The Molecular Mechanism of Quercetin Improving Cerebral Ischemia Injury by Regulating NLRP3 Inflammasome Mediated Pyroptosis Axis

doi:10.21203/rs.3.rs-5330142/v1

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Research Article

The Molecular Mechanism of Quercetin Improving Cerebral Ischemia Injury by Regulating NLRP3 Inflammasome Mediated Pyroptosis Axis

https://doi.org/10.21203/rs.3.rs-5330142/v1

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Background

In the pathological process of cerebral ischemia, the neuroinflammation triggered by NLRP3 inflammasome-mediated neuronal pyroptosis is considered a crucial factor contributing to brain damage. Although, previous research has revealed the anti-inflammatory and neuroprotective effects of quercetin (Que), its precise mechanisms in intervening and alleviating neuroinflammation and the pyroptosis axis triggered by cerebral ischemia remain to be fully elucidated.

Objective

To investigate the role and mechanism of Que in alleviating cerebral ischemia injury by modulating the NLRP3 inflammasome-mediated pyroptosis axis.

Methods

The oxygen-glucose deprivation and reperfusion (OGD/R) model with BV2 microglial cells and the photothrombosis (PT) model with C57/BL male mice were utilized. The study explored the neuroprotective effect of Que against cerebral ischemia injury through the regulation of the pyroptosis axis via the NLRP3 inflammasome, assessed through experiments including reactive oxygen species measurement, cell pyroptosis rate detection, behavioral tests, HE staining, Nissl staining, and examination of pyroptosis-related proteins and inflammatory factors.

Results

Que alleviated cell damage and pyroptosis induced by OGD/R, reduced intracellular reactive oxygen species levels, improved behavioral impairment in mice after modeling, decreased brain injury area, maintained neuronal morphology, and lowered the expression of pyroptosis axis-related proteins such as NLRP3, Caspase-1, ASC, GSDMD, as well as inflammatory factors IL-18, IL-1β, TNF-α.

Conclusion

Que alleviated cerebral ischemia injury by regulating the pyroptosis axis through inhibiting the activation of NLRP3 inflammasome. This provides robust experimental evidence for the anti-neuroinflammatory effects of Que and the development of neuroprotective drugs.

Quercetin

Cerebral Ischemia

Neuroinflammation

Pyroptosis Axis

Inflammasome

Cerebral ischemia refers to a condition where inadequate blood supply to a specific part of the brain leads to impaired brain function and may result in complications such as cerebral thrombosis. This disease poses a serious threat to human life and health. Among the various mechanisms contributing to cerebral ischemia injury, inflammation reaction is the most notable (Lambertsen et al., 2019,Joy, Carmichael, 2021). Therefore, the search for effective anti-inflammatory drugs to treat cerebral ischemia is currently a primary concern.

Pyroptosis is a form of programmed cell death closely associated with inflammatory reactions (Lyden, 2021). Pyroptosis of microglial cells is closely linked to the inflammatory cascade response in cerebral ischemia. Upon activation of microglial cells, the NOD-like receptor pyrin domain-containing 3 (NLRP3) receptor recognizes and activates NLRP3. After activation, NLRP3 recruits the adaptor protein apoptosis-associated speck-like protein containing CARD (ASC) and the precursor protein of cysteinyl aspartate specific proteinase-1 (caspase-1), Pro-Caspase-1, through the NACHT domain, forming the NLRP3 inflammasome. The NLRP3 inflammasome can rapidly activate Caspase-1, forming the NLRP3/Caspase-1/GSDMD pyroptosis axis, inducing pyroptosis (Gou et al., 2021).GSDMD is cleaved into GSDMD-N-terminal, which binds to the cell membrane, forming pores, causing cell membrane rupture, releasing inflammatory cytokines, and ultimately triggering an inflammatory cascade reaction (Shi et al., 2015). At present, clinical and animal experiments show that inhibiting the activation of microglial cell NLRP3 inflammasomes and their regulated pyroptotic axis has a protective effect on neurons after cerebral ischemia injury (Ma et al., 2021). For example, the expression of pyroptosis axis-related proteins such as NLRP3, ASC, Caspase-1, and GSDMD increased (Fann et al., 2013) after cerebral ischemia-hypoxia in both in vitro and in vivo experiments. The activation of NLRP3 inflammasomes was observed. However, inhibition of Caspase-1 restrained this phenomenon, allvating cerebral ischemia injury. As a result, regulating the pyroptosis axis through NLRP3 inflammasome holds great potential as a therapeutic approach for treating cerebral ischemia injury (Robinson et al., 2019).

