JNK‐IN‐8 treatment improves ARDS‐induced cognitive impairment by inhibiting JNK/NF‐κB‐mediated NLRP3 inflammasome

Abstract Purpose Cognitive impairment is a critical complication of acute respiratory distress syndrome (ARDS). However, effective interventions are lacking. Growing evidence demonstrates that c‐Jun N‐terminal kinase (JNK)‐mediated neuroinflammation is involved in the development of ARDS. Therefore, we hypothesized that the JNK pathway is involved in ARDS‐induced cognitive impairment. Methods An in vivo rat model of ARDS was established by treating it with lipopolysaccharide. The cognitive function was assessed by behavioral tests. The levels of pro‐inflammatory cytokines, JNK and NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3) were analyzed by enzyme‐linked immunosorbent assay, western blot, or immunohistochemical analysis. Results We found that JNK inhibitor 8 (JNK‐IN‐8) alleviated cognitive impairment, neuroinflammation, and NLRP3 inflammasome activation in the ARDS rat model. Additionally, an in vivo study showed that the protective effect of JNK‐IN‐8 on cognitive impairment was blocked by nigericin, an NLRP3 activator. Conclusions Our data suggest that JNK‐IN‐8 treatment improves ARDS‐induced cognitive impairment by inhibiting the JNK/nuclear factor‐κB‐mediated NLRP3 inflammasome.

impairment and other neurological deficits (Ermis et al., 2021;Graf et al., 1990). Although great attention has been paid to ARDS in recent decades, patients with ARDS remain at a high risk of long-term cognitive impairment, and the related mechanisms remain unclear (Brown et al., 2019;Thompson et al., 2017;Torbic & Duggal, 2018).
A previous study JNK-IN-8 improves functional recovery by inhibiting neuroinflammation in ischemic stroke (Zheng et al., 2020). Thus, we hypothesized that JNK-IN-8 could improve ARDS-induced cognitive impairment by suppressing JNK/NF-κB signaling. We established an LPS-induced ARDS rat model and investigated the effects of JNK-IN-8 on ARDS-induced cognitive impairment and neuroinflammation.

Animals
All animal procedures were performed according to the ARRIVE guidelines to minimize the number of animals used and their suffering.

LPS-induced ARDS rat model and drug administration
Rats were anesthetized by inhalation of 2% isoflurane (Sigma-Aldrich, MO, USA) and then subjected to intratracheal instillation of LPS (5 mg/kg) from Escherichia coli O111:B4 (Sigma-Aldrich) to establish an experimental ARDS model, as previously described Rocha et al., 2021). Sham rats were subjected to intratracheal instillation of phosphate-buffered saline (PBS, 100 μL).
To investigate the role of the JNK pathway in ARDS-induced cognitive impairment, 36 rats were randomly assigned to 3 groups as

Behavioral tests
The Morris water maze (MWM) test was used to assess the cognitive impairment level of each group. The rats were forced to search for the platform (d = 18 cm; 2 cm below the water level) in a rounded water tank (d = 188 cm). The tank was filled with opaque water at a temperature of 25 ± 2 • C. For the first week, the rats were trained to search for the platform four times a day. The rats were randomly released into one F I G U R E 2 c-Jun N-terminal kinase inhibitor 8 (JNK-IN-8) inhibited microglial activation and neuroinflammation in vivo following acute respiratory distress syndrome (ARDS) induction: (A and B) microglial activation in the brain tissue of ARDS rats was analyzed by immunofluorescence. Scale bar = 50 μm; (C) tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β expression levels in the brain tissue of ARDS rats were detected by enzyme-linked immunosorbent assay (ELISA). *p < .05, **p < .01, and ***p < .001.

F I G U R E 3 c-Jun N-terminal kinase inhibitor 8 (JNK-IN-8) suppressed JNK and nuclear factor (NF)-κB pathway activation: (A and B) western
blotting and semiquantitative analysis for phosphorylated (p)-JNK and JNK in the brain tissue of acute respiratory distress syndrome (ARDS) rats; (C and D) western blotting and semiquantitative analysis for p-p65 and p65 in the brain tissue of ARDS rats. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *p < .05, **p < .01, and ***p < .001.
quadrant of the water. The rats were permitted to rest for 20 s upon reaching the platform. If none of the rats reached the platform within 90 s, they were placed on it for 20 s. After the last training session on the seventh day, the rats were subjected to drug or vehicle administra-

Immunohistochemistry (IHC) and immunofluorescence
At 24 h after ARDS induction, three rats from each group were deeply anesthetized with pentobarbital sodium (40 mg/kg), followed by transcardial perfusion with 100 mL PBS and 300 mL 4% paraformaldehyde (PFA; C104190; Aladdin, Shanghai, China). The hippocampal and cortex tissues were fixed in 4% PFA for 12 h and embedded in optimal cutting temperature compound (Sakura, CA, USA). Hippocampal tissue slices (50 μm) were renatured in citrate buffer (pH 6.0) at 108 • C for 5 min. For immunohistochemistry (IHC), the renatured slices were pretreated with 1% H 2 O 2 at room temperature (RT) for 15 min. After three PBS washes, the slices were masked with 10% NGS for 1 h in PBS.

Western blot
Three rats from each group were euthanized, as previously described, and their hippocampal tissues were collected. The tissue was homoge-

Statistical analysis
All digital image data were quantified using the ImageJ software.
Western blotting and ELISA were performed in triplicates. All values are displayed as the mean ± standard error of the mean. The differences between two groups were verified via Student's t-test or one-way ANOVA using SPSS 20.0. Statistical significance was set at p < .05.

