Activation of LRP1 ameliorates cerebral ischemia/reperfusion injury and cognitive decline by suppressing neuroinflammation and oxidative stress through TXNIP/NLRP3 signaling pathway in mice

1. Introduction

Cerebral ischemia/reperfusion (I/R) injury is a potentially life-threatening disorder with high morbidity and mortality [1, 2]. Primary ischemic insult is initiated due to insufficient oxygen and glucose delivery that supports cellular homeostasis [3]. On the other hand, restoring blood flow to the brain may cause a series of secondary pathological cascades, including excitotoxicity, neuroinflammation, oxidative stress, apoptosis, and death, or developing severe neurological deficits within the first several weeks due to delayed neuronal cell death [4, 5]. This phenomenon suggested that preventing and treating delayed neuronal necrosis are essential in addition to restoring cerebral blood flow in the treatment of cerebral I/R injury. However, a lack of specific neuroprotective agents for cerebral I/R injury necessitates the investigation of the underlying mechanism and finding effective therapeutic strategies.

Microglia are innate immune cells in the brain and homologous to macrophages in the peripheral system. Microglia play a vital role in the neuroinflammation of brain injury. Depending on the type of stimulus, activated microglia have distinct functions related to the M1/M2 microglial polarization [6]. The M1 phenotypes of microglia are proinflammatory, releasing tumor necrosis factor-a (TNF-α), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS). Conversely, the M2 phenotypes of microglia are anti-inflammatory and conducive to the production of IL-10, transforming growth factor-β (TGF-β), and arginase-1 (Arg-1) [7]. Thus, microglial polarization modulation towards the anti-inflammatory M2 phenotype might be a promising therapeutic target, which ameliorates cerebral I/R injury.

Low-density lipoprotein receptor-related protein-1 (LRP1) is a multifunctional receptor with two main biological functions in endocytosis and in the regulation of cell signaling pathways, which is widely expressed in neurons and glial cells in the central nervous system (CNS) [8–10]. Apolipoprotein E (APOE = gene, apoE = protein), one of the ligands of LRP1, is synthesized by astrocytes and oligodendrocytes in the brain [11]. Human APOE consists of three different isoforms: APOEε2, APOEε3, and APOEε4. Previous studies demonstrated that both human apoE3 and murine apoE exert neuroprotection by interacting with its receptor LRP1, while the APOEe4 isoform exhibits a poor prognosis in acute and chronic neurological diseases [12–14]. ApoE protein is 34 kilodaltons (kDa) and cannot permeate through the blood-brain barrier, making it difficult to use clinically. COG1410, a small molecule ApoE-mimetic peptide, is derived from the apoE receptor binding region [15]. Some studies showed that COG1410 has multiple neuroprotective properties, including anti-inflammation, anti-oxidation, and anti-apoptosis, by activating its receptor LRP1 [16–18]. However, whether LRP1 activation with COG1410 can relieve I/R-induced cerebral damage and its underlying mechanism is yet unknown.

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is composed of oligomerized NLRP3, apoptosis-associated speck-like protein (ASC), and procaspase-1 and is widely distributed in the microglia [19]. Previous studies found that NLRP3 inflammasomes promote inflammation and apoptosis by activating microglia in the cerebral I/R injury [20]. Moreover, oxidative stress is one of the crucial factors in cerebral I/R injury. Thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of thioredoxin (TRX), plays a vital role in maintaining cellular redox balance [21]. Previous studies demonstrated that TXNIP aggravates oxidative stress and promotes inflammatory response by activating the NLRP3 inflammasome. Therefore, TXNIP is also considered a potential target for the treatment of some I/R injuries [22, 23]. Nevertheless, the role of LRP1 and the TXNIP/NLRP3 signaling pathway in the microglial polarization modulation after cerebral I/R injury is yet to be elucidated.

In the present study, we hypothesized that activation of LRP1 with COG1410 could ameliorate neurological defcits and alleviate neuroinfammation and oxidative stress through TXNIP/NLRP3 signaling pathway after cerebral I/R in mice.

2. Materials And Methods

2.1. Animals

The current study was approved by the Animal Care Committee of Southwest Medical University (China). A total of 353 male C57BL/6 mice (weighing 18–22 g) were purchased from Chengdu Dossy Experimental Animals Co., Ltd (China). The animals were housed in cages in a temperature-controlled room (22 ± 2 °C) with alternating 12-h light/dark cycle and acclimatized for one week. Before the study, the mice were fasted for 8 h but allowed water ad libitum. All procedures were performed according to the National Institutes of Health guidelines for the use of experimental animals.

2.2. Experimental design

The experimental flow diagram is illustrated in Figure 1.

2.2.1. Experiment 1

To evaluate the time course of endogenous LRP1, TXNIP, NLRP3, and cleaved caspase-1 expression and histopathological changes in the hippocampal tissues of mice after cerebral I/R, 70 mice were randomized into five groups: sham, 6, 12, 24, and 72 h after I/R (n = 14/group). Western blot analysis was performed to assess the protein level of LRP1, TXNIP, NLRP3, and cleaved caspase-1. Then, hematoxylin and eosin (HE) staining was used for histopathological examination of the hippocampal tissues. Additionally, the cellular localization of LRP1 with neurons (Neuronal Nuclei, NeuN), astrocytes (glial fibrillary acidic protein, GFAP), or microglia (calcium-binding adaptor molecule 1, Iba-1) was evaluated by double immunofluorescence staining in the Sham and I/R-72 h group (n = 3/group).

2.2.2. Experiment 2

To determine the beneficial effects of LRP1 activation on neurobehavioral functions, brain water content (BWC), and histopathological changes at 72 h after cerebral I/R, 120 mice were randomized into five groups: sham, I/R + Vehicle, I/R + COG1410 (0.5 mg/kg), I/R + COG1410 (1.0 mg/kg), and I/R + COG1410 (2.0 mg/kg) (n = 24/group). The BWC and histopathological changes were tested at 72 h after reperfusion, while Morris water maze (MWM) was performed for neurobehavior tests on days 3–8 after cerebral I/R.