Quercetin (Que) (Singh et al., 2021) is a natural flavonoid compound known for its significant effects on inhibiting oxidative stress signaling pathways and suppressing the generation of inflammatory factors (Batiha et al., 2020). In a study investigating the neuroprotective effects of Que on MCAO model rats, it was found that Que significantly reduced cerebral infarction volume, neurological deficits, blood-brain barrier permeability, and ROS production through the Sirt1/Nrf2/HO-1 signaling pathway (Yang et al., 2021). Likewise, another study demonstrated that pretreatment with Que (25 mg/kg) alleviated TNF-α and IL-1β mRNA expression in the rat hippocampus, as well as Caspase-3 activity, confirming the anti-inflammatory and anti-apoptotic properties of Que (Wang et al., 2020). However, the relationship between Que and the pyroptosis axis regulated by NLRP3 inflammasome, as well as the molecular mechanisms through which Que alleviates cerebral ischemia injury, remains unclear. Thus, this study aims to investigate the neuroprotective effects of Que against cerebral ischemia injury through the regulation of the NLRP3 inflammasome-mediated pyroptosis axis. The oxygen-glucose deprivation and reperfusion (OGD/R) model with BV2 microglial cells (Hu et al., 2020) and the photothrombosis (PT) model with C57/BL male mice were utilized (Conti et al., 2023). This study focused on NLRP3 inflammasome and the pyroptosis axis to validate the therapeutic efficacy and potential applications of Que in cerebral ischemia diseases.

2.1 Materials

Que was purchased from Shanghai Jizhi Biochemical Technology Co., Ltd.; MCC950 inhibitor was obtained from Shandong ScienCell Biotechnology Co., Ltd.; reactive oxygen species detection kit, MTT cell proliferation and cytotoxicity assay kit, lactate dehydrogenase cytotoxicity assay kit were all purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; ELISA assay kit was acquired from Xingbo Sheng Biological Technology Co., Ltd.; Hoechst33342/PI double staining kit was obtained from Beijing Solarbio Science & Technology Co., Ltd.; DMEM high glucose/low glucose culture media, PBS were purchased from Hangzhou Situofan Biological Technology Co., Ltd.; fetal bovine serum was obtained from Inner Mongolia Op-sci Biotechnology Co., Ltd.; trypsin digestion solution was purchased from Thermo Fisher Scientific; GAPDH, ASC, HRP-labeled goat anti-rabbit IgG antibody were purchased from Jiangsu Qinko Biomedical Research Center Co., Ltd.; NLRP3, Caspase-1 antibodies were obtained from Huaan Biological Technology Co., Ltd.; GSDMD antibody was purchased from Abcam Technologies Co., Ltd.

Male SPF C57BL/6J mice, aged 6–8 weeks, weighing approximately 20–25 g, were purchased from Shanghai SLAC Laboratory Animal Co., Ltd [Experimental Animal Production License Number: SCXK (Hu) 2022-0004]. The experimental animals were raised at the Zhejiang University of Medicine Animal Experiment Center [Experimental Animal Use License Number: SYXK (Zhe) 2021-0012]. During the housing period, the experimental animals were provided with adequate food and water, and they were kept in a well-maintained environment with a 12 h light/12 h dark cycle. All experimental procedures were conducted following the ethical guidelines of the Zhejiang Chinese Medical University Animal Experiment and Research Center [Ethical Approval Number: IACUC-20220725-06].

