JNK inhibition mitigates sepsis-associated encephalopathy via attenuation of neuroinflammation, oxidative stress and apoptosis

doi:10.21203/rs.3.rs-4872005/v1

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JNK inhibition mitigates sepsis-associated encephalopathy via attenuation of neuroinflammation, oxidative stress and apoptosis

https://doi.org/10.21203/rs.3.rs-4872005/v1

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Sepsis-associated encephalopathy (SAE) is a severe complication of sepsis, leading to cognitive dysfunction and neuronal damage. C-Jun N-terminal kinases (JNKs), a subset of the MAP kinase family, have attracted substantial interest for their role in cellular events during sepsis conditions. Previous investigations have established the involvement of JNK signaling against memory impairment and abnormal synaptic plasticity. However, the present study is the first to investigate the effects of JNK inhibition in sepsis-associated cerebral injury and cognitive impairments. This study investigated the neuroprotective effects of SP600125, a selective JNK inhibitor, in cecal ligation and puncture (CLP) mouse model of sepsis. CLP-induced sepsis resulted in significant cognitive impairments, as assessed by the open field test, inhibitory avoidance test, morris water maze, and novel object recognition test. Additionally, septic mice exhibited increased serum levels of neuronal injury markers (S100B and NSE), pro-inflammatory cytokines (TNF-α and IL-1β), and oxidative stress markers (MDA), along with decreased antioxidant levels (GSH, SOD, and CAT). Histological analysis revealed neuronal pyknosis, degeneration, and loss of Nissl bodies in the cortex and hippocampus of septic mice. Furthermore, sepsis-induced blood-brain barrier dysfunction was evident from increased cerebral edema. Treatment with SP600125 (10, 30, and 50 mg/kg) significantly attenuated CLP-induced cognitive deficits, neuronal injury, neuroinflammation, oxidative stress, and apoptosis in a dose-dependent manner. The present study provides preliminary evidence that JNK inhibition by SP600125 exerts neuroprotective effects against sepsis-induced encephalopathy in vivo via suppression of neuroinflammation, oxidative stress, and apoptosis.

Sepsis

SAE

SP600125

inflammation

cognition

JNK

CLP

Sepsis is a life-threatening condition characterized by a widespread inflammatory response to infection. It can progress to septic shock and multiple organ failure, leading to a high risk of mortality in intensive care settings (Singer et al., 2016; Matsuda et al., 2024). According to the latest WHO report from June 2022, sepsis-associated mortality has increased from 20–26.7% of all deaths since 2017. The total burden of sepsis has also risen from 189 to 276 per 100,000 population over the past decade, marking a 46% increase (Fleischmann-Struzek et al., 2020). Recent studies have demonstrated the potential of machine learning in identifying genes linked to multiple organ dysfunctions (Atreya et al., 2024). Despite recent progress, the treatments for sepsis remain limited to antibiotics and fluid resuscitation (Lozano-Rodríguez et al., 2023). However, the increasing use of antibiotics with high resistance levels has raised significant concerns about the efficacy of sepsis treatment. Moreover, the prevalence of treatment paradox underscores the need for a profound understanding of the cellular and molecular mechanisms underlying sepsis-associated multiple organ failure.

The evidence from both preclinical and clinical studies sheds light on the key factors contributing to sepsis, including apoptosis, inflammation, cytokine release, oxidative stress, and systemic stress (Sidheeque Hassan et al., 2023; Bajgai et al., 2024). These factors lead to leukocyte infiltration and multiorgan dysfunction, causing observed phenotypic alterations in sepsis cases (Bode et al., 2023). Notably, the changes observed during sepsis conditions might be attributed to an altered signal transduction pathway, which plays a critical role in cell proliferation, migration, and apoptosis. C-Jun N-terminal kinases (JNKs), a subset of the MAP kinase family, has gained considerable interest for its contribution to cellular events under sepsis conditions (Liu & Lin, 2005; Huang et al., 2024). The MAPK family comprises three genes, JNK1, JNK2, and JNK3, producing ten splice variants. JNK1 and JNK2 are widely expressed in various tissues, including the lungs, liver, and intestine, whereas JNK3 is localized to neurons. The JNK pathway is an essential pathway for signal transduction of many cytokines in the pathogenesis of sepsis, such as TNF-α and IL-1β (Chen et al., 2021; Kim et al., 2023). Once activated, JNK triggers apoptosis and contributes to tissue damage and organ dysfunction (Bloch et al., 2014; Lee et al., 2024). In addition, activation of JNK is critical for a variety of inflammatory disorders (Kumar et al., 2015; Yan et al., 2024). Furthermore, the pharmacological inhibition of the JNK effectively prevents sepsis-associated lung injury (Yang et al., 2023; Gao et al., 2023; Nie et al., 2023), liver injury (Gu et al., 2023; Arunachalam et al., 2023), intestinal injury (Zou et al., 2019; Izadparast et al., 2022), cardiac injury (Huang et al., 2018; Li et al., 2021; Hadi et al., 2023) and kidney injury (Ibrahim et al., 2020; Yang et al., 2023). Studies have shown that pre-treatment with JNK inhibitors has a significant impact on signaling pathways, including JNK/c-Myc (Zhang et al., 2019), Mst1-JNK (Ouyang et al., 2020; Zhu et al., 2022), MKK4-JNK (Gu et al., 2023), JNK-dependent autophagy (Zheng et al., 2020), and Sirt1/FoxO3a (Lee et al., 2018; Zou et al., 2019). Previous studies demonstrated the involvement of JNK signaling against memory deficit and synaptic plasticity (Morel et al., 2018). Considering the beneficial effects exerted by JNK inhibitors in sepsis, a positive therapeutic outcome can be expected by targeting the JNK signaling in the treatment of CLP-induced cerebral injury and cognitive impairment.

