INTRODUCTION
Leflunomide inhibits the mitochondrial dihydroorotate dehydrogenase, as fourth
enzyme step of pyrimidine synthesis pathway (Breedveld
and Dayer, 2000). It has immunomodulatory effect and anti-inflammatory activity
which was suitable to treat rheumatoid arthritis in 1998 (Alcorn
et al., 2009). Drugs metabolized by CYPs to reactive metabolites
consequantly react with cellular macromolecules and subsequently disable hepatocellular
homeostasis (Lammert et al., 2010). Although,
leflunomide is metabolized in vivo in both humans and experimental animals,
its metabolic pathways and the relationship to hepatotoxicity is indistinct
(Rozman, 2002).
Silymarin purithougfied extract is widely used to treat toxic effects in the
liver. Further purification into individual compounds is difficult, expensive
and therefore not commercially available (Sonnenbichler et
al., 1996). The aim of the present study was for identifying the risk
factors and understanding the mechanisms of leflunomide-induced liver injury
are of critical importance. Therefore to minimize hepatic toxicity of leflunomide
by using silymarin, to provide more detailed evaluation of the anti-inflammatory
and anti-arthritic effects produced by silymarin in the rat.
MATERIALS AND METHODS
Animals: Adult albino wistar rats, weighting 120-130 grams, were obtained
from the animal house colony in National Research Center (Giza, Egypt). Animals
were housed in a conditioned room at 25±2°C, standard diet and tap
water source were supplied ad libitum.
Drugs: Leflunomide, N-[4-trifluoro-methylphenyl]-5-methylisoxazol-4-carboxamide;
was obtained from Aventis Pharma, (Cairo, Egypt). Silybum marianum was a provided
from Sedico Pharmaceutical Company (Cairo, Egypt) as pure powder. The dose was
calculated according to Paget and Barnes (1964).
Methods
Adjuvant-induced arthritis: It was done according to Newbould
(1963) method animals were divided into six groups, each consisting of 8
rats. One group acts as control normal and in five groups, adjuvant arthritis
was induced in the right hind paw by subplantar injection of 0.1 mL Freund's
Complete Adjuvant (FCA) on zero day to induce inflammation. Drugs were orally
adminstration according to the following scheme starting daily from 10th day
till 39th day. Group (1): Received saline and served as arthritic control, group
(2): Received leflunomide in dose of (10 mg kg-1), group (3): Received
silymarin in dose of (100 mg kg-1), group (4): Received combination
of leflunomide and silymarin, finally group at the same time and group (5):
Received silymarin for two weeks followed by leflunomide for another two weeks,
at the end of experiment.
Estimation of stable oxidation products of nitric oxide (NO): Reduction
of nitrate by vanadium (III) combined with detection by the acidic Griess reaction
according to Miranda et al. (2001). method, using
an plate reader (Bio-Tek Instruments, Inc.).
Assessment of liver function: The activity of serum Glutamate-Oxalate
Transaminase (sGOT) and Glutamate-pyruvate-Transaminase (sGPT) were measured
according to the method described by Reitman and Frankel
(1957).
Assessment of antioxidant properties: Lipid peroxides were determined
according to the method described by Ruiz-Larrea et al.
(1994) and expressed as nmol g-1 wet tissue and expressed as
nmol g-1 wet tissue. Lipid peroxidation products were estimated by
the determination of the level of TBARS that were measured as malondialdehyde
(MDA). The latter is the decomposition product of the process of lipid peroxidation
and is used as an indicator of this process. The principle of the assay depends
on the colorimetric determination of a pink pigment product, resulting from
the reaction of TBARS with thiobarbituric acid (TBA) in an acidic medium, at
high temperature. Reduced glutathione (GSH) content was determined in stomach
homogenates according to the method of Ellman (1959).
The data were expressed as μmol g-1 tissue. The method
depends on the fact that both protein and non-protein thiol (SH-) groups (mainly
GSH) react with Ellman’s reagent [5,5'-dithiobis (2-nitrobenzoic acid)]
to form a stable yellow color of 5-mercapto-2-nitrobenzoic acid, which can be
measured colorimetrically at 412 nm.
Statistical analysis: Values were expressed as Means±SE. Results
experiments were analyzed using one way ANOVA followed by Least Significant
Difference (LSD) multiple comparisons test. p<0.05 was accepted as being
significant in all types of statistical tests. Statistical analysis of results,
were done using software SPSS 14.
RESULTS
Effect on paw edema induced by FCA: Induction of arthritis by 0.1 mL FCA
was accompanied with significant increased in paw edema reaching about 106%
of the normal value. Oral treatment of leflunomide and silymarin starting from
day 10th of induction succeeded to cause decrease in paw edema. Moreover, the
combination of them decreased paw edema significantly compared to arthritic
group (Fig. 1).
Adjuvant arthritis was induced in the right hind paw by subplantar injection
of 0.1 mL FCA. The magnitude of swelling of the injected hind paw was measured
and paw volume % change was calculated. Saline was administrated in one group
of animals orally and the group served as control. LEF (10 mg kg-1)
and SIL (100 mg kg-1) and their combination were administrated orally
starting from day 10th of induction. Paw volume was duplicated measured just
prior to adjuvant injection and every three days for 39 days after adjuvant
injection using water displacement plethysmometer and the mean values were recorded.
Results are expressed as Means±SEM (n = 8). Significantly different from
arthritic group at p<0.05. Statistical analysis was done using one way ANOVA
followed by LSD for multiple comparisons, respectively.
Effect on serum nitric oxide: There was a significant increase in NO
level was observed in arthritic groups by 92% compared to normal group. Conversely,
leflunomide, silymarin and their compination are normalized serum nitric oxide
(Fig. 2).
Adjuvant arthritis was induced in the right hind paw by subplantar injection
of 0.1 mL FCA. The magnitude of swelling of the injected hind paw was measured
and paw volume % change was calculated.
|
| Fig. 1: |
Effects of leflunomide, silymarin and their combination on
freund’s complete adjuvant-induced paw edema in rats |
|
| Fig. 2: |
Effects of leflunomide, silymarin and their combination on
serum nitric oxide level of adjuvant-induced arthritic rats |
|
| Fig. 3: |
Effects of leflunomide, silymarin and their combination on
serum AST level of adjuvant-induced arthritic rats |
Saline was administrated in one group of animals orally and the group served
as control. LEF (10 mg kg-1) and SIL (100 mg kg-1) and
their combination were administrated orally starting from day 10th of induction.
At the end of experiment period, blood was collected from the retro-orbital
venus plexus. Then serum nitric oxide level was measure. Results are expressed
as Means±SEM (n = 8). An (a)-Significant difference from normal rats
p<0.05 and (b) Significant difference from hyperglycaemic rats p<0.05.
Effect on liver function
Effect on serum AST: Induction of arthritis by 0.1 mL FCA did not
show any change in the serum AST level. However, treatment with leflunomide
(10 mg kg-1 day-1 o.p.) showed an increase in the serum
AST level reaching about 22% of the normal value. On the other hand, combination
of leflunomide and silymarin did not change in the serum AST level (Fig.
3).
|
| Fig. 4: |
Effects of leflunomide, silymarin and their combination on
serum ALT level of adjuvant-induced arthritic rats |
Adjuvant arthritis was induced in the right hind paw by subplantar injection
of 0.1 mL FCA. The magnitude of swelling of the injected hind paw was measured
and paw volume % change was calculated. Saline was administrated in one group
of animals orally and the group served as control. LEF (10 mg kg-1)
and SIL (100 mg kg-1) and their combination were administrated orally
starting from day 10 till 39th of induction. Blood samples from animals were
withdrawn using heparinized capillary tubes and serum was used for AST determination
after 24 and 39 consecutive days of FCA injection. Results are expressed as
Means±SEM (n = 8). An (a) Significant difference from normal rats p<0.05
and (b) Significant difference from hyperglycaemic rats p<0.05.
Effect on serum ALT: Induction of arthritis by 0.1 mL FCA did not show
any change in the serum ALT level. However, treatment with leflunomide (10 mg
kg-1 day-1 o.p.) showed an increase in the serum ALT level
reaching about 48% of the normal value. Whereas silymarin in combination with
leflunomide was normalized the elevated serum ALT level (Fig.
4).
Adjuvant arthritis was induced in the right hind paw by subplantar injection
of 0.1 mL FCA. The magnitude of swelling of the injected hind paw was measured
and paw volume % change was calculated. Saline was administrated in one group
of animals orally and the group served as control. LEF (10 mg kg-1)
and SIL (100 mg kg-1) and their combination were administrated orally
starting from day 10th till 39th of induction. Blood samples from animals were
withdrawn using heparinized capillary tubes and serum was used for ALT determination
after 24 and 39 consecutive days of FCA injection. Results are expressed as
Means±SEM (n = 8). An (a) Significant difference from normal rats p<0.05
and (b) Significant difference from hyperglycaemic rats p<0.05.
Effect on liver homogenate MDA: Induction of arthritis by 0.1 mL FCA
showed by 24% increase in the liver MDA level. Furthermore, treatment with leflunomide
(10 mg kg-1 day-1 o.p.) showed an increase in the liver
MDA level reaching about 35% of the normal value. Conversely, treatment with
silymarin leflunomide decreased the liver MDA level of the normal value (Fig.