2.2.3. Experiment 3

To assess the roles of LRP1 activation on microglia, neuroinflammation, and apoptosis at 72 h after cerebral I/R, 36 mice were randomly divided into three groups for western blot, enzyme-linked immunosorbent assay (ELISA), immunofluorescence and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining: sham, I/R + Vehicle, and I/R + COG1410 (n = 12/group).

2.2.4. Experiment 4

In order to investigate the underlying mechanism of TXNIP/NLRP3 pathway in LRP1 mediated microglia polarization modulating, 70 mice were randomized into five groups: sham, I/R + Vehicle, I/R + COG1410, I/R + COG1410 + AAV-NC, I/R + COG1410 + AAV-TXNIP (n = 14/group). The sample for sham, I/R + Vehicle, and I/R + COG1410 groups were shared from experiment 3, and an additional 34 mice were used for the experiment. Moreover, to verify the efficacy of AAV-TXNIP on TXNIP overexpression, 18 mice were randomly divided into three groups: naïve, naïve + AAV-NC, naïve + AAV-TXNIP (n = 6/group). Histopathological changes and the level of oxidative stress were examined, and western blot analysis was performed at 72 h after cerebral I/R.

2.2.5. Experiment 5

To explore the underlying mechanism of TXNIP/NLRP3 pathway in LRP1 mediated long-term neuroprotective effect, 75 mice were randomly divided into five groups: sham, I/R + Vehicle, I/R + COG1410, I/R + COG1410 + AAV-NC, I/R + COG1410 + AAV-TXNIP (n = 15/group). The survival rates of different groups were monitored at 28 days after cerebral I/R, and WMW was conducted on days 28–33 after cerebral I/R.

2.3. Cerebral I/R model

The model of cerebral I/R was established using bilateral common carotid artery occlusion (BCCAO) as described previously [24]. Briefly, mice were anesthetized with pentobarbital sodium (45 mg/kg, intraperitoneally). Then, the trachea was exposed by a 0.5 cm midline neck incision, and the bilateral common carotid arteries (CCAs) were separated from the vagus nerve. Subsequently, the bilateral CCAs were occluded with the microvascular clamp, with the right CCA clamped first. After 30 min of ischemia, both CCAs were reperfused. In the sham operation group mice, the same procedures were performed without CCA occlusion. After infiltration with 0.25% bupivacaine, the wound was closed by sterile sutures. During the surgery, the body temperature of mice was maintained with a heating pad.

2.4. Intraperitoneal administration

COG1410 (acetyl-AS-Aib-LRKL-Aib-KRLL-amide synthesized by GL Biochem, Shanghai, China) was diluted with saline to different concentrations (0.5 mg/kg, 1.0 mg/kg, and 2.0 mg/kg), as described previously (Laskowitz et al., 2007). A volume of 100 μL COG1410 was administered intraperitoneally at 30 min, 24 h, and 48 h after reperfusion. The mice in the sham and I/R + Vehicle groups received equal volumes of saline at the same time point.

2.5. AAV injection

The TXNIP overexpression mouse model was established on day 28 before BCCAO, as described previously [25]. Briefly, the adeno-associated virus vectors (AAV-TXNIP and AAV-NC) were constructed by Syngentech Co., Ltd (Beijing, China). Before exposing the skull, the mice were anesthetized with pentobarbital sodium in a stereotaxic apparatus (RWD, Shenzhen, China). A volume of 1.5 µL of the AAV vectors was injected stereotaxically into the bilateral hippocampal CA1 area (AP-2.0 mm; ML±1.0 mm; DV 2.0 mm) at a rate of 0.5 μL/min, following which the needle was kept in place for 5 min and screwed out slowly. The skin incision was sutured, and the mice were housed in a single cage after they awoke from anesthesia.

2.6. Specimen collection

The mice were sacrificed at 6, 12, 24, or 72 h after reperfusion under deep anesthesia. Some animals were perfused transcardially with ice-cold saline, and then the hippocampal tissues were separated on ice, frozen in liquid nitrogen, and stored at -80 °C for Western blot, ELISA, detection of oxidative stress, and RT-PCR. The other mice were perfused transcardially with 4% paraformaldehyde for HE, TUNEL, and immunofluorescence staining.

2.7. Morris water maze test

The function of spatial learning and memory for mice was assessed using the Morris water maze (MWM) test [26]. The maze (Xinruan, Shanghai, China) consisted of a round tank (height 50 cm, diameter 120 cm), a platform (diameter 10 cm), and a camera analysis system. The tank was divided into four quadrants, filled up to a depth of 30 cm with mixed milk water (22 ± 1 °C). The mice were trained for four trials per day for five consecutive days during the place navigation test. In every trial, mice were placed in a different start quadrant and allowed to find the platform submerged below 1 cm of the water surface within a maximum duration of 60 s; the escape latency was recorded. On day 6, in the probe trial, the platform was withdrawn, and the mice were allowed to navigate freely in water for 60 s. The number of times crosses over the removed platform, duration in the target quarter (%), and swimming speed was recorded during the trial.

2.8. Histopathological examination

Whole-brain tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 3-μm-thick coronal sections. The sections were stained with HE and observed under a light microscope (Olympus, Tokyo, Japan) by two experienced pathologists blinded to the study. The number of normal neurons per square millimeter (cells/mm²) was calculated according to the size of the CA1 subregion in four high-magnification fields of each section, and the average was considered.

2.9. Brain water content

The brain water content (BWC) was estimated by the wet-to-dry brain weight ratio as described previously [27]. Briefly, the whole brains were harvested under deep anesthesia and immediately weighed to obtain the wet weight. Then, the brain tissues were baked at 65 ℃ for 48 h to obtain the dry weight. BWC = (wet weight − dry weight) / wet weight × 100%.

