Alleviating the NF-κB/NLRP3 pathway-mediated pyroptosis and ameliorating the cognitive function of aged mice post partial hepatectomy by increasing the Bmal1 level via subanesthetic doses of ketamine

2.1 Animals and grouping for testing

This survey drew on research methods reported in the literature [32]. The Laboratory Animal Center of the First Affiliated Hospital of PLA General Hospital (Beijing, China) provided 40 SPF-grade female mice numbered C57BL/6 (specifications: 15-month old; 25–35 g). These mice were randomly grouped into six groups (ten mice per group): a group receiving sham surgery (sham group), a group injected with normal saline (NS group), a group receiving injection with anesthetic doses of ketamine (anesthetic group), a group receiving injection with subanesthetic doses of ketamine (subanesthetic group), a group receiving injection with subanesthetic doses of ketamine + NF-κB agonists BAY 11-7085 (subanesthetic + BAY group), and a group receiving injection with subanesthetic doses of ketamine + NLRP3 agonist Nigericin (subanesthetic + Nig group). Except for the sham group, the remaining five model groups underwent partial hepatectomy (PH). One hour before surgery, the sham group and the NS group were injected with 1 mL of normal saline; the anesthetic and subanesthetic groups were administered intraperitoneally with 1 and 0.1 mg/kg of ketamine, respectively. One hour after operation, 5 μg/kg BAY 11-70851 and 1 mL/kg Nigericin were injected intraperitoneally in the subanesthetic + BAY group and subanesthetic + Nig group, respectively.

To assess the way Bmal1 affects aged mice’s cognitive functions, 30 female mice numbered C57BL/6 with identical identifications were selected from the Chinese Academy of Sciences (Shanghai, China) affiliated Laboratory Animal Center. For facilitating the tests, the 30 mice were classified equally into three groups: a group of wild-type mice with active Bmal1 gene (WT group), a group of mice with Bmal1 gene deactivated (Bmal1-KO group), and a group of mice with Bmal1 gene deactivated and receiving injection with subanesthetic doses of ketamine (Bmal1-KO + subanesthetic group). All three groups experienced PH. One hour prior to surgery, the WT and Bmal1-KO groups received an intraperitoneal injection of 1 mL normal saline; the Bmal1-KO + subanesthetic group received an intraperitoneal injection of 0.1 mg/kg ketamine. The mice were accommodated in a quiet environment with adequate ventilation and air filtration systems, at the following settings: 20–22°C (room temperature); around 50% relative humidity; and alternated 12-h light and 12-h dark. The mice had free access to water and food, and their padding and cages were changed once every two days.

2.2 PH mice modeling

All groups of mice were narcotized using 1% sodium pentobarbital (40 mg/kg). Next, the sham group received laparotomy only, and the remaining three model groups were subjected to both laparotomy and subsequent PH. The operation steps are as follows: (1) fix the narcotized mice properly and an incision on the lower edge of the right upper abdominal costal arch; (2) enter the abdominal cavity and mobilize the liver; (3) use a surgical thread to ligate the vessels at the portal area of the left lateral lobe of the liver and use a high-frequency electrocautery to cut off the left lateral lobe; (4) suture the abdomen after ensuring that there was no significant bleeding or damage. After entering the stage of recovery from anesthesia, the mice were placed in an incubator (settings: 37℃; constant temperature) for ventilation till the mice recovered and behaved normally and actively. After the surgery, an EMLA cream mixture (ingredients: 2.5% lidocaine and 2.5% prilocaine) was applied to the wound site at an interval of 8 h for easing pains. Note: all mice were inoculated with the same doses of drugs for anesthesia and analgesic treatment.

2.3 Tests in Morris water maze (MWM)

All groups of mice were tested in a MWM on days 1, 3, and 7 after the surgery successively. This MWM system (DMS-2) was manufactured by the Institute of Materia Medica, affiliated with the Chinese Academy of Medical Sciences (Beijing, China). In this system, a circular stainless-steel water tank was provided. This tank was 120 cm in diameter and 50 cm in height. The tank was equally divided into four quadrants. In each quadrant, different colored papers were fixed at the midpoint of the inner wall and above the water surface to serve as visual markers for the mice to locate the platform. A circular platform (9 cm in diameter; 20 cm in height) was positioned at the center point of the target quadrant and 1 cm below the water surface. The entire water tank was surrounded by a blackout curtain to avoid light completely. Before starting the test, the tank was filled with water, which was then mixed with titanium dioxide (a white dye) and stirred till the water became milky white. In the testing process, the incandescent lamp outside the water tank was turned on, with its light directly shining on the curtain and evenly reflecting across the tank. Right above the maze, a camera was mounted and connected to a display system to simultaneously record the mice’s movement paths. In the end, the obtained outcomes were processed using an MWM data collection and analysis system.

