protective role of curcumin, glucosamine and vitamin c on
TRANSCRIPT
182
Alexandria Journal of Veterinary Sciences
www.alexjvs.com
AJVS. Vol. 62(1): 182-193 July 2019
DOI: 10.5455/ajvs.51618
Protective Role of Curcumin, Glucosamine and Vitamin C on Oxidative Stress,
Biochemical and Histopathological Alterations Induced By Levofloxacin
Hosny Abd Elfadil1, Ahmed A. Mohamed1, Amany Behairy2, Heba M. Abdel-Ghany 3, Eman M.
Amaym 1 1 Department of Pharmacology, Faculty of Veterinary Medicine, Zagazig University, Egypt.
2 Department of Physiology, Faculty of Veterinary Medicine, Zagazig University, Egypt. 3 Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Egypt.
1. INTRODUCTION
Fluoroquinolones (FQs) antibiotics are widely used
for the treatment of various infections. The limited use of
FQs is attributed to arthropathy and articular- epiphyseal
cartilage complexes in several species of juvenile animals
(Liu, 2010; Burkhardt et al., 1990) and in children (Liu,
2010).
Levofloxacin is a third-generation fluoroquinolone
that fights against most Gram-negative and Gram-
positive organisms (Al-Soufi and Al-Rekabi, 2019).
Levofloxacin is used widely in veterinary medicine, in
poultry (chicken, duck, goose) and pigs bacterial
infection by E. coli, staphylococcus infection, ovarian
inflammation, Bacillus pyogenes infection, yellow and
white dysentery, pericarditis, enteritis and infection
caused by cholera etc (Kyuchukova et al., 2013).
Levofloxacin likes other fluoroquinolone
antibacterials acts via blocking bacterial DNA replication
pathway and the inhibition of DNA gyrase (Bano et al.,
2014), topoisomerase IV (Noel, 2009) (enzymes involved
in DNA replication, transcription, repair and
recombination). Despite the popularity of levofloxacin
usage, it may cause several side effects on the central
nervous system, caused gastrointestinal disturbances such
as nausea, vomiting, and constipation (Carbon, 2001) and
affect dermatological and the hepatic systems (Stahlmann
and Lode 2013). In addition, it induced tendinitis, and
ABSTRACT
Key words: Levofloxacin, Immature
rats, Curcumin,
Glucosamine
hydrochloride, Vitamin C
Levofloxacin is considered a strong weapon against several bacterial infections but its
serious side effects limit its use. Curcumin, glucosamine hydrochloride (Gls) and vitamin C
(Vit C) have strong effective properties. Hence, the study was carried out to assess their
protective roles on levofloxacin adverse effects in immature albino rats. Fifty immature male
albino rats were divided into five equal groups: I- Control group (received sterile saline
solution) orally; II- Levofloxacin group (10mg/kg b.wt) intraperitoneally; III- Levofloxacin +
curcumin (200mg/kg b.wt) orally; IV- Levofloxacin + Gls (500mg/kg b.wt) orally; V-
Levofloxacin + Vit C (100mg/kg b.wt) intraperitoneally. Rats were euthanized on zero day
and 14 days post treatment. The degree of protection was measured using calcium (Ca),
phosphorus (P), alkaline phosphatase (ALP) and oxidative biomarkers levels. Furthermore,
specimens from tendon, joint, ligament and cartilage were collected and subjected to routine
histological technique. Levofloxacin significantly decreased Ca concentration and increased
P level and ALP activity. Co-administration of curcumin, Gls and/or Vit C restored these
biochemical alterations and improved antioxidant defense system. Levofloxacin produced
degeneration of collagen fibers of tendons, necrosis of chondrocytes and destruction to
lacunae of articular cartilage of joint, degeneration of collagen bundles of ligaments.
Curcumin, Gls and/or Vit C were also markedly ameliorated histopathological effect of
levofloxacin. In conclusion, curcumin, Gls and/or Vit C exerted protective actions against
levofloxacin adverse effects.
*Corresponding to: Amanybehairy25688@gma
il.com
Article History Received: 01 May 2019
Revised: 30 May 2019
Accepted: 15 June 2019
Abd Elfadil et al. 2019. AJVS. 62 (1) 182-193
183
tendon rupture particularly in juvenile animals (Akali and
Niranjan, 2008).
So, we are in a greet need for finding protective
substances to decrease levofloxacin side effects.