2.10. Assessment of apoptosis

The brains were dehydrated with 30% sucrose and sliced into 8-μm-thick coronal frozen sections using a cryostat (Leica, Germany). The apoptosis of cells in the hippocampus was detected using the TUNEL staining, according to the manufacturer’s protocol (#12156792910, Roche, Basel, Switzerland) [28]. The numbers of TUNEL-positive cells were counted in the CA1 subregion of the hippocampus. Four random high-magnification fields per section over a microscopic field of 400x magnification were averaged. Data were presented as the ratio of TUNEL-positive cells (%).

2.11. Immunofluorescence staining

Immunofluorescence staining was performed as described previously [29]. Briefly, the frozen sections were incubated with primary antibody at 4 °C overnight: mouse anti-Iba1 (1:200, #GT10312, Invitrogen, USA), mouse anti-GFAP (1:200, #3670S, Cell Signaling Technology (CST), USA), mouse anti-NeuN (1:200, #ab104224, Abcam, USA), rabbit anti-LRP1 (1:200, #64099S, CST, USA), rabbit anti-TXNIP (1:200, #14715S, CST, USA), and rabbit anti-NLRP3 (1:200, #15101S, CST, USA). After washing with phosphate-buffered saline (PBS), the sections were incubated with the corresponding secondary antibody (Cy3-labeled goat anti-rabbit IgG, FITC-labeled goat anti-mouse IgG) (1:200, #S0011, #0007, Affinity, USA) at room temperature for 1 h, followed by DAPI (#C0060, Solarbio, China) staining. Finally, the sections were visualized, and images were captured using a fluorescence microscope.

2.12. Detection of inflammatory factors

Hippocampal tissue homogenates were prepared and clarified by centrifugation at 12000 rpm at 4 °C for 15 min. TNF-α, IL-6, IL-1β, and IL-18 levels were measured in the supernatants of hippocampal tissues using the ELISA kits (#MM-0132M1, #MM-0163M1, #MM-0040M1, #MM-0169M1, Meimian Jiangsu, China) by the manufacturer’s instruction as described previously [30].

2.13. Detection of oxidative stress

Hippocampal tissue homogenates were prepared, and lysates were collected by centrifugation at 3000 rpm at 4 °C for 10 min. The MDA content and SOD activity in the supernatant of hippocampal tissues were detected by commercial biochemical kits (#A003-1-2, #A001-3-2, Nanjing Jiancheng, China) as described previously [31].

2.14. qRT-PCR

Total RNA was extracted from the hippocampal tissues using an RNA simple total RNA kit (#DP419, Tiangen, Beijing, China) following the standard protocol. This isolated RNA was reverse-transcribed into cDNA using the ReverTra Ace qPCR RT Master Mix (#FSQ-201, Toyobo, Japan) according to the manufacturer’s protocol. Then, the cDNA was amplified using SuperReal PreMix Plus (SYBR Green) (#FP205-02, Tiangen, Beijing, China) on a real-time PCR system (Roche). The amplification parameters were as follows: 95 °C for 15 min, followed by five cycles of 95 °C for 10 s and 60 °C for 32 s. β-actin was used as an internal reference for the quantification of the TXNIP gene expression level. The relative mRNA expression level in the naive group (target mRNA/β-actin value) was set as the normalization to calculate the fold-changes of the mRNA level in other groups. The primer sequences are listed in Table 1.

Table 1

The PCR primers sequences.

Genes	Primer sequences
TXNIP	Forward: 5’-ATACTCCTTGCTGATCTACG-3’
	Reverse: 5’-TGGGGTATCTGGGATGTTTA-3’
β-actin	Forward: 5’-TTTGCAGCTCCTTCGTTGC-3’
	Reverse: 5’-TCGTCATCCATGGCGAACT-3’

Abbreviation: TXNIP, thioredoxin-interacting protein.

2.15. Western blot analysis

The protein in the hippocampal tissue sample was estimated using an enhanced BCA protein assay kit (#P0012, Beyotime, Shanghai, China). An equivalent of 30 µg protein was separated on the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride membrane (PVDF; Amersham Biosciences, NJ, USA) or a nitrocellulose membrane (Beyotime). Then, the membrane was blocked with 5% non-fat milk at room temperature for 1 h and probed with the primary antibodies at 4 ℃ overnight: anti-LRP1 (1:1000, #64099S, CST), anti-TXNIP (1:1000, #14715S, CST), anti-NLRP3 (1:1000, #15101S, CST), anti-cleaved caspase-1 (1:200, #SC-56036, Santa Cruz, USA), anti-Iba-1 (1:1000, #17198S, CST), anti-iNOS (1:1000, #AF0199, Affinity, USA), anti-Arg-1 (1:1000, #DF6657, Affinity), anti-Bcl-2 (1:1000, #ab59348, Abcam, USA), anti-Bax (1:1000, #50599-2-Ig, Proteintech, China), anti-cleaved caspase-3 (1:1000, #19677-1-AP, Proteintech), and anti-β-actin (1:5000, #6609-1-Ig, Proteintech), followed by incubation with the appropriate horseradish peroxidase (HRP)-labeled secondary antibody (1:5000, #SE131, #SE134, Solarbio, China) at room temperature for 1 h. Then, the membranes were developed using enhanced chemiluminescence (ECL; Amersham, Buckinghamshire, UK). Finally, the immunoreactive bands were analyzed using Image J software (version 1.31; National Institutes of Health (NIH), Bethesda, MD, USA).

2.16. Statistical analysis

The GraphPad Prism 8.3 statistical software (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. All data were expressed as the mean and standard error of the mean (mean ± SEM). One-way analysis of variance (ANOVA) and Tukey’s post hoc test was used for comparisons. For the training phase of the WMW test, the escape latency over time was analyzed by two-way repeated-measures ANOVA. Kaplan-Meier survival curves were analyzed using the log-rank test. P < 0.05 was considered statistically significant.