Place Navigation Test: The platform was fixed in the target quadrant and remained in the same position throughout the tests. Mice were trained within the same period, four times daily. Every day, the quadrant for mice to enter water for the first time was selected randomly. On the same day, each mouse was trained in a different quadrant, respectively. When entering the same quadrant, the mice entered the water from the same position. Each swimming cycle was 90 s. If a mouse found the preset platform and stayed for more than 3 s on the platform, it was judged that this mouse found the platform successfully. The time a mouse took from entering water to finding the platform was treated as the escape latent period. If a mouse failed to find the platform within 90 s, the mouse would be guided to the platform, staying for 15 s. In this case, the mouse’s escape latent period was recorded as 90 s. This test was conducted on days 1, 3, and 7 post-surgery, respectively, with the escape latent period and total swimming distance recorded within 90 s.

Spatial Search Test: Following the above test, the platform was removed from the target quadrant. The mouse was placed in a randomly chosen quadrant, with the image collection and analysis system automatically recording the search time the mouse stayed in the quadrant where the platform was originally positioned, the search distance, and the frequency of accurate crossings over this quadrant within 90 s.

2.4 Collection and preparation of samples

Aged mice were euthanized with the cardiac puncture method on day 3 post-PH, followed by separation of the hippocampal tissue using the following procedures: (1) fix the mice narcotized with 1% sodium pentobarbital (P3761) prepared by Merck Millipore, Billerica (MA, USA); (2) use scissors to cut open the skin and ribs of the thoracic cavity, exposing the heart and liver; (3) insert an injection needle into the left ventricle of the mouse’s heart and cut open the right atrial appendage to allow blood to flow out; (4) perfuse the heart with pre-cooled phosphate-buffered saline on ice for blood replacement till the fluid flowing out of the right atrial appendage was no longer visibly red; (5) cut open the scalp and skull of the mouse in turn, exposing the brain tissue; (6) cut off the medulla oblongata, obtaining the whole brain tissue; (7) have the brain tissue samples for histopathological observation of hippocampal neurons through Nissl staining, TUNEL staining, and immunohistochemical assays placed in 4% paraformaldehyde (P1110) supplied by Solarbio (Beijing, China) and those for western blot and ELISA analyses placed in labeled pre-cooled microcentrifuge tubes without nucleases and stored at −80°C.

2.5 Nissl staining for hippocampal tissue

Referring to the research method adopted by Hu et al. [33], we carried out the following procedures for this assay: (1) immerse the hippocampal tissue samples of each experimental mouse (five mice from each group) in 4% paraformaldehyde and culture overnight in a 4°C refrigerator; (2) embed the hippocampal tissue samples in paraffin, then cut the paraffin blocks into slices (thickness: 5 µm) and dewax the slices; (3) dehydrate the slices in a graded series of ethanol, immerse in xylene, and then re-hydrate in gradient concentrations of ethanol; (4) add 1% thionine (G3668) supplied by Solarbio to the slices, followed by 30 min of hydration at 60℃; (5) hydrate the slices immersed in gradient concentrations of ethanol for dehydration, clean in xylene, and then mount with neutral balsam (G8590) sourced from Solarbio; and (6) observe and photograph the hippocampal forms under a light microscope (BX43) provided by Olympus (Tokyo, Japan).

