Keywords
sepsis - molecular docking - tofacitinib - multiple organ dysfunction - cecal ligation
puncture - histopathology
Highlights
-
Sepsis is an uncontrolled response to infection which leads to Multiple organ
dysfunction syndrome (MODS)
-
Tofacitinib (TOFA) is an FDA-approved JAK inhibitor used in autoimmune
diseases
-
Tissue damage in sepsis induced MODS was reduced by Tofacitinib
-
However, a higher dose of Tofacitinib can cause toxicity in kidney and
liver
-
Tofacitnib can be a potential drug candidate against sepsis induced MODS
Introduction
Sepsis, a life threatening condition, emerges from severe viral, bacterial, or fungal
infections, triggering a systemic inflammatory response syndrome (SIRS). It
manifests symptoms like fever, reduced blood pressure (hypotension), and possibly
fatal multiple organ dysfunction or failure, that can even cause mortality [1]. In 2017, an estimated 49 million people
were impacted, contributing to around 11 million potentially preventable global
deaths (WHO). Information from India remains limited, predominantly focusing on
infection epidemiology (both community and hospital acquired), rather than sepsis
as
a host’s infection response [2].
The progression of sepsis unfolds across three stages: SIRS, severe sepsis, and
septic shock. Initial suspicion of sepsis arises from noticeable shifts in body
temperature (extremely high or low), leukopenia, elevated respiratory and heart
rates. These manifestations collectively signify SIRS, which can escalate to sepsis
if an infection is present. As the infection disseminates, it triggers acute organ
dysfunction, leading to more severe outcomes like laboured breathing, reduced
platelet count, decreased urine production, and heightened bilirubin levels in the
liver-hallmarks of “severe sepsis.” Ultimately, this cascade culminates in septic
shock—the most critical sepsis stage. Septic shock is characterized by profound
circulatory, metabolic, and cellular anomalies, accompanied by hypotension
necessitating vasopressor intervention. This stage carries a higher mortality risk
compared to sepsis alone [3]. However,
sepsis-induced immunosuppression through Compensatory Anti-Inflammatory Response
Syndrome (CARS) contributes to secondary infections and delayed mortality [4]. Importantly, both SIRS and CARS
responses in sepsis are orchestrated via the JAK-STAT signalling pathway.
Sepsis leading to multiple organ dysfunction syndrome (MODS) is one of the primary
causes of death in sepsis patients. During sepsis, the kidneys, lungs, and liver are
more susceptible to damage and functional impairment [5]. While most septic patients begin with
the failure of one organ system, such as acute kidney damage requiring renal
replacement treatment, the failure of another organ system frequently follows, and
MODS ensues. There is a substantial correlation between the number of malfunctioning
organ systems and patient outcome [6]. The
exact pathogenic processes that contribute to the development of MODS during sepsis
are still unknown.
Ruxolitinib, Barcitinib, Fligocitinib, Tofacitinib (TOFA) etc. are among some of the
JAK inhibitors which were tested for clinical trials for the regulation of immune
mediated diseases [7]. Tofacitinib is a FDA
(Food and Drug Administered) approved inhibitor of JAK, being used in the treatment
of rheumatoid arthritis which obstructs the signal transduction of inflammatory
cytokines by blocking the JAK STAT signalling pathway [8]. Among the four classes of JAK family
(JAK1, JAK2, JAK3 and TYK2) TOFA preferentially inhibits JAK1 and JAK3 [9]. Earlier, studies showed that the TOFA
has been an effective drug in inflammation related disorders such as ulcerative
colitis [10], psoriasis [11], pemphigus vulgaris [12] etc. However, the potential benefits of
TOFA treatment in the context of sepsis induced multi-organ failure remain
relatively unexplored. Present study is an attempt to investigate the protective
role of TOFA against the multiple organ dysfunction (liver, kidney, lungs and
spleen) in the CLP model of sepsis.