3. Results

3.1. Time course of endogenous protein levels of LRP1, TXNIP, NLRP3, cleaved caspase-1, and histopathological changes in the hippocampus after cerebral I/R

HE staining was performed to determine the histopathological changes in the hippocampal tissues on the animal models. The results showed that the total normal cells of the CA1 region started decreasing at 6 h and were the lowest at 72 h after cerebral I/R injury (Fig. 2A-B). Furthermore, Western blot analysis showed that the protein level of LRP1 was significantly increased in the I/R group compared to the sham group at 12-72 h after surgery. The protein level of TXNIP was significantly increased in the I/R group compared to the sham group at 6 h and 72 h post-surgery. The protein level of NLRP3 was significantly increased in the I/R group compared to the sham group at 72 h after surgery. The protein level of cleaved caspase-1 was significantly increased in the I/R group compared to the sham group at 24-72 h after surgery (Fig. 2C–G).

Double immunofluorescence staining of LRP1 with microglia (Iba-1), neurons (NeuN), and astrocytes (GFAP) was performed in the sham group and I/R-72 h group. The cellular colocalization of LRP1 with Iba-1, NeuN, and GFAP was detected in the CA1 subregion of the hippocampus and the number of LRP1-positive microglia, neurons, and astrocytes was increased at 72 h after cerebral I/R (Fig. 2H).

3.2. COG1410 ameliorated short-term neurological deficits, brain edema, and histopathological damage at 72 h after cerebral I/R

In the place navigation test, the escape latency for the mice to find the platform was significantly prolonged in the I/R + Vehicle group compared to the sham group. However, a significantly shorter escape latency was observed on training days 4-5 in the I/R + COG1410 (1.0 mg/kg and 2.0 mg/kg) groups (Fig. 3A). In the probe trial, the mice in the I/R + Vehicle group required fewer platform crossings and less time percentage in the target quarter compared to the sham group, while the reference memory deficits were improved by COG1410 treatment (1.0 mg/kg and 2.0 mg/kg). Also, no significant differences were observed in the swimming speed among each group (Fig. 3B–E).

The results of HE staining showed that the structure of the hippocampal CA1 region was regular morphology in the sham group. However, many injured neurons, characterized by shrunken and stained dark, were observed in the hippocampal CA1 region of the mice after cerebral I/R. Moreover, fewer normal neurons of the hippocampal CA1 area and higher BWC of the whole brain tissues were observed in the I/R + Vehicle group compared to the sham group mice at 72 h after cerebral I/R. Compared to the animals treated with vehicles, administration of 1.0 mg/kg and 2.0 mg/kg COG1410 significantly attenuated the histopathological damage and brain edema (Fig. 4A–C). Based on the MWM test, histopathological results, and BWC, the dose of 1.0 mg/kg was selected for subsequent experiments.

3.3. COG1410 reduced apoptosis of hippocampal cells at 72 h after cerebral I/R

The cell apoptosis of the hippocampal CA1 region in mice was evaluated by the TUNEL method. Apoptotic cells were not detected in the sham group. On the other hand, after 72 h of reperfusion, the number of apoptotic cells was significantly increased in the hippocampal CA1 region. These changes were alleviated by COG1410 treatment, manifested as a decreased number of TUNEL-positive cells (Fig. 5A, C).

Furthermore, the Western blot data indicated that the protein levels of cleaved caspase-3 and Bax increased in the I/R + Vehicle group compared to the sham group, while that decreased significantly in the I/R + COG group compared to the I/R + Vehicle group (Fig. 5B, D-E). In addition, the protein levels of Bcl-2 were significantly lower in the I/R + Vehicle group compared to the sham group but were increased in the I/R + COG group compared to the I/R + Vehicle group (Fig. 5B, F).

3.4. COG1410 promoted M1-to-M2 phenotypic polarization of microglia and alleviated inflammatory response

Immunofluorescence staining with Iba-1 antibody was performed at 72 h after cerebral I/R. The sham group showed fewer Iba-1-positive cells with ramified thin processes. However, compared to the sham group, the number of Iba-1-positive cells was significantly increased, and the microglial cells showed considerable branch retraction, thickening, amoeba-like morphology in the I/R + Vehicle group, while the I/R + COG group showed less microglial activation than the I/R + Vehicle group (Fig. 6A).

Upon activation, microglial polarized to M1 or M2 phenotypes with distinct proinflammatory and immunosuppressive functions, among which iNOS was an M1 phenotype marker and Arg-1 was an M2 phenotype marker. The Western blot data indicated that the protein levels of Iba-1 and iNOS were upregulated in the I/R + Vehicle group compared to the sham group but were significantly lower in the I/R + COG group compared to the I/R + Vehicle group (Fig. 6B–D). Moreover, the protein level of Arg-1 was increased in the I/R + Vehicle group compared to the sham group and was significantly higher in the I/R + COG group compared to the I/R + Vehicle group (Fig. 6B, E).

Next, we detected the expression of inflammatory factors, including IL-1β, IL-18, TNF-α, and IL-6 in each group by ELISA. As shown in Fig. 6F–I, the levels of inflammatory factors were significantly increased in the I/R + Vehicle group compared to the sham group, while a significantly decreased expression of the inflammatory factors was observed in the I/R + COG group compared to the I/R + Vehicle group.

3.5. COG1410 inhibited TXNIP/NLRP3 signaling pathway in the hippocampus after cerebral I/R

Western blot was used to detect the levels of LRP1, TXNIP, NLRP3, and cleaved caspase-1 in the hippocampus of mice at 72 h after cerebral I/R. The results showed that the levels of these proteins increased in the I/R + Vehicle group compared to the sham group. Interestingly, the expression levels of TXNIP, NLRP3, and cleaved caspase-1 decreased significantly in the I/R + COG group compared to the I/R + Vehicle group. However, no statistically significant difference was observed between the I/R + Vehicle group and the I/R + COG group concerning the LRP1 level (Fig. 7B, E-H).

Double immunofluorescence staining was performed to verify the cellular localization of the TXNIP/NLRP3 signaling pathway after cerebral I/R. The results showed that TXNIP/NLRP3 signaling pathway was co-localized with microglia in the hippocampus of mice at 72 h after cerebral I/R. Moreover, quantitative analysis showed that the number of TXNIP-positive and NLRP3-positive microglia were increased significantly in the I/R + Vehicle group compared to the sham group, while the number of the TXNIP-positive and NLRP3-positive microglia was significantly decreased in the I/R + COG group compared to the I/R + Vehicle group (Fig. 7A, C-D).