2.6 Tunel staining for hippocampal tissue

For this assay, TUNEL assay kits (No.: C1091) were provided by Beyotime Biotechnology (Shanghai, China) and DAPI solution. The apoptotic status of neuronal cells in hippocampal tissue was determined using the TUNEL assay kits as per their user manuals. The detailed steps are as follows: (1) dewax the hippocampal tissue slices, dehydrate the slices using a graded series of ethanol, and clean the slices using xylene; (2) rinse the slices thrice using PBS; (3) add 20 µg/mL proteinase K solution onto the slices, culturing for 30 min at room temperature; (4) rinse the slices with PBS and then put in 3% H₂O₂, and incubate for 15 min; (5) rinse the slices with PBS again and administer 50 µL of TUNEL reaction mixture and incubate 60 min in the dark with 37℃ for reaction; (6) rewash the slices using PBS and add DAPI solution for staining, keeping in the dark and at the room temperature for 5 min incubation; (7) use PBS to wash the slices to get rid of DAPI; (8) use an anti-fade mounting medium to seal the remaining matter; and (9) observe five random high-power fields of the seal sheet under a microscope and quantify the TUNEL-positive cells.

2.7 Immunofluorescence for hippocampal tissue

The steps of this experiment are as follows: (1) the paraffin sections of the hippocampus were dewaxed and hydrated and placed in a citric acid solution, and microwave treatment was performed to expose the antigen; (2) the sections were dripped with blocking solution to cover the tissue for 30 min; (3) NeuN (ab104224 and IBA-1 (ab178847) antibodies were added and incubated overnight at 4℃; (4) the sections were washed with PBS and incubated with fluorescent secondary antibody (ab150077) for 1 h; and (5) after sealing the film, a microscope was used to observe and take images.

2.8 Western blot assay

In this assay, Tris-buffered saline-Tween (TBST), polyvinylidene fluoride membranes (Merck Millipore Corp., Billerica, MA, USA), the anti-NeuN (ab177487; prepared by Abcam, Waltham, MA, USA), anti-p-p65 (ab76302), anti-p65 (ab32536), anti-NLRP3 (ab263899), anti-iNOS (ab3523), anti-CD206 (ab300621), anti-cleaved caspase-1 (ab256469), anti-caspase-1 (ab138483), anti-GSDMD (ab219800), anti-GSDMD-N (ab215203), anti-Bmal1 (ab235577), anti-β-actin (ab8227), anti-N-methyl-d-aspartate receptor (NMDAR) subunit 2B (NR2B, ab254356), anti-brain-derived neurotrophic factor (BDNF, ab216443) and horseradish peroxidase-labeled secondary antibody (ab6721) were used. Enhanced chemiluminescence (ECL) substrate (a Pierce ECL western blotting substrate) was provided by Thermo Scientific. The computer picture analysis system ChemiDoc XRS+ was provided by Bio-Rad (Hercules, CA, USA). The assay steps are detailed as follows: (1) extract total proteins from the hippocampal tissues of each mouse group (100 µg from each group) using RIPA lysis buffer; (2) perform SDS-based polyacrylamide gel electrophoresis on the extracted proteins and then transfer the proteins to polyvinylidene fluoride membranes; (3) block the membranes for 2 h at room temperature in 0.1% TBST solution containing 5% bovine serum albumin; (4) add primary antibodies anti-NeuN (diluted to 1:1,000), anti-p-p65 (diluted to 1:5,000), anti-p65 (diluted to 1:1,000), anti-iNOS (diluted to 1:1,000), anti-CD206 (diluted to 1:1,000), anti-NLRP3 (diluted to 1:1,000), anti-Cleaved caspase-1 (diluted to 1:1,000), anti-caspase-1 (diluted to 1:1,000), anti-GSDMD (diluted to 1:1,000), anti-GSDMD-N (diluted to 1:1,000), anti-Bmal1 (diluted to 1:1,000), and anti-β-actin (diluted to 1:1,000) to the solution, incubating overnight at 4°C; (5) wash the two groups of cells for three repetitions using TBST and add horseradish peroxidase-labeled second antibody (diluted to 1:2,000) to the obtained protein strips, incubating at the room temperature for 2 h; (6) dropwise add ECL substrate to the protein strips, wait 1–3 min for the reaction; and (7) use the ChemiDoc XRS+ to measure taking β-actin as an internal reference.

