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Effect of
diclofenac sodium on systemic enzymes activity and their role on
pro-inflammatory situation in brain environment was studied to understand
corresponding drug induced changes. Male Wistar rats Rattus norvegicus were assigned to three groups like Control,
FCA+FIA and DS. The activities of ACP, ALP, LDH, AST and ALT were estimated
from brain tissue. The result shows reduced ACP activity in FCA+FIA and DS
group and one fold increased ALP activity in FCA+FIA group and reduction in the
DS group. The LDH and AST in DS group shows reduced activity and also in the
FCA+FIA group, but ALT activity was found to be marginally elevated in DS group
when compared to FCA+FIA. Reduced ACP activity in DS and FCA+FIA correlated to
dynamic inflammatory relevant functional metabolism. Significantly decreased
ACP, ALP, LDH, AST and ALT in DS treatment are mainly attributed to
inflammatory changes and active bone lesion/turnover. The decreased enzymes
activity in the DS acknowledged to the fundamental alteration in mitochondrial
function.
Keywords: Arthritogenesis, ACP, ALP, LDH, AST, ALT, FCA,
FIA
Abbreviations: FCA: Freund's Complete Adjuvant; FIA: Freund's Incomplete Adjuvant; DS: Diclofenac sodium treatment; ACP: Acid phosphatase; ALP: Alkaline Phosphatases; LDH: Lactate Dehydrogenases; AST: Glutamate-Oxaloacetate transaminase; ALT: Glutamate-Pyruvate Transaminase; NSAID: Nonsteroidal Anti-Inflammatory Drugs
INTRODUCTION
Diclofenac sodium (DS) shows neurotoxic effects through various metabolic pathways which leads to region-specific oxidative damages and more specific endangered drug induced toxicity
in the central
nervous system (CNS) [1,2]. Free radicals released from
different regions of CNS can induce mitochondrial damage, suppressed ATP production that resulted in mitochondrial permeability to cytochrome-C release and followed by caspases involved apoptosis in the surrounding
environment [3]. DS toxicity can
induce cyclooxygenase
(COX)
activity,
prostaglandin synthesis, increased lipid
oxidation by decreasing glutathione production, increasing free radical formation induced oxidative stress, massive genomic DNA fragmentation
and
apoptosis. DS increased LDH,
AST and ALT enzymes activities
noted in this present study. This study engaged to understand ACP, ALP, LDH,
AST and ALTs participation in the cascading events behind the DS toxic
manifestation since the brain region is highly privileged.
MATERIALS AND METHODS
Animals
This present study used eighteen male Wistar
rats Rattus norvegicus weighing between 180-220 g procured from King
Institute, Chennai, India and followed the India Animal Ethical committee
animal use protocol. All
animals were housed in polypropylene cages in a temperature controlled room at
24 ± 4°C. The animals were fed with
pelleted rat feed with free access to water throughout the experiment. They
were acclimatized at least 3 weeks before starting the experiments. All studies were carried out
using 6 rats in each group, assigned to three groups (Control, Freund's Complete
Adjuvant+Freund's Incomplete Adjuvant (FCA+FIA), Diclofenac Sodium treatment
(DS). All studies
were carried out using 6 rats in each group. This work has got clearance from
the Institutional Animal Ethical Committee (IAEC).
Drug administration
Control rats were injected with 0.2 ml of deionized
water at right leg footpad. In FCA rats, on
day ‘0’, 0.2ml of FCA was
injected in the right leg footpad and 0.2 ml of FIA was injected with FCA rats’ right leg footpad on day ‘7’ and this subsequent dosage was taken as booster dosage. DS rats are treated with 10µl Diclofenac Sodium/day (50µl/5 days)/intramuscularly on days
‘0’, ‘5’, ‘10’ and ‘15’ in FCA+FIA
rats.
Biochemical parameters estimation
All group rats were kept to 21 days under constant observation. They are animals
mildly anesthetized using chloroform to collect brain tissue. The Capac
estimated by Tenniswood et al. [4] and the ALP was done by Tietz et al. [5]. The LDH, AST and ALT activities were
estimated by [6-8].
Statistical analysis
All data were analyzed with one-way
ANOVA and the difference between test groups and the control was considered
significant when P<0.01.
RESULTS AND DISCUSSION
Present study
observed reduced ACP activity in DS treated and elevated ALP activity in
FCA+FIA group with more than one fold increase. While the DS rats show reduced
ALP activity compared to control and FCA+FIA. Reduced LDH activity was noted in
FCA+FIA and DS groups. In DS treatment, the LDH activity found two fold
decreases to control rats (Table 1).
Table 2 shows reduced AST and ALT
activity in FCA+FIA and DT. The DS treated rats showed marginally increased
activity to that of FCA+FIA rats but still found below control rats.