3.6. Overexpression of TXNIP reversed the effects of COG1410 on M2 microglial polarization, apoptosis, and oxidative stress

Hippocampal injection of AAV vectors with green fluorescence 28 days before BCCAO significantly increased the fluorescence intensity of the hippocampus in the AAV vector groups compared to the naïve group, which confirmed the expression efficacy of the vectors (Fig. 8B). Furthermore, the overexpression efficacy of AAV-TXNIP was verified by Western blot and qRT-PCR. The results showed that both the mRNA and protein levels of TXNIP were significantly increased in the AAV-TXNIP group compared to the AAV-NC group (Fig. 8A, C).

Strikingly, compared to vehicle treatment after cerebral I/R, COG1410 treatment had no effect on the protein level of LRP1, but it significantly decreased the protein levels of TXNIP, NLRP3, cleaved caspase-1, M1 microglial phenotype marker Iba-1 and iNOS, and pro-apoptotic proteins cleaved caspase-3 and Bax and increased the levels of M2 microglial phenotype marker Arg-1 and anti-apoptotic protein Bcl-2. When the TXNIP protein was overexpressed in the I/R + COG + AAV-TXNIP group, the effects of COG1410 on the TXNIP/NLRP3 signaling pathway inhibition, M2 microglial polarization, and anti-apoptosis were reversed compared to the I/R + COG + AAV-NC group (Fig. 8D–N).

The results of HE staining showed that the normal neuronal cells in the hippocampus were significantly increased in the I/R + COG group compared to the I/R + Vehicle group and that in the I/R + COG + AAV-TXNIP group was also decreased compared to the I/R + COG + AAV-NC group (Fig. 9A, B). Since TXNIP plays a vital role in maintaining the balance of cellular redox, we assessed the degree of oxidative stress via MDA content and SOD activity and found that the degree of oxidative stress was significantly reduced with COG1410 treatment after cerebral I/R. Nevertheless, the anti-oxidative effect of COG1410 was abolished when the TXNIP protein was overexpressed (Fig. 9C, D).

3.7. COG1410 improved survival rate and long-term neurological functions through the TXNIP/NLRP3 signaling pathway after cerebral I/R

To evaluate the long-term effects of COG1410 on cerebral I/R mice, a 28-day survival rate of different groups was observed. Interestingly, the survival rate was significantly decreased in the I/R + Vehicle group compared to the sham group, while that of the cerebral I/R mice was improved by COG1410 treatment. However, the effect of COG1410 on improving the survival rate of cerebral I/R mice was abolished after overexpression of TXNIP (Fig. 10A). The results of the MWM test showed that compared to the sham group, the learning and memory function were impaired in the mice 28 days after cerebral I/R. The neurological deficits of cerebral I/R mice were alleviated with COG1410 treatment, while the neuroprotective function of COG1410 was reversed by TXNIP overexpression (Fig. 10B–F).

4. Discussion

In the current study, we first investigated the beneficial effects of LRP1 activation and explored its underlying mechanism in the cerebral I/R mouse model. Next, we demonstrated that LRP1 activation with COG1410 regulated microglia polarization, decreased brain edema, reduced oxidative stress, suppressed apoptosis in the hippocampus, and improved both short- and long-term neurological functions after cerebral I/R. In addition, LRP1 activation was associated with the downregulation of TXNIP, NLRP3, cleaved caspase-1, Iba-1, iNOS, IL-1β, IL-18, IL-6, TNF-α, cleaved caspase-3, Bax, and MDA and the upregulation of Arg-1, Bcl-2, and SOD after cerebral I/R. However, overexpression of TXNIP reversed the beneficial effects of LRP1 activation on microglial polarization, oxidative stress, apoptosis, and neurological functions. These results suggested that LRP1 activation attenuates neuroinflammation, apoptosis, and oxidative stress after cerebral I/R, which was, at least partially, related to microglial polarization mediated by the inhibition of TXNIP/NLRP3 signaling pathway.

In the present study, a BCCAO-induced cerebral I/R mice model was used. The hippocampal neurons are related to the functions of spatial learning and memory, of which the CA1 neurons are most vulnerable to ischemic damage. Transient BCCAO delays hippocampal neuron death and is often used to study the mechanism of cerebral I/R injury [32]. However, the BCCAO model shows great variability due to the presence of posterior communicating arteries (PcomA). Previous studies have confirmed that C57BL/6 mice have less PcomA patency, which is ideal for establishing a BCCAO animal model [33–35]. In this study, HE staining was used to determine the success of the mice model. The results of the histopathological analysis showed that the number of normal hippocampal pyramid cells were significantly decreased 72 h after BCCAO, which was consistent with previous studies [36, 37].

Microglia, a hallmark of neuroinflammation, plays a critical role in the delayed neuronal death caused by cerebral I/R. Activated microglia causes endocytosis of cell debris and participates in tissue repair in the early stage of cerebral I/R. However, overactivated brain microglia release inflammatory factors, such as TNF-α and IL-6, and participate in chronic inflammation, causing delayed neuronal death, brain edema, and neurological deficits [38, 39]. In this study, the results of double immunofluorescence staining showed that the LRP1 was co-localized with microglia, neurons, and astrocytes in the CA1 subregion of the hippocampus at 72 h after cerebral I/R. Previous studies indicated that direct protein-protein binding effects of apoE with its signal receptor LRP1 enable microglia to exert immunosuppressive and neuroprotective effects in neuroinflammation caused by hemorrhagic stroke, traumatic brain injury, and multiple sclerosis [40]. Another study has shown that activation of LRP1 with COG1410 promotes microglial M1 to M2 phenotypic shift, and LRP1 was expressed in M2 phenotypes but not in M1 phenotypes of activated microglia in subarachnoid hemorrhage (SAH) mice [41]. In the present study, we found that the activation of LRP1 by COG1410 significantly decreased the iNOS-positive M1 phenotypes but increased the Arg-1 positive M2 phenotypes of activated microglia. Moreover, apoE-mimic peptide COG1410 treatment significantly inhibited neuroinflammation, reduced apoptosis in the hippocampal CA1 area, decreased brain edema, and improved short- and long-term neurological function after cerebral I/R in mice.