Curcumin was investigated mainly for its anti-
inflammatory and anti-oxidant potency (Tejada et al.,
2016). It was shown to modulate human tendonitis
(Buhrmann et al. 2011) by the inhibition of
Cyclooxygenase-2 (COX-2) via its effect on nuclear
factor kappa-light-chain-enhancer of activated B cells
(NF-κB). Interestingly, curcumin is involved in bone
health, particularly associated with improved bone mass
density (BMD) and mechanical properties, suggesting
potential usage of curcumin for bone disorders treatment
(Rohanizadeh et al., 2016).
Glucosamine is an endogenous amino sugar that
is necessary for glycoproteins and glycosaminoglycans
synthesis. It is found in synovial fluids, tendons,
ligaments, cartilage and other joint structures (Bee and
Liew, 2010). Glucosamine hydrochloride (Gls) is one of
the main compounds including glucosamine (Anderson et
al., 2005).
Exogenous glucosamine is produced synthetically or
obtained from marine exoskeletons of shrimps, lobsters
and crabs. Glucosamine stimulates chondrocytes
metabolism in the articular cartilage (Bee and Liew, 2010)
via enhancing proteoglycan synthesis, inhibition of
collagen degradation, suppression of IL-1 induced
activation of NF-κB activity (Largo et al., 2003). Hua et
al. (2005) reported that high doses of glucosamine (300
mg kg) reduced both arthritic score and synovitis in
arthritic rats.
Vitamin C (Vit C) prevents bone resorption by its
anti-oxidative properties and plays a role in collagen
synthesis (Sahni et al., 2016). Dietary intake of Vit C was
associated with a reduction in the risk of cartilage loss and
osteoarthritis in humans via its antioxidant activity (Li et
al., 2016). Vitamin C deficiencies impair bone mass
cartilage (Kipp et al., 1996) and inhibit collagen synthesis
(De Arruda et al., 2014). In the light of these explanations,
the present study aimed to use curcumin, Gls or Vit C as
a promising therapeutic approach in a trial to relieve
levofloxacin irreversible adverse effects on Ca, P, ALP
activity and oxidative status and to ameliorate
levofloxacin drawbacks on tendon, joint, ligament and
cartilage.Therefore, biochemical, antioxidant markers
and histopathological studies were performed.
2. MATERIALS AND METHODS
2.1. Drugs:
• Levofloxacin, commercial form (levoflotanet),
Ampoule, 30ml, contains 750mg levofloxacin. It was
distributed by Arab Company for Medical products for
TriStar Medical Group, Egypt.
• Curcumin was purchased from SIGMA Company,
Egypt.
• Glucosamine hydrochloride (Gls) was obtained from
ROTH Company, Egypt.
• Vitamin C was purchased as commercial form
(Cevarol) from Memphis Co. for Pharm. & Chemical
Ind. Egypt. Each ampoule, 5 ml, contains 1000 mg Vit
C.
Curcumin (Edrees et al., 2018) and Gls (Gibson et al.,
2014) were dissolved in sterile normal saline solution.
2.2. Animal care and housing:
Fifty juvenile male albino rats (5 weeks old)
weighing 70-90 gm/each were used in this study.
Immature rats were obtained from Laboratory Animal
House, Faculty of Vet. Medicine, Zagazig University. All
animals were kept in clean properly ventilated separate
cages under similar environmental conditions and fed on
balanced ration composed of bread, milk, barley and
water ad libitum. All rats were kept for one week for
acclimatization before experimental study. All
experiments were approved by the institutional Animal
Care and Use Committee of the Faculty of Veterinary
Medicine, Zagazig University, Egypt.
2.3. Experimental design:
Rats were allocated into five equal groups (ten for
each).
Group I: Control group; received sterile saline solution.
Group II: Levofloxacin group; injected intraperitoneally
with levofloxacin (10mg/kg b.wt) (Olayinka et al., 2015)
for 14 sequential days.
Group III: Levofloxacin + curcumin group; received
Levofloxacin with curcumin orally (200mg/kg b.wt) (Ali
et al., 2005) for 14 sequential days.
Group IV: Levofloxacin + Gls group; administered
Levofloxacin and Gls orally (500mg/kg b.wt) (Barrientos
et al., 2010) for 14 sequential days.
Group V: Levofloxacin + Vit C group; injected
intraperitoneally with both Levofloxacin and Vit C
(100mg/kg b.wt) (Adeneye and Olagunju, 2008) for 14
sequential days.
2.4. Sampling:
After 14 days of treatment, five rats from each
group were euthanized after 12 hours fasting on zero day
and 14 days post treatment. Blood samples were collected
in plain tubes without anticoagulant, allowed to clot.