2.2 Methods

2.2.1 PT Stroke Model Preparation and Animal Grouping

C57BL/6J male mice were randomly assigned to the following groups: Sham group, Stroke group, Stroke + Que group, and Stroke + MCC950 group. Within 24 h after modeling, mice were intraperitoneally injected with Que (100mg/kg) and MCC950 (10mg/kg). After intraperitoneal injection of 1% pentobarbital sodium (70mg/kg) for anesthesia, mice were fixed on a stereotaxic apparatus. The head fur was shaved, and the head skin was disinfected with iodine before incision to expose the skull. Using Bregma as the origin, the modeling point was set at 2 mm to the right of the sagittal suture and 2 mm posterior to the coronal suture. While grinding the skull, sterile physiological saline was added dropwise until the cerebral vessels were clearly visible. Subsequently, mice were intraperitoneally injected with rose bengal (100 mg/kg), and after 5 min, the modeling point was illuminated with a cold light source (532 nm) for 20 min. After illumination, the scalp of the mice was disinfected and sutured, and they were placed on a heating pad to maintain a temperature of 37°C until they regained consciousness and were returned to their cages. Subsequent experiments were conducted 24 h after modeling.

2.2.2 Cell Culture

Mouse microglia (BV2) were purchased from the Cell Bank of the Chinese Academy of Sciences. Cells were cultured with complete medium (95% high sugar DMEM medium + 5% fetal bovine serum + 0.5% dual antibody) and placed in 37℃, 5% CO2 incubator. Cells were grown to cover 80% area of the culture flask for passaging.

2.2.3 OGD/R Model Preparation and Grouping

Cells were divided into Ctrl group, OGD/R group, OGD/R + Que group, OGD/R + MCC950 group. OGD/R group: the culture medium was discarded and washed by adding PBS for 1–2 times, replaced with Eagle's sugar-free medium, and put the cells in the hypoxia device and then continuously ventilated with 95% N2 and 5% CO2, and then closed the air inlet and outlet for 5 min. Put the oxygen-deficient device into 37℃ constant temperature incubator, after 8 h of hypoxia and glucose deprivation, take it out, add DMEM high-sugar medium and put it back into 5% CO2 incubator to reoxygenate for 16 h. OGD/R + Que group: after 8 h of hypoxia and glucose deprivation, add pre-prepared DMEM high-sugar medium containing Que and put it back into 5% CO2 incubator to reoxygenate for 16 h. OGD/R + MCC950 group: after 8 h of hypoxia and glucose deprivationy, add pre-prepared DMEM high-sugar medium containing MCC950 and put it back into 5% CO2 incubator to reoxygenate for 16 h.

2.2.3 MTT Assay

BV2 cells were uniformly inoculated in 96-well plates at 1×10⁴/well, and 6 replicate wells were set in each group. Cultivate according to the above grouping, when the cell density reached about 70%-80%, discard the culture medium, wash with PBS for 1–2 times, and carry out oxygen-sugar deprivation/complex-sugar reoxygenation treatment; add 10µL of MTT culture medium to each well, and incubate at a constant temperature of 37℃ for 4 h; discard the culture medium, add 100µL of dimethylsulfoxide (DMSO) to the wall, protect from light, and shake the shaker at a low speed for 15 min; measure the absorbance at the wavelength of 490 mm by using an enzyme marker and calculate the viability value. Absorbance was measured at 490 mm and the viability value was calculated.

2.2.4 ROS Assay

After the cells were treated according to the above grouping, the probes were first loaded, and the DCFH-DA was diluted with serum-free culture medium 1:1000 to a final concentration of 10µM, the old medium was removed, and the cells were incubated for 20 min in 70µL per well, protected from light; the serum-free culture medium was washed for three times in order to sufficiently remove the DCFH-DA that had not entered into the cells; the cells were observed and photographed under the fluorescent inverted microscope (Axio Observer. A1). observation and photographed for recording.

