# Reversing Postcardiopulmonary Bypass Associated Cognitive Dysfunction Usingk-Opioid Receptor Agonists to Regulate Microglial Polarization via the NLRP3/Caspase-1 Pathway

Authors: Pei Song; Zhuo Yi; Yiji Fu; Dandan Song; Keyan Chen; Jingjing Zheng; Yingjie Sun; Yugang Diao
Journal: Journal of Healthcare Engineering
 (2021)

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## Abstract

Cardiopulmonary bypass (CPB) is mainly used during cardiac surgeries that treat ischemic, valvular, or congenital heart disease and aortic dissections. The disorders of central nervous system (CNS) that occur after cardiopulmonary bypass are attracting considerable interest. Postoperative neurocognitive disorders (PND) have been reported as the leading cause of patients’ disability and death following CPB. Thek-opioid receptor (KOR) agonists (U50488H) have been suggested to be vital in the treatment of surgically induced CNS neuroinflammatory responses. In this article, the transitions between the M1 and M2 microglial polarization state phenotypes were hypothesized to significantly affect the regulatory mechanisms of KOR agonists on postcardiopulmonary bypass (post-CPB) neuroinflammation. We investigated the effects of U50488H on neuroinflammation and microglia polarization in rats exposed to CPB and explored the method of the NLRP3/caspase-1 pathway. Thirty SD rats were randomly divided into three groups: sham operation group, cardiopulmonary bypass model group, and CPB+ k-opioid receptor agonist (U50488H) group, with ten rats in each group. The Morris water maze was used to evaluate the changes in the cognitive function of CPB rats. Hematoxylin and eosin (HE) staining and TUNEL were performed to assess the rats’ hippocampal damage. Enzyme-Linked Immunosorbent Assay (ELISA) was used to detect changes in brain injury markers and inflammatory factors. Furthermore, immunofluorescence was used to observe the expression of microglia polarization and NLRP3 followed by Western blots to detect the expression of the NLRP3/caspase-1 pathway and microglia polarization-related proteins. Rat microglia were cultured in vitro, with LPS stimulation, and treated with U50488H and a caspase-1 antagonist to evaluate the effects and mechanism of action of U50488H. KORs alleviated hippocampal damage caused by CPB and improved PND. CPB activated the NLRP3 inflammasome and upregulated pro-caspase-1 expression which promoted the expression of pro-IL-lβ and pro-IL-18 and resulted in increased inflammation. However, KORs also inhibited NLRP3 and transformed microglia from the M1 to the M2 state. Caspase-1 inhibitor treatment reduced the microglial polarization induced by KORs. The κ-opioid receptor agonists inhibited the inflammation mediated by microglia and improved PND through the NLRP3/caspase-1 signaling pathway.


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## Body

## 1. Introduction

Cardiopulmonary bypass (CPB) is primarily used during cardiac surgeries that treat ischemic, valvular, or congenital heart disease and aortic dissections. However, post-CPB central nervous system- (CNS-) related complications have gained considerable attention, of which postoperative neurocognitive disorders (PND) have become a leading cause of disability and death of patients [1]. Studies have suggested that CPB-induced inflammatory responses are the primary cause of PND. Thus, reducing PND and mortality through suppressing immunological stress-induced CNS inflammatory responses has tremendous clinical significance [2].CNS inflammation is highly correlated with CPB use during surgery [3]. Several clinical studies have indicated that CPB surgical trauma might lead to immune system activation, and numerous inflammatory factors can be detected in peripheral blood after surgery [4–6]. Microglia (MI) are a type of CNS neuroglial cells and they have functions equivalent to macrophages. Microglia act as immune cells in the CNS and constantly remove damaged and dead neurons, plaque, cellular debris, and pathogens in the CNS [7–9]. MI typically reside in a resting state but are rapidly activated when the CNS is stressed, a process called MI activation [10]. MI activation includes morphological changes that result in rapid transition from a branched form to an amoeba-like shape and increased cell size [11]. In CNS, MI can be activated directly by endogenous harmful stimuli to produce inflammatory factors such as IL-1β, resulting in the production of oxygen free radicals that affect the activity and synapse plasticity of neurons [12, 13]. These factors adversely impact CNS functions, resulting in impaired cognitive function and other dysfunctions. Accumulated evidence in rat studies has suggested that elevated expression of IL-1β impaired spatial learning ability [14, 15]. These studies suggested that surgical trauma activated the immune system, resulting in increased IL-1β content in the hippocampus, which decreased the learning abilities and memory in the affected rats [16].IL-1β is produced through continuous MI activation, primarily by the reactions that take place in the MI cytoplasm [17]. When MI are stimulated by pathogenic bacteria and related factors, damage, and stress, nuclear factor kappa-B (NF-κB) is activated by interleukin-1 receptor 1 (IL-1R1), Toll-like receptors (TLRs), and other receptors or different signaling pathways [18–20]. Activation of these factors promotes transcription of NLRP3, pro-IL-1β, and pro-IL-18 in the cytoplasm, resulting in the activation and functionalization of the NLRP3 inflammasome in the cytoplasm and reduction of the activation domain of the NLRP3 inflammasome [21]. The activated NLRP3 undergoes oligomerization, recruits the linker protein, ASC, and further activates caspase-1 through interactions between the PYD and CARD domains, thereby processing pro-IL-1β into mature IL-1β, which undergoes extracellular release to carry out its functions [22, 23].Opioids are presently the most extensively used analgesic drugs in clinical practice. There are types of typical opioid receptors includingδ-opioid receptors, μ-opioid receptors, and κ-opioid receptors (KORs). The KORs are abundantly expressed in the prefrontal cortex and other brain regions and are reported to carry out various functions, including reducing brain tissue damage and improving functional recovery in animal models of systemic and local cerebral ischemia [24]. KORs agonists are broadly applied in perioperative analgesia due to their robust analgesic effects. They also control emotional and cognitive functions. The KOR agonist, U50488H, was found to decrease cognitive impairment significantly. Pharmacological studies also propose that KORs contribute to the control of chemical, mechanical, and thermal pain, mainly at the spinal level. KORs can alleviate brain damage and increase the restoration of functions in systemic and regional cerebral ischemia in animals. The KOR agonist U50488H was administered in the hippocampus during nerve injury induced by ischemia and the results indicated a substantial reduction in cognitive impairment [25]. However, its exact controlling functions remain to be elucidated. Charron et al. [26] verified that U50488H, a KOR agonist, can stimulate hippocampal cholinergic neurons by motivating KORs on hippocampal nerve cells, which can considerably lessen hippocampal nerve impairment produced by ischemia. Accumulated evidence has revealed that U50488H, a KOR agonist, can rescue choline dysfunction-caused cognitive decline in mice, especially scopolamine-induced cognitive impairment [26]. This effect is related to the cholinergic system and can be reversed by nor-BNI, a selective KOR antagonist.KORs are expected to be a possible treatment for PND caused by CPB used during cardiac surgery. In this study, a CPB rat model was established, and the experimental rats were given the KORs agonist, U50488H, to assess its ability to reduce cognitive dysfunction and hippocampal neuronal damage in mice. Additional investigation was carried out on the inhibitory effects of KORs on the NLRP3/caspase-1 signaling pathway, which suggested a probable procedure for the prevention and treatment of post-CPB PND.The rest of the paper is organized as follows: Section2 provides a detailed explanation of the proposed method and experimental process. In Section 3, different results are presented. Section 4 is about discussion and Section 5 presents the conclusion.