ACP
Acid phosphatases (ACP) activity is much
important for
cerebellar development and also they participate in neurodegenerative disorders. Activated lysosomes show their
content to
outflow to excrete exocytosis and increased functional ability to fuse with cell membranes to produce
different activities like axon growth, neuronal
migration,
vesicular traffic [9], MHC II molecules up-regulation and also involved
in neuronal death [10,11]. Glial cells are noted that, it leaks lysosomal acid
phosphatase through its intact cell membranes at low osmatic pressure. The secreted ACPs were found to hydrolyze phosphomono ester metabolites and release inorganic phosphates (Pi), which are used to synthesize ATPs, phospholipids, proteins and participated in signaling processes. Squirrel monkey brain study documented
that, ACP was more concentrated
in different
regions namely, in nuclei
of basalis Meynert, diagonalis band
of Broca, magnocellular hypothalamic, corpus mammillaris, thalamus, motor
neurons of cranial nerve nuclei, giant pyramidal cells of cerebral cortex and in the Purkinje cells of cerebellar
cortex
[12]. Lipid phosphatases are involved in the nerve cells proliferation,
survival, apoptosis, migration and also in the development and maintenance of
the central nervous system [5]. The protein phosphatases are found to
participate in the N-methyl-D-aspartate receptors (NMDARs) mediated
excitotoxicity-induced neuronal death [13]. In the brain region, the ACP activity is attributed to more
related static cell metabolism
than to dynamic activity [12]. DS induced toxicity shows
reduced ACP and elevated ALP, AST, ALT activities
accompanied with increased TNF-α, uric acid levels and TNF-α gene expressions. This has been attributed to active bone lesion [14-16]. Similarly,
DS caused imbalanced antioxidant system, lipid
peroxidation, pro-inflammatory
responses were
also noted. The elevated ALP, AST, ALT, LDH activities
are attributed to
oxidative stress induced tissue
damage
or death [17]. The oral DS administration showed increased
uric acid, ALT and decreased total protein may be associated with the reactive
metabolites production via hepatic cytochrome P450 [18,19]. Although, the breakdown of the antioxidant
system may
be the result of pro-inflammatory
responses.
In this present study, reduced ACP activity in DS treatment and marginally
altered in FCA+FIA group has been noted (Table
1). This may be taken as, no vertical imbalance in the anti-oxidative or lipid peroxidation systems and the
reduced activity in DS treatment below Control rats may be taken as
corresponding toxicity in the brain oxidative metabolism and may be attributed
to tissue damage followed by
inflammation induced dynamic
functional metabolism.
ALP
ALP is the enzyme that primarily participates in liver, kidneys and brain endothelial cell activities. They
also play more important and
fundamental roles in neurogenesis, plasticity development, neuronal tissues cortical functions and purinergic
signalling mechanism during inflammation [20,21]. The ALP purinergic signalling
activity has been found to accomplish with ATP-nucleotide receptors complex
involved inflammatory activity after an acute stress event. The elevated ALP
activity is implicated in neuronal death through increased tau
dephosphorylation, high-sensitivity C-Reactive Protein (hs-CRP) and disease
severity prediction dementia patients [22-24]. In concurrence to the above,
three times greater ALP activity was noted to higher oxidative stress,
ischaemia patients mortality and vascular smooth muscle calcification followed
by arteriosclerosis, ischemic cerebral stroke, leukodystrophy, and intracranial
hemorrhage [25,26]. In contrast, extracellular ATP degradation can reduce
inflammatory signals and can induce adenine receptors involved
anti-inflammation responses [27,28]. Similarly, ALP treated
lippopolysaccharides showed reduced capacity to induce inflammation through T Cell Receptor
through lipid
Ahydrolysis, which is
central in TLR4 recognition [29]. In
addition, elevated
ALT, AST, ALP, urea and creatinine levels found reduced by DS administration added with cytochrome p-450 inhibitors to the culture [30]. In this
present study, more than one fold increased ALP activity in the FCA+FIA rats
and nearly two fold decreased activity in DS treatment compared to control rats
is observed. The increased ALP activity in the FCA+FIA group may result from the central nervous system injury by increased
activity of dephosphorylation. The decreased activity with DS treatment can be
taken as oxidative
stress.
Since, variable
metabolic pathways
profoundly can lead to
region-specific accumulation of oxidative damage in different regions of the
CNS and
followed by specific brain region to become more vulnerable to drug toxicity.
LDH
LDH gets encoded by two distinct genes, LDH-A
and B. Increased LDH-A gene is more
associated to HIF-1
direct transcriptional regulation and c-myc expression. DS treatment is found to inhibit COX-2 activity by c-myc expression followed by decrease in glucose transporter 1(GLUT-1) gene
expression, and lactate secretion [31,32]. DS inhibited STAT-3
phosphorylation, c-myc gene expression, G2/M phasecell cycle arrest, reduced lactate levels in cell culture and inhibited
LDH activity was also documented
by Verena et al. [33] and Desmet et al. [34].