Materials and Methods
Molecular Docking
Molecular docking is a structural and computational tool to find out the
ligand-protein binding conformations. In the present study docking was used to
predict the binding energy of JAK1 and JAK3 with tofacitinib. The chemical
structures and associated data of Tofacitinib (ligand), was downloaded from
Pubchem in SDF format [13]. Further,
SDF format was converted into PDB format by using the open babel tool [14]. And finally, it was changed into
PDBQT format. Structures of the receptor JAK1 (6N7A) and JAK3 (5LWM) were
downloaded from the PDB database [15]
[16]
[17]. Missing loops in the structures
were modulated by modeller which is a plugin of chimera molecular visualization
tool [18]
[19]. JAK1 and JAK3 were converted into
PDBQT format from PDB format. This was achieved by adding charge and H bonds
using AutoDock-4 [20]. Now, both ligand
and receptor were compatible for docking by Autodock vina [21]. Furthermore, tofacitinib was docked
against JAK1 and JAK3 individually with 32 exhaustiveness after the preparation
of the grid which enabled the selection of docking sites. Further, the H bonds,
binding energy and interaction of amino acids in JAK1 and JAK3 with tofacitinib
were recorded. Tofacitinib docked location within the active site was
represented by ribbon, hydrophobic surface images, and interaction with JAK1 and
JAK3 amino acids is illustrated by 2D ligplot graph [22].
Mice study in CLP model
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Reagents and equipments
Reagents employed in this study included Dimethyl sulphoxide (DMSO),
Tofacitinib Citrate, Phosphate buffer saline (PBS), Tween 20, Ketamine
(95 mg/kg body weight), Xylazine (12.5 mg/kg body weight), Sterile
saline solution (0.9% (wt/vol) saline), as well as protective gear such
as latex gloves and a face mask. Additional items included were Hair
removal cream, Autoclaved gauze pads, Non-absorbable surgical suture,
Surgical blades, Syringes, and a set of Surgical instruments comprising
dissection scissors, microdissection scissors, straight surgical
forceps, straight anatomical forceps, needle holder, Wax tray and an
Oral gavage 20 were also utilized throughout this investigation.
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Animals
Swiss albino mice were purchased from NIPER (National institute of pharmaceutical
education & research) Mohali, (Punjab) and were acclimatized at room
temperature (25–27°C) with 12-h light and dark cycles for 1 week before surgery.
The mice were supplied with standard laboratory feed and water. The experimental
procedures used in the current study were approved by the Departmental Ethical
Committee, University of Rajasthan, Jaipur.
CLP (Cecal ligation puncture) model creation
Swiss albino mice of about seven to nine weeks, weighing 30–35 g were
anaesthetized by using ketamine (95 mg/kg) and xylazine (12.5 mg/kg) via
intraperitoneal injection and fixed on a wax tray. After disinfecting the
abdominal skin with 70% ethanol, hair removal cream was applied and left for
2–3 minutes. Then cream was removed with a cotton swab and a longitudinal
incision of approximately 1.5–2 cm was made in the skin with a scalpel along the
vertical line above the pubic bone and further with the use of small scissors,
peritoneal cavity was reached, the cecum was identified and was carefully taken
out with the use of blunt anatomical forceps. Cecum was punctured using a
sterilized needle and it was then squeezed gently in order to expel a small
amount of faeces from the site of perforation. The cecum was placed back to the
peritoneal cavity and incision was stitched with the help of non-absorbable
suture. Saline was injected to the mice for resuscitation. Mice were then placed
back into the cages with proper water and food availability.
Experimental Design
Twenty Swiss albino mice weighing between 30–35 g were randomly allocated into
four groups, each consisting of five mice: the control group, the CLP group
(mice subjected to cecal ligation puncture), the 150 mg/kg TOFA group (CLP mice
treated with 150 mg/kg of tofacitinib), and the 300 mg/kg TOFA group (CLP mice
treated with 300 mg/kg of tofacitinib). In the treatment groups, mice were
administered with the first dose of TOFA (dissolved in PBS and tween 80)
150 mg/kg or 300 mg/kg via oral gavage, six hours after the CLP surgery.
Subsequently, three additional doses were administered at 12-hour intervals.
Throughout the experiment, the body weights of mice were recorded and their
short term survival were monitored. Following a six-hour interval from the last
TOFA dose, the experimental mice were euthanized, blood was collected by the
heart puncture method from all the groups, and tissues (Liver, lungs, kidney and
spleen) were collected and stored in 10% formalin for further histopathological
examination ([Fig. 1]).
Fig. 1 The schematic illustration of experimental design.