2.9 Immunohistochemical assay

In this assay, the citrate antigen retrieval solution (C1032) was provided by Solarbio; the primary antibody anti-Bmal1 (ab230822) and the secondary antibody IgG (ab6721) were supplied by Abcam; diaminobenzidine (DAB, Numbered 24002) was prepared by Thermo Scientific (Waltham, MA, USA); microscope Eclipse 80i was obtained from Nikon (Chiyoda-ku, Tokyo, Japan); and the ImageJ 1.47n software was developed by Wayne Rasband (National Institute of Health, Bethesda, MD, USA). This assay was conducted using a method appropriately modified as per the method reported in the literature [34,35]. The specific steps are as follows: (1) place the hippocampal tissue samples of mice in 4% paraformaldehyde and storing in a refrigerator set at 4°C overnight; (2) dehydrate the samples, embed them in paraffin, and then slice the block into pieces (thickness: 5 µm); (3) perform antigen retrieval using citrate antigen retrieval solution and use 0.01 M PBS (comprising 5% bovine serum albumin and 0.1% Triton X-100) to block the slices for 1 h at the room temperature; (4) incubate the slices overnight at 4 °C using the anti-Bmal1 (diluted to 1:1,000); (5) administer IgG (diluted to 1:2,000) to the slices and incubate for 2 h; (6) add DAB to develop the images of the slices; (7) use the microscope Eclipse 80i to observe and photograph and the staining status on the slices; and (8) analyze the density using the Image J 1.47n software.

2.10 Enzyme-linked immunosorbent assay

Hippocampal tissue samples were extracted with the method given in Section 2.4. Then, the samples were tested using the IL-1β ELISA kit (Numbered ab197742) and TNF-α ELISA kit (Numbered ab208348) supplied by Abcam, respectively, in accordance with the user manuals of the kits. Finally, the optical densities of these two inflammatory cytokines were determined at a wavelength of 450 nm, and their expression levels were compared.

2.11 Analysis of the obtained data

The obtained data were analyzed statistically using the SPSS 26.0 designed by SPSS Inc., Chicago, IL, USA. All the data were processed using the Kolmogorov–Smirnov method to examine their normal distribution. All the measured data satisfying normal distribution are expressed as the mean ± standard error (i.e., x ¯ ± s). The comparison between two groups of samples was conducted through unpaired t-tests, while comparisons among at least three groups were conducted using the one-way ANOVA method. *P < 0.05 indicates a large difference from the control group; ^# P < 0.05 signifies that there is a marked difference from the NS group.

1. Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals. The study was approved by the Ethics Committee of Shaanxi Provincial Cancer Hospital.

3.1 Subanesthetic dose improved the cognitive function of ketamine in PH-aged mice

To ascertain how different doses of ketamine affect the memory and learning ability of post-PH-aged mice, we conducted the MWM tests on mice on days 1, 3, and 7 post-surgery, respectively. Figure 1a illustrates the test methods and flowchart of this survey. The behavioral test results reveal the following: First, compared to the sham group, post-PH mice in the other three groups exhibited significantly prolonged escape latent period on days 1, 3, and 7 post-operation (Figure 1b); the frequency of crossings over the platform and the time staying in the target quadrant both showed varying degrees of decline (Figure 1c and d). This outcome indicates that the mice’s memory and learning ability were impaired by PH. Hence, an animal model with cognitive dysfunction was successfully established. Second, in comparison with the NS group, mice in the anesthetic group experienced a longer escape latent period, stayed a shorter time in the target quadrant, and crossed over the platform less frequently, while the mice in the subanesthetic group displayed the opposite behaviors (Figure 1b–d). In addition, this study also found that PH significantly reduced the expression of p-NR2B and BDNF proteins, which further indicated that mice had postoperative cognitive dysfunction. Compared with the NS group, the expression of p-NR2B and BDNF protein in the anesthesia group was significantly decreased, and the expression of p-NR2B and BDNF proteins in the sub-anesthesia group was significantly increased (Figure 1e–g). These phenomena prove that subanesthetic doses of ketamine helped ameliorate the postoperative cognitive functions in aged mice.

Figure 1

Subanesthetic dose improved the cognitive function of ketamine in PH-aged mice (*signifies a comparison with the sham group; #signifies a comparison with the normal saline group; &signifies a comparison with the subanesthetic group). (a) Timeline design for the test. (b–d): Escape latent period, duration staying in the target quadrant, and frequency of crossings over the platform. (e–g) p-NR2B and BDNF protein levels expressed in the hippocampus as tested by western blotting.