JNK inhibition has shown its adequacy in several disorders, nevertheless, its preventive role against sepsis-associated enecphalopathy remains elusive. Consequently, the current study attemps to explore the effects of SP600125, a selective JNK inhibitor, on cerebral injury in an experimental mouse model of sepsis.

EXPERIMENTAL ANIMALS

Swiss albino mice weighing 25–30 grams of either sex were employed in the present study. Animals were acquired from Lala Lajpat Rai University of Veterinary & Animal Sciences (LUVAS) in Hisar (Haryana). The experimental protocol was approved by the Institutional Animal Ethical Committee (IAEC), Central University of Punjab, Bathinda (CUPB/IAEC/2023/15). All animals were housed in three per cage under a controlled temperature of 22–25°C, 20–30% humidity, and a 12-hour light/dark cycle with unlimited access to food and water. The experimental work was carried out in compliance with the Committee for Control and Supervision of Experimental Animals (CCSEA) guidelines and under IAEC supervision (2140/GO/Re/S/21/CCSEA).

DRUG AND CHEMICALS

SP600125 (JNK inhibitor) was acquired from BLD Pharmatech Pvt Ltd (Hyderabad, Telangana, India) and dissolved in 2% DMSO in Saline (0.9% normal saline). The mouse TNF-α, IL-1β, caspase-3, NSE (Neuron Specific Enolase), and S100β ELISA kit were purchased from G-Biosciences Pvt. Ltd (St. Louis, USA). Nissl staining chemical, Cresyl violet stain, was brought from HIMEDIA Laboratories Pvt. Ltd (Maharashtra, India). The dose of SP600125 (10 mg/kg, 30 mg/kg, and 50 mg/kg) was selected based on previously published data (Liang et al., 2020; Urrutia et al., 2014; Xie et al., 2015)

SEPSIS INDUCTION BY CECAL LIGATION AND PUNCTURE

Mice were subjected to Cecal Ligation and Puncture (CLP) surgery for induction of sepsis (Zang, 2010; Toscano et al., 2011; Sidheeque Hassan et al., 2023). The mice were fasted for 24 hours before surgery, and on the day of surgery, the animals were initially anesthetized with a cocktail of Ketamine (0.9 ml) and Xylazine (0.5 ml). Under anesthesia, a 2-cm midline incision was made to expose the cecum, which was then ligated at the base with a 3 − 0 silk suture. Then, the cecum is punctured with an 18G needle, and feces is extruded from the punctured site. Afterward, the cecum was repositioned back in the abdomen cavity, and the incision was closed using surgical sutures. To prevent infection after surgery, dressing with an antiseptic solution of povidone-iodine and neomycin powder as an antibiotic. In addition, Buprenorphine (0.05mg/kg, S.C.) was administered as post-operative analgesia to reduce the after-surgery pain, and fluid resuscitation was done using Normal Saline (37°C, 5ml/100gm, S.C.). Following that, mice were placed in a temperature-controlled room (22°C) with a 12-hour light and dark cycle with free access to food and water.

EXPERIMENTAL PROTOCOL

In the present study, 30 mice of either sex (15 male, 15 female) were used and divided into five groups (n = 6), as follows: (1) the sham group (2) the CLP group (3) the CLP + SP600125(10mg/kg, i.p.) (4) the CLP + SP600125 (30mg/kg, i.p.) (5) the CLP + SP600125 (50mg/kg, i.p) (Table 1). The Sham and CLP group animals received normal saline per their body weight. SP600125 was freshly prepared on the day of treatment and administered intraperitoneally once a day for six days following surgery. The following behavioral and memory parameters were performed on day seven and from day eight to twelve, respectively. After twelve days, animals were sacrificed via cervical dislocation, and blood was collected using cardiac puncture. The levels of S100β, NSE, Caspase-3, Bcl₂, TNFα, and IL-1β were measured by using specific ELISA kits. MDA, reduced GSH, SOD, and Catalase were measured using a specific assay method, while the wet/dry ratio method was used to estimate brain edema. The cerebral morphology was evaluated using Nissl staining. (Fig. 1)

BEHAVIORAL EVALUATION

Six days following surgery, the animals were subjected to four distinct behavioral assessments in a separate room with low lighting and silence. One animal underwent only one test at a time, and the animal's behavioral were monitored using video tracking software (Maze Master software-version:5). All behavioral procedures were carried out between 7:00 AM to 2:00 PM.