## 2. Material and Methods

### 2.1. Experimental Groups and Animals

In this experiment, thirty male SPF-grade Sprague Dawley rats, weighing 350–450 g, were obtained from the Department of Laboratory Animal Sciences of the General Hospital of Northern Theater Command (laboratory animal user license no. SYXK (Military) 20120007; laboratory animal production license no. SCXK (Military) 20120006). The rats were randomly divided into the three following groups with each group having ten rats: Sham operation group, CPB model group, and a group of CPB rats given U50488H. The animal experiments in this study were approved by the IGDB-IACUC of the General Hospital of Northern Theater Command (IACUC no. 2020013).


### 2.2. Establishment of the CPB Rat Model

Rats were anesthetized with an intraperitoneal injection (30 mg/kg) of pentobarbital sodium (#57-33-0, Sigma-Aldrich, USA) and were positioned on the operating table in the supine position. The rat was intubated and connected to a small animal ventilator and an anesthesia machine. During the surgery, rats were continuously anesthetized with 2% isoflurane (#26675-46-7, Sigma-Aldrich). A 24 G trocar needle was placed into the right femoral artery to infuse 6% hydroxyethyl starch (#9005-27-0, Merck, USA) at a rate of 0.5 ml/h. A 22 G trocar needle was placed onto the left femoral artery to monitor real-time arterial pressure. CPB was established by placing a 22 G trocar needle into the tail artery and another 18 G trocar needle was positioned in the right jugular vein. Venous blood was drained to a blood storage tank after each tube was connected and fixed in place. The blood was returned to the body via the tail artery after diverting the blood via a peristaltic pump and oxygenated with an external oxygenator. The anesthetic level was adjusted as needed based on the blood gas analysis. The physiological parameters of the rats were maintained at MAP > 60 mmHg, pH levels in a normal range, PaCO2 at 35–45 mmHg, and the base excess (BE) at 0 ± 3 mmol/L. Because the isovolemic hemodilution was targeted to achieve an Hct level of 25 ± 1% during the CPB, the flow could be gradually stopped, and mechanical ventilation resumed. With continuous maintenance of the mechanical ventilation, the tubes were removed, and the remaining blood in the reservoir was slowly infused back to the body to maintain circulation stability. Successful establishment of the CPB rat model was characterized by the survival of rats at 6 h after surgery. The rats in the KORA group were injected intravenously 30 min before the CPB procedure with U50488H (#D8040, Sigma-Aldrich) at a concentration of 1.5 mg/kg [27, 28].


### 2.3. Cell Culture

Rat microglial (RM) cells were purchased from ScienCell™ (#R1900, ScienCell™ Research Laboratories, USA), which had been isolated from the cerebral cortex of two-day-old rat pups. Cells were delivered frozen as a secondary culture. When initiating the subculture from the cryopreserved cells, the cells were cultured in an incubator at 5% CO2 and 37°C, using microglia medium (#1901, ScienCell™ Research Laboratories). Then the cells were seeded on coverslips in 12-well plates at a concentration of 5 × 106 cells per well that contained phenol red-free DMEM (HyClone, USA). The RM cells were treated with 10 ng/mL lipopolysaccharide (LPS) (#L6529, Sigma-Aldrich) for 24 h. This in vitro study included the following groups: control RM cell group (RM), LPS-treated RM cell group (LPS), LPS + U50488H RM cell group (LU), LPS + Ac-YVAD-cmk (SML0429, Sigma-Aldrich) RM cell group (LAC), and LPS + U50488H + RS09 (#5928, TOCRIS, UK) RM cell group (LUR). The cells were treated with 100 ng/mL LPS for 24 h, followed by exposure to 1 μmol/L of U50448H for 30 min. Ac-YVAD-cmk was dissolved in 500 μl of DMSO, and the working concentration was 50 μM (27 μg/ml), which was added to the cells for 30 min before the LPS stimulation.


### 2.4. Morris Water Maze

In each group, the rats were trained and tested in the Morris water maze starting on the 21st postoperative day to observe changes in cognitive function. Rats were placed into the maze from a randomized quadrant and facing the pool wall. Each rat was allowed 90 seconds for free swimming to find the hidden platform. The escape latency period was recorded according to the duration of time each rat searched and then the hidden platform located. The Morris water maze experiments were carried out for five consecutive days. The results from the first four days were considered acquisition training, and the test scores obtained on the fifth day were recorded as the spatial learning and memory scores for the rats in each group. The submerged platform was removed 24 h after completion of the hidden platform test. The rats were placed into the water maze at the same entry points as previously indicated. The swimming paths for each rat were recorded for 60 s. The amount of time the rats resided in the target quadrant and the number of times the platform location was crossed were recorded. The data were analyzed using the Morris imaging analysis system.


### 2.5. Tissue Sample Collection

After the Morris water maze experiments were completed, rats in each group were anesthetized with 2% isoflurane and euthanized by exsanguination due to transection of the abdominal aorta. Blood samples were collected and centrifuged at 2,000 ×g for 5 min to obtain the plasma. The plasma samples were stored at −80°C. The brains were dissected, and the tissue samples were separated. A subset of the hippocampus samples were fixed in 4% paraformaldehyde, and the remaining hippocampus samples were stored, unfixed, at −80°C.


### 2.6. Hematoxylin and Eosin (HE) Staining

Paraformaldehyde-fixed brain tissue samples were dehydrated in a graded ethanol series (from 70% to 100%) and then cleared in two changes of xylene. The tissue samples were embedded in paraffin, and 4μm sections were obtained using a microtome. The sections were placed on glass microscope slides, deparaffinized, and stained with hematoxylin stain for 5 min. Before the sections were differentiated with 1% HCl-ethanol, they were rinsed with phosphate-buffered saline (PBS, pH 7.4). The eosin stain was applied to the sections for 30 s, followed by dehydration with a graded ethanol series, cleared with xylene, and then cover-slipped with a neutral resin. Histopathological changes were observed under a light microscope.


### 2.7. Pyroptosis and Apoptosis Detection

Detection of the cell apoptosis rate was performed using TUNEL staining provided by thein situ cell apoptosis assay kit (C10619, Invitrogen, USA). GSDMD antibody (NBP2-80427, NOVUS, USA) staining was used to detect cellular pyroptosis, as GSDMD is the pyroptotic factor [29]. The cells were counterstained with TUNEL. Paraffinized hippocampus tissues were cut into 5 μm sections, placed on glass microscope slides, and treated with TUNEL working reagent, as well as a diluted GSDMD primary antibody solution by incubating the slices at 37°C for 1 h while being protected from light. The sections were incubated with a streptavidin-HRP working reagent in darkness for 30 min. DAPI staining was performed to stain the cell nuclei. Subsequently, the sections were dehydrated with a graded ethanol series and sealed with coverslips using an antifade mounting medium. Images were captured under a fluorescence microscope for visual observations of pyroptosis and apoptosis. The rates of these processes were determined for each group.