3.2 Subanesthetic dose of ketamine improved the pathological damage of hippocampal neurons in PH-aged mice

Through Nissl staining, Tunel staining, immunofluorescence, and western blot assays, the pathological damage to hippocampal neurons in mice with cognitive dysfunction was examined. The Nissl staining results show that the sham group had clear and intact neurons with abundant Nissl bodies. In contrast with the sham group, mice in the other three model groups exhibited fewer Nissl-positive cells and lighter staining of Nissl bodies in the hippocampal neurons: mice in the NS group had disordered neurons and shrunken nuclei, along with a substantial loss of Nissl bodies; exacerbated neuronal damage was observed in the anesthetic group in comparison with the NS group; the conditions in the NS group were seen relieved evidently in the subanesthetic group (Figure 2a and b). The Tunel staining results indicate a higher number of Tunel-positive cells in the other three model groups than the sham group, suggesting an increment in the hippocampal neuronal apoptotic rate in aged mice post-PH. Notably, this rate in the subanesthetic group was significantly lower than that in the NS group (Figure 2c and d). Additionally, the results of testing on the neuronal cell marker protein NeuN (Figure 2e–h) also confirmed that there were more NeuN-positive cells in the hippocampus of the sham group, and the cells were multi-layered and closely arranged; the NeuN-positive cells in the hippocampus of the other three model groups were significantly reduced, the structure was loose (Figure 2e and f), and the level of NeuN protein was also significantly reduced (Figure 2g and h). However, the NeuN-positive cells and protein levels in the subanesthetic group were significantly higher than those in the NS group, indicating that, contrary to the normal anesthetic dose, the subanesthetic dose of ketamine can effectively mitigate postoperative neuronal injuries in aged mice.

Figure 2

Subanesthetic dose of ketamine improved the pathological damage of hippocampal neurons in PH-aged mice. (a) and (b) Nissl-stained hippocampus (×40, 50 μm). (c) and (d) The hippocampal neuronal apoptotic rate tested by tunnel staining (×40, 50 μm). (e) and (f) The number of NeuN-positive neurons in the hippocampus was detected by immunofluorescence (×40, 50 μm). (g) and (h) NeuN protein levels expressed in the hippocampus as tested by western blotting.

3.3 Subanesthetic dose of ketamine improved hippocampal neuroinflammation in PH-aged mice

Neuroinflammation associated with surgical trauma is an important pathogenic factor of postoperative cognitive dysfunction, and microglia play an important role in it. Immunofluorescence results showed that IBA-1-positive cells in the hippocampal CA1 region of PH mice were significantly increased, and the number of IBA-1-positive cells in the anesthesia group was further increased, while the IBA-1-positive cells in the sub-anesthesia group were significantly reduced (Figure 3a and b), indicating that subanesthetic doses of ketamine inhibited microglial activation. Moreover, the M1 polarization marker iNOS in the hippocampus was significantly increased after PH, further increased after the intervention of anesthetic dose of ketamine, and significantly decreased after the intervention of subanesthetic dose of ketamine. However, there was no difference in the M2 polarization marker CD206 among the four groups (Figure 3c–e), indicating that neuroinflammation caused by PH might be related to M1 microglia. Finally, ELISA results also confirmed that a subanesthetic dose of ketamine could alleviate postoperative hippocampal neuroinflammation in elderly mice. The contents of TNF-α and IL-1β increased significantly after PH, but decreased significantly in the subanesthetic group (Figure 3f and g).

Figure 3

Subanesthetic dose of ketamine improved hippocampal neuroinflammation in PH-aged mice. (a) and (b) The expression of IBA-1 in hippocampus (CA1 region) was detected by immunofluorescence (×40, 50 μm), (c)–(e) Western blot was used to detect the expression of iNOS and CD206 in hippocampus, and (f) and (g) the levels of TNF-α and IL-1β in hippocampus were detected by ELISA.