4.5.1 Open Field Test

After six days of dosing, i.e., on the seventh day after surgery, animals were evaluated in the open-field test. This test was performed in a wooden box measuring 45 cm x 45 cm with a height of 25 cm. The test was used to evaluate locomotor and exploratory ability. The box floor was divided into sixteen black and white squares alternatively. Each trial lasted for 5 min, and during each trial number of crossings, poking, and rearing was recorded to determine the exploratory behavior. The number of lines crossed in the arena denoted the total number of crossings. In contrast, a number of poking and rearing was checked by observing the head touching of animals towards walls and elevation on rear paws, denoting exploratory behavioral (Bali & Jaggi, 2015; Sree et al., 2023).

4.5.2 Step-down Inhibitory Avoidance Test

This task evaluates the aversive memory. For training, mice were placed on a platform measuring 10 cm x 7 cm x 2 cm in the center of an inhibitory avoidance box. The box floor was made up of conductive metal grids, spread 1cm apart, and connected to a current source with a measurement of 38 cm x 25 cm x 30 cm. When mice tried to step down the platform via four paws, a foot shock of 0.7mA was induced at once for 2 seconds. Twenty-four hours after the training, mice were placed on the platform with four paws recorded. Five minutes was used as a ceiling time for the test session (Zhou et al., 2021).

4.6 MEMORY EVALUATION

4.6.1 Morris Water Maze

From day eight to twelfth, the MWM test was performed in a circular pool with a 180 cm diameter; this test was conducted to assess spatial learning and memory. The platform was kept in quadrant 1, one of four equally sized quadrants of the pool. In a water maze, animals often find the hidden platform within 60 seconds; however, if an animal cannot do so, it is escorted to the platform and permitted to stay there for 30 seconds. We kept track of how much time each animal spent in the platform quadrant throughout the trial. During the retention experiment, the hidden platform was removed from the pool, and mice had 120 seconds to locate it. The time spent swimming in pool quadrant 1, where the platform had been installed, was recorded to assess spatial memory (Morris, 1984).

4.6.2 Novel Object Recognition Test

From the eighth to the twelfth day after the surgery, this test was performed in an open-field box of 45 cm x 45 cm x 25 cm. For the test, two similar objects, say (A), and two different objects (B & C) were used. Firstly, two similar objects (A) were placed at two adjacent corners of the box, and mice were placed in the center and allowed to explore freely for 5 minutes. One and a half hours later, short-term memory was evaluated by replacing one similar object (A) with a new object (B), and the mice were placed in the box's center again for 5 minutes. Further, on the next day or after 24 hours, long-term memory was evaluated by replacing another familiar object (A) with a new object (C), and the mice were again placed in the box for 5 minutes. All objects (A, B, C) were of the same colour, size, and texture but had different shapes, i.e., square, triangular, and cylindrical. To prevent displacement due to the exploratory behaviour of animals during test sessions, objects were taped to the box of the open field apparatus. Sniffing (exploring the object from 3–5 cm away) or touching the object with the nose or forepaws during the test was considered a criterion to check the exploration (Leger et al., 2013; Lueptow, 2017). The discrimination index was calculated for each animal as,

Recognition index (R.I) = (TB)/(TB + TA) * 100

where TA – Familiar object (A) exploration time

TB – Novel object (B) exploration time

4.7 CEREBRAL EDEMA ASSAY

Brain edema assay was carried out using the wet-dry method to determine the amount of water in the brain. The fresh brain weight was estimated after the animal sacrifice; this weight was referred to as the wet weight. The complete brain was dried in an oven at 100°C for 24 hours to determine the dry weight (Ghasemloo et al., 2021).

The following equation was used to determine the brain’s water content in different groups;

Cerebral Edema = [(wet weight) - (dry weight)]/ (wet weight) x 100

4.8 PREPARATION OF BRAIN TISSUE HOMOGENATE

The brains were removed via cervical dislocation under anesthesia of ketamine/xylazine and homogenized in 10% phosphate buffer (pH 7.4) using a homogenizer (REMI Motors Pvt. Ltd). After this, the homogenate was centrifuged at 10000 rpm for 10 minutes at 4°C was performed, and the clear supernatant was used to estimate MDA, GSH, SOD, and CAT.

4.8.1 Protein Estimation

The total protein content was determined by using the Lowry method (Lowry et al., 1951) using bovine serum albumin (BSA) as a standard.