Then, serum was separated by centrifugation on 3000 rpm
for 10 minutes for determination of calcium (Ca),
phosphorus (P), alkaline phosphatase (ALP), lipid
peroxidation level and antioxidant enzymes activities.
Tendon, joint, ligament and cartilage specimens were
excised for histopathological examination.
2.5. Biochemical analysis:
Abd Elfadil et al. 2019. AJVS. 62 (1) 182-193
184
Serum ca and P were performed by using
electrolyte analyzer (EasyLyte, Medica, USA) as
described by (Tietiz, 1971). ALP was measured
colorimetrically according to (Bowers and McComb,
1972). Catalase (CAT), glutathione peroxidase (GPX)
and malondialdehyde (MDA) were measured using
commercial ELISA kits (CUSABIO BIOTECH CO. Ltd.,
China) according to, (Aebi, 1984), (Paglia and Valentine,
1967) and (Ohkawa et al., 1979) respectively.
2.6. Histopathological examination:
The collected specimens were fixed in 10%
buffered neutral formalin solution, dehydrated in
ascending grades of ethyl alcohol (70-100%), cleared in
xylene and embedded in paraffin wax. Five micron
thickness of paraffin sections were prepared and stained
with hematoxylin and eosin dyes according to (Bancroft
and Stevens, 1996) and then examined microscopically.
2.7. Statistical analysis:
The results were represented as mean± SE for
each group. The variation between groups were
statistically analyzed using one-way analysis of variance
(ANOVA) followed by Duncan’s multiple ranges post
hoc test for multiple comparisons. Data were considered
significant at P ≤ 0.05 (Tamhane and Dunlop, 2000).
3. RESULTS
3.1. Effect of levofloxacin, curcumin, Gls
and/or Vit C on Ca and P levels:
As showed in table (1), intraperitoneal
administration of levofloxacin revealed a significant
decrease in Ca level with a significant increase in P level
at zero day post treatment compared with control group.
Co-administration of levofloxacin with curcumin and/or
Gls displayed a significant increase in Ca level with a
significant decrease in P level on zero day post treatment
compared with levofloxacin group.
Comparing with control group, Ca & P levels
improved on 14th day post treatment but Ca level still
significantly decreased with a significant increase in P
level in levofloxacin group. A statistically significant
increase in Ca level with a significant decrease in P level
on 14th day post treatment in immature rats administered
levofloxacin concurrently with curcumin, Gls or Vit C
compared with levofloxacin group.
3.2. Effect of levofloxacin, curcumin, Gls and/or Vit C
on ALP activity:
Levofloxacin treated immature rats showed a
significant increase in ALP activity on zero & 14 days
post treatment in comparison with control group.
Administration of curcumin and/or Gls concurrently with
levofloxacin showed a significant decrease in ALP on
zero day post treatment when compared with levofloxacin
group. ALP activity ameliorated by treatment with
curcumin and/or Gls or Vit C on 14th day post treatment
comparing with levofloxacin group (Table 1).
3.3. Effect of levofloxacin, curcumin, Gls and/or Vit
C on antioxidant / oxidant status:
Individual administration of levofloxacin to
immature rats revealed a significant decrease in CAT and
GPX activities with a significant increase in MDA
concentration on zero &14th day post treatment
comparing with control group. On zero day post
treatment, co-treated rats of curcumin, Gls or Vit C with
levofloxacin represented a significant increase in CAT
and GPX activities with a significant decrease in MDA
level comparing with levofloxacin only- treated rats
(Table 2). On 14 days post treatment CAT, GPX activities
and MDA concentration were ameliorated in co-treated
groups of curcumin, Gls or Vit C with levofloxacin but
CAT, GPX activities still significantly decreased with a
significant increase in MDA concentration.
3.4. Histopathological findings:
3.4.1. Tendon:
Light microscopic examination revealed
abundant collagen fibers with elongated flattened
tenocytes which packed between bundles of collagen
fibers and outer layer of tendon (paratenon) of control
group (fig. 1a). Acute paratenonitis represented by
infiltration of mononuclear inflammatory cells with
congestion of blood vessel besides hyaline thickening of
some blood vessels walls (fig. 2a) were detected in
levofloxacin treated rats on zero day post treatment. In
addition, degenerated tenocytes with separation collagen
bundles from each other were detected at 14 days post
treatment (fig. 3a). Newly vascularized blood vessels in
paratenon of tendon were observed in co- treated
curcumin rats at zero day post treatment (fig. 4a) with
partial necrosis to collagen bundles and loss of nuclei at
14 days post treatment (Necro-tendenitis) (fig. 5a).