### 2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

The expression levels of S-100β (CSB-E08066r, CUSABIO, USA), NSE (CSB-E07963r, CUSABIO), M-CSF (CSB-E07424r, CUSABIO), IL-6 (CSB-E04640r, CUSABIO), TNF-ɑ (CSB-E11987r, CUSABIO), IL-1β (CSB-E08055r, CUSABIO), and IL-18 (CSB-E04610r, CUSABIO) in the rat hippocampi were determined using ELISA kits. The procedures described in the manufacturer’s protocol were strictly followed. Briefly, 100 μl of the standards and diluted samples was added to the enzyme-linked wells in each ELISA microplate, followed by incubation at 37°C for 2 h. Then the solutions were removed, the wells were rinsed with three changes of PBS-Tween 20, and biotin-labeled antibodies were added to each well at 37°C for 1 h. 100 μl of an HRP-avidin solution was added to the wells after the secondary antibodies were removed. 90 μl of a TMB substrate solution was added to each well, and the plates were incubated for 15 min at 37°C while protected from light. 50 μl of a stop solution of 0.2 M H2SO4 was added to each well. Optical density (OD) values were obtained by reading the plates at 450 nm with a microplate reader. The concentration of each indicator in the rat hippocampi was calculated based on the obtained OD values and standard curves.


### 2.9. Immunofluorescence (IF)

5μm deparaffinized hippocampus tissue sections were rehydrated through a graded ethanol series and treated with a 3% H2O2 solution to quench endogenous peroxidases. The sections were immersed in a 0.1 M sodium citrate solution for antigen retrieval, followed by thorough rinsing with PBS (pH 7.4). The sections were blocked by incubation in 5% goat serum at 37°C for 30 min. After removal of the goat serum, the ROS probe (#88-5930-74, Invitrogen) and primary antibodies against IBA-1 (MABN92-AF647, Sigma-Aldrich), iNOS (#PA1-036, Invitrogen), arg-1 (#PA5-85267, Invitrogen), and NLRP3 (#MA5-32255, Invitrogen) were applied to the sections. The sections were counterstained with iNOS/IBA-1, arg-1/IBA-1, and NLRP3/IBA-1. Specifically, sections were incubated with the above probe and antibodies at 4°C for approximately 12 h. Then, the ROS probe and primary antibodies were removed, and the sections were incubated with fluorescence-labeled secondary antibody at 37°C for 30 min. DAPI staining (#62248, Thermo Scientific, USA) was used to label the cell nuclei. The stained sections were cover-slipped with an antifade mounting medium, and the fluorescent staining was visualized using a fluorescence microscope. Relative expression levels were analyzed using ImageJ software.


### 2.10. Western Blots

The frozen rat hippocampal tissues were removed from −80°C and the protein samples were extracted using RIPA lysis buffer (#20–188, Millipore, USA) containing 1% PMSF (#93482, Sigma-Aldrich) and 2% protease inhibitors (P9599, Sigma-Aldrich). Tissue suspensions were homogenized and centrifuged at 12,000 rpm at 4°C for 10 min. The extracted protein samples were obtained from the centrifuged supernatants and were stored at −80°C for later use. The protein concentration for each sample was determined using a BCA protein quantitation assay kit (#23227, Thermo Scientific). The protein samples were loaded onto 15-well SDS-PAGE gels for electrophoresis and then transferred onto 0.2μm PVDF membranes (GE10600022, Amersham Hybond, USA). The membranes were blocked in 3% (m/v) skim milk (#70166, Millipore) overnight at 4°C. Subsequently the blocked membranes were incubated for 3 h at room temperature on a shaking incubator with primary antibodies against GSDMD (ab219800, Abcam, UK), GSDMD-NT (#93709, CST, USA), NLRP3 (ab263899, Abcam, USA), pro-caspase-1 (ab179515, Abcam, USA), pro-IL-1β (ab205924, Abcam, USA), pro-IL-18 (ab191860, Abcam, USA), iNOS (ab15323, Abcam, USA), arg-1 (ab233548, Abcam, USA), and IBA-1 (ab178846, Abcam, USA). GAPDH (ab9485, Abcam, USA) was used as the internal loading control. The membranes were thoroughly washed with TBS-Tween 20 to remove the primary antibodies. Then the membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody at room temperature for 1.5 h. ECL (RPN2209, GE Healthcare, USA) was applied to the membranes to visualize the protein bands. A gray analysis was performed using ImageJ software.


### 2.11. Statistical Analysis

The data were analyzed with SPSS version 22.0 (IBM SPSS Statistics Software) and represented. Experimental data are expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) was performed followed by Tukey’s post hoc test for comparison tests. One-way ANOVA with Tukey’s post hoc test and Mann-WhitneyU test were used where appropriate. Differences between groups were determined to be statistically significant when the p value was less than 0.05.


## 2.1. Experimental Groups and Animals

In this experiment, thirty male SPF-grade Sprague Dawley rats, weighing 350–450 g, were obtained from the Department of Laboratory Animal Sciences of the General Hospital of Northern Theater Command (laboratory animal user license no. SYXK (Military) 20120007; laboratory animal production license no. SCXK (Military) 20120006). The rats were randomly divided into the three following groups with each group having ten rats: Sham operation group, CPB model group, and a group of CPB rats given U50488H. The animal experiments in this study were approved by the IGDB-IACUC of the General Hospital of Northern Theater Command (IACUC no. 2020013).


## 2.2. Establishment of the CPB Rat Model

Rats were anesthetized with an intraperitoneal injection (30 mg/kg) of pentobarbital sodium (#57-33-0, Sigma-Aldrich, USA) and were positioned on the operating table in the supine position. The rat was intubated and connected to a small animal ventilator and an anesthesia machine. During the surgery, rats were continuously anesthetized with 2% isoflurane (#26675-46-7, Sigma-Aldrich). A 24 G trocar needle was placed into the right femoral artery to infuse 6% hydroxyethyl starch (#9005-27-0, Merck, USA) at a rate of 0.5 ml/h. A 22 G trocar needle was placed onto the left femoral artery to monitor real-time arterial pressure. CPB was established by placing a 22 G trocar needle into the tail artery and another 18 G trocar needle was positioned in the right jugular vein. Venous blood was drained to a blood storage tank after each tube was connected and fixed in place. The blood was returned to the body via the tail artery after diverting the blood via a peristaltic pump and oxygenated with an external oxygenator. The anesthetic level was adjusted as needed based on the blood gas analysis. The physiological parameters of the rats were maintained at MAP > 60 mmHg, pH levels in a normal range, PaCO2 at 35–45 mmHg, and the base excess (BE) at 0 ± 3 mmol/L. Because the isovolemic hemodilution was targeted to achieve an Hct level of 25 ± 1% during the CPB, the flow could be gradually stopped, and mechanical ventilation resumed. With continuous maintenance of the mechanical ventilation, the tubes were removed, and the remaining blood in the reservoir was slowly infused back to the body to maintain circulation stability. Successful establishment of the CPB rat model was characterized by the survival of rats at 6 h after surgery. The rats in the KORA group were injected intravenously 30 min before the CPB procedure with U50488H (#D8040, Sigma-Aldrich) at a concentration of 1.5 mg/kg [27, 28].