Histological slide preparation and analysis
To facilitate histological analysis, all collected tissues (including the liver,
lung, spleen, and kidney) underwent embedding in molten wax to create paraffin
wax blocks. These blocks were prepared following a dehydration process,
involving a progressive alcohol concentration series to remove formalin and
water from the tissues. Subsequently, organic solvent (xylene) was employed to
eliminate residual alcohol during the clearing process, allowing for effective
infiltration of paraffin wax. Once solidified, the paraffin blocks were
meticulously sectioned into 5μm-thin sections using a microtome. These thin
sections were then carefully transferred onto slides and subjected to staining
with haematoxylin and eosin. The resulting stained slides were subsequently
examined under a light microscope for detailed histological analysis.
To quantify the availability of alveolar airspace, we employed ImageJ software
[23]. This software allowed for a
detailed analysis by separating the specific area representing alveolar
airspaces. We achieved this through color deconvolution, which essentially
isolates the channel containing only the white color – the color typically used
to represent airspaces in these images. Following this isolation, a threshold
was applied to define a clear distinction between the airspaces and surrounding
tissue. This created a mask, effectively filtering out any unwanted signal.
Finally, the image occupied by this mask (representing the white airspace area)
was measured. This process was repeated for each image, and the mean value was
calculated to provide a quantitative measure of alveolar air space availability
across the samples.
Statistical Analysis
GraphPad Prism5 software was utilized for data analysis and figure preparation.
To evaluate statistical differences among the different treatment groups,
one-way ANOVA with post hoc Tukey’s test was employed, and the differences
between the groups were considered to be statistically significant when P value
was less than 0.05 (95% confidence level).
Results
-
Molecular Docking
The binding energies of tofacitinib with JAK1 and JAK3 were − 8.0 and − 7.9
Kcal/mol, respectively ([Table
1]). Tofacitinib formed an H-bond with leu 109 at 3.07 Å and a
hydrophobic contact with Phe (108), Ala (56), Gly (112), Leu (31), Gly (32),
Arg (137), Asn (158), Asp (171), Lys (58), Val (39), Leu (160) residues in
JAK1’s active site. Tofa formed two hydrogen bonds with JAK3 protein, one
with Lya (42) and the other with Asp (54) that had bond lengths of 3.21 Å
and 3.18 Å, respectively. The amino acids Gly (156), Glu (58), Gln (51), Phe
(20), Gln (175), Tyr (181), Pro (177), Leu (157), and Asp (154) acted
hydrophobically ([Table 1]).
Ribbon and hydrophobic pictures demonstrate that Tofa fits into the active
site of JAK1,JAK3, and interacts with it. The DAF motif binding has a
function in modulating enzyme kinase activity ([Fig. 2]).
-
Granulocyte and Lymphocyte Percentage
Granulocyte and lymphocyte counts were estimated from the blood samples
obtained during autopsy using the Accurex Automated Double Chamber
Haematology Analyzer CBC-360 Plus blood analyzer. A significant increase in
granulocyte percentage was observed in both the CLP and 300 mg/kg TOFA
groups when compared to the control group (P<0.05) ([Fig. 3a]). Interestingly, the
administration of TOFA at a concentration of 150 mg/kg mitigated this
increase in granulocyte levels when compared with control and decreased when
compared with CLP group, but these findings were not statistically
significant. Conversely, no significant change was seen in the impact of
TOFA on agranulocytes, particularly lymphocytes, across all groups ([Fig. 3b]).
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Experimental observations
When compared to the control group, mice in the CLP groups demonstrated
symptoms such as shivering and jerky movements following CLP surgery which
indicates the successful creation of the model. However, in treatment
groups, these symptoms have decreased. Beside this, during organ collection
via dissection, fragile spleen and liver were found in the CLP group. In
addition, spleen enlargement was seen in the CLP group. Body weight
measurements were also recorded for all three days of the experiment,
plotted on graph and analyzed using one-way ANOVA. The results demonstrated
a significant decrease in body weight on both days 1 and 3 of the experiment
(P<0.05) across all groups. [Fig.
3c] illustrates the decline in body weight throughout the duration
of the experiment in the CLP group and as well as in treatment groups which
is attributed to sepsis.