2.2.5 LDH Assay

Cells were inoculated with 1×10⁴/well in 96-well plates and grouped as above. Take the culture medium of each well into a 1.5mL centrifuge tube, 1500r/5min, take the supernatant, and transfer it into a new 96-well plate; add 30µL LDH working solution to each well and mix it well (prepared according to the proportion of the instruction manual); shaking the bed for 30 min at room temperature, avoiding the light, and detecting the absorbance at 490 nm on the enzyme marker.

2.2.6 Hoechst33342/PI Staining

To detect the cell scorch death, cells were inoculated at 2.5×10⁵/well in a 6-well plate and treated in groups as described above. Add 5µL of Hoechst staining solution and 5µL of PI staining solution to each well, mix well; ice bath for 30 min, wash with PBS for 1–2 times; observe under fluorescence inverted microscope (Axio Observer. A1) and take photos to record.

2.2.7 ELISA Assay

BV2 cells were inoculated in 6-well plates at a density of 2.5×10⁵/well, processed in groups as described above, and the supernatant was collected. Blood was taken from the orbits of mice after anesthesia and centrifuged for half an hour to collect the supernatant. Take the kit out of the refrigerator, equilibrate at room temperature for 30 min, and operate according to the instructions of the kit. Turn on the zymograph 20 min in advance, zero the blank wells, and set the wavelength of 450 nm to measure the absorbance (OD value) of each well. The concentrations of IL-1β, IL-18, TNF-α and TGF-β in each sample were calculated according to the standard curve.

2.2.8 Western Blotting

Cells were inoculated in 6-well plates at 2.5×10⁵/well, processed according to the above grouping, trypsin digested the cells and collected. Mice were anesthetized and the brain was removed, the cortical injury site and surrounding brain tissues were isolated, weighed and transferred into 1.5mL EP tubes. Cells and brain tissues were lysed for BCA protein concentration determination. Protein samples were separated by 10% or 12% SDS-PAGE and electroblotted onto polyvinylidene difluoride (PVDF) membranes, which were closed with 5% skimmed milk at room temperature for 2 h. The membranes were incubated with NLRP3 (1:1000), GSDMD (1:1000), ASC (1:1000), Caspase-1 (1:1000), GAPDH (1:3000) Primary antibodies were treated overnight at 4°C, washed 3 times with TBST, then treated with HRP-labeled goat anti-rabbit IgG (1:20,000) for 2 h at room temperature, washed again with TBST, and protein bands were displayed using a fluorescence and chemiluminescence imaging system (ChemiScope6200). Protein bands were quantitatively analyzed using Image J software.

2.2.9 Behavioral Tests

Grid experiment: The mice were first placed on a horizontal metal grid (12cm×12cm, with a grid spacing of 1cm), and a camera was placed underneath the grid to record the support of the mice's limbs while crawling. The camera was filmed for 3 min and played back at 1/5 of the normal speed to record the number of times the mouse's limbs stepped out of place during crawling. Calculate the rate of missteps = (number of missed steps on the left side/100)*100%. Cylinder Experiment: Mice were placed in a 10cm x 15cm (diameter x height) transparent Plexiglas cylinder, and when the mice stood up in the cylinder, they would use one or both of their forelimbs to support their bodies by placing them on the wall of the cylinder. A camera was placed on top of the cylinder to record the mice's forelimbs against the wall. The camera was filmed for 5 min and played back at 1/5 of the normal speed, recording the number of times the right limb, the left limb, and the left and right limbs of the mouse were placed on the wall of the cylinder at the same time. Calculate the asymmetry rate = (R-L)/(R + L + B) × 100%. r = the number of times the right forelimb was attached to the wall, L = the number of times the left forelimb was attached to the wall, and B = the number of times the forelimbs on both sides were attached to the wall.

