arthritis.