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Short Term Survival Assessment Throughout the Experiment
Throughout the experiment, we closely monitored all groups to assess the
effectiveness of this sepsis model development process and the impact of
TOFA treatment. Notably, all mice in the control and 300 mg/kg TOFA groups
survived throughout the investigation. Conversely, in both the CLP and
150 mg/kg TOFA groups, one mouse per group succumbed, resulting in a
mortality rate of 20% in both of these groups.
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Histological Analysis
Fig. 2 Interactions of compounds dext-linkng molecular docking
(a) JAK1 and Tofacitinib (b) JAK3 and Tofacitinib and (I)
Ribbon surface image (II) 2D ligplot interaction (III) Hydrophobic surface
image.
Fig. 3 Effect of Tofacitinib on CLP induced sepsis in mice. a)
Percentage of granulocytes (P=0.0044) b) Percentage of Lymphocytes
c) Comparison of body weight dext-linkng the experiment. *(P ˂
0.05) vs control group. Statistical significance was assessed by one-way
analysis of variance (ANOVA) with post hoc tukey’s test. Data were
represented as mean±standard deviation (SD), n=5.
Table 1 Docking binding energy and amino acid interaction
with Tofacitinib through H-bond and hydrophobic interaction within
binding pocket.
|
Docked Complex
|
Binding Energy
|
H-bond
|
Hydrophobic Interaction
|
|
JAK1_Tofa
|
− 8.0
|
Leu (109)
|
Phe (108), Ala (56), Gly (112), Leu (31), Gly (32), Arg (137),
Asn (158), Asp (171), Lys (58), Val (39), Leu (160)
|
|
JAK3_Tofa
|
− 7.9
|
Lys (42), Asp (54)
|
Gly (156), Glu (58), Gln (51), Phe (20), Gln (175), Tyr (181),
Pro (177), Leu (157), Asp (154)
|
After CLP surgery, mice were administered tofacitinib at two different
concentrations, and tissue samples (lungs, liver, kidney, and spleen) were harvested
at 48 hours after the treatment. These tissue specimens were processed and stained
with hematoxylin-eosin. Subsequently, the stained slides of these tissue specimens
were observed under light microscope for histological examination to investigate the
influence of tofacitinib on CLP induced septic mouse models.
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Lungs
In the control group, normal airways with thin alveolar lining and no
neutrophil infiltration were observed. On the other hand, the CLP group
exhibited patchy neutrophil infiltration, thickened alveolar walls, and
signs of congestion. Remarkably, the 150 mg/kg TOFA group displayed a
reduction in neutrophil infiltration, alveolar thickness, and reduced
congestion in comparison to the CLP group. At the 300 mg/kg TOFA
concentration, neutrophil infiltration decreased, while alveolar thickness
remained relatively constant ([Fig.
4]). Further, semi-quantitative analysis of alveolar airspace
availability using ImageJ software revealed a significant decrease in
alveolar air space in the CLP group compared to the control group.
Conversely, treatment with TOFA (150 mg/kg and 300 mg/kg) in the CLP group
resulted in a notable increase in available alveolar airspace, as shown in
[Fig. 5].
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Liver
Liver tissue from the control group mice showed a normal hepatic
architecture. In contrast, the CLP-induced group displayed cytoplasmic
degradation, neutrophil infiltration, and reduced hepatocyte regeneration.
The 150 mg/kg TOFA group showed a lower degree of degeneration compared to
its regeneration, along with decreased neutrophil infiltration. However, the
300 mg/kg TOFA group displayed increased hepatocyte degeneration
(hepatocellular apoptosis) relative to its regeneration, coupled with
neutrophil infiltration ([Fig.
6]).
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Spleen
The splenic tissue of the control group exhibited a normal red and white pulp
architecture. In the CLP group, cytoplasmic degeneration was evident, and
during autopsy, spleen dilation and enlargement were also observed in the
CLP group. In the 300 mg/kg TOFA group, degeneration decreased, but clear
differentiation between red and white pulp was not discernible. In contrast,
the 150 mg/kg TOFA group exhibited a distinct partition between red and
white pulp, and there was an increase in the area of red pulp compared to
the CLP group ([Fig. 7]).
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Kidney
Fig. 4 Histological changes in lung parenchyma in septic mice after 48
hrs of treatment by Tofacitinib (H&E stain, 100X).