Glucosamine hydrochloride was less effective where
mononuclear cells infiltration, extravasated erythrocytes
with congestion of the blood vessel and hyaline
thickening in the wall of other blood vessels in the tendon
sheath were showed (figs. 6a, 7a). Mild thickening of
tendon sheath was observed in Vit C co-treated rats (figs.
8a, 9a).
Abd Elfadil et al. 2019. AJVS. 62 (1) 182-193
185
Table (1): Effect of levofloxacin, curcumin, Gls and/ or Vit C on Ca and P levels and ALP activity.
Groups
Ca (mg/dl) P (mg/dl) ALP (IU/L)
Zero day
Post
treatment
14 days post
treatment
Zero day
Post
treatment
14 days post
treatment
Zero day
Post
treatment
14 days post
treatment
Control 8.90a±0.33 9.70a±0.24 2.65c±0.17 2.93d±0.19 212.57c±5.58 193.37d±3.50
Levofloxacin 2.05d±0.22 5.35e±0.16 8.37a±0.27 6.25a±0.13 303.83a±5.53 246.95a±2.65
levofloxacin+
Curcumin
6.50b±0.42 9.08b±0.09 3.13bc±0.39 3.28d±0.11 227.17bc±11.01 199.77cd±5.18
Levofloxacin+ Gls 4.73c±0.46 8.00c±0.15 3.85bc±0.62 4.28c±0.14 247.15b±8.68 204.40c±2.20
Levofloxacin+ Vit
C
3.00d±0.50 6.43d±0.17 4.75b±1.04 5.33b±0.15 282.75a±2.78 218.77b±3.15
Data are expressed as (mean ± S.E). Means within the same column in each category carrying different superscription letters are significant at p ≤ 0.05. (n
=5)
Table (2): Effect of levofloxacin, curcumin, Gls and/ or Vit C on antioxidant/ oxidant status.
Groups
CAT (U/L) GPX (mU/ml) MDA (nmol/ml)
Zero day Post
treatment
14 days post
treatment
Zero day
Post
treatment
14 days post
treatment
Zero day Post
treatment
14 days post
treatment
Control 112.28a±6.55 132.88a±3.01 1.36a±0.47 2.49a±0.20 4.88c±0.16 6.04d±0.48
Levofloxacin 39.15e±3.12 83.70c±2.82 0.39e±0.01 0.71d±0.01 37.68a±3.32 25.05a±0.86
levofloxacin+
Curcumin
90.95b±4.10 127.65a±3.51 1.08b±0.04 1.83b±0.15 6.45c±0.95 9.85c±0.41
Levofloxacin+ Gls 74.85c±2.33 108.80b±3.63 0.83c±0.03 1.12c±0.05 9.76c±2.68 11.90c±0.65
Levofloxacin+ Vit C 60.53d±2.85 92.55c±4.61 0.63d±0.03 1.06cd±0.05 22.35b±4.34 19.05b±1.14
Data are expressed as (mean ± S.E). Means within the same column in each category carrying different superscription letters are significant
at p ≤ 0.05. (n =5)
3.4.2. Joint
Normal histological picture of joint was seen
in control rats (fig.1b). Levofloxacin administration
revealed thickening of synovial membrane due to
hyperplasia of fat cells. Destruction to lacunae of articular
cartilage and replaced by RBCS was present. Thickening
of joint capsule were noticed (figs. 2b, 3b). Co- treated
curcumin rats showed normal appearance of synovial
membrane with hypoplasia of articular cartilage (fig. 4b).
Conversely, thickening of synovial membrane with
infiltration of inflammatory cells and mild thickening of
joint capsule were detected in co-treated glucosamine
group at zero, 14th day post treatment (figs. 6b, 7b).
Microscopy observation of Vit C co- treated rats revealed
mild thickening of perichondrium with proliferation of
chondrocytes and newly vascularized blood vessels (figs.
8b, 9b).
3.4.3. Ligament
Ligament sections of control group
exhibited parallel arrangement of collagen fibers (fig. 1c)
compared to levofloxacin treated group which showed
acute inflammation of ligament represented by
mononuclear cells infiltration among collagen fibers and
congested blood vessels with present mild edema between
collagen bundles (figs. 2c, 3c). Both rats co- administered
curcumin (figs. 4c, 5c) and/or Vit C (figs. 8c, 9c) showed
regular arrangement of collagen bundles with present few
infiltration of mononuclear inflammatory them while Gls
administered concurrently with levofloxacin revealed
erosion of ligament with round cells infiltration among
collagen bundles and newly vascularized blood vessels
beside separation of collagen fibers from each other (figs.