3.4 Subanesthetic dose of ketamine-mediated neuronal pyroptosis in PH-aged mice by inhibiting the NF-κB/NLRP3 pathway and improving postoperative cognitive function

Neurocognitive functions are critically regulated by neuroinflammation, as widely acknowledged in scientific research [36]. NLRP3 inflammasome, if activated, may trigger its downstream inflammatory cytokines, leading to neuroinflammation and subsequent pyroptosis of cells. To investigate the involvement of ketamine in post-PH-aged mice’s neuroinflammation, hippocampal tissues were collected from aged mice after ketamine, NF-κB agonist BAY 11-7085, and NLRP3 agonist Nigericin intervention, and the expressed levels of cytokines involved in the NF-κB/NLRP3 pathway were assessed. As revealed through western blotting, compared with the sham group, the hippocampal tissues of mice in the other three model groups encountered upregulated levels of pathway protein p-p65/p65, NLRP3, cleaved caspase-1, and GSDMD-N. Compared with the NS group, the protein level of the anesthesia group was further upregulated, while that of the subanesthesia group was significantly reduced; after the application of BAY 11-7085 and Nigericin on the basis of subanesthesia, these protein levels were significantly increased (Figure 4a–f). Moreover, the MWM experiment showed that compared with the subanesthesia group, the escape latency of mice was significantly increased after the application of BAY 11-7085 and Nigericin, the target quadrant residence time and the number of crossing platforms were significantly reduced (Figure 4g–i), and the levels of p-NR2B and BDNF proteins were also significantly reduced (Figure 4j–l). It is suggested that a subanesthetic dose of ketamine inhibited the NF-κB/NLRP3 signaling pathway and inhibited neuronal pyroptosis, which explains why a subanesthetic dose of ketamine can improve postoperative cognitive function in aged mice.

Figure 4

Subanesthetic dose of ketamine mediated neuronal pyroptosis in PH-aged mice by inhibiting NF-κB/NLRP3 pathway and improving postoperative cognitive function. (a)–(f) The p-NF-κB p65, NF-κB p65, NLRP3, cleaved caspase-1, caspase-1, GSDMD, and GSDMD-N levels expressed in the hippocampus, as tested by western blotting. (g)–(i) Escape latent period, duration staying in the target quadrant, and frequency of crossings over the platform in the MWM experiment. (j)–(l) p-NR2B and BDNF protein levels expressed in hippocampus, as tested by western blotting.

3.5 Subanesthetic dose of ketamine upregulated the expression of Bmal1 in the hippocampus of PH-aged mice

Immunohistochemical and western blot assays reveal that the Bmal1 gene in aged mice post-PH was downregulated. Regarding the number of Bmal1-positive cells, the three model groups ranked as follows: subanesthetic group > NS group > anesthetic group (Figure 5a); a similar trend was exhibited in the levels of Bmal1 protein (Figure 5b and c). These outcomes match the findings from the author’s previous study [32]. Above all, subanesthetic doses of ketamine appeared to upregulate the level of the Bmal1 gene and suppress the NF-κB/NLRP3 signaling pathway. This action contributed to the recovery of the postoperative cognitive functions in aged mice. Hence, it was hypothesized that there was a reciprocal regulatory relationship between Bmal1 and NF-κB/NLRP3, and subanesthetic doses of ketamine might inhibit hippocampal neuronal pyroptosis by modulating the NF-κB/NLRP3 signaling pathway via the Bmal1 gene.

Figure 5

Subanesthetic dose of ketamine upregulated the expression of Bmal1 in the hippocampus of PH-aged mice. (a) The expressed level of Bmal1 in the hippocampus as tested immunohistochemically (×40, 50 μm). (b) and (c) The expressed level of Bmal1 in the hippocampus, as tested by western blotting.

3.6 Subanesthetic dose of ketamine attenuated cognitive impairment and hippocampal neuronal pathological damage induced by Bmal1 knockout in PH-aged mice