4.9 ASSESSMENT OF OXIDATIVE STRESS MARKERS

4.9.1 Estimation of MDA level

The estimation of total MDA level by Thiobarbituric acid reactions (Ohkawa et al., 1979). 25 µl of sample solution along with 25 µl of 8.1% sodium dodecyl sulfate (SDS) (Sigma-Aldrich, USA), 187.5 µl of 0.8% of thiobarbituric acid (TBA) (Sigma-Aldrich, USA), 187.5µl of 20% acetic acid (Sigma-Aldrich, USA), and 75µl of distilled water was incubated at 95°C in the water bath for 1 hour and further cooled with tap water. Then, the mixture is centrifuged at 10,000 rpm for 10 minutes. The absorbance was measured at 532nm by separating the upper layer of the mixture. For standard, tetramethoxypropane was used in different concentrations as per protocol. MDA levels were expressed as ng/mg protein (Choubey et al., 2019).

4.9.2 Estimation of Reduced Glutathione level

The reduced GSH level in brain tissue was estimated using Ellman’s method, 1959 (Ellman, 1959). Briefly, the homogenate was mixed with an equal volume of 50% TCA and vortexed for 10 min. The mixture was allowed to stand for 5 min before centrifugation for 15 min at 10000 rpm. The supernatant (66 µl) was then transferred to a new set of test tubes, 131 µl of Tris buffer was added, and then 3 µl of the Ellman’s reagent (DTNB) (0.1 mM) was added and prepared in 0.3 M phosphate buffer. The absorbance of the solution was measured at 405 nm against blank, and the amount of reduced GSH was expressed as per mg of protein.

4.9.3 Estimation of Superoxide Dismutase Activity

The estimation of SOD level by Marklund,1974 method (Marklund & Marklund, 1974; Gao et al., 1998). 100 µl of cytosolic supernatant was mixed with Tris Hcl buffer, and the volume was made up to 3 ml. Then, 25 µl of pyrogallol was added to it. The change in absorbance after the addition of pyrogallol in each sample was measured at 30-second intervals for 3 min at 420 nm using a spectrophotometer. The SOD activity was expressed as ng/mg protein.

4.9.4 Estimation of Catalase Activity

The estimation of catalase by Luck, 1963 method (H, 1963). 100 µl of the supernatant was added to 1.9 ml of 50mM phosphate buffer (pH 7.0) in the cuvette. The addition of 1 ml of freshly prepared 30 mM H₂O₂ initiated the reaction. The rate of decomposition of H₂O₂ was measured spectrometrically, and the alteration in absorbance was done for 2 min at 30-second intervals at 240 nm. The catalase activity was expressed as U/mg protein.

4.10 BLOOD SAMPLE PREPARATION

Blood samples were taken via cardiac puncture on the twelfth day of the experimental protocol. Then, serum was immediately extracted with the help of a refrigerated centrifuge at (10000 rpm for 10min at 4°C temperature) and stored at -80°C till further investigation.

4.10.1 Biochemical Markers of Cerebral Injury

The serum levels of S100β and NSE were measured using ELISA kits per the manufacturer’s instructions.

4.10.2 Cytokines measurement

The level of TNF-α and IL-1β in the serum was determined in the supernatant obtained after the process of centrifugation of blood by using ELISA detection kits as per the manufacturer’s instruction.

4.10.3 Estimation of Apoptotic markers

The level of caspases-3 (pro-apoptotic marker) and Bcl-2 (anti-apoptotic marker) in the serum was determined in the supernatant obtained after the process of centrifugation of blood by using ELISA detection kits as per the manufacturer’s instruction.

4.11 HISTOPATHOLOGY

The brain tissues were removed after sacrifice and immediately preserved in 10% buffered formalin for 3–4 days. The next day, tissues were fixed in paraffin wax, and the block was prepared. After that, the blocks were sectioned into 5µm thick pieces for further staining procedure.

5.11.1. Nissl staining:

For the Nissl staining procedure, tissues were first dipped in xylene (5 minutes) before exposure to the altered decreasing concentrations of 100%, 90%, and 70% alcohol (20 seconds in each). After that, tissues were treated with the Cresyl violet solution for (1 minute). Tissue was thoroughly cleaned with ultra-pure water before and after staining with Cresyl violet for 5 minutes, followed by treated with acetone for 10 seconds, then submerged in 90% and 100% ethyl alcohol (20 seconds) and xylene (5 minutes). Finally, slides were coated with DPx mountant and observed using a light microscope (Nikon Eclipse Ei) at 100x magnification (Rousselet et al., 2012; Zhao et al., 2024).

4.12 STATISTICAL ANALYSIS

In the present study, statistical analysis was performed using Graph Pad Prism (Graph Pad Software Inc., La Jolla, CA, USA). One-way analysis of variance (ANOVA) followed by post hoc Tukey’s test was used for comparing the statistical differences among different groups. The results were expressed as mean ± SD, and the value p < 0.05 was considered statistically significant.

Effect of SP600125 on CLP-induced motor performance and non-associative memory deficits:

In the open field test (OFT), parameters such as the number of line crossings, latency to the central area, rearing, and total distance traveled were measured to assess motor performance and non-associative memory deficits. CLP-treated mice spent more time in corners of the open field apparatus and crossed fewer lines, as well as reduced rearing behavior and total distance traveled, indicating impairment in cognitive function. In contrast, treatment of SP600125 (10, 30, and 50 mg/kg) effectively reversed the CLP-induced exploratory activity and memory impairment (Fig. 2).

Effect of SP600125 on CLP-induced recognition memory impairment:

Mice were exposed to novel object recognition tests at separate time intervals, i.e., at 1.5 hours (short-term memory) and at 24 hours (long-term memory), to evaluate their memory and learning ability. Regarding short-term memory assessment, CLP-treated mice spent similar time exploring new and familiar objects, symptomatic of learning deficits. However, animals in the SP600125 (10, 30, and 50 mg/kg) treatment group spent more time exploring the new objects, indicative of improved recognition memory compared to the CLP-treated mice (Fig. 3). Again, while assessing long-term memory, CLP-treated animals spent similar time exploring both new and familiar objects, while SP600125 treated animals explored more time the new object, signifying the preservation of recognition memory during SP600125 treatment.

Effect of SP600125 on CLP-induced impairments in spatial learning and memory:

In the Morris water maze test, parameters such as escape latency (from days 8–11) and time spent in the target quadrant (day 12) were conducted to evaluate the spatial memory and learning ability of mice. On day 4 of the test, once the platform had been removed, it was discovered that mice in the CLP group had considerably decreased retention times in the target quadrant while their escape latency had increased. On day 3 of the test, in comparison to the CLP group. SP600125 treatment, however, significantly increased time spent in the target quadrant while decreasing day 3 escape latency, demonstrating that sepsis-induced cognitive dysfunction was reversed after SP600125 treatment (Fig. 4).

Effect of SP600125 on CLP-induced step-down latency deficits:

The purpose of this test was to determine if mice trained in an inhibitory avoidance apparatus could reliably memorize foot-shock events. Mice exposed to CLP had considerably lower step-down latencies as compared to the sham group, which is a symptom of memory suppression. Whereas those who received the drug SP600125 (10, 30 and 50 mg/kg) displayed improved memory preservation as seen by improved contextual learning and memory and step-down latency (Fig. 5).

Effect of SP600125 on the CLP-induced brain edema:

An intense increase in brain water content was observed in the CLP-subjected mice, confirming that sepsis leads to blood-brain barrier (BBB) impairment. Administration of SP600125 (10, 30, and 50 mg/kg) significantly attenuated the content of brain water in CLP-treated mice (Fig. 6).

Effect of SP600125 on oxidative stress:

By analyzing the amounts of MDA (malondialdehyde), GSH (reduced glutathione), SOD (sodium dismutase), and CAT (catalase) as a measure of oxidative stress, the effect of SP600125 on oxidative stress was determined. MDA level was found to be increased in CLP-treated mice, while GSH, SOD, and CAT significantly diminished as compared to the sham group. Treatment with SP600125 (10, 30, and 50 mg/kg) modulated the level of given biomarkers, such as decrease in the level of MDA along with the up-regulation of GSH, SOD, and CAT content, suggesting the protective effect in the brain by inhibiting the release of oxidative stress markers (Fig. 7).

Effect of SP600125 on CLP-induced cerebral injury:

The serum levels of neuronal injury markers, S100B (S100 calcium-binding protein B), and neuron-specific enolase (NSE) were evaluated via respective kits to assess the extent of neuronal damage. In the CLP group, expression levels of S100B and NSE were considerably higher suggesting that the mice in this group had greater brain tissue damage compared to sham group animals. Treatment of SP600125 (10, 30, and 50 mg/kg) significantly attenuated the levels of S100B and NSE, indicating a decline in neuronal deficit (Fig. 8).

Effect of SP600125 on the CLP-induced alteration of inflammatory cytokine:

The tumor necrosis factor (TNF-α) and Interleukin-1 beta (IL-1β) levels in mice brains were measured in order to assess any inflammation. TNF-α and IL-1β level was found to significantly increase in the CLP-treated mice, indicating that CLP causes the release of inflammatory factors in the brain (Figure pathway). While treatment with SP600125 (10, 30, and 50 mg/kg) treatment attenuated CLP-induced cytokine release (Fig. 9).

Effect of SP600125 on the CLP-induced alteration of apoptotic marker:

The caspase-3 (pro-apoptotic marker) and Bcl-2(anti-apoptotic marker) levels in mice brains were measured to assess apoptosis. Caspase-3 level was significantly increased whereas the Bcl-2 level was increased dramatically in the CLP-treated mice, indicating that CLP causes apoptosis in the brain. SP600125 (10, 30, and 50 mg/kg) treatment modulates the level of apoptotic markers by attenuating the caspase-3 and increasing the Bcl-2 level in a dose-dependent manner, suggesting the protective effect of SP600125 in the brain by regulating CLP-induced apoptosis (Fig. 10).