2.2.10 HE Staining and Nissl Staining

Mice were anesthetized and their brains were removed by severing their heads and placed in 4% paraformaldehyde for 24 h after fixation, then the brain tissues were dehydrated and paraffin-embedded. The embedded brain tissue was paraffin sectioned at a thickness of 4µm. After dewaxing and rehydration, the sections were placed in hematoxylin staining solution for 5 min, 1% hydrochloric acid alcohol for rapid differentiation, ammonia to return to the blue, and rinsed in running water for about 1 min, and then placed in eosin staining solution to re-stain the sections for 1–3 min. Sections were sequentially put into gradient alcohol, xylene I and II in rows of dehydration, neutral gum sealing, using digital pathology section scanning system C13210-01 to observe pathological changes; the sections were stained with 1% toluidine blue, 60 ℃ oven for 30 min. After washing the floating color with distilled water, the sections were dehydrated in 95% as well as 100% ethanol, transparent in xylene, sealed with neutral gum, and the pathological changes were observed using Digital Pathology Slice Scanning System C13210-01.

2.2.11 Immunohistochemical Detection

Paraffin sections of brain tissue were taken, antigen repaired, closed, NLRP3 primary antibody (1:100) was added dropwise, incubated overnight at 4°C, secondary antibody incubated, DAB color developed, dehydrated and sealed, and then NLRP3-positive cells were observed and counted using Digital Pathology Slice Scanning System C13210-01.

2.2.12 Statistical Analysis

All experimental data were expressed as mean (Mean) ± standard deviation (SD) (n ≥ 3). Statistical analyses were performed using Graph Pad Prism 8.0 software with one-way ANOVA (one-way ANOVA) and Tukey, multiple comparisons. p < 0.05 was considered statistically significant.

3.1 Effect of Different Concentrations of Que on OGD/R Cell Viability

Within a certain concentration range, the BV2 cell viability increased with the increasing concentration of quercetin, indicating that quercetin could alleviate cell damage induced by OGD/R. When the concentration of quercetin was 100µmol/L, the cell viability was significantly higher compared to the OGD/R group. This concentration was selected for subsequent experiments in this study (Fig. 1A).

3.2 Que Inhibits OGD/R-Induced Microglial Cell Damage

When the cell membrane ruptures, LDH is released into the extracellular space. Hence, assessing the LDH content in the cell culture supernatant can infer the integrity of the cell membrane, thereby evaluating the occurrence of pyroptosis. The results showed that compared to the Ctrl group, the LDH content significantly increased in the OGD/R group. However, after quercetin treatment, the LDH content decreased, showing a similar effect to MCC950 (Fig. 1B). The increase in intracellular reactive oxygen species (ROS) is one of the markers of BV2 cell activation, prompting BV2 cells to secrete more pro-inflammatory factors and exacerbate inflammation. To determine whether quercetin reduces ROS production induced by OGD/R, ROS detection was performed. The results showed that the ROS levels increased in the OGD/R group, while Que treatment significantly inhibited intracellular ROS production (Fig. 1C-D).

3.3 Que Inhibited Pyroptosis Induced by OGD/R

Hoechst can penetrate the cell membrane, stain the cell nucleus, and PI is a nucleic acid dye that does not stain normal or apoptotic cells but colors necrotic cells. Therefore, this study used Hoechst33342/PI staining to verify whether Que can inhibit pyroptosis. The results showed that compared to the Ctrl group, OGD/R group cells exhibited nuclear condensation and an increase in pyroptotic cells, while treatment with Que and MCC950 significantly reduced nuclear condensation and pyroptotic cells (Fig. 2A-B).

3.4 Que Reduced the Expression of Pyroptotic Inflammatory Factors

After BV2 cells undergo pyroptosis, they release a large number of pro-inflammatory factors, such as IL-1β, IL-18, TNF-α, etc., and the release of the anti-inflammatory factor TGF-β decreases. IL-18 and IL-1β play a crucial role in pyroptosis, while TNF-α, as an inflammatory factor in the brain, can enhance the central nervous system's inflammatory response by affecting blood-brain barrier permeability, leading to central invasion and inflammation. ELISA results showed that compared to the Ctrl group, the OGD/R group exhibited an increase in IL-1β, IL-18, TNF-α levels, and a decrease in TGF-β levels. After quercetin treatment, the levels of pro-inflammatory factors decreased, and anti-inflammatory factors increased, with a similar effect to the MCC950 inhibitor (Fig. 2C-F). Thus,the results indicated that Que could reduce the expression of inflammatory factors after pyroptosis occurred.