Fig. 5 Quantification of available airspace in the lungs of all the
groups. Statistical significance was assessed by one-way analysis of
variance (ANOVA) with post hoc tukey’s test. Data were represented as
mean±standard deviation (SD), n=5.
Fig. 6 Histological changes in liver parenchyma in septic mice after
48 hrs of treatment by Tofacitinib (H&E stain, 100X).
Fig. 7 Histological changes in spleen parenchyma in septic mice after
48 hrs of treatment by Tofacitinib (H&E stain, 100X).
The control group displayed a normal renal histological appearance without signs of
congestion or inflammatory infiltration. In the CLP group, renal tubular changes,
glomerular atrophy, and necrosis were observed. Notably, the 150 mg/kg TOFA group
showed some tubular dilation with tubular vacuolization, while the glomerular
structure remained intact compared to the CLP group. However, the 300 mg/kg TOFA
group exhibited a higher degree of degeneration and necrosis relative to all other
groups, along with glomerular atrophy ([Fig.
8]).
Fig. 8 Histological changes in kidney parenchyma in septic mice after
48 hrs of treatment by Tofacitinib (H&E stain, 200X).
Discussion
Sepsis, a life-threatening condition leading to multi-organ damage, remains a major
concern despite advancements in research focused on its treatment [24]. Tofacitinib, an FDA-approved JAK
inhibitor for rheumatoid arthritis [8], has
shown promising protecting effects against sepsis. Recent studies have highlighted
its potential benefits in sepsis, such as its preventive effect against LPS-induced
acute kidney injury [25] and its ability to
reduce acute lung injury and improved survival, in septic rats via JAK/STAT
signalling pathway targeting [26]. Few
previous studies have demonstrated the multifaceted effects of tofacitinib.
Jarneborn et al. observed that tofacitinib pretreatment significantly improved
survival in a staphylococcal sepsis mice model, highlighting its potential role in
sepsis treatment [27]. Additionally,
tofacitinib’s immunomodulatory properties through JAK1 inhibition have been widely
explored in regulating inflammatory bowel disease (IBD) [28]. These findings collectively suggest the
therapeutic potential of tofacitinib against various immunological disorders by
regulating inflammation.
However, the current understanding of tofacitinib’s efficacy in established sepsis
with multiple organ dysfunction syndrome (MODS) remains limited. This study
addresses this gap by investigating the protective effects of orally administered
tofacitinib in a CLP-induced septic mice model, focusing on its ability to mitigate
MODS and improve survival. While previous studies have documented the safety and
efficacy of lower-dose tofacitinib regimens (twice or once daily) in reducing
inflammation, we chose a higher dose specifically for patients with established
MODS, a critical factor contributing to sepsis mortality. This investigation aimed
to explore the potential of higher-dose tofacitinib in regulating tissue damage and
prolonging survival in this severe sepsis scenario. By analyzing tissue
histopathology at two different tofacitinib concentrations, we sought to elucidate
its role in combating MODS and improving outcomes in septic mice. So, 150 mg/kg or
300 mg/kg TOFA doses were administered four times to septic mice, at an interval of
12 hrs in order to evaluate its effect. Six hours after the last dose, tissue
samples were collected for histological analyses.
An elevation in the total white blood cell (WBC) count is often associated with
inflammation and infection. This count can fluctuate in various clinical scenarios,
including non-infectious inflammatory conditions such as rheumatoid arthritis,
lupus, and cancer [29]
[30]. However, in certain sepsis cases, the
WBC count may remain within normal range or even decrease. Consequently, the total
WBC count exhibits low specificity, which limits its effectiveness as a sepsis
biomarker [31]. In addition, several
studies have reported that lymphocyte counts tend to decline during the initial
phase of sepsis and are associated with adverse outcomes [32]
[33]. Therefore, we conducted an analysis of lymphocyte and granulocyte
percentages in each group.
Our study revealed elevated granulocyte percentages in the CLP group and 300 mg/kg
TOFA group, indicating persistent infection and potential TOFA-related hazards at
this dose respectively. Conversely, a decrease in granulocyte percentage at the
150 mg/kg TOFA concentration suggests TOFA’s potential in reducing infection.
Lymphocyte count patterns showed no significant changes among groups, except for a
slight decrease in lymphocytes in all groups compared to the control. Additionally,
a significant drop in mouse body weight was observed on days 1 and 3 of the
experiment.
The surgical CLP procedure induced tissue damage in the lung, liver, spleen, and
kidney, affirming the successful establishment of the septic model. Lung tissue from
the control group served as a representation of normal pulmonary physiology in the
absence of infection. Microscopic comparison between the control and CLP groups
revealed notable neutrophil infiltration in the alveolar spaces of the lungs,
resulting in a reduction of the functional surface area for gaseous exchange.
Conversely, the other two groups receiving 150 mg/kg TOFA and 300 mg/kg TOFA
exhibited remarkably diminished neutrophil infiltration and reduced pathological
severity. Interestingly, earlier studies [26] have also reported comparable effects of TOFA on CLP-induced acute
lung injury in septic rat models, further supporting our findings. It can be
inferred that TOFA administration plays a role in mitigating neutrophil infiltration
at both concentrations. However, it is noteworthy that at the 300 mg/kg
concentration, an unexpected thickening of the alveolar walls with dilated airspaces
was observed. This increased alveolar thickness can potentially impair the diffusion
capacity of gases between the bloodstream and air, leading to respiratory
difficulties.
Comparatively, the CLP group exhibited signs of acute liver injury characterized by
cytoplasmic degeneration, hepatocellular degeneration, and regeneration when
contrasted with the control group. The liver possesses remarkable regenerative
capabilities, allowing it to recover and repair following damage. However, in the
CLP group, the extent of hepatocyte degeneration surpassed that of regeneration,
indicative of liver damage. Additionally, the presence of a substantial influx of
neutrophils in the CLP group corroborates the occurrence of infection, aligning with
previous studies that elucidate the pathogenesis of liver injury in sepsis.
Muftuoglu et al.’s histological findings have also underscored hepatocellular damage
in CLP-induced septic models [34].
Conversely, in the 150 mg/kg TOFA group, reduced neutrophil infiltration into the
liver sinusoidal cavities suggests a reduction in infection, possibly attributable
to the influence of TOFA. However, at the 300 mg/kg TOFA concentration, a notable
increase in hepatocellular apoptosis was observed. This phenomenon may be explained
as a combined effect of sepsis and the potential toxicity of tofacitinib.”
The spleen plays a pivotal role in bolstering phagocytosis and serves as a vital
defense against infections in a healthy body. Individuals with compromised spleen
function are notably more susceptible to developing fulminant sepsis, an ailment
characterized by a high fatality rate [35].
Sepsis patients often exhibit significant apoptosis in spleen tissue, a detrimental
occurrence that, if prevented, could potentially enhance survival rates [36]. In line with the findings of our
current study, the splenic tissue of the CLP group displayed cytoplasmic
degeneration, coupled with an indistinct separation of the red and white pulp,
indicative of the sepsis-induced infection. Conversely, the 150 mg/kg TOFA group
exhibited a clear demarcation between the red and white pulp, signifying TOFA’s role
in re-establishing the marginal zone and showcasing its protective function.
However, within the 300 mg/kg TOFA group, there was a limited recovery of the
marginal zone. Notably, aside from these adjustments, there was minimal observable
tissue damage within the spleen.
The association between sepsis and acute kidney injury (AKI) has been thoroughly
documented and extensively studied. Sepsis is linked to up to 50% of AKI cases, and
as many as 60% of sepsis patients may concurrently experience AKI [37]. In our present study, we observed
several effects indicative of AKI within the CLP group, including renal tubular
dilation, tubular vacuolization, and glomerular atrophy. Renal tubular dilation can
result from pathogenic processes that interfere with absorption and secretion.
Furthermore, cytoplasmic vacuolization in renal tubules is generally associated with
degeneration. Similar histological changes have been observed in a study by Tiwari
et al. conducted on a C57BL/6 mice model of LPS-induced sepsis, where LPS induced
tubular degeneration, mild brush-border loss, and vacuolization in the early
segments of proximal tubules of the kidney [38]. Additionally, glomerular atrophy, possibly attributed to infection,
was also noted within the CLP group. All these forms of tissue damage were notably
reduced in the 150 mg/kg TOFA group, which exhibited a relatively normal renal
architecture when compared to the CLP group. In stark contrast, the 300 mg/kg TOFA
group displayed more severe tissue damage than the CLP group, suggesting that at
this concentration, TOFA may induce kidney toxicity.