6c, 7c).
3.4.4. Cartilage
Cartilages of normal rats consisted of
perichondrium and chondrocytes inside lacunae with
hyaline matrix between them (fig. 1d). Microscopically,
cartilage sections of levofloxacin treated rats exhibited
erosions with destruction of bone matrix and mild
thickening of perichondrium with hypoplasia of articular
cartilage (figs. 2d, 3d). Interestingly, regular parallel
arrangement of collagen fibers with few amount of
mononuclear inflammatory cells between collagen
bundles were observed in co-treated curcumin group on
zero day post treatment (fig. 4d).Cartilage specimens of
rats administered Gls with levofloxacin showed
hyperplasia of chondrocytes of articular cartilage with
normal arrangement of calcified bone (figs. 6d, 7d). Mild
thickening of perichondrium with normal appearance of
articular cartilage in Vit C co-treated rats (figs. 8d, 9d).
Abd Elfadil et al. 2019. AJVS. 62 (1) 182-193
186
Figure 1: Normal Histology of the control group. A- Section of tendon showing abundant collagen fibers (arrow) with elongated flattened tendocytes and
paratenon (arrow head). B- Section of joint showing bone (white arrow), bone marrow (oblique arrow), articular cartilage (arrow head), joint space (thin
arrow) and synovial membrane (thick arrow). C- Section of ligament showing parallel arrangement of collagen fibers with elongated fibroblast (arrow). D-
Section of cartilage consisted of perichondrium (oblique arrow) and chondrocytes inside lacunae with hyaline matrix between them (arrow) with present bone
(arrow head) and bone marrow (white arrow) (H&E, bar 100 µm for images A-D)
Figure 2: Histopathological changes in levofloxacin group on zero day post-treatment. A- Section of tendon showing acute paratenonitis (inflammation of
tendon sheath) represented by mononuclear cells infiltration (arrow head) intercellular edema and congestion of blood vessel (arrow) beside hyaline thickening
of some blood vessel walls (oblique arrow). B- Section of joint with higher magnification showing an empty lacunae of articular cartilage from chondrocytes
(oblique arrow) with destruction to lacunae of articular cartilage and diffuse hemorrhage (arrow). C- Section of ligament showing Enthesitis (acute
inflammation of ligament) represented by mononuclear cells infiltration among collagen fibers (white arrow) and congested blood vessels (arrow head) with
present mild edema between collagen bundles (arrow). D- Section of cartilage showing destruction of lacunae of articular cartilage with necrosis of
chondrocytes (arrow head) beside arthritis represented by destruction of bone which replaced by mononuclear inflammatory cells (oblique arrow) (H&E, bar
20 µm in image B, bar 100 µm in others).
Figure 3: Histopathological changes in levofloxacin group on 14th day post-treatment. A- Section of tendon showing degenerated tenocytes which become
oval or round not elongated as usual (arrow) with separation collagen bundles from each other (arrow head).B- Section of joint showing formation of
granulation tissues on joint surface (arrow) with thickening of perichondrium tissue (arrow head). C- Section of ligament showing hyaline degeneration of
collagen bundles which fused together (arrow) with present (lymphocytes& macrophages) among them (arrow head).D- Section of cartilage showing necrosis
of chondrocytes which represented by karyorrhexis of nucleus (arrow) with present RBCS in some lacunae (arrow head) while other lacunae without
chondrocytes (oblique arrow) (H&E, bar 100 µm in image B, bar 20 µm in others).
Abd Elfadil et al. 2019. AJVS. 62 (1) 182-193
187
4. DISCUSSION:
Arthropathy, tendon disorders and cartilage
degeneration have been reported to be toxic effects for
FQs in juvenile animals which restrict their therapeutic
use, and the patient should not be rechallenged with FQS
treatment (Burkhardt et al., 1993; Mehlhorn and Brown,
2007; Durey et al., 2010). In this study, we recommend to
use either curcumin, Gls or Vit C as a trial to relieve the
adverse effects of levofloxacin.
Calcium is engaged in cytoskeletal remodeling,
cell hyperpolarization, and cell death (Li et al., 2016), it
is involved in physiological and pathological processes of
chondrocyte. Phosphorus is essential for cellular
functions and skeletal mineralization (Fontes- Pereira et
al., 2018) and it is associated strongly with Ca level
(Endres and Rude, 1999). Calcium phosphate is major
mineral constituent of bone, forming hydroxyapatite
crystals which necessary for stiffness and resistance to
compression (Fontes- Pereira et al., 2018). The
calcium/phosphorus ratio in the blood is a very
important biomarker during bone formation (Ganguly,
2017).Intraperitoneal injection of levofloxacin revealed a
significant decrease in Ca level with a significant increase
in P level (Table 1) at zero day post treatment compared
with control group. Fluoroquinolones interact with
multivalent cations such as calcium, magnesium and iron
and form non-absorbable ion-quinolone chelates in the
stomach and small intestine (Lomaestro and Bailie,
1995).Hence, this mechanism enables them to exert their
effects. Our findings agree with Walwadkar et al. (2006)
who reported that rheumatoid arthritis causes oxidative
stress with a decrease in calcium/ phosphorus level in
blood and this decrease indicates an altered calcium and
phosphorous metabolism. The elevation of phosphorous
is attributed to tissue hypoxia and increase of ATP
degradation that consequently lead to the release of
inorganic phosphorous from cells to extracellular pool.
Osteoarthritis causes degeneration of articular cartilage,
which is considered as an avascular tissue and obtains its
oxygen and nutrients during joint movement via the
dynamic flow of synovial fluid (Urban, 1994). The
oxygen tensions are further decreased in osteoarthritic
joints (Schneider et al., 1996).
A significant increase in Ca level with a
significant decrease in P level in immature rats
administered levofloxacin concurrently with curcumin
was observed (Table 1). Consistent with Al-Bogami et al.
(2016) who reported the ability of curcumin to increase
the absorption of Ca and decrease its loss. Curcumin
adjusts regulation of bone turnover. The observed
increase in serum and bone calcium content represents
decrease in rate of bone turnover (Rangrez et al., 2011).
Rats that co- treated levofloxacin with Vit C showed a
significant increase in Ca level with a significant decrease
in P level. These findings are agreed with Doan, (2000)
who investigated higher serum Ca levels with the Vit C-
supplemented diet compared with control diet.
Metabolism of calcium affected by dietary Vit C (Orban
et al., 1993), it influences the activity of 25(OH) D3-1-
hydroxylase and converts 25(OH) D3 to 1, 25 (OH) 2D3
that necessary for intestinal absorption of calcium (Sahni
et al., 2016) and for the bones mineralization (Lohakare
et al., 2005).
ALP is a biomarker used to evaluate bone
metabolism (Moran et al., 2012). Levofloxacin treated
immature rats showed a significant increase in ALP
activity on zero & 14 days post treatment in comparison
with control group (Table 1). Elevated ALP levels can be
seen with worsening magnitude of bone turnover (Freethi
et al., 2016). Elevated serum ALP activity with normal
serum Ca concentration was reported in patients suffer
from bone diseases (Saraç and Saygılı, 2007). ALP is
important for bone mineralization by hydrolyzing
pyrophosphate in the extracellular milieu, produced by
osteoblasts in bone tissue in response to decreased
calcium levels (Freethi et al., 2016).ALP activity
ameliorated by treatment with curcumin and/or Gls or Vit
C. Lower ALP activity was detected in curcumin-
stimulated human osteoblast-like cells (MG-63 cells),
confirming an effect on osteoblast differentiation (Moran
et al., 2012). Glucosamine prevented the rise of ALP
(Melnykovych and Costlow, 1971).Vit C regulates the
energy status of maturing chondrocytes, plus related ALP
activity (Omata et al., 2007). Serum ALP activity was
higher in chicks fed the diet not supplemented with Vit C
compared with those fed a supplemented diet (Lohakare
et al., 2005).
Negative correlation between calcium/phosphorus ratio
and lipid peroxide recommends excess generation of
reactive oxygen species (ROS) which important in the
bone resorption associated with inflammatory diseases
(Garrett et al., 1990). Enzymatic antioxidants such as
SOD, CAT, and GPX protect from ROS action (Gupta et
al., 2007). Individual administration of levofloxacin to
immature rats revealed a significant decrease in CAT and
GPX activities with a significant increase in MDA
concentration on zero &14th day post treatment
comparing with control group (Table 2).
Fluoroquinolones treatments resulted in a progressive
increase of these oxidative products over time (Afolabi
and Oyewo, 2014). Disruption of the oxidant- antioxidant
status known to occur in rheumatic diseases (Dwivedi et
al., 2016).A significant decrease was observed in the GPX
activity from the 3rd day till the 14th day compared to
control when studying the effect of levofloxacin
Abd Elfadil et al. 2019. AJVS. 62 (1) 182-193
188
Figure 4: Histopathological changes in levofloxacin + curcumin group on zero day post-treatment. A- Section of tendon showing newly vascularized blood
vessels in paratenon (arrow) with mononuclear cells infiltration (arrow head). B- Section of joint showing normal appearance of synovial membrane (arrow)
with hypoplasia of articular cartilage (arrow head). C- Section of ligament showing parallel arrangement of collagen fibers with mononuclear inflammatory
cells between collagen bundles. D- Section of cartilage showing normal arrangement of chondrocytes inside lacunae (arrow) with regular hyaline matrix between
them (arrow head) (H&E, bar 20 µm in image D, bar 100 µm in others).
Figure 5: Histopathological alterations due to co-administration of levofloxacin + curcumin group on 14th day post-treatment. A- Section of tendon showing
partial necrosis of collagen bundles with with nuclear disappearance (arrow).B-Section of joint showing thickening of synovial membrane with mononuclear
cells infiltration (arrow head) besides thickening of joint capsule (black arrow) and partial loss of cartilage (white arrow) with fragmentation of bone matrix
(oblique arrow). C-Section of ligament with higher magnification showing newly vascularized blood vessels (arrow) with few lymphocytes infiltration (arrow
head). D- Section of cartilage showing highly thickening of perichondrium (arrow) (H&E, bar 20 µm in images A and C, bar 100 µm in images B and D).
Figure 6: Histopathological abnormalities in case of levofloxacin + Gls group on zero day post-treatment. A- Section of tendon with higher magnification
showing mild mononuclear cells infiltration (arrow head), minute foci of extravasated erythrocytes (oblique arrow) and hyaline thickening of paratenon blood
vessel (arrow). B- Section of joint showing thickening of synovial membrane with infiltration of inflammatory cells (arrow). C- Section of ligament showing
erosion of ligament (arrow) with round cells infiltration among collagen bundles (arrow head) and newly vascularized blood vessels (white arrow) beside
dissociation of collagen fibers from each other. D- Section of cartilage showing hyperplasia of chondrocytes of articular cartilage (arrow) with present calcified
bone (arrow head) (H&E, bar 20 µm in image A, bar 100 µm in others).
Abd Elfadil et al. 2019. AJVS. 62 (1) 182-193
189
Figure 7: Histopathological changes in levofloxacin + Gls group on 14th day post-treatment. A- Section of tendon showing mononuclear cells infiltration among
collagen bundles (arrow) with newly vascularized blood vessels in paratenon (arrow head). B- Section of joint showing thickening of joint capsule (arrow) with
erosion of articular cartilage (arrow head). C- Section of ligament showing hyperplasia of adipocytes in ligament sheath (arrow) with congestion of blood vessel
(oblique arrow) beside degeneration of collagen. D- Section of cartilage showing erosion and desquamation of perichondrium tissue (arrow) and degenerated
perichondrium is replaced by mononuclear inflammatory cells (arrow head) beside newly vascularized blood vessels (oblique arrow) (H&E, bar 100 µm for
images A-D).
Figure 8: Histopathological changes in levofloxacin + Vit C group on zero day post-treatment. A- Section of tendon with higher magnification showing newly
vascularized blood vessels in paratenon (arrow) with mononuclear cells infiltration (arrow head). B- Section of joint showing mild thickening of perichondrium
(arrow) with proliferation of chondrocytes and each one present singly inside lacunae (arrow head). C- Section of ligament showing regular arrangement of
collagen bundles (arrow head) with present few infiltration of mononuclear inflammatory cells (arrow). D- Section of cartilage with higher magnification
showing thickening of perichondrium (arrow) and some lacunae free from chondrocytes (arrow head) (H&E, bar 100 µm in image B, bar 20 µm in others).
Figure 9: Histopathological changes in levofloxacin + Vit C group on 14th day post-treatment. A- Section of tendon showing mononuclear cell infiltration
and fibroblasts cells (arrow) among degenerated collagen bundles (arrow head) with mild edema between them (oblique arrow). B- Section of joint showing
newly vascularized blood vessels (arrow) beside extravasated erythrocytes in paratenon (arrow head). C- Section of ligament showing newly vascularized
blood vessels with endotheliosis (arrow) beside infiltration of few mononuclear cells (arrow head) together with hyaline degeneration of the collagen fibers
(oblique arrow). D- Section of cartilage showing mild thickening of perichondrium (arrow) with normal appearance of articular cartilage (arrow head)
(H&E, bar 20 µm in images A and C, bar 100 µm in images B and D).
Abd Elfadil et al. 2019. AJVS. 62 (1) 182-193
190
administration on brain regions of male albino rats (Rawi
et al., 2011). In the present study, CAT, GPX activities
and MDA concentration were improved in levofloxacin
co-treated groups with curcumin, Gls or Vit C at 14th days
post treatment.
Similarly, Jiang et al. (2016) demonstrated a
significant decrease in tissue MDA levels and elevated
SOD activity in curcumin treated groups. Curcumin may
exert a chondroprotective effect via its anti-inflammatory,
anti-oxidative stress, and anti-catabolic activity which
mitigate osteoarthritis pathogenesis (Zhang et al., 2016).
Curcumin can reduce the formation of ROS that
responsible for cartilage degradation (Jancinova et al.,
2009). Glucosamine treatment reduced the MDA level
and increased the SOD, CAT, GPX and GSH content
compared to rats with rheumatoid arthritis (Dai et al.,
2018).Vitamin C is highly effective antioxidant, reacts
with numerous aqueous free radicals and ROS preventing
bone resorption (Padayatty et al., 2003).
This study revealed acute paratenonitis,
destruction of lacunae of articular cartilage with necrosis
of chondrocytes and arthritis represented by destruction
of bone which replaced by mononuclear inflammatory
cells in rats administered 10mg/kg b.wt levofloxacin for
14 consecutive days (figs 2,3). Arthropathy and tendon
disorders occurred with or following fluoroquinolones
treatment induced via chelation of magnesium in cartilage
which impaired integrin function and disturbance of
chondrocyte - extracellular matrix interaction, after that
formation of ROS, apoptosis, and tissue damage (Sendzik
et al., 2009). Chondrocytes oxidative injury induced by
quinolones has been reported by (Hayem et al., 1994)
through inhibition of chondrocytes DNA synthesis and
compromised mitochondrial integrity (Mont et al., 1996).
Quinolones -induced changes were reported in immature
articular cartilage of weight-bearing joints of rats (Kato
and Onodera, 1988).
Co-treatment of levofloxacin with curcumin, Gls
and/or Vit C reduced adverse effect of levofloxacin on
zero day post treatment. Anti-inflammatory and
antioxidant agents protect cells against the oxidative
damage induced by inflammation and help tendon healing
(Jeong et al., 2018). Experimently, when curcumin
administered topically on dermal wounds, it increased
granulation tissues formation, accelerated wound
maturation, and increased tensile strength (Kant et al.,
2014). Another study of Jiang et al. (2016) demonstrated
that oral administration of curcumin for 14 days
increased expression of collagen type I and III and
concentration of hydroxyproline in rat tendon.
Curcumin administration resulted in more collagen
deposition and remodelling (Behfar et al., 2018).
Glucosamine has anti-catabolic and anti-inflammatory
effects, used as a chondroprotective agent in osteoarthritis
to alleviate disease progression (Ilic et al., 2008).Vitamin
C prevents bone resorption and helps in collagen
formation (Sahni et al., 2016).
The present study histologically
demonstrates persisting of certain lesions at 14th post
treatment with levofloxacin, consistent with (Cavusoglu
et al. 2000) who reported great erosions in the articular
cartilage even in the fourth month following the
termination of the 7-days treatment. Kato and Onodera,
(1988) found that the surface of repaired articular
cartilage was irregular and the matrix did not contain any
chondrocyte at 10 weeks after 7 days treatment of juvenile
rats with Ofloxacin. The limited vascularity and
relatively poor cellularity make tendon repair
challenging and slow (Docheva et al., 2015) and the
healed tendon does not gain the structure and
mechanical function of intact tendon (Behfar et al.,
2018).
5. CONCLUSIONS:
Levofloxacin administration induced
oxidative stress which inversely affected
calcium/phosphorus ratio and altered ALP activity. It also
induced progressive irreversible lesions in the tendon,
articular joint, ligament and cartilage of juvenile rats
which persist even after termination of 14 days therapy.
Curcumin, Gls and Vit C improved biochemical and
oxidative parameters. Histopathological changes
minimized by curcumin and /or Vit C treatment while Gls
is not entirely consistent and further studies are still
required to prove their efficacy.
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