To verify Bmal1’s close connection to postoperative cognitive dysfunction in aged mice, subsequent experiments were conducted on aged mice with a congenitally inactive Bmal1 gene. Similarly, such mice were also tested in the MWM on days 1, 3, and 7 after surgery. The results are as follows: First, compared with the WT group, aged mice in the Bmal1-KO group on these 3 days post-operation presented significantly longer escape latent periods (Figure 6a), shortened duration staying in the target quadrant (Figure 6b), and reduced frequency of crossings over the platform (Figure 6c). In addition, p-NR2B and BDNF protein levels were also significantly reduced (Figure 6d–f). This phenomenon indicates that the mice’s memory and learning ability were impaired with the inactivation of the Bmal1 gene. When the aged mice with inactive Bmal1 gene were inoculated with subanesthetic doses of ketamine, some of their cognitive functions were recovered (Figure 6a–f). Thereby, it is verified that Bmal1 is a crucial target by which subanesthetic doses of ketamine repair the postoperative cognitive dysfunction of aged patients. As proved above, subanesthetic doses of ketamine can effectively mitigate neuronal injuries in aged mice post-surgery. By knocking the Bmal1 gene out, how subanesthetic doses of ketamine acted in repairing pathological injuries of hippocampal neurons in post-PH-aged mice was further validated. The results are as follows: First, compared to the WT group, the Bmal1-KO group of mice manifested reduced Nissl-positive cells in their hippocampal neurons (Figure 6g and h), increased Tunel-positive cells (Figure 6i and j), and lowered NeuN-positive cells (Figure 6k and l) and protein levels (Figure 6m and n), indicating an elevated rate of neuronal apoptosis in hippocampal neurons of post-PH-aged mice with Bmal1 gene deactivated. Second, mice in the Bmal1-KO + subanesthetic group showed clearly weakened neuronal apoptosis in hippocampal neurons (Figure 6g–n). These experimental results demonstrate that subanesthetic doses of ketamine can alleviate hippocampal neuronal injuries through neuronal apoptosis, diminishing by elevating the level of the Bmal1 gene.

Figure 6

Subanesthetic dose of ketamine attenuated cognitive impairment and hippocampal neuronal pathological damage induced by Bmal1 knockout in aged PH mice (*signifies a comparison with the WT group; #signifies a comparison with the Bmal1-KO group). (a)–(c) Escape latent period, duration staying in the target quadrant, and frequency of crossings over the platform. (d)–(f) p-NR2B and BDNF protein levels expressed in hippocampus, as tested by western blotting. (g) and (h) Nissl-stained hippocampus (×40, 50 μm). (i) and (j) The hippocampal neuronal apoptotic rate tested by tunnel staining (×40, 50 μm). (k) and (l) The number of NeuN positive neurons in the hippocampus was detected by immunofluorescence (×40, 50 μm). (m) and (n) NeuN protein levels expressed in the hippocampus, as tested by western blotting.

3.7 Subanesthetic dose of ketamine attenuates neuroinflammation and neuronal pyroptosis in the hippocampus of Bmal1 knockout-induced PH-aged mice

For validating the previous hypothesis regarding the regulatory relationship between Bmal1 and NF-κB/NLRP3, the expressed levels of cytokines related to the NF-κB/NLRP3 pathway were examined following the deactivation of the Bmal1 gene. Consequently, the following outcomes were obtained: First, in the Bmal1-KO group, the levels of IBA-1-positive cells (Figure 7a and b), M1 polarization marker iNOS (Figure 7c and d) and inflammatory cytokines TNF-α and IL-1β (Figure 7f and g) in the hippocampal region were significantly increased, but there was no significant difference in the level of M2 polarization marker CD206 (Figure 7c and e). Second, the protein expression levels of p-p65, NLRP3, cleaved caspase-1, and GSDMD-N were increased (Figure 7h–l). However, in the Bmal1-KO + subanesthesia group, there was still no significant difference in CD206 levels, and the levels of the remaining indicators were effectively restored (Figure 7a–l). In summary, the Bmal1 gene is suppressive for the NF-κB/NLRP3 pathway. This confirms that subanesthetic doses of ketamine can restrain the NF-κB/NLRP3 signaling pathway by elevating the volume of the Bmal1 gene, and this restraining further leads to alleviation of hippocampal neuronal pyroptosis, thus improving the aged mice’s cognitive dysfunction post-surgery.

Figure 7

Subanesthetic doses of ketamine ease the NF-κB/NLRP3 pathway-mediated post-PH neuronal pyroptosis in aged mice due to Bmal1 knockout. (a) and (b) The expression of IBA-1 in hippocampus (CA1 region) was detected by immunofluorescence (×40, 50 μm). (c)–(e) Western blotting was used to detect the expression of iNOS and CD206 in the hippocampus. (f) and (g) The levels of TNF-α and IL-1β in the hippocampus were detected by ELISA. (h–l) The p-NF-κB p65, NF-κB p65, NLRP3, cleaved caspase-1, caspase-1, GSDMD, and GSDMD-N levels expressed in the hippocampus, as tested by western blotting.