3.5 Inhibition of NLRP3 Inflammasome Activation by Que Attenuates OGD/R-induced BV2 Cell Pyroptosis

NLRP3 inflammasome activation triggers the focal death execution protein GSDMD to execute focal death. So, NLRP3 inflammasome activation is a key factor in inducing microglia to undergo focal death. The Hoechst33342/PI Staining initially verified that Que could attenuate microglial cell pyroptosis, and further studies were needed to investigate whether Que could attenuate OGD/R-induced BV2 cell pyroptosis by inhibiting NLRP3 inflammasome activation.The Western blot results showed that the expression of pyroptosis-related proteins, NLRP3, Caspase-1, and GSDMD was significantly elevated in the OGD/R group, as compared with that in the Ctrl group, ASC expression was significantly elevated; MCC950, a specific inhibitor of NLRP3 inflammatory vesicles, significantly inhibited NLRP3 inflammatory vesicle activation. Obviously, after the treatment with Que and MCC950, the expression of NLRP3, Caspase-1, GSDMD, and ASC were significantly reduced (Fig. 2G-K). Consequently, these results indicated that Que attenuated OGD/R-induced pyroptosis by regulating the pyroptosis axis through inhibiting the activation of NLRP3 inflammasome.

Figure 2 Que attenuated OGD/R-induced pyroptosis by regulating the pyroptosis axis through inhibiting the activation of NLRP3 inflammasome (A) Representative images of Hoechst/PI staining, scale bar = 50µm.(B) Quantitative analysis of Hoechst/PI staining. (C-F) ELISA for the expression levels of IL-1β, IL-18, TNF-α, and TGF-β in cell supernatants. (G) Representative Western blot images of NLRP3, Caspase-1, ASC, and GSDMD; (H-K) Quantitative protein analysis of NLRP3, Caspase-1, ASC, and GSDMD. Compared with Ctrl group, ^**P < 0.01; compared with OGD/R group, ^##P < 0.01; Mean ± SD, n = 3.

3.6 Que Ameliorated Motor Dysfunction and Neuronal Cell Injury in PT Mice

In order to verify the protective effect of Que on motor function impairment in mice with cerebral ischemia injury, the cerebral ischemia model was established by PT in this study. The results showed that the rate of missteps and forelimb asymmetry in the Stroke group was significantly higher compared with that before modeling, suggesting that the mice suffered from motor dysfunction after cerebral ischemia injury; the rate of missteps and forelimb asymmetry were significantly decreased after treatment with Que and MCC950 (Fig. 3A-B). The results indicated that Que attenuated the motor dysfunction in PT mice. The Nissl staining results showed that, compared to the Sham group, the Stroke group exhibited a significant decrease in the number of Nissl bodies, sparse distribution, and an enlarged area of brain injury. However, after the treatment with Que and MCC950, the number of Nissl bodies increased, and the area of brain injury decreased (Fig. 3C-D). HE staining showed that the neuronal cells in the Sham group were structurally intact and tightly packed, while the nuclei of neuronal cells in the Stroke group were solidified and deeply stained, with increased cellular interspaces. The neurons in the Stroke group showed nuclear consolidation, deep staining, increased cell gaps and cavities; the neuronal damage was slowed down after the treatment with Que and MCC950 (Fig. 3E). The above results indicate that Que could improve post-ischemic brain injury and had a protective effect on neurons.