Similarly, DS administration inhibited Stat3 gene expression,
IL-10 transactivation and IL-12 gene expression blockade are noted [35,36]
DS administration induced Cox-2 upregulation and PGE2 synthesis favoured IL12 production from dentritic
cells has also been documented
[37]. DS toxicity from different experiments are documented for long decades of
study. The DS administration is found to increase TNF-ɑ cytotoxicity in HepG2 and Hepa1c1c7
cells
and TNF-ɑ release from activated Kupffer cells [38,39]. The TNF-ɑ is found to involve
in both pro-inflammatory and anti-inflammatory conditions. The DS associated reduced inflammation is attributed to NFκB pathway and followed by the transcriptional factors gene expression activated [40]. In this present study the LDH activity found
reduced with DS treatment and unaltered in FCA+FIA group. The reduced LDH
activity in DS is positively implicated upon its role on the anti-inflammatory
activity as discussed.
AST and ALT
AST and ALT enzymes activity has important
roles in brain region glucose
metabolism, neurotransmitters production, synapses maintenance, amyloid-β and tau accumulation.The reduced ALT and AST activity is implicated to reduced pyruvate production, altered gluconeogenesis and CSF glutamate
levels [41]. The increased ALT activity is related to glucose metabolism, especially in the bilateral frontal, parietal and temporal lobes. In contrast, the
higher ALT activity is implicated to reduce glucose metabolism in the bilateral frontal, parietal and
temporal lobes [42] and but which is associated to elevated alkaline phosphatase activity. This study
also found that, the increased AST to
ALT ratio is associated to reduced CSF
amyloid-βaccumulation, higher CSF p-tau
and t-tau deposition. In addition, AST reactionare found to induce α-ketoglutarate, aspartate and the altered ammonia
generation in the neuronal mitochondria environment. Those metabolites are largely utilized by astrocytes during normal and pathogenic situations. Neuron lack beta-oxidation enzymes and
profoundly uses amino acids, ketone bodies, citric acid cycle intermediates, pyruvate
and lactate for energy purpose. The
large change which
takes
places in amino acids, glutamine, glutamate, aspartate,
alanine
levels in the inflammatory brain must be considered to be the driving force for the inflammatory progression. These precursors are found to be taken by aminotransferases immediately [43]. In
this present study is decreased AST and ALT in FCA+FIA was observed and elevated
activity was seen in DT treatment. The elevated AST and ALT in DS treatment is
positively correlated to the glutame levels in the brain region and may be associated with the increased glucose metabolism
especially in the bilateral frontal, parietal, and temporal lobes [44]. The decreased levels of
ALT found with FCA+FIA treatment may implicate greater
amyloid-β deposition and stress environment in the brain region.
Similarly, the reduced ALT and the elevated alkaline phosphatase activity in the
FCA+FIA group were also attributed to the brain inflammatory situation persist
in the present investigation [45,46].
CONCLUSION
In this present study, reduced ACP activity in
DS treatment and unaltered in FCA+FIA group is taken as no vertical imbalance in the anti-oxidative and lipid peroxidation system. The reduced
ACP activity in DS group below Control rats may implicate upon corresponding
toxicity in the brain oxidative metabolism and can be attributed to be active bone lesion/turnover. The
increased ALP activity in the FCA+FIA group may result from the central nervous system injury. The decreased
activity with DS treatment can be taken as oxidative stress. Since, variable metabolic pathways will lead to region-specific accumulation of
oxidative damage in different regions of the CNS and can cause specific brain regions
to become more vulnerable to drug toxicity. In this present study the LDH
activity was found reduced with diclofenac treatment and was unaltered in
FCA+FIA group. The reduced LDH activity in diclofenac is positively implicated
upon its role on the anti-inflammatory activity as discussed. The elevated AST
and ALT in DS treatment is positively correlated to the glutamate levels in the
brain region and may be associated
with the increased glucose metabolism
especially in the bilateral frontal, parietal, and temporal lobes. The decreased levels of
ALT found with FCA+FIA treatment may implicate upon greater
amyloid-β deposition and stress situation in the brain region.
Similarly, the reduced ALT and the elevated alkaline phosphatase activity in the
FCA+FIA group is also attributed to the brain inflammatory situation persist in
the present investigation.
AUTHOR CONTRIBUTION STATEMENT
SS conceived, designed research, conducted experiments, analyzed data, wrote the manuscript and Institute central Laboratory has contributed reagents and analytical tools. The author has read and approved the
manuscript.
COMPLIANCE WITH ETHICAL STANDARDS
All applicable international, national,
and institutional guidelines for the care and use of animals were followed.
CONFLICT OF INTERESTS
The authors
declare that they have no conflicts of interest.
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