Furthermore, how LRP1 regulates microglial M1 to M2 phenotypic shift after cerebral I/R is yet to be elucidated. The potential mechanism involves the cleavage of the intracellular domain of the LRP1 after interacting with its ligand. The cytoplasmic fragment of LRP1 may have functions in the cytoplasm or the nucleus via protein cascades or transcriptional regulation[42]. A previous study showed that the C-terminal fragment of LRP1 regulates microglial activation via inhibited JNK phosphorylation after the binding between apoE-mimic peptide COG1410 and LRP1 [43]. Another study found that stimulation of LRP1 by apoE-mimic peptide COG1410 modulated microglial polarization through Shc1/PI3K/Akt signaling pathway [41]. Nonetheless, the mechanisms of LRP1 underlying microglial phenotypic shift in cerebral I/R are yet to be elucidated. TRX is a vital intracellular antioxidant substance that exists as TRX/TXNIP under physiological conditions. The generation of a large number of reactive oxygen species (ROS) increases the expression of TXNIP and activates the NLRP3 inflammasome during the post-ischemic reperfusion phase [44]. On the other hand, some studies demonstrated that the NLRP3 inflammasome and its downstream IL-1β activate the microglial M1 phenotypes [45, 46]. The activated M1 microglia, in turn, promotes inflammation and oxidative stress, thus forming a vicious circle [47, 48]. Therefore, the TXNIP/NLRP3 signaling pathway is speculated to play a major role in regulating the polarization of microglia. The current study found that microglial M1 phenotypes are elevated, accompanied by TXNIP, NLRP3, and cleaved caspase-1 upregulation after cerebral I/R, while increased microglial M2 phenotypes are accompanied by TXNIP, NLRP3, and cleaved caspase-1 downregulation after COG1410 treatment. In addition, the results of double immunofluorescence staining showed that the number of TXNIP-positive and NLRP3-positive microglia were increased in the hippocampus of mice after cerebral I/R, while these types of cells were decreased after COG1410 treatment. Also, hippocampal apoptosis induced by cerebral I/R was observed, as indicated by increased positive cells in TUNEL staining, upregulated pro-apoptotic proteins cleaved caspase-3 and Bax, and downregulated anti-apoptotic protein Bcl-2. Previous studies have shown that cleaved caspase-1 catalyzed by activated NLRP3 inflammasomes promotes the mature release of IL-1β and IL-18 to regulate inflammation and apoptosis [49]. Some studies also found that TXNIP promotes apoptosis by activating pro-apoptotic protein signal-regulating kinase (ASK-1) and inducing the release of cytochrome-C from mitochondria [50]. Therefore, it could be deduced that apoptosis is also significantly reduced following the inhibition of the TXNIP/NLRP3 signaling pathway by activating LRP1 in this study.

To further verify the correlation between LRP1 and the TXNIP/NLRP3 signaling pathway, the TXNIP protein was overexpressed through the hippocampal injection of AAV vectors in this study. TXNIP overexpression with AAV-TXNIP significantly reversed the effects of LRP1 activation on TXNIP/NLRP3 pathway inhibition, M2 microglial polarization, anti-apoptosis, and antioxidant, while the LRP1 levels remained unchanged. These data suggested that LRP1 activation promotes M2 microglial polarization by inhibiting the TXNIP/NLRP3 pathway after cerebral I/R in mice.

Nevertheless, the present study had some limitations. Firstly, we only found that the activation of LRP1 affects the TXNIP/NLRP3 signaling pathway; however, the specific mechanism needs further study. Secondly, apoE is bound and internalized via receptor-mediated endocytosis by some receptors, such as the low-density lipoprotein receptor (LDLR), apoE receptor 2 (apoEr2), and triggering the receptor expressed on myeloid cells 2 (TREM2) in the CNS. How the apoE-mimic peptide COG1410 affects these receptors and the related pathways need to be investigated further. Finally, the clinical translation of this study needs to be further elucidated.

References

Virani S, Alonso A, Aparicio H, Benjamin E, Bittencourt M, Callaway C, Carson A, Chamberlain A, Cheng S, Delling F, et al. Heart disease and stroke statistics-2021 update: a report from the American heart association. Circulation. 2021;143:e254-e743.
Madathil R, Hira R, Stoeckl M, Sterz F, Elrod J, Nichol G. Ischemia reperfusion injury as a modifiable therapeutic target for cardioprotection or neuroprotection in patients undergoing cardiopulmonary resuscitation. Resuscitation. 2016;105:85-91.
Khoshnam SE, Sarkaki A, Khorsandi L, Winlow W, Badavi M, Moghaddam HF, Farbood Y. Vanillic acid attenuates effects of transient bilateral common carotid occlusion and reperfusion in rats. Biomed Pharmacother. 2017;96:667-674.
Gupta V, Dhull DK, Joshi J, Kaur S, Kumar A. Neuroprotective potential of azilsartan against cerebral ischemic injury: possible involvement of mitochondrial mechanisms. Neurochem Int. 2020;132:104604.
Spray S, Edvinsson L. Improved assessment of outcomes following transient global cerebral ischemia in mice. Exp Brain Res. 2016;234:1925-1934.
Lu Y, Zhou M, Li Y, Li Y, Hua Y, Fan Y. Minocycline promotes functional recovery in ischemic stroke by modulating microglia polarization through STAT1/STAT6 pathways. Biochem Pharmacol. 2021;186:114464.
Xiong X, Liu L, Yang Q. Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke. Prog Neurobiol. 2016;142:23-44.
Brifault C, Gilder A, Laudati E, Banki M, Gonias S. Shedding of membrane-associated LDL receptor-related protein-1 from microglia amplifies and sustains neuroinflammation. J Biol Chem. 2017;292:18699-18712.
Wang G, Manaenko A, Shao A, Ou Y, Yang P, Budbazar E, Nowrangi D, Zhang JH, Tang J. Low-density lipoprotein receptor-related protein-1 facilitates heme scavenging after intracerebral hemorrhage in mice. J Cereb Blood Flow Metab. 2017;37:1299-1310.
Sun R, Peng M, Xu P, Huang F, Xie Y, Li J, Hong Y, Guo H, Liu Q, Zhu W. Low-density lipoprotein receptor (LDLR) regulates NLRP3-mediated neuronal pyroptosis following cerebral ischemia/reperfusion injury. J Neuroinflammation. 2020;17:330.
Qin X, You H, Cao F, Wu Y, Peng J, Pang J, Xu H, Chen Y, Chen L, Vitek M, et al. Apolipoprotein E mimetic peptide increases cerebral glucose uptake by reducing blood-brain barrier disruption after controlled cortical impact in mice: an F-fluorodeoxyglucose PET/CT study. J Neurotrauma. 2017;34:943-951.
Peng J, Qin X, Pang J, Wu Y, Dong J, Huang C, Wan W, Yang X, Sun X, Chen L, Jiang Y. Apolipoprotein E ε4: a possible risk factor of intracranial pressure and white matter perfusion in good-grade aneurysmal subarachnoid hemorrhage patients at early stage. Front Neurol. 2017;8:150.
Wu H, Ruan J, Zhang X, Xia H, Jiang Y, Sun XJBr. Association of promoter polymorphism of apolipoprotein E gene with cerebral vasospasm after spontaneous SAH. Brain Res. 2010;1362:112-116.
Wilkins H, Koppel S, Bothwell R, Mahnken J, Burns J, Swerdlow R. Platelet cytochrome oxidase and citrate synthase activities in APOE ε4 carrier and non-carrier Alzheimer's disease patients. Redox Biol. 2017;12:828-832.
Chen S, Peng J, Sherchan P, Ma Y, Xiang S, Yan F, Zhao H, Jiang Y, Wang N, Zhang JH, Zhang H. TREM2 activation attenuates neuroinflammation and neuronal apoptosis via PI3K/Akt pathway after intracerebral hemorrhage in mice. J Neuroinflammation. 2020;17:168.
Laskowitz DT, McKenna SE, Song P, Wang H, Durham L, Yeung N, Christensen D, Vitek MP. COG1410, a novel apolipoprotein E-based peptide, improves functional recovery in a murine model of traumatic brain injury. J Neurotrauma. 2007;24:1093-1107.
Laskowitz DT, Lei B, Dawson HN, Wang H, Bellows ST, Christensen DJ, Vitek MP, James ML. The apoE-mimetic peptide, COG1410, improves functional recovery in a murine model of intracerebral hemorrhage. Neurocrit Care. 2012;16:316-326.
Wu Y, Pang J, Peng J, Cao F, Vitek MP, Li F, Jiang Y, Sun X. An apoE-derived mimic peptide, COG1410, alleviates early brain injury via reducing apoptosis and neuroinflammation in a mouse model of subarachnoid hemorrhage. Neurosci Lett. 2016;627:92-99.
Slusarczyk J, Trojan E, Wydra K, Glombik K, Chamera K, Kucharczyk M, Budziszewska B, Kubera M, Lason W, Filip M, Basta-Kaim A. Beneficial impact of intracerebroventricular fractalkine administration on behavioral and biochemical changes induced by prenatal stress in adult rats: possible role of NLRP3 inflammasome pathway. Biochem Pharmacol. 2016;113:45-56.
Heneka M, McManus R, Latz E. Inflammasome signalling in brain function and neurodegenerative disease. Nat Rev Neurosci. 2018;19:610-621.
Nyandwi JB, Ko YS, Jin H, Yun SP, Park SW, Kim HJ. Rosmarinic acid inhibits oxLDL-induced inflammasome activation under high-glucose conditions through downregulating the p38-FOXO1-TXNIP pathway. Biochem Pharmacol. 2020;182:114246.
Yan YR, Zhang L, Lin YN, Sun XW, Ding YJ, Li N, Li HP, Li SQ, Zhou JP, Li QY. Chronic intermittent hypoxia-induced mitochondrial dysfunction mediates endothelial injury via the TXNIP/NLRP3/IL-1β signaling pathway. Free Radic Biol Med. 2021;165:401-410.
Jia Y, Cui R, Wang C, Feng Y, Li Z, Tong Y, Qu K, Liu C, Zhang J. Metformin protects against intestinal ischemia-reperfusion injury and cell pyroptosis via TXNIP-NLRP3-GSDMD pathway. Redox Biol. 2020;32:101534.
Li L, Deng B, Wang S, Zhong H, Liu Z, Jin W, Jiang T, Xiao Z, Wang Q. Asynchronous therapy targeting Nogo-A enhances neurobehavioral recovery by reducing neuronal loss and promoting neurite outgrowth after cerebral ischemia in mice. J Drug Target. 2016;24:13-23.