3.7 Que Inhibited Inflammasome Activation to Alleviate Pyroptosis Induced by Cerebral Ischemia

Importantly, the activation of the NLRP3 inflammasome is the primary cause of regulating the pyroptosis axis and triggering inflammatory responses.Thus, this study employed immunohistochemistry to detect the expression of the key protein NLRP3 involved in inflammasome activation in the cortex. The results showed a significant increase in NLRP3-positive cells in the Stroke group compared to the Sham group. However, the Stroke + Que and Stroke + MCC950 groups exhibited a reduction in NLRP3-positive cells compared to the Stroke group (Fig. 4A-B). After pyroptosis occurred, inflammatory factors such as IL-1β, IL-18, and TNF-α were rapidly released into the extracellular space, triggering a cascade of inflammatory reactions through cell membrane pores. ELISA results demonstrated a significant increase in the expression of IL-1β, IL-18, and TNF-α in the Stroke group compared to the Sham group. However, the Stroke + Que and Stroke + MCC950 groups showed a notable decrease in the expression levels of these inflammatory factors compared to the Stroke group (Fig. 4C-D). Western Blot results revealed a significant elevation in the expression levels of pyroptosis-related proteins NLRP3, Caspase-1, GSDMD, and ASC proteins in the Stroke group compared to the Sham group. In contrast, the Stroke + Que and Stroke + MCC950 groups displayed a significant reduction in the expression levels of these proteins compared to the Stroke group (Fig. 4F-J). The above data suggested that Que regulated the pyroptosis axis through the NLRP3 inflammasome, reducing the expression of pyroptosis-related proteins and inflammatory factors in PT mice.

Figure 4 Que inhibited pyroptosis in PT mice by regulating the NLRP3 inflammasome and the pyroptosis axis (A) Immunohistochemistry was used to detect the expression of NLRP3-positive cells in the cortex of mice in each group treated with Que. Scale bar = 50µm; (B) Quantitative analysis of NLRP3-positive cells. (D) ELISA was performed to measure the expression levels of IL-1β in the serum of mice in each group; (E) ELISA was conducted to determine the expression levels of IL-18 in the serum of mice in each group. (F) ELISA was carried out to assess the expression levels of TNF-α in the serum of mice in each group. (G) Representative images of Western Blot analysis of pyroptosis-related proteins in the cortical tissues of mice in each group; (H-J) Quantitative analysis of the expression levels of pyroptosis-related proteins. Compared with Sham group ^**P < 0.01; compared with Stroke group, ^##P < 0.01; Mean ± SD, n = 3.

Existing literature reported that cerebral ischemia injury can lead to increased vascular permeability, vascular-origin brain edema formation, resulting in infarction, and the manifestation of abnormal neurological behavior(Schaller, Graf, 2004). For example, Que (50 mg/kg) was disclosed to be effective in improving the neurological functional damage in hypoxic-ischemic brain injury (HIBI) mice and has a protective effect on neuronal cell damage(Le et al., 2020).Through behavioral analysis, HE staining, and Nissl staining results, this experiment observed an improvement in motor dysfunction in mice with cerebral ischemia injury after Que treatment. Additionally, there was a significant increase in Nissl body staining in the brain injury area, neuronal morphology gradually recovered.

Pyroptosis is an atypical form of programmed cell death that occurs under inflammatory conditions(Ma et al., 2020). During the process of pyroptosis, cells continuously swell until the cell membrane ruptures. Before membrane rupture, numerous vesicular structures, known as pyroptotic bodies, form inside the cell. Subsequently, the cell membrane ruptures, forming pores of 10–15 nm, through which inflammatory cytokines such as LDH, IL-1β, and IL-18 are released in large quantities. If a PI dye that can penetrate the damaged cell membrane is applied at this time, it can enter the nucleus of pyroptotic cells, and the staining result will be positive(Gao et al., 2018,Man et al., 2017). Currently, the study of the pyroptosis axis in cerebral ischemia injury remains a hot topic. Interventions targeting the pyroptosis axis may become a potential therapeutic approach, offering hope for alleviating the inflammatory response and reducing cerebral ischemia injury. In this study, LDH release test results demonstrated that Que reduced the LDH content in the cell supernatant after OGD/R. Hoechst33342/PI staining showed that Que reduced the PI-positive rate after OGD/R in BV2 cells. Clearly, both results confirmed that Que could inhibit pyroptosis in BV2 cells after OGD/R.