Effect of SP600125 on the extent of CLP-induced brain injury

To evaluate the extent of brain injury and whether SP600125 abrogated sepsis-associated brain injury, Nissl staining was performed to label alive neurons in the hippocampus. Results showed that the CLP group showed serious neuronal degeneration in the cerebral cortex in the form of highly stained nuclei appearance in the cortex region and the Nissl bodies in the hippocampal CA1 and CA3 region disappeared compared with sham control. Moreover, SP600125 treatment (10, 30, and 50 mg/kg) the morphology of neurons in the hippocampus of mice were improved to a certain extent by reappearance of nissl bodies, and cells in the cerebral cortex region shown to significantly reduced pyknosis, suggesting the protective effect of SP600125 in the brain injury (Fig. 11).

In the present study, induction of sepsis by cecal ligation and puncture produced a significant sepsis-associated cerebral injury and cognitive dysfunction. A significant decline in motor activity exploratory behavior was observed, assessed in terms of the total distance traveled, line crossing, frequency of rearing, and time spent in the central area during the open field test (OFT) performed 7th day post-sepsis. Also, a significant decline in aversive memory (aversive stimulus) was noted as decreased step-down latency in the inhibitory avoidance test (IAT) on the 7th day post-sepsis. Additionally, a significant decline in the spatial learning and memory functions was observed by the escape latency from day 8th to day 11th, and on the 12th day, the time spent in the target quadrant was also decreased in CLP-subjected mice in the Morris water maze (MWM). Also, a significant impairment in the recognition memory was observed by the exploration time for the novel object in the Novel object recognition test (NORT) compared to the sham control group. CLP is the considered gold standard model in experimental sepsis research, as it mimics closely human sepsis conditions. In this model, ligation (which causes trauma and activates inflammatory mediators) and puncture (which increases the distribution of gram-negative bacteria such as E. coli in the abdomen) cause pathophysiological phenomena in the body that partially mimic sepsis-associated encephalopathy and result in neurobehavioral changes (Dejager et al., 2011). Several studies have employed this model to assess sepsis-associated encephalopathy (Ref). Further, OFT (Belviranlı & Okudan, 2015), IAT (Roesler et al., 1999), MWM (Barnhart et al., 2015), and NORT (Szentes et al., 2019) are standardized models to assess the non-associative, aversive, spatial, and recognition memory which is altogether responsible for locomotor and cognitive function. These behavioral research findings demonstrate that sepsis causes severe behavioral changes, including impairment in locomotor and cognitive functions, which closely resemble the clinical state of septic survivors (Barichello et al. 2007; Giridharan et al. 2022).

Further, CLP significantly increased the neuronal injury assessed via elevated S100B and NSE serum levels compared with sham control. S100β and NSE are sepsis-specific markers that suggest damage to glial cells and neurons. S100B is a calcium-binding protein found in the astrocytes of the brain, whereas NSE is located in the cytoplasm of the neuron(Y. Huang et al., 2021). Previous findings have also reported increased levels of these neuronal injury markers which further results in apoptotic cell death and brain damage in CLP (Tiainen et al., 2003; Yao et al., 2014; Y. Huang et al., 2021; Elgendy et al., 2024). Clinical studies have also addressed the clinical significance of higher serum levels of S100β and NSE in sepsis-associated encephalopathy (Hu et al., 2023). These results are consistent with the previous evidence suggesting that sepsis-induced neuronal damage contributes significantly to cognitive impairment (Qin et al., 2021; Zhang et al., 2021).

In a current study, a notable increase in the serum level of TNF-α and IL-1β was observed after CLP, indicative of neuroinflammation. In addition, mice exposed to sepsis exhibited a significantly elevated level of oxidative stress markers including Malondialdehyde (MDA) (a marker of lipid peroxidation), with decreased antioxidant levels such as reduced glutathione (GSH), Superoxide Dismutase (SOD), and Catalase (CAT), indicating that increased oxidative stress contributes to sepsis pathophysiology(Xu et al., 2024). Cytokine activation plays an important function in the onset of inflammation in several inflammatory and neurological diseases (Dias-Carvalho et al., 2024). Sepsis causes a considerable rise in inflammation and oxidative stress (Keshani et al., 2024). Previous studies have also reported that TNF-α, IL-1β, and IL-6 are known to induce neuroinflammation, which has been linked to the development of cerebral injury (Wu et al., 2024). Thus, it is conceivable to infer that activation of the JNK pathway enhances the host inflammatory response and oxidative stress, which in turn induces pathophysiological abnormalities in septic encephalopathy.