## 2.3. Cell Culture

Rat microglial (RM) cells were purchased from ScienCell™ (#R1900, ScienCell™ Research Laboratories, USA), which had been isolated from the cerebral cortex of two-day-old rat pups. Cells were delivered frozen as a secondary culture. When initiating the subculture from the cryopreserved cells, the cells were cultured in an incubator at 5% CO2 and 37°C, using microglia medium (#1901, ScienCell™ Research Laboratories). Then the cells were seeded on coverslips in 12-well plates at a concentration of 5 × 106 cells per well that contained phenol red-free DMEM (HyClone, USA). The RM cells were treated with 10 ng/mL lipopolysaccharide (LPS) (#L6529, Sigma-Aldrich) for 24 h. This in vitro study included the following groups: control RM cell group (RM), LPS-treated RM cell group (LPS), LPS + U50488H RM cell group (LU), LPS + Ac-YVAD-cmk (SML0429, Sigma-Aldrich) RM cell group (LAC), and LPS + U50488H + RS09 (#5928, TOCRIS, UK) RM cell group (LUR). The cells were treated with 100 ng/mL LPS for 24 h, followed by exposure to 1 μmol/L of U50448H for 30 min. Ac-YVAD-cmk was dissolved in 500 μl of DMSO, and the working concentration was 50 μM (27 μg/ml), which was added to the cells for 30 min before the LPS stimulation.


## 2.4. Morris Water Maze

In each group, the rats were trained and tested in the Morris water maze starting on the 21st postoperative day to observe changes in cognitive function. Rats were placed into the maze from a randomized quadrant and facing the pool wall. Each rat was allowed 90 seconds for free swimming to find the hidden platform. The escape latency period was recorded according to the duration of time each rat searched and then the hidden platform located. The Morris water maze experiments were carried out for five consecutive days. The results from the first four days were considered acquisition training, and the test scores obtained on the fifth day were recorded as the spatial learning and memory scores for the rats in each group. The submerged platform was removed 24 h after completion of the hidden platform test. The rats were placed into the water maze at the same entry points as previously indicated. The swimming paths for each rat were recorded for 60 s. The amount of time the rats resided in the target quadrant and the number of times the platform location was crossed were recorded. The data were analyzed using the Morris imaging analysis system.


## 2.5. Tissue Sample Collection

After the Morris water maze experiments were completed, rats in each group were anesthetized with 2% isoflurane and euthanized by exsanguination due to transection of the abdominal aorta. Blood samples were collected and centrifuged at 2,000 ×g for 5 min to obtain the plasma. The plasma samples were stored at −80°C. The brains were dissected, and the tissue samples were separated. A subset of the hippocampus samples were fixed in 4% paraformaldehyde, and the remaining hippocampus samples were stored, unfixed, at −80°C.


## 2.6. Hematoxylin and Eosin (HE) Staining

Paraformaldehyde-fixed brain tissue samples were dehydrated in a graded ethanol series (from 70% to 100%) and then cleared in two changes of xylene. The tissue samples were embedded in paraffin, and 4μm sections were obtained using a microtome. The sections were placed on glass microscope slides, deparaffinized, and stained with hematoxylin stain for 5 min. Before the sections were differentiated with 1% HCl-ethanol, they were rinsed with phosphate-buffered saline (PBS, pH 7.4). The eosin stain was applied to the sections for 30 s, followed by dehydration with a graded ethanol series, cleared with xylene, and then cover-slipped with a neutral resin. Histopathological changes were observed under a light microscope.


## 2.7. Pyroptosis and Apoptosis Detection

Detection of the cell apoptosis rate was performed using TUNEL staining provided by thein situ cell apoptosis assay kit (C10619, Invitrogen, USA). GSDMD antibody (NBP2-80427, NOVUS, USA) staining was used to detect cellular pyroptosis, as GSDMD is the pyroptotic factor [29]. The cells were counterstained with TUNEL. Paraffinized hippocampus tissues were cut into 5 μm sections, placed on glass microscope slides, and treated with TUNEL working reagent, as well as a diluted GSDMD primary antibody solution by incubating the slices at 37°C for 1 h while being protected from light. The sections were incubated with a streptavidin-HRP working reagent in darkness for 30 min. DAPI staining was performed to stain the cell nuclei. Subsequently, the sections were dehydrated with a graded ethanol series and sealed with coverslips using an antifade mounting medium. Images were captured under a fluorescence microscope for visual observations of pyroptosis and apoptosis. The rates of these processes were determined for each group.


## 2.8. Enzyme-Linked Immunosorbent Assay (ELISA)

The expression levels of S-100β (CSB-E08066r, CUSABIO, USA), NSE (CSB-E07963r, CUSABIO), M-CSF (CSB-E07424r, CUSABIO), IL-6 (CSB-E04640r, CUSABIO), TNF-ɑ (CSB-E11987r, CUSABIO), IL-1β (CSB-E08055r, CUSABIO), and IL-18 (CSB-E04610r, CUSABIO) in the rat hippocampi were determined using ELISA kits. The procedures described in the manufacturer’s protocol were strictly followed. Briefly, 100 μl of the standards and diluted samples was added to the enzyme-linked wells in each ELISA microplate, followed by incubation at 37°C for 2 h. Then the solutions were removed, the wells were rinsed with three changes of PBS-Tween 20, and biotin-labeled antibodies were added to each well at 37°C for 1 h. 100 μl of an HRP-avidin solution was added to the wells after the secondary antibodies were removed. 90 μl of a TMB substrate solution was added to each well, and the plates were incubated for 15 min at 37°C while protected from light. 50 μl of a stop solution of 0.2 M H2SO4 was added to each well. Optical density (OD) values were obtained by reading the plates at 450 nm with a microplate reader. The concentration of each indicator in the rat hippocampi was calculated based on the obtained OD values and standard curves.


## 2.9. Immunofluorescence (IF)

5μm deparaffinized hippocampus tissue sections were rehydrated through a graded ethanol series and treated with a 3% H2O2 solution to quench endogenous peroxidases. The sections were immersed in a 0.1 M sodium citrate solution for antigen retrieval, followed by thorough rinsing with PBS (pH 7.4). The sections were blocked by incubation in 5% goat serum at 37°C for 30 min. After removal of the goat serum, the ROS probe (#88-5930-74, Invitrogen) and primary antibodies against IBA-1 (MABN92-AF647, Sigma-Aldrich), iNOS (#PA1-036, Invitrogen), arg-1 (#PA5-85267, Invitrogen), and NLRP3 (#MA5-32255, Invitrogen) were applied to the sections. The sections were counterstained with iNOS/IBA-1, arg-1/IBA-1, and NLRP3/IBA-1. Specifically, sections were incubated with the above probe and antibodies at 4°C for approximately 12 h. Then, the ROS probe and primary antibodies were removed, and the sections were incubated with fluorescence-labeled secondary antibody at 37°C for 30 min. DAPI staining (#62248, Thermo Scientific, USA) was used to label the cell nuclei. The stained sections were cover-slipped with an antifade mounting medium, and the fluorescent staining was visualized using a fluorescence microscope. Relative expression levels were analyzed using ImageJ software.