Upon comprehensive analysis of the results, it is evident that tofacitinib exhibits
a
protective effect against multiple organ damage at a concentration of 150 mg/kg.
Nevertheless, at the higher dosage of 300 mg/kg, tofacitinib’s toxicological impact
on the liver and kidney was observed, rendering it unsuitable for further
evaluation. Intriguingly, despite these observed toxic effects, no fatality was
observed within the 300 mg/kg TOFA group, though the underlying explanation for this
phenomenon remains unclear. Considering the results, it is quite difficult to say
whether the toxicity is against septic models or in overall healthy mice. In order
to claim that TOFA is toxic at 300 mg/kg concentration in sepsis models further
investigation is required in comparison with mice only administered with TOFA
without CLP surgery.
The molecular docking of tofacitinib against JAK1 and JAK3 shows that Tofa binding
with JAK3 has two H-bonds, although JAK-1 only has one H-bond in the docked position
and TOFA interacts with the DFG motif in both the JAK1 and JAK3 enzymes. DFG-motif
is the activation loop (A-loop), a stretch of 20–30 residues, which serves as the
regulator of kinase activities [39]. Thus,
our molecular docking study indicates that TOFA’s protective effects are due to its
inhibitory interactions with JAK1 and JAK3, which regulate the JAK/STAT inflammatory
signalling pathway and lower cytokine production by inhibiting JAKs [26].
The JAK-STAT signalling pathway holds immense significance in various pathological
conditions such as cancer [40],
immunological disorders [24], and
infectious diseases like sepsis. Moreover, the association and the identification
of
the multiple organ failure in sepsis patients has been previously documented in a
study [41]. Inflammatory cytokines (IL-6,
IL-10, and TNF-α) activate the JAK/STAT pathway, initiating receptor dimerization
and JAK phosphorylation. Subsequently, phosphorylated JAK in turn activates the STAT
by adding phosphate to it and then two phosphorylated STAT binds to each other to
form a dimer. This dimer translocates to the nucleus, regulating gene expression
([Fig. 9]). Targeting JAK receptors
can limit JAK/STAT activation, rendering it a potential therapeutic strategy for
autoimmune and inflammatory conditions, including sepsis [42].
Fig. 9 Binding of inflammatory cytokines to the receptor leads to the
dimerization of the receptor and phosphorylation of JAK. Activated JAK then
phosphorylates the STAT and phosphorylated STAT then forms a dimer which
enters to the nucleus in order to regulate gene expression by binding to the
promoter.
The present study underscores the protective potential of TOFA at a concentration
of
150 mg/kg, suggesting its ability to modulate the JAK/STAT signalling pathway.
Future research employing immunohistochemistry and western blotting can offer deeper
insights into CLP induced cytokine rise in sepsis and TOFA’s role in reducing this
cytokine production. Assessing cytokine expression levels will provide a more
comprehensive understanding of TOFA’s mechanism of action within the JAK/STAT
signalling cascade during sepsis. Additionally, to evaluate TOFA’s long-term effects
and survival outcomes in this sepsis model, it is imperative to maintain and monitor
the experimental mice over an extended period.
Conclusion
In conclusion, the results of this study showed that administering TOFA at a dosage
of 150 mg/kg can effectively mitigate tissue damage in CLP induced septic mice.
TOFA, on the other hand, has toxic effects in the liver and kidney at concentrations
of 300 mg/kg. It was shown that TOFA plays a protective role in the multiple organ
dysfunction in CLP-induced sepsis models. Given TOFA’s proven status as an
FDA-approved JAK inhibitor, these findings suggest that the drug is a good
therapeutic candidate for the treatment of MODS in sepsis.
Ethics approval and consent to participate
Ethics approval and consent to participate
Study is approved by the Departmental Ethical Committee.
CRediT Author Statement
Vaishnavi Singh: Experimentation, Methodology, Writing- Original draft preparation.
Kavita Joshi: Experimentation, Methodology, Writing- Original draft preparation.
Samit Chatterjee: Methodology, Reviewing and Editing. Sameer Qureshi: Data curation,
Investigation. Snigdha Siddh: Data curation, Investigation. Vandana Nunia:
Conceptualization, Supervision, Methodology.