JNK-IN-8 improved ARDS-induced cognitive impairment
To examine the effect of JNK-IN-8 on ARDS-induced cognitive impairment, we performed the MWM test. In particular, we evaluated the cognitive function of ARDS rats with or without JNK-IN-8 treatment.
In the hidden platform test (Figure 1a

JNK-IN-8 suppressed microglia-mediated neuroinflammation after ARDS induction
We assessed microglial activation using an immunofluorescence assay to explore the underlying mechanism by which JNK-IN-8 improved ARDS-induced cognitive impairment. Figure 2a,b shows that the microglial activation was markedly increased in ARDS rats, as evidenced by intensive Iba-1-positive staining, whereas the effect was significantly suppressed by JNK-IN-8.
Given that neuroinflammation plays an important role in the development of cognitive impairment, we used ELISA to assess the production of pro-inflammatory cytokines in the brain tissues. As shown in Figure 2c, the IL-1β, IL-6, and TNF-α expressions were markedly upregulated in the ARDS group, and this effect was markedly

JNK-IN-8 suppressed JNK and NF-κB pathway activation
We assessed JNK pathway activation by western blotting. Figure 3a,b shows that p-JNK levels in the brain tissue of ARDS rats were significantly increased compared with those of control rats, and this effect was blocked by JNK-IN-8.
A previous study has suggested that JNK-IN-8 can inhibit NF-κB activation in LPS-induced acute lung injury (Du et al., 2021). NF-κB is a key upstream inducer of pro-inflammatory cytokines in microglia.
As a result, we looked into whether inhibiting JNK-IN-8-induced neuroinflammation in ARDS rats is connected to the activation of NF-κB pathway. As shown in Figure 3c,d, p-p65 levels in ARDS rats were significantly increased compared with those in control rats, and this effect was blocked by JNK-IN-8.

JNK-IN-8 inhibited NLRP3 inflammasome activation
Many studies have suggested that NF-κB plays an important role in NLRP3 activation, and that the NLRP3 activation in macrophage is considered a key mediator of neuroinflammation. Consistent with these conclusions, our results found that the activation of NLRP3 inflammasome was mainly localized in macrophages in ARDS mice model ( Figure 4a). Next, we evaluated NLRP3 inflammasome activation in the brain tissue of ARDS rats. As shown in Figure 4b,c, NLRP3, ASC, caspase-1, and IL-1β expressions were markedly increased in ARDS rats, and this effect was blocked by JNK-IN-8. More importantly, in addition, IHC staining for NLRP3 and caspase-1 in the cortex of brain tissue further confirmed this conclusion (Figure 4d). Therefore, our results indicate that JNK-IN-8 treatment inhibited NLRP3 inflammasome activation in ARDS rat brain tissue.

JNK-IN-8 improved ARDS-induced cognitive impairment by mediating NLRP3 inflammasome activation
To confirm that the treatment effects of JNK-IN-8 are dependent on NLRP3 activation, we used nigericin, an NLRP3 activator, to regulate NLRP3-mediated inflammation. We performed the MWM test to investigate the cognitive function of ARDS rats treated with JNK-IN-8 with or without nigericin. Figure 5a,b shows that in the hidden platform test, the escape time prolonged by ARDS was markedly decreased by JNK-IN-8 treatment; however, this effect was reversed by nigericin treatment. Consistently, in the space exploration experiment, the platform crossing times decreased by ARDS were markedly increased by JNK-IN-8 treatment; however, this effect was reversed by nigericin treatment (Figure 5c,d). Together, these data suggest that JNK-IN-8 improves ARDS-induced cognitive impairment by mediating NLRP3 inflammasome activation.

DISCUSSION
ARDS is a common and serious acute lung injury, with high morbidity and mortality rates. Furthermore, cognitive impairment is Previous research has revealed that ARDS-induced cognitive dysfunction may be caused by a variety of factors. One of these factors is hypoxemia, which is often seen in ARDS patients due to impaired alveolar gas exchange and mechanical ventilation. Prolonged hypoxemia can lead to cerebral hypoxia and ultimately result in cognitive impairment. Additionally, the inflammatory response associated with ARDS, which can spread throughout the body, including the brain, may also contribute to cognitive dysfunction by damaging brain cells.
Another factor is hypotension, which is frequently experienced by ARDS patients and can lead to inadequate cerebral blood flow, causing hypoxia and nutrient deprivation that can further impact cognitive function. Furthermore, medications used to treat ARDS, such as sedatives and muscle relaxants, may also affect the brain and contribute to cognitive impairment (Sasannejad et al., 2019). Among these factors, neuroinflammation has been identified as a significant pathogenic mechanism of ARDS-induced cognitive impairment. However, despite the recognition of this mechanism, appropriate interventions to suppress neuroinflammation remain lacking. JNK signaling is associated with microglial activation, neuroinflammation, and the development of cognitive impairment. In addition, NF-κB signaling, a key upstream inducer of pro-inflammatory cytokines (Cheng et al., 2022), is activated by JNK. Therefore, several JNK inhibitors have been used to improve cognitive function. Carboni et al. (2008)  A growing number of studies have shown that NLRP3 inflammasomes are regulated by NF-κB signaling (Wang et al., 2018;Yu et al., 2017). Furthermore, the NLRP3 inflammasomes contribute to the microglia-mediated neuroinflammation (Heneka et al., 2014). Therefore, we hypothesized that JNK-IN-8 inhibits neuroinflammation by regulating NLRP3 inflammasome activation. As expected, our data showed that the NLRP3 inflammasome was activated in the ARDS rat brain, and that this effect was blocked by JNK-IN-8 treatment.
More importantly, the MWM study showed that the protective effect of JNK-IN-8 on cognitive impairment was blocked by the NLRP3 activator nigericin, suggesting that JNK-IN-8 treatment improved ARDS-induced cognitive impairment by suppressing the JNK/NF-κBmediated NLRP3 inflammasome.