## 2.10. Western Blots

The frozen rat hippocampal tissues were removed from −80°C and the protein samples were extracted using RIPA lysis buffer (#20–188, Millipore, USA) containing 1% PMSF (#93482, Sigma-Aldrich) and 2% protease inhibitors (P9599, Sigma-Aldrich). Tissue suspensions were homogenized and centrifuged at 12,000 rpm at 4°C for 10 min. The extracted protein samples were obtained from the centrifuged supernatants and were stored at −80°C for later use. The protein concentration for each sample was determined using a BCA protein quantitation assay kit (#23227, Thermo Scientific). The protein samples were loaded onto 15-well SDS-PAGE gels for electrophoresis and then transferred onto 0.2μm PVDF membranes (GE10600022, Amersham Hybond, USA). The membranes were blocked in 3% (m/v) skim milk (#70166, Millipore) overnight at 4°C. Subsequently the blocked membranes were incubated for 3 h at room temperature on a shaking incubator with primary antibodies against GSDMD (ab219800, Abcam, UK), GSDMD-NT (#93709, CST, USA), NLRP3 (ab263899, Abcam, USA), pro-caspase-1 (ab179515, Abcam, USA), pro-IL-1β (ab205924, Abcam, USA), pro-IL-18 (ab191860, Abcam, USA), iNOS (ab15323, Abcam, USA), arg-1 (ab233548, Abcam, USA), and IBA-1 (ab178846, Abcam, USA). GAPDH (ab9485, Abcam, USA) was used as the internal loading control. The membranes were thoroughly washed with TBS-Tween 20 to remove the primary antibodies. Then the membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibody at room temperature for 1.5 h. ECL (RPN2209, GE Healthcare, USA) was applied to the membranes to visualize the protein bands. A gray analysis was performed using ImageJ software.


## 2.11. Statistical Analysis

The data were analyzed with SPSS version 22.0 (IBM SPSS Statistics Software) and represented. Experimental data are expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) was performed followed by Tukey’s post hoc test for comparison tests. One-way ANOVA with Tukey’s post hoc test and Mann-WhitneyU test were used where appropriate. Differences between groups were determined to be statistically significant when the p value was less than 0.05.


## 3. Results

### 3.1. KORs Reduced Cerebral Damage and PND in CPB-Treated Rats

To investigate the effect of the KOR agonist, the Morris water maze experiment was carried out to evaluate cognitive functions in the rats used in this study. In the hidden platform training test, CPB rats exhibited increased time needed to find the hidden platform, reduced swimming distances, and time residing in the target quadrant. However, these results were reversed in rats treated with the KOR agonist. The results are shown in Figure1(a).Figure 1
KORs improve cerebral damage and rescue PND in CPB rats. (a) Morris water maze experiments were carried out 3 days after the operation for detection of rats’ cognitive functions. Escape latency for reaching hidden platforms, platform-crossing times, and residence duration in target quadrant for rats in each group (n = 10/group) was recorded on the 5th day of the Morris water maze experiment as the first 4 days were of acquisition training. (b) Histopathological changes in rats’ hippocampal CA1 region were observed using HE staining (scale bar = 50 μm). (c) Contents of S-100β (pg/ml) and NSE (ng/ml) in rats’ brain tissue samples were determined using ELISA. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.
(a)(b)(c)Based on the histopathological assessments, the CPB rats’ hippocampi exhibited severe damage, disordered arrangement of cells, and increased spaces between cells (Figure1(b)). To further determine the extent of the cerebral damage, the expression levels of relevant cerebral damage markers were quantified. Results showed that NSE and S-100β concentrations were elevated in CPB rats (Figure 1(c)). However, the administration of U50488H significantly reduced the histopathological changes observed in the treated rats and decreased the degree of cerebral damage. These findings suggested that KORs could decrease CPB-induced cerebral damage and PND in rats.


### 3.2. KORs Regulated the MI Polarization State in CPB Rats

It has been reported that MI act as CNS immunocytes, and their activation plays an essential role in the development and progression of CNS inflammation [21]. In this study, IBA-1 was used to label MI cells in the CA1 area of the hippocampus. The IBA-1-labeled MI cells were counterstained with M1 and M2 markers, iNOS, and arg-1, respectively. The results demonstrated that CPB induced excessive polarization of MI toward the M1 classification, resulting in significantly elevated expression levels of iNOS. When U50488H was administered to CPB rats, the MI polarization transition from M1 to M2 type cells was observed (Figure 2(a)). It was also determined that the overactivated M1 type MI cells secreted considerable amounts of inflammatory factors, leading to decreased M-CSF and increased levels of IL-6 and TNF-ɑ (Figure 2(b)). Just as the MI polarization transition was detected previously, the concentrations of the inflammatory factors were reversed with the administration of U50488H. This observation indicated that KORs regulated the transition of the MI polarization state from an M1 type to an M2 type and exhibited protective effects in CPB rats.Figure 2
KORs regulate MI polarization state in CPB rats. (a) For detection of MI polarization state transitions, immunofluorescent counterstaining of iNOS/IBA-1 and arg-1/IBA-1 was carried out (scale bar = 50μm). Accumulated IBA-1 expressions were detected when CPB occurred, and the MI polarization transition from M1 toward M2 phenotype was also observed when U50488H was applied to CPB rats. (b) Contents of inflammatory factors, M-CSF (pg/ml), IL-6 (pg/ml), and TNF-ɑ (pg/ml), in rats’ brain tissue samples were determined using ELISA. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.
(a)(b)


### 3.3. KORs Inhibited Post-CPB NLRP3 Inflammasome Expression in Rat Brain Tissue

The initiation of inflammatory responses requires the involvement of the inflammasome protein complex. It has been reported that CPB can cause significant upregulation of NLRP3 inflammasome expression, leading to a decrease in thein vivo antioxidant capacity and excessive release of ROS, which may induce biofilm lipid peroxidation and DNA damage, resulting in cell death, tissue damage, and disease occurrence. In our study, the elevated expressions of NLRP3 (Figure 3(a)) and ROS accumulation (Figure 3(b)) were detected after the CPB protocol. The activated NLRP3 inflammasome catalyzed the upregulation of pro-caspase-1 expression to promote the expression of pro-IL-1β and pro-IL-18 (Figure 3(c)), which transitioned into IL-1β and IL-18 with bioactivity by activated NLRP3 (Figure 3(d)). The administration of U50488H suppressed the accumulation of ROS, downregulated the expression levels of NLRP3, simultaneously increased the protein expression of pro-caspase-1, pro-IL-1β, and pro-IL-18, and inhibited the secretion of IL-1β and IL-18. These results indicated that KORs suppressed the NLRP3 inflammasome expression in brain tissues of CPB rats.Figure 3
KORs inhibit post-CPB NLRP3 inflammasome expression in rats’ brain tissue. (a) NLRP3 inflammasome expression level was detected using western blotting. (b) Release of ROS in a different group of rats was visualized with IF (scale bar = 50μm). (c) Expression levels of preform inflammatory proteins, pro-Caspase-1, pro-IL-1β, and pro-IL-18, were detected with western blotting. (d) Contents of mature IL-1β (pg/ml) and IL-18 (ng/ml) in rats’ brain tissue samples were determined using ELISA. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.
(a)(b)(c)(d)