Liu B, Cao Y, Shi F, Wang L, Li N, Cheng X, Du J, Tian Q, Zhou X. The overexpression of RBM3 alleviates TBI-induced behaviour impairment and AD-like tauopathy in mice. J Cell Mol Med. 2020;24:9176-9188.
Song D, Chen Y, Chen C, Chen L, Cheng O. GABAB receptor antagonist promotes hippocampal neurogenesis and facilitates cognitive function recovery following acute cerebral ischemia in mice. Stem Cell Res Ther. 2021;12:22.
Yang B, Zhang LY, Chen Y, Bai YP, Jia J, Feng JG, Liu KX, Zhou J. Melatonin alleviates intestinal injury, neuroinflammation and cognitive dysfunction caused by intestinal ischemia/reperfusion. Int Immunopharmacol. 2020;85:106596.
Chen Y, Lei Y, Mo L, Li J, Wang M, Wei J, Zhou J. Electroacupuncture pretreatment with different waveforms prevents brain injury in rats subjected to cecal ligation and puncture via inhibiting microglial activation, and attenuating inflammation, oxidative stress and apoptosis. 2016;127:248-259.
Zhang Y, Tan SL, Du J, Chen Y, Jia J, Feng JG, Liu KX, Zhou J. Dexmedetomidine alleviates neuroinflammation, restores sleep disorders and neurobehavioral abnormalities in rats with minimal hepatic encephalopathy. Int Immunopharmacol. 2021;96:107795.
Yang L, Guo Y, Fan X, Chen Y, Yang B, Liu KX, Zhou J. Amelioration of coagulation disorders and inflammation by hydrogen-rich solution reduces intestinal ischemia/reperfusion injury in rats through NF-κB/NLRP3 pathway. Mediators Inflamm. 2020;2020:4359305.
Jia Y, Li Z, Feng Y, Cui R, Dong Y, Zhang X, Xiang X, Qu K, Liu C, Zhang J. Methane-rich saline ameliorates sepsis-induced acute kidney injury through anti-inflammation, antioxidative, and antiapoptosis effects by regulating endoplasmic reticulum stress. Oxid Med Cell Longev. 2018;2018:4756846.
Cai M, Phan P, Hong J, Kim D, Kim J, Park S, Liu X, Han J, Park H, Choi J, Ryu J. The neuroprotective effect of eupatilin against ischemia/reperfusion-induced delayed neuronal damage in mice. Eur J Pharmacol. 2012;689:104-110.
Barone F, Knudsen D, Nelson A, Feuerstein G, Willette R. Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy. J Cereb Blood Flow Metab. 1993;13:683-692.
Kitagawa K, Matsumoto M, Yang G, Mabuchi T, Yagita Y, Hori M, Yanagihara T. Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery. J Cereb Blood Flow Metab. 1998;18:570-579.
Zhen G, Dore S. Optimized protocol to reduce variable outcomes for the bilateral common carotid artery occlusion model in mice. J Neurosci Methods. 2007;166:73-80.
Tajiri S, Oyadomari S, Yano S, Morioka M, Gotoh T, Hamada JI, Ushio Y, Mori M. Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP. Cell Death Differ. 2004;11:403-415.
Lei X, Lei L, Zhang Z, Cheng Y. Neuroprotective effects of lycopene pretreatment on transient global cerebral ischemia/reperfusion in rats: The role of the Nrf2/HO1 signaling pathway. Mol Med Rep. 2016;13:412-418.
Park J, Park J, Ahn J, Kim Y, Kang I, Won M, Lee C. Transient cerebral ischemia induces albumin expression in microglia only in the CA1 region of the gerbil hippocampus. Mol Med Rep. 2017;16:661-665.
He B, Wang X, He Y, Li H, Yang Y, Shi Z, Liu Q, Wu M, Sun H, Xie J, et al. Gamma ray-induced glial activation and neuronal loss occur before the delayed onset of brain necrosis. FASEB J. 2020;34:13361-13375.
Chuang T, Guo Y, Seki S, Rosen A, Johanson D, Mandell J, Lucchinetti C, Gaultier A. LRP1 expression in microglia is protective during CNS autoimmunity. Acta Neuropathol Commun. 2016;4:68.
Peng J, Pang J, Huang L, Enkhjargal B, Zhang T, Mo J, Wu P, Xu W, Zuo Y, Peng J, et al. LRP1 activation attenuates white matter injury by modulating microglial polarization through Shc1/PI3K/Akt pathway after subarachnoid hemorrhage in rats. Redox Biol. 2019;21:101121.
Boucher P, Herz J. Signaling through LRP1: protection from atherosclerosis and beyond. Biochem Pharmacol. 2011;81:1-5.
Yang L, Liu C, Zheng H, Kanekiyo T, Atagi Y, Jia L, Wang D, N'songo A, Can D, Xu H, et al. LRP1 modulates the microglial immune response via regulation of JNK and NF-κB signaling pathways. J Neuroinflammation. 2016;13:304.
Nasoohi S, Ismael S, Ishrat T. Thioredoxin-interacting protein (TXNIP) in cerebrovascular and neurodegenerative diseases: regulation and implication. Mol Neurobiol. 2018;55:7900-7920.
Cao X, Wang Y, Gao L. CHRFAM7A overexpression attenuates cerebral ischemia-reperfusion injury via inhibiting microglia pyroptosis mediated by the NLRP3/Caspase-1 pathway. Inflammation. 2021;44:1023-1034.
Campagno K, Mitchell C. The P2X receptor in microglial cells modulates the endolysosomal axis, autophagy, and phagocytosis. Front Cell Neurosci. 2021;15:645244.
Catani M, Gasperi V, Bisogno T, Maccarrone M. Essential dietary bioactive lipids in neuroinflammatory diseases. Antioxid Redox Signal. 2018;29:37-60.
Du SQ, Wang XR, Zhu W, Ye Y, Yang JW, Ma SM, Ji CS, Liu CZ. Acupuncture inhibits TXNIP-associated oxidative stress and inflammation to attenuate cognitive impairment in vascular dementia rats. CNS Neurosci Ther. 2018;24:39-46.
Wang K, Lv Q, Miao YM, Qiao SM, Dai Y, Wei ZF. Cardamonin, a natural flavone, alleviates inflammatory bowel disease by the inhibition of NLRP3 inflammasome activation via an AhR/Nrf2/NQO1 pathway. Biochem Pharmacol. 2018;155:494-509.
Lu J, Holmgren A. Thioredoxin system in cell death progression. Antioxid Redox Signal. 2012;17:1738-1747.

Activation of LRP1 ameliorates cerebral ischemia/reperfusion injury and cognitive decline by suppressing neuroinflammation and oxidative stress through TXNIP/NLRP3 signaling pathway in mice

Abstract

1. Introduction

2. Materials And Methods

3. Results

4. Discussion

5. Conclusions

Abbreviations

Declarations

References