In the current study, SAE mice showed increased caspases-3 (a pro-apoptotic marker) and a reduction in Bcl-2 (an anti-apoptotic marker), indicating enhanced apoptosis and neurodegeneration. Furthermore, apoptosis has been linked to immune cell death as a result of increased ROS generation. In addition to ROS, heat, radiation, and proinflammatory cytokines, CLP can trigger apoptosis (Zhang et al., 2014; Li et al., 2024). Previous research in sepsis has revealed that caspases-3 and Bcl-2 are key pathogenic indicators for neuronal apoptosis and play an important role in mediating sepsis and that inhibiting cell death further reduces neuronal damage and SAE.

In CLP-subjected brain tissue, cresyl violet (Nissl staining) staining revealed severe alteration in the structural integrity of the septic brain, including neuronal pyknosis, and degeneration throughout the cortex and hippocampus along with the disappearance of Nissl bodies in the hippocampal part. In addition to this, loss of BBB integrity is a key cause of sepsis-induced cerebral dysfunction and subsequent systemic damage (Gu et al., 2021). Brain water content was measured to better understand BBB function. When compared to the sham control group, CLP-exposed mice showed a substantial rise in cerebral edema.

To further explore the role of JNK signaling, SP600125 is a specific inhibitor of c-Jun N-terminal kinase (JNK), a type of mitogen-activated protein kinase (MAPK). JNKs are involved in regulating various cellular processes such as inflammation, apoptosis, and cell differentiation and was employed in the present study. In the current investigation, treatment with SP600125 at doses of 10, 30, and 50 mg/kg significantly prevented CLP-induced cerebral injury. Further, SP600125-treated septic mice showed a significant increase in total distance traveled, line crossing, frequency of rearing, and time spent in the central area in an open field test in a dose-dependent manner. In the inhibitory avoidance test, SP600125 treatment showed a significant increase in the step-down latency time indicating improvement in aversive memory as compared to the CLP-subjected group. Further, the SP600125 treatment improves the recognition and spatial memory by significantly improving the recognition index, escape latency, and time spent in the target quadrant in dose-dependent manner compared to the CLP group and indicating improvement in locomotor activity and memory function. Previous preclinical studies reported the notable effect of SP600125 in reversing the behavioral abnormalities and improved cognitive deficits associated with β-amyloid (Aβ)-induced memory deficit (Ramin et al., 2011; Solas et al., 2023).

In the present study, SP600125 treatment at dosages of 10, 30, and 50 mg/kg in CLP-subjected mice significantly attenuated the neuronal injury in terms of decreased serum levels of S100B and NSE, indicating the protective role of SP600125 in sepsis-induced brain injury. Several studies have also found that SP600125 treatment improves inflammatory disorders (Kim et al., 2023; Rajan et al., 2024; Sahu & Rawal, 2024). The current investigation found SP600125 treatment (10, 30 and 50 mg/kg) significantly reduced CLP-induced elevated serum TNF-α and IL-1β levels. Additionally, previous studies also found that SP600125 reduced inflammatory factors such as TNF-α, IL-1β, IL-6, and IFN-γ in septic rats (Zhang et al., 2023). Further, lipid peroxidation markers including MDA have been significantly attenuated in SP600125-treated septic mice with elevated levels of antioxidants GSH, SOD, and Catalase, indicating attenuated CLP-induced oxidative stress. Earlier studies have also reported the anti-inflammatory and anti-oxidant potential of SP600125 and also examined the ability of SP600125 to reduce oxidative stress and inflammation in neurological disorders such as Alzheimer's disease (Colombo et al., 2009; Ploia et al., 2011; Zyuzkov et al., 2024), Parkinson’s disease (Crocker et al., 2011; Pragati & Sarkar, 2024; Vasudevan Sajini et al., 2024).

In the present study, SP600125 treatment at 10, 30, and 50 mg/kg significantly decreased the caspases-3 level with improved Bcl-2 level compared with the CLP group, signifying the protective role of SP600125 against sepsis-induced apoptosis. An earlier study also reported the protection of SP600125 against apoptosis (Patil et al., 2024). Loss of BBB integrity is a key cause of sepsis-induced cerebral dysfunction and subsequent systemic damage (Gu et al., 2021). Brain water content was measured to better understand BBB function. When compared to the sham control group, CLP-exposed mice showed a substantial rise in cerebral edema. Thus, treatment of SP600125 at dosages of 10, 30, and 50 mg/kg considerably reduces CLP-induced cerebral edema in the brain. In contrast to animals who were subjected to CLP, the deteriorated brain morphology of both cortex and hippocampus was found to be improved with SP600125 treatment (10, 30, and 50 mg/kg) and showed significantly reduced pyknosis with reappearance of nissl bodies in the hippocampal CA1 and CA3 region. Thus, suggesting SP600125 restores the brain morphology compared to the CLP group.

Based on the present findings, it is plausible to suggest that sepsis activates JNK, which causes neuronal injury by triggering the production of pro-inflammatory cytokines, as well as oxidative stress and apoptosis, resulting in blood-brain barrier dysfunction. SP600125 treatment significantly protects against the sepsis-associated cerebral injury and cognitive deficits via inhibiting JNK activation and further reduction of neuronal damage, inflammation, oxidative stress, apoptosis, and brain edema.

Based on the above findings, it can be inferred that, during sepsis, the JNK pathway is activated, resulting in sustained production and release of inflammatory cytokines, oxidative stress, and pathophysiological abnormalities in septic encephalopathy. Moreover, the administration of SP600125 treatment exhibits a significant improvement in cerebral injury and cognitive impairment. The neuroprotective effects of SP600125 were associated with decreased release of inflammatory cytokines (TNF-, IL-1β), apoptosis (Caspase-3, Bcl-2), oxidative stress (MDA), neuronal injury (S100B, NSE), and improved morphological characteristics of brain tissue in sepsis-induced mice. This supports the hypothesis that JNK inhibition could be a promsising approach management of septic encephalopathy.

Ethical approval: The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC), Central University of Punjab, Bathinda with approval number CUPB/IAEC/2023/15.

Consent to publication: The authors agree to publish the paper in subscription

Consent to participate: All the authors contributed equally.

Author’s contribution: Riya Gagnani: conducted the animal study and wrote the manuscript, Harshita Singh and Manisha Suri: conducted the biochemical study and interpret results, Anjana Bali: designed the experiment, analyzed results and finalized the manuscript. All authors read and approved the manuscript and all data were generated in-house and that no paper mill was used.

Funding: The authors are also grateful to the Science and Engineering Research Board (SERB), New Delhi for funding EEQ project-EEQ/2022/000997 and DST-INSPIRE for funding INSPIRE research fellowship (Reg. No. IF220193).

Acknowledgement: The authors are grateful to Department of Pharmacology, Central university of Punjab, Bathinda and Animal House, CUPB Bathinda for providing the facility to conduct the research.

Competing interest: The authors declare no conflict interest.

Availability of data and materials: The data will be made available on demand.

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Table 1: Experimental Grouping

Sr. No.	Groups	No of animal
1	Sham	6
2	CLP	6
3	CLP + SP600125 (10mg/kg, i.p)	6
4	CLP + SP600125 (30mg/kg, i.p)	6
5	CLP + SP600125 (50mg/kg, i.p)	6
Total Animals		30

No competing interests reported.

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JNK inhibition mitigates sepsis-associated encephalopathy via attenuation of neuroinflammation, oxidative stress and apoptosis

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Abstract

Figures

INTRODUCTION

METHODOLOGY

EXPERIMENTAL ANIMALS

DRUG AND CHEMICALS

SEPSIS INDUCTION BY CECAL LIGATION AND PUNCTURE

EXPERIMENTAL PROTOCOL

BEHAVIORAL EVALUATION

4.5.1 Open Field Test

4.5.2 Step-down Inhibitory Avoidance Test

4.6 MEMORY EVALUATION

4.6.1 Morris Water Maze

4.6.2 Novel Object Recognition Test

Recognition index (R.I) = (TB)/(TB + TA) * 100

4.7 CEREBRAL EDEMA ASSAY

Cerebral Edema = [(wet weight) - (dry weight)]/ (wet weight) x 100

4.8 PREPARATION OF BRAIN TISSUE HOMOGENATE

4.8.1 Protein Estimation

4.9 ASSESSMENT OF OXIDATIVE STRESS MARKERS

4.9.1 Estimation of MDA level

4.9.2 Estimation of Reduced Glutathione level

4.9.3 Estimation of Superoxide Dismutase Activity

4.9.4 Estimation of Catalase Activity

4.10 BLOOD SAMPLE PREPARATION

4.10.1 Biochemical Markers of Cerebral Injury

4.10.2 Cytokines measurement

4.10.3 Estimation of Apoptotic markers

4.11 HISTOPATHOLOGY

5.11.1. Nissl staining:

4.12 STATISTICAL ANALYSIS

RESULTS

Effect of SP600125 on CLP-induced motor performance and non-associative memory deficits:

Effect of SP600125 on CLP-induced recognition memory impairment:

Effect of SP600125 on CLP-induced impairments in spatial learning and memory:

Effect of SP600125 on CLP-induced step-down latency deficits:

Effect of SP600125 on the CLP-induced brain edema:

Effect of SP600125 on oxidative stress:

Effect of SP600125 on CLP-induced cerebral injury:

Effect of SP600125 on the CLP-induced alteration of inflammatory cytokine:

Effect of SP600125 on the CLP-induced alteration of apoptotic marker:

Effect of SP600125 on the extent of CLP-induced brain injury

DISCUSSION

CONCLUSION

Declarations

References

Table 1

Additional Declarations

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