### 3.4. KORs Inhibited Cell Pyroptosis through Mediation of Cerebral Pyrophosphorylation in CPB Rats

The close correlation between NLRP3 inflammasome activation and pyrophosphorylation has been reported previously. We investigated the occurrence of pyrophosphorylation and its effects on the hippocampus. GSDMD-NT regulates the essential process of pyroptosis, and the cleavage of GSDMD is the critical indicator used to evaluate pyroptosis. The expression and cleavage of GSDMD were significantly increased in CPB rats, and KOR agonists reversed these changes considerably (Figure4(a)). Based on the counterstaining of GSDMD and TUNEL in the hippocampal CA1 regions, it was observed that CPB induced pyroptosis and apoptosis. These findings suggested that CPB-induced hippocampal neuronal damage was not entirely due to cell apoptosis but also involved cell pyroptosis. The KOR agonists demonstrated protective effects for the damaged hippocampal neurons. Therefore, KOR agonists inhibited the rate of apoptosis and simultaneously suppressed cell pyroptosis (Figure 4(b)), suggesting that the pyrophosphorylated cell death might play a central role in the process of CPB-induced hippocampal neuronal damage associated with cell pyroptosis.Figure 4
KORs inhibit cell pyroptosis through mediating cerebral pyrophosphorylation in CPB rats. (a) Expression levels of GSDMD and cleaved GSDMD-N-terminal in rats’ brain tissue samples were quantified with western blotting. (b) Counterstaining of GSDMD and TUNEL (scale bar = 50μm) was carried out for evaluation of pyrophoric and apoptotic rate, respectively. Relative pyroptotic and apoptotic rates were represented in the form of a bar chart. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.
(a)(b)


### 3.5. KORs Suppressed the Activation of the NLRP3 Inflammasome in MI

To explore the correlation between MI and the NLRP3 inflammasome, IF counterstaining with IBA-1 and NLRP3 was carried out. The results demonstrated increased coexpression of IBA-1 and NLRP3 in the CPB rats’ hippocampi, and KOR agonists inhibited the expression levels of NLRP3 (Figure5(a)). To further investigate these interactions, RM cells from the rat MI cell line were selected and cultivated in an in vitro experiment. The RM cells were stimulated by LPS, which activated the NLRP3 inflammasome and upregulated pro-caspase-1, thereby increasing the expression levels of pro-IL-1β and pro-IL-18 (Figure 5(b)). Consequently, the expression levels of NLRP3 and its downstream proteins were inhibited by the administration of U50488H, which suggested that KOR agonists suppressed the activation of the NLRP3 inflammasome in MI cells.Figure 5
KORs suppress the activation of NLRP3 inflammasome in MI. (a) Immunofluorescent counterstaining of NLRP3/IBA-1 (scale bar = 50μm) in rats’ hippocampal CA1 regions was performed. (b) Expression levels of NLRP3/caspase-1 pathway-related proteins and their downstream proteins in RM cells were detected using western blotting. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.
(a)(b)


### 3.6. KORs Suppressed MI Activation through Regulation of the NLRP3/Caspase-1 Pathway

KOR agonists have been reported to mediate MI-associated neuroinflammation and suppress NLRP3 inflammasome activation. In this study, we applied caspase-1 inhibitors to LPS-stimulated RM cells to explore the mechanisms of action associated with the anti-inflammatory properties of KOR agonists via the NLRP3/caspase-1 signaling pathway. It was observed that LPS still activated the NLRP3 inflammasome, but the pro-caspase-1 expression was not catalyzed due to the addition of the caspase-1 inhibitor, AC-YVAD-cmk [30] (Figure 6(a)). To investigate the correlation between the regulatory mechanisms of KOR agonists and the NLRP3/caspase-1 signaling pathway, U50488H and RS09 were administered simultaneously to LPS-stimulated RM cells. U50488H and RS09 are TLR4 agonists, and it has been reported that these compounds have significant activation effects on the NLRP3 inflammasome and can serve as an alternate mechanism of inflammasome activation [31].Figure 6
KORs suppress MI activation by regulating the NLRP3/caspase-1 pathway. (a) Western blotting was carried out to determine the protein expression levels of NLRP3 inflammasome and pro-caspase-1 in LPS-stimulated RM cells treated with caspase-1 inhibitor (AC-YVAD-cmk). LPS-stimulated RM cells were coadministered with the KOR agonist (U50488H) and excessively activated NLRP3 triggered by TLR4 agonist (RS09). The relative expression levels of NLRP3/caspase-1 pathway-related proteins (b) and MI-polarization-associated proteins (c) were quantified with western blotting. (d) Levels of inflammatory factors, M-CSF (pg/ml), IL-6 (pg/ml), and TNF-ɑ (pg/ml), in RM cell supernatant were determined using ELISA. All data in charts were represented as mean ± standard deviation; “ns” was labeled when the comparison between groups (p value) was greater than 0.05, while “∗” was labeled when the p value was less than 0.05.
(a)(b)(c)(d)It was revealed that excessive activation of the NLRP3 inflammasome eliminated the protective effects of the KOR agonists and enhanced the inflammatory responses (Figure6(b)). Moreover, RS09 administration blocked the regulatory mechanisms of the KOR agonists on the MI polarization state, which led to an elevated accumulation of M1 type MI cells, resulting in increased expression of iNOS and reduced expression of arg-1 (Figure 6(c)). It also was observed that the M-CSF content in the cellular supernatant was decreased, and the levels of IL-6 and TNF-ɑ were increased when RS09 was administered (Figure 6(d)). These results illustrated that the KOR agonists suppressed MI-mediated inflammatory responses, thereby improving PND via regulation of the NLRP3/caspase-1 pathway.


## 3.1. KORs Reduced Cerebral Damage and PND in CPB-Treated Rats

To investigate the effect of the KOR agonist, the Morris water maze experiment was carried out to evaluate cognitive functions in the rats used in this study. In the hidden platform training test, CPB rats exhibited increased time needed to find the hidden platform, reduced swimming distances, and time residing in the target quadrant. However, these results were reversed in rats treated with the KOR agonist. The results are shown in Figure1(a).Figure 1
KORs improve cerebral damage and rescue PND in CPB rats. (a) Morris water maze experiments were carried out 3 days after the operation for detection of rats’ cognitive functions. Escape latency for reaching hidden platforms, platform-crossing times, and residence duration in target quadrant for rats in each group (n = 10/group) was recorded on the 5th day of the Morris water maze experiment as the first 4 days were of acquisition training. (b) Histopathological changes in rats’ hippocampal CA1 region were observed using HE staining (scale bar = 50 μm). (c) Contents of S-100β (pg/ml) and NSE (ng/ml) in rats’ brain tissue samples were determined using ELISA. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.
(a)(b)(c)Based on the histopathological assessments, the CPB rats’ hippocampi exhibited severe damage, disordered arrangement of cells, and increased spaces between cells (Figure1(b)). To further determine the extent of the cerebral damage, the expression levels of relevant cerebral damage markers were quantified. Results showed that NSE and S-100β concentrations were elevated in CPB rats (Figure 1(c)). However, the administration of U50488H significantly reduced the histopathological changes observed in the treated rats and decreased the degree of cerebral damage. These findings suggested that KORs could decrease CPB-induced cerebral damage and PND in rats.


## 3.2. KORs Regulated the MI Polarization State in CPB Rats

It has been reported that MI act as CNS immunocytes, and their activation plays an essential role in the development and progression of CNS inflammation [21]. In this study, IBA-1 was used to label MI cells in the CA1 area of the hippocampus. The IBA-1-labeled MI cells were counterstained with M1 and M2 markers, iNOS, and arg-1, respectively. The results demonstrated that CPB induced excessive polarization of MI toward the M1 classification, resulting in significantly elevated expression levels of iNOS. When U50488H was administered to CPB rats, the MI polarization transition from M1 to M2 type cells was observed (Figure 2(a)). It was also determined that the overactivated M1 type MI cells secreted considerable amounts of inflammatory factors, leading to decreased M-CSF and increased levels of IL-6 and TNF-ɑ (Figure 2(b)). Just as the MI polarization transition was detected previously, the concentrations of the inflammatory factors were reversed with the administration of U50488H. This observation indicated that KORs regulated the transition of the MI polarization state from an M1 type to an M2 type and exhibited protective effects in CPB rats.Figure 2
KORs regulate MI polarization state in CPB rats. (a) For detection of MI polarization state transitions, immunofluorescent counterstaining of iNOS/IBA-1 and arg-1/IBA-1 was carried out (scale bar = 50μm). Accumulated IBA-1 expressions were detected when CPB occurred, and the MI polarization transition from M1 toward M2 phenotype was also observed when U50488H was applied to CPB rats. (b) Contents of inflammatory factors, M-CSF (pg/ml), IL-6 (pg/ml), and TNF-ɑ (pg/ml), in rats’ brain tissue samples were determined using ELISA. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.
(a)(b)


## 3.3. KORs Inhibited Post-CPB NLRP3 Inflammasome Expression in Rat Brain Tissue

The initiation of inflammatory responses requires the involvement of the inflammasome protein complex. It has been reported that CPB can cause significant upregulation of NLRP3 inflammasome expression, leading to a decrease in thein vivo antioxidant capacity and excessive release of ROS, which may induce biofilm lipid peroxidation and DNA damage, resulting in cell death, tissue damage, and disease occurrence. In our study, the elevated expressions of NLRP3 (Figure 3(a)) and ROS accumulation (Figure 3(b)) were detected after the CPB protocol. The activated NLRP3 inflammasome catalyzed the upregulation of pro-caspase-1 expression to promote the expression of pro-IL-1β and pro-IL-18 (Figure 3(c)), which transitioned into IL-1β and IL-18 with bioactivity by activated NLRP3 (Figure 3(d)). The administration of U50488H suppressed the accumulation of ROS, downregulated the expression levels of NLRP3, simultaneously increased the protein expression of pro-caspase-1, pro-IL-1β, and pro-IL-18, and inhibited the secretion of IL-1β and IL-18. These results indicated that KORs suppressed the NLRP3 inflammasome expression in brain tissues of CPB rats.Figure 3
KORs inhibit post-CPB NLRP3 inflammasome expression in rats’ brain tissue. (a) NLRP3 inflammasome expression level was detected using western blotting. (b) Release of ROS in a different group of rats was visualized with IF (scale bar = 50μm). (c) Expression levels of preform inflammatory proteins, pro-Caspase-1, pro-IL-1β, and pro-IL-18, were detected with western blotting. (d) Contents of mature IL-1β (pg/ml) and IL-18 (ng/ml) in rats’ brain tissue samples were determined using ELISA. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.
(a)(b)(c)(d)


## 3.4. KORs Inhibited Cell Pyroptosis through Mediation of Cerebral Pyrophosphorylation in CPB Rats

The close correlation between NLRP3 inflammasome activation and pyrophosphorylation has been reported previously. We investigated the occurrence of pyrophosphorylation and its effects on the hippocampus. GSDMD-NT regulates the essential process of pyroptosis, and the cleavage of GSDMD is the critical indicator used to evaluate pyroptosis. The expression and cleavage of GSDMD were significantly increased in CPB rats, and KOR agonists reversed these changes considerably (Figure4(a)). Based on the counterstaining of GSDMD and TUNEL in the hippocampal CA1 regions, it was observed that CPB induced pyroptosis and apoptosis. These findings suggested that CPB-induced hippocampal neuronal damage was not entirely due to cell apoptosis but also involved cell pyroptosis. The KOR agonists demonstrated protective effects for the damaged hippocampal neurons. Therefore, KOR agonists inhibited the rate of apoptosis and simultaneously suppressed cell pyroptosis (Figure 4(b)), suggesting that the pyrophosphorylated cell death might play a central role in the process of CPB-induced hippocampal neuronal damage associated with cell pyroptosis.Figure 4
KORs inhibit cell pyroptosis through mediating cerebral pyrophosphorylation in CPB rats. (a) Expression levels of GSDMD and cleaved GSDMD-N-terminal in rats’ brain tissue samples were quantified with western blotting. (b) Counterstaining of GSDMD and TUNEL (scale bar = 50μm) was carried out for evaluation of pyrophoric and apoptotic rate, respectively. Relative pyroptotic and apoptotic rates were represented in the form of a bar chart. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.
(a)(b)


## 3.5. KORs Suppressed the Activation of the NLRP3 Inflammasome in MI

To explore the correlation between MI and the NLRP3 inflammasome, IF counterstaining with IBA-1 and NLRP3 was carried out. The results demonstrated increased coexpression of IBA-1 and NLRP3 in the CPB rats’ hippocampi, and KOR agonists inhibited the expression levels of NLRP3 (Figure5(a)). To further investigate these interactions, RM cells from the rat MI cell line were selected and cultivated in an in vitro experiment. The RM cells were stimulated by LPS, which activated the NLRP3 inflammasome and upregulated pro-caspase-1, thereby increasing the expression levels of pro-IL-1β and pro-IL-18 (Figure 5(b)). Consequently, the expression levels of NLRP3 and its downstream proteins were inhibited by the administration of U50488H, which suggested that KOR agonists suppressed the activation of the NLRP3 inflammasome in MI cells.Figure 5
KORs suppress the activation of NLRP3 inflammasome in MI. (a) Immunofluorescent counterstaining of NLRP3/IBA-1 (scale bar = 50μm) in rats’ hippocampal CA1 regions was performed. (b) Expression levels of NLRP3/caspase-1 pathway-related proteins and their downstream proteins in RM cells were detected using western blotting. All data in charts were represented as mean ± standard deviation; “∗” was labeled when the comparative significance between groups (p value) was less than 0.05.
(a)(b)