In the cerebral ischemia model, the interruption of blood supply and the subsequent reperfusion lead to the generation of a large amount of reactive oxygen species, inflammatory mediators, and intracellular calcium influx, among other factors, activating complexes such as the NLRP3 inflammasome(Han et al., 2023). The NLRP3 inflammasome, composed of NLRP3, ASC, and Pro-Caspase-1, can further activate Caspase-1. Activated Caspase-1 induces the formation of the pyroptosis axis through GSDMD(Shi et al., 2017,Gaidt, Hornung, 2016). Meanwhile, it promotes the maturation and release of IL-1β and IL-18, ultimately resulting in neuronal pyroptosis and triggering an inflammatory cascade(Sun et al., 2020). Recent studies indicated that the NLRP3 inflammasome is a crucial pathway in regulating the pyroptosis axis, participating in the pathogenesis of cerebral ischemia injury(Silverman et al., 2009). For instance, research by Fann et al.(Fann et al., 2013) demonstrated that administering a Caspase-1 inhibitor reduces the expression of NLRP3 inflammasome and pyroptosis axis-related proteins, inhibiting neuronal pyroptosis. Similarly, a study found that inhibiting the NLRP3 inflammasome reduced neuronal apoptosis induced by cerebral ischemia injury(Yang et al., 2014). In this study, we observed that the levels of various inflammatory factors such as IL-1β, IL-18, TNF-α in the serum of OGD/R BV2 cells and cerebral ischemia mice treated with Que were significantly higher than those in the normal group but significantly lower than those in the model group. Simutaneously, the protein levels of NLRP3, Caspase-1, ASC, and GSDMD in the damaged cortical area of the brain showed a similar phenomenon, being significantly higher than the normal group and significantly lower than the model group, with effects comparable to MCC950. This indicates that Que may inhibit the formation and maturation of the inflammasome by directly or indirectly acting on key components of the NLRP3 inflammasome. It prevented the cleavage of GSDMD to form GSDMD-N-terminal, reduced the formation of pores on the cell membrane, thereby lowering the release of inflammatory factors in the brain, interrupting the cascade of pyroptosis, and alleviating brain damage. This result further validated the significant intervention effect of Que through the regulation of the pyroptosis axis via the NLRP3 inflammasome.

In addition, ROS is one of the agonists that trigger the activation of the NLRP3 inflammasome, and reducing the expression of ROS can prevent the activation of the NLRP3 inflammasome(Hou et al., 2018,Letteria et al., 2016). Que reduced cerebral ischemic injury by alleviating oxidative stress and inflammatory reactions, as well as inhibiting neuronal apoptosis(Chang et al., 2014).In this study, it was found that Que reduced the production of intracellular ROS after OGD/R, indicating that Que effectively scavenged reactive oxygen species generated during the OGD/R process, maintaining the redox balance within cells. This effect helped alleviate pathological changes caused by oxidative stress, such as lipid peroxidation, DNA damage, and protein dysfunction, providing a more favorable environment for the survival of damaged neurons.

The above research results all indicated that Que regulated the pyroptosis axis through the NLRP3 inflammasome, alleviated neuroinflammation, and exerted a protective effect against cerebral ischemia injury. Nonetheless, future research needs to delve into how Que specifically acts on various components of the NLRP3 inflammasome and explore its pharmacokinetic characteristics and potential side effects in the in vivo environment, providing better guidance for clinical application.

In conclusion, this study demonstrated that Que exerted multiple biological effects in cerebral ischemia injury, including inhibiting the assembly of the NLRP3 inflammasome and its downstream pyroptosis signal transduction, downregulating the expression of pro-inflammatory factors, and improving oxidative stress status. These combined effects collectively constitute the potