INTRODUCTION:
Nephrotoxicity is kidney condition in which
the kidney do not work properly due to the damage or destruction by toxicants
such as chemicals or medications which may affect glomerular hemodynamics,
tubular cell toxicity, inflammation, crystal nephropathy, rhabdomyolysis, and
thrombotic microangiopathy1. Ibuprofen is a non-steroidal
anti-inflammatory drugs (NSAIDs) that is the most commonly used medication
worldwideand may be abused2. It is a non-selective inhibitor of
cyclooxygenase - 1 (COX-1) and cyclooxygenase-2 (COX-2)3. Prolonged
use of ibuprofen may affect the kidneys and lead to acute renal failure,
interstitial nephritis, and nephritic syndrome4.
Several studies have reported that olive leaves
extract and their contents possess a wide range of biological properties
including antiarrhythmic, spasmolytic, immune-stimulant, anti-atherosclerotic,
hypotensive, anti-inflammatory, antioxidant, anti-thrombic and hypoglycemic effects were represented5,6. Olive leave
extract, rich in bio phenols showed good levels of antioxidant activity and a
source of radical scavenging activity7. The major biophenols of the
olive leaf ethanolic extracts was the secoiridoid oleuropein8.
Oleuropein is believed to be responsible for many of the therapeutic properties
of olive leaf extracts.
Ocimum basilicum contains phenolic and
flavonoids compounds such as cinnamic acid, caffeic acid, sinapic acid, and
ferulic acid9. The ethanolic extract of its leaves
display potent antioxidant effects10. Basil extracts of various
parts of plants have been used for their antibacterial, anticancer,
anticonvulsant, antidiabetic, antihyperlipidemic, anti-inflammatory,
antioxidant, antistress, hepato protective, anti-aging effects and immune
modulatory properties11. In the present study, we aim to investigate
if olive leave extract or basil leave extract could prevent the development of
ibuprofen-induced nephrotoxicity.
MATERIAL
AND METHODS:
Animals:
A total of 36 male albino rats weighting (110-140g)
were housed in plastic cages and maintained under standard conditions of
temperature (25˚C±5˚C), humidity and 12 h light/dark cycle along the
experimental period. Animal care and experimental procedures were in accordance
with the ethical guidelines adopted by the Guide for the Care and Use of
Laboratory Animals (8th edition, National Academies Press).
Ibuprofen and preparation of extracts:
The Ibuprofen (BRUFEN 600mg) was purchased
from kahira pharmaceuticals and chemical industries company under licence of
ABBOTT laboratories-USA as 600mg granules powder.The olive or basil leave extracts
were prepared according to the method of Mohagheghi et al.12
and kandil et al.13 respectively.
Experimental designs and doses:
Thirty-six rats were divided equally into
6 groups (each group contains 6rats) as following:
· Control group,
feed on normal diet.
· Ibuprofen group,
received ibuprofen dissolved in 0.9% saline in a single dose of 15mg/kg body
weight, by gastric intubation14.
· Olive group,
received ethanolic extract of olive leave.
· Basil group,
received ethanolic extract of basil leave.
· Ibuprofen + olive
group, received ibuprofen followed by ethanolic extract of olive leave.
· Ibuprofen + basil
group, received ibuprofen followed by ethanolic extract of basil.
Olive and basil leaves extract were
administered by gastric intubation in a dose of 100mg/ kg body weight/ day for
4 weeks15.
Blood and tissue Sampling:
By the end of the experimental period (4
weeks), animals were overnight fasted for 10-12 hours before sacrificing under
light ether anesthesia. Serums obtained were used to measure all
biochemicalparameters needed. Subsequently, kidney was homogenized at certain
rates in phosphate buffer (pH7.4) and were centrifuged at 2000 r.p.m. Following
centrifugation, the clear non haemolysed supernatant was used for further
oxidative stress and antioxidant analysis.
Serum samples were analyzed for urea kits
purchased from Diamond diagnostics chemical company (Germany), uric acid kits
was purchased from spinreact company (Spain), creatinine, total protein and
albumin kits were purchased from Bioscope Diagnostics Chemical Company (Egypt),
erythropoietin (EPO) by using reagent kits purchased from Kamiya Biochemical
Company (U.S.A), Interleukin -1 b
(IL-1b) by using reagent
kits purchased from Thermo Scientific Company (U.S.A) and Interleukin 10
(IL-10) by using reagent kits purchased from RandD Systems International
Company (U.S.A).
Kidney homogenate was used to assay for lipid
peroxidation by the method of Preuss et al.16, catalase (CAT)
by the method of Cohen et al.17, superoxide dismutase (SOD)
by Kakkar et al.18 and Glutathione (GT) by the method of
Beutler et al.19. Data was analyzed by SPSS statistical package
version 20.One Way ANOVA followed by tukey test was used to declare the
significant difference between groups at p<0.05. Results were expressed as
mean ± standard error (SE).
RESULTS:
The experimental data that obtained from the Ibuprofen
administrated rats group revealed a significant (P < 0.001) decrease in
serum total protein, albumin, erythropoietin and Interleukin 10 concentration
levels and also showed a significant (P < 0.001) increase in serum urea,
creatinine, uric acid and interleukin 1-b as compared to
those of the control group. When olive leave extract or basil leave extract was
administrated with Ibuprofen, it significantly (P < 0.001) ameliorate these
changeswhen compared with ibuprofen administrated rat group (table 1, fig
1).They also recorded a significant improve of the increased renal oxidation
recorded in ibuprofen group (table2).
Figure 1. The effect of olive leave
extract or basil leave extract against the toxicity induced by ibuprofen on inflammatory
biomarkers of administrated rats.
Table
1.The effect of olive leave extract
or basil leave extract against the nephrotoxicity induced by ibuprofen
administrated rats:
|
 Group
Parameter
|
Control
|
Ibuprofen
|
Olive leave extract
|
Basil leave extract
|
Ibuprofen+ Olive leave
extract
|
Ibuprofen + Basil leave
extract
|
|
Urea (mg/ dl)
|
27.22 ±0.32a
|
39.13 ± 1.56c
|
22.08 ±0.71a
|
21.92 ±0.84a
|
28.17 ±1.60b
|
29.50 ±0.89b
|
|
Creatinine (mg/ dl)
|
0.60 ± 0.37a
|
1.18 ± 0.16b
|
0.59 ± 0.01a
|
0.61 ± 0.04a
|
0.62 ± 0.02a
|
0.63 ± 0.03a
|
|
Uric acid (mg/ dl)
|
1.71 ± 0.13a
|
2.74 ± 0.23b
|
1.57 ± 0.17a
|
1.63 ± 0.12a
|
1.72 ± 0.12a
|
1.78 ± 0.11a
|
|
Total proteins (g/ dl)
|
7.52 ± 0.08b
|
5.97 ± 0.25a
|
7.63 ± 0.13b
|
7.57 ± 0.09b
|
7.33 ± 0.44b
|
7.10 ± 0.16b
|
|
Albumin (g/ dl)
|
4.27 ± 0.11b
|
2.50 ± 0.12a
|
4.47 ± 0.91b
|
4.47 ± 0.15b
|
3.76 ± 0.20b
|
3.77 ± 0.27b
|
|
Erythropoietin (Iu/ L)
|
30.50 ± 0.84c
|
13.60 ± 1.02a
|
32.75 ± 1.33c
|
33.97 ± 0.51c
|
20.15 ± 0.35b
|
22.65± 0.68b
|
Data are expressed as mean ± standard
error. Number of animals in each group is six.Means, which have the same
superscript symbol(s), are not significantly different.F-probability:
P<0.001.
Table 2.The effect of olive
leave extract or basil leave extract against the renal oxidation system of
ibuprofen administrated rats:
|
 Group
Parameter
|
Control
|
Ibuprofen
|
Olive leave extract
|
Basil leave extract
|
Ibuprofen + Olive leave
extract
|
Ibuprofen + Basil leave
extract
|
|
Lipid peroxidation
(nmole/gm)
|
343.14 ± 8.70b
|
410.32 ± 3.54c
|
303.30 ± 3.45a
|
305.36 ± 2.36a
|
368.45 ± 10.01b
|
351.54 ± 7.98b
|
|
Catalase(k*100)
|
84.99 ± 4.66c
|
27.37±1.16a
|
91.70± 11.61c
|
87.28 ± 2.28c
|
51.61±1.30b
|
50.93±2.79b
|
|
Superoxide dismutase
(U /g)
|
89.75 ± 2.34c
|
38.60 ± 2.40a
|
84.40 ± 1.61c
|
89.33 ± 2.03c
|
78.44 ± 3.61b
|
76.00 ± 1.44b
|
|
Glutathione
(nmole/gm)
|
45.38 ± 0.46b
|
33.43±0.52a
|
49.53 ± 3.46b
|
50.61 ± 2.89b
|
43.64 ±0.34b
|
44.57± 1.11b
|
Data are expressed as mean ± standard
error.Number of animals in each group is six.Means, which have the same
superscript symbol(s), are not significantly different.F-probability: P<0.00.
DISCUSSION:
Nephrotoxicity
is often recognized by several metabolic disorders including the increase in
serum creatinine and urea 20. Uric acid is a marker for decreased
renal function and has a causal role in hypertension and vascular disease 21.
The ibuprofen administration showed marked increase in urea, creatinine and
uric acid concentration levels, these data are in accordance with those
obtained by Gholamiet al. 22. The administration of ibuprofen
decreased the levels of total protein and albumin that is due to the proximal
tubular toxicity 23.
Ibuprofen
administration induced imbalance between oxidative stresses in administrated
rats resulting in formation of more reactive oxygen species, clarified by the
increased lipid peroxidation and decreased antioxidants enzymes activities and
glutathione content. These results are in accordance with Ali and Mahboob24.
The lipid peroxidation elevation was due to mitochondrion damage and oxidative
phosphorylation which increased ROS production as result of ibuprofen
administration25. As result of oxidative stress direct damage to
proteins or alteration of their amino acids occur which lead to cell
dysfunction26. SOD directs antioxidant defense system by converting
the reactive superoxide anion into H2O2 and, if not regulated,
causes many types of cell damage. Decreased renal catalase levels inhibited the
conversion of H2O2 into water and oxygen and lead to
increasing ROS and cell damage23. Parlakpinar et al.27
supported that; the Lowered levels of GSH facing increased amount of H2O2
and hydroxyl radicals which are responsible to initiate ibuprofen
nephrotoxicity. Ibuprofen inhibited ROS scavenging activity and increased
oxygen free radicals’ generation leading to excessive accumulation of ROS
resulting in disruption of the balance inside and outside of cells and finally
cell damage28.
The
protective effect of olive leave extractis evidenced by Hedeab et al.29 who
recorded this ameliorating effect on cyclosporine induced nephrotoxicity. The
protective effects of the olive leave extract are probably due to its content
from oleuropein, hydroxytyrosol, and tyrosol which have strong free-radical
scavenging capacity30. Treatment
with O. basilicum in ibuprofen-induced toxicity confers significant improvement
to the kidneys tissues by reducing lipid peroxidation and increase SOD, CAT and
glutathione level, suggesting the role of O. basilicum extract in quenching ROS
formed. Several studies represented that O. basilicum exhibited protection
against xenobiotics-induced oxidative damage31. The O. basilicum
extract showed antioxidant effects as shown by its significant anti-lipid peroxidation
effects and radical scavenging activities19. The ameliorative effect
against ibuprofen of basil leave extract may be referred to the presence of
flavonoids and compounds of high antioxidant activities that scavenge the
produced super oxide anion and hydroxyl radicals32.
Erythropoietin (EPO) is hemopoietic hormone mainly
synthesized by the kidney in adults. EPO is a cytokine that regulates the erythropoiesis
by promoting the survival, proliferation, and differentiation of erythroid
progenitor cells. In response to hypoxia, EPO increases the red cell mass,
thereby improving tissue oxygenation33. It is expressed in the
cortical peritubular cells, glomerular, mesangial and interstitial cells and
also produced in proximal tubules34. The ibuprofen nephrotoxicity is
mostly in renal glomeruli and proximal convoluted tubules. Some studies
demonstrated that drugs such as cisplatin and 5-flurouracil which give rise to
nephrotoxicity due to renal proximal tubules destruction produced significant
decreased in serum EPO level35.
Inflammation is a complex response of the immune
system induced by a microbial infection or tissue injury: ischemic, toxic or
autoimmune. This process represents a complex of interactions between soluble
factors and cells. Activated macrophages secrete several mediators such as
cytokines with pro-inflammatory effect, as tumor necrosis factor,
interleukin-1b, IL-6 and anti-inflammatory, as IL-1036. IL-1b is a
pro inflammatory cytokine that acts as an endogenous pyrogen and it has diverse
effects on cell proliferation, differentiation, and function of many innate and
specific immune competent cells. IL-1b mediates many inflammatory diseases by
initiating and potentiating immune and inflammatory responses37.
IL-10 is an anti-inflammatory cytokine that limits the activation of leukocytes
and inhibits the production of pro-inflammatory cytokines including IL-1b and
chemokines38. In this study, ibuprofen significantly elevated serum
IL-1b levels and decreased IL-10 levels these data are in accordance with
Shafeek et al.39and Kumar et al.40. Considering
the results presented here, olive leave extract and basil leave extract showed
an important anti-inflammatory activity, due to the inhibition of pro-inflammatory
cytokines and enhancing of anti-inflammatory cytokines. Arranz et al.36
indicated that basil essential oil presented important anti-inflammatory
properties by decreasing IL-1b and increasing IL-10 levels. Moreover, Mueller et
al.41 also reported that extracts obtained from several plants,
including basil was able to enhance the production of IL-10 in a
lipopolysaccharide stimulated macrophages model.Gong et al.42 represented
that hydroxyltyrosol-20 from olive
leave extract has anti-inflammatory and antinociceptive effects by
inhibiting the production of IL-1b and tumor necrosis factor.Generally the
increased production of anti-inflammatory cytokine IL-10 may counterbalance the
production of pro-inflammatory cytokines. Polyphenols of olive extract had anti-inflammatory
effect as inhibit tumor necrosis factor and enhance IL-10 production43.
CONCLUSION:
The obtained results showed that Olive leave or basil
leave ethanolic extract bears therapeutic potential againstnephrotoxicity
caused by ibuprofen. A long-term treatment with ibuprofen could be safe if
olive leave extract or basil leave extract is taken during the treatment.
However, further pharmacological and medical studies are required to support
these findings.
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