## 3.6. KORs Suppressed MI Activation through Regulation of the NLRP3/Caspase-1 Pathway

KOR agonists have been reported to mediate MI-associated neuroinflammation and suppress NLRP3 inflammasome activation. In this study, we applied caspase-1 inhibitors to LPS-stimulated RM cells to explore the mechanisms of action associated with the anti-inflammatory properties of KOR agonists via the NLRP3/caspase-1 signaling pathway. It was observed that LPS still activated the NLRP3 inflammasome, but the pro-caspase-1 expression was not catalyzed due to the addition of the caspase-1 inhibitor, AC-YVAD-cmk [30] (Figure 6(a)). To investigate the correlation between the regulatory mechanisms of KOR agonists and the NLRP3/caspase-1 signaling pathway, U50488H and RS09 were administered simultaneously to LPS-stimulated RM cells. U50488H and RS09 are TLR4 agonists, and it has been reported that these compounds have significant activation effects on the NLRP3 inflammasome and can serve as an alternate mechanism of inflammasome activation [31].Figure 6
KORs suppress MI activation by regulating the NLRP3/caspase-1 pathway. (a) Western blotting was carried out to determine the protein expression levels of NLRP3 inflammasome and pro-caspase-1 in LPS-stimulated RM cells treated with caspase-1 inhibitor (AC-YVAD-cmk). LPS-stimulated RM cells were coadministered with the KOR agonist (U50488H) and excessively activated NLRP3 triggered by TLR4 agonist (RS09). The relative expression levels of NLRP3/caspase-1 pathway-related proteins (b) and MI-polarization-associated proteins (c) were quantified with western blotting. (d) Levels of inflammatory factors, M-CSF (pg/ml), IL-6 (pg/ml), and TNF-ɑ (pg/ml), in RM cell supernatant were determined using ELISA. All data in charts were represented as mean ± standard deviation; “ns” was labeled when the comparison between groups (p value) was greater than 0.05, while “∗” was labeled when the p value was less than 0.05.
(a)(b)(c)(d)It was revealed that excessive activation of the NLRP3 inflammasome eliminated the protective effects of the KOR agonists and enhanced the inflammatory responses (Figure6(b)). Moreover, RS09 administration blocked the regulatory mechanisms of the KOR agonists on the MI polarization state, which led to an elevated accumulation of M1 type MI cells, resulting in increased expression of iNOS and reduced expression of arg-1 (Figure 6(c)). It also was observed that the M-CSF content in the cellular supernatant was decreased, and the levels of IL-6 and TNF-ɑ were increased when RS09 was administered (Figure 6(d)). These results illustrated that the KOR agonists suppressed MI-mediated inflammatory responses, thereby improving PND via regulation of the NLRP3/caspase-1 pathway.


## 4. Discussion

PND is a common complication that occurs after cardiac surgery. It is caused by low perfusion or low average arterial pressure, hemodynamic instability, cerebral thrombosis, systemic inflammation, anemia, hyperglycemia, and trauma caused by CPB, resulting in reduced neurocognitive functions. In this study, a CPB rat model was established, and the effects of U50488H treatment were assessed. The results confirmed that U50488H reduced PND in CPB rats, inhibited inflammatory responses, and protected brain tissue from damage. We observed that cell pyroptosis also played an essential role in PND. U50488H inhibited the NLRP3 inflammasome-triggered pyrophosphorylated cell death, and its mechanism of action was related to the microglial polarization regulated by the NLRP3/caspase-1 signaling pathway.NLRP3 participates in inflammatory responses as an intracellular signal recognition receptor and plays critical roles in the innate immune system and microglia-mediated neuroinflammation [32]. Cao et al. [33] proved that NLRP3-mediated neuroinflammation in patients with Parkinson’s disease was closely associated with the loss of midbrain substantia nigra neurons. In a mouse model stimulated with neurotoxic substances, increased numbers of surviving neurons and improved motor functions were observed when the expression level of the NLRP3 gene was suppressed or the activation of caspase-1 was blocked. In this study, we confirmed that KORs suppressed the expression of NLRP3 and inhibited the expression of pro-caspase-1 and its downstream proteins. NLRP3, NLRP1, and other relevant inflammasomes can chemotactically activate caspase-1 when the caspase-1-mediated classical cell pyroptosis pathway is triggered through extracellular stimulation [22]. Simultaneously, activated caspase-1 can cleave GSDMD at specific sites to release the pore-active GSDMD-N-terminal domain, which can accumulate inside the cell membrane and form nonselective pores, leading to increased permeability of the cell membrane to cause cell pyroptosis [34, 35]. Our study reported that neuronal death was not entirely due to apoptosis, but cells died via pyroptosis. It also was speculated that pyrophosphorylated cardiomyocytes also might have crucial roles. These observations were substantiated by performing counterstaining for GSDMD and TUNEL.In a study on Parkinson’s disease, it was found that activation of the NLRP3 inflammasome also could be induced by ROS accumulation and the accumulation of pathologicalα-synuclein in mitochondria [36]. Our research found that the inflammatory responses of rat brain tissues were substantially increased after CPB, and increased accumulation of ROS was also detected. These observations led to the speculation that changes in the cerebral microenvironment acted as a signal source to activate the NLRP3 inflammasome and stimulate neuroinflammation. Moreover, we also reported that MI played an essential role in regulating the immunomicroenvironment through immune defense mechanisms, which are influenced by damaged neurons, plaques, and infectious substances, leading to the expression of cerebral neuroinflammation and damage. It was found that excessive neuroinflammation might induce the MI polarization toward the M1 cell classification, which can induce an increased release of cellular inflammatory factors and neurotoxic substances, leading to neuronal damage. However, the administration of KORs induced the MI polarization transition from the M1 to the M2 cell type, which exhibits protective effects. Thus, inhibiting MI activation or inducing the MI polarization transition toward the M2 cell type is hypothesized to be a novel potential treatment pathway for rescuing PND.Published reports have suggested that KORs can significantly reduce ischemia-induced hippocampal damage that results in cognitive, learning, and memory impairments [37]. Among the several existing KORs, U50488H has high selectivity for the K receptor and less affinity for the μ and δ receptors (24). U50488H can rescue hippocampal damage in the CA1 region and improve learning and memory deficits. U50488H is widely used in the study of KORs [26]. In our study, we observed that KORs exhibited neuroprotective effects on post-CPB brain tissues through suppression of microglial activation. Moreover, we detected through counterstaining with NLRP3 and IBA-1 that KOR agonists inhibited the expression levels of the NLRP3 inflammasome. Furthermore, a regulatory relationship was observed through the in vitro study, in which LPS-stimulated RM cells were selected to produce neuroinflammation. The results demonstrated that KOR agonists suppressed the expression of NLRP3, and increased activation of the NLRP3 inflammasome blocked its protective effects. In addition, the MI polarization transition from M1 type to M2 type cells was observed. These results illustrated that KOR agonists inhibited MI-mediated inflammatory responses through the NLRP3/caspase-1 signaling pathway to rescue PND.There were some limitations in this study.μ-Opioid receptors have been proposed to play a critical role in modulating social behavior, learning, and memory in humans and animals. Thus, we need to investigate the relationship of μ-opioid receptor agonists and postcardiopulmonary bypass cognitive dysfunctions.


## 5. Conclusion

This study explored the effects of KOR agonist on microglia polarization and neuroinflammation in mice exposed to CPB and investigated the method of the NLRP3/caspase-1 pathway. Morris water maze was used to evaluate the changes in the cognitive function of CPB rats and hematoxylin and eosin (HE) staining and TUNEL were made to measure the rats’ hippocampal damage. ELISA was used to predict the changes in brain injury. In addition, IF was used to detect the expression of microglia polarization and NLRP3 followed by western blots to identify the expression of the NLRP3/caspase-1 pathway and microglia polarization-related proteins. KORs alleviated the hippocampal damage caused by CPB and improved PND. Likewise, KORs also inhibited NLRP3 and transformed microglia from the M1 to the M2 state. Theκ-opioid receptor agonists inhibited the inflammation mediated by microglia and improved PND through the NLRP3/caspase-1 signaling pathway. Thus, the present study demonstrated that KOR agonists provided neuroprotective effects against brain damage in mice.



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*Source: 3048383-2021-10-01.xml*