5).
|
| Fig. 5: |
Effects of leflunomide, silymarin and their combination on
liver thiobarbituric acid reactive substances (MDA) content of adjuvant-induced
arthritic rats |
Adjuvant arthritis was induced in the right hind paw by subplantar injection
of 0.1 mL FCA. The magnitude of swelling of the injected hind paw was measured
and paw volume % change was calculated. Saline was administrated in one group
of animals orally and the group served as control. LEF (10 mg kg-1)
and SIL (100 mg kg-1) and their combination were administrated orally
starting from day 10th of induction. At the end of experiment period, animals
were sacrificed by cervical dislocation under ether anesthesia. Liver were dissected
and part of each liver was homogenized and the homogenate was used for the determination
of liver thiobarbituric acid reactive substances (MDA) content. Each value represents
the mean of 8 rats±SE. Statistical analysis was carried out by one way
analysis of variance (ANOVA) followed by Least Significant Difference (LSD)
multiple comparisons test:
| • |
Significantly different from normal group at p<0.05 |
| • |
Significantly different from control group at p<0.05 |
Effect on liver homogenate GSH: Induction of arthritis by 0.1 mL FCA
showed 17% in the liver GSH level. Furthermore, treatment with leflunomide (10
mg kg-1 day-1 o.p.) showed decrease in the liver GSH level
reaching about 28% of the normal value. Conversely, treatment with silymarin
reversed the decrease in the liver GSH content elicited by FCA or leflunomide
when compared to the control group (Fig. 6).
Adjuvant arthritis was induced in the right hind paw by subplantar injection
of 0.1 mL FCA. The magnitude of swelling of the injected hind paw was measured
and paw volume % change was calculated. Saline was administrated in one group
of animals orally and the group served as control. LEF (10 mg kg-1)
and SIL (100 mg kg-1) and their combination were administrated orally
starting from day 10th of induction. At the end of experiment period, animals
were sacrificed by cervical dislocation under ether anesthesia. Liver were dissected
and part of each liver was homogenized and the homogenate was used for the determination
of liver reduced glutathione content (GSH) content. Each value represents the
mean of 8 rats±SE.
|
| Fig. 6: |
Effects of lefunomide, silymarin and their combination on
serem GSH mevel of adjuvant induced arthiritic rats |
Statistical analysis was carried out by one way analysis of variance (ANOVA)
followed by Least Significant Difference (LSD) multiple comparisons test:
| • |
Significantly different from normal group at p<0.05 |
| • |
Significantly different from control group at p<0.05 |
DISCUSSION
Leflunomide converted to active metabolite (malononitrilamide) (Schuna
and Megeff, 2000), that excreted in the bile, but it is efficiently reabsorbed
by enterohepatic recirculation (Bartlett et al.,
1996).
In rheumatic diseases, oxidative stress resulted from migration of active phagocytes
and leukocytes into synovial and periarticular tissues (Aaseth
et al., 1998). Therefore, supplementation of antioxidants resulted
in benefit effect in rheumatoid arthritis.
Findings of the present study indicate that leflunomide in a dose of 10 mg
kg-1 showed significant decreased in rat paw volume in comparison
to control group; silymarin had less effect than leflunomide. Additionally,
their co-administration showed same manner like leflunomide on FCA model.
The anti-arthritic activity of leflunomide may be attributed to inhibition
of de novo pyrimidine synthesis in activated T cells by selective inhibition
of DHODH (Fox et al., 1999). It has also other
activities as inhibition of tyrosine kinase is one of the most important mechanisms
of action of leflunomide (Xu et al., 1997).
Leflunomide also inhibits the expression of cell adhesion molecules, which facilitate
cellular interactions involved in antigen presentation, secretion of cytokines
and production of matrix metalloproteinases that degrade articular cartilage
and bone (Kraan et al., 2000).
Leflunomide significantly decreased serum nitric oxide level in arthritic rats,
may be due to inhibition of iNOS activation (Jankovic et
al., 2000; Miljkovic et al., 2001).
Furthermore, treatment with silymarin showed a significant decrease on serum
nitric oxide level in comparison to arthritic group.
Silymarin was less active and only at the highest concentration in modifying
iNOS expression their inhibitory activity on NO-2 production. Effect
of co-administration of both leflunomide and silymarin on the same parameters
showed improvement in liver enzymes. While pre-treatment of silymarin to leflunomide
did not show significant improvement in liver enzymes.
According to the results, leflunomide significantly increased liver lipid peroxides
content, while it was significantly decreased GSH represented in liver homogenate
of arthritic rats. Peroxidation of lipids is a common consequence of tissue
damage by free radicals and the presence of the reaction product malondialdehyde
(MDA) is an indication that such process has taken place (Furuno
et al., 1998).
On the other hand, silymarin normalized lipid peroxides contents and GSH content
in liver homogenate of arthritic rats as silymarin was used pretreatment with
leflunomide or even together.
CONCLUSION
Finding of the present study suggest that silymarin has anti arthritic activity
which may be related to its anti oxidant effect and decreasing of NO as one
of the most important mediators of inflammation. The present study indicates
that it is better to use leflunomide with silymarin that control the liver toxicity
resulted from leflunomide treatment in arthritic rats.
ACKNOWLEDGMENT
This study was supported by the National Research Center in Cairo, Egypt (No.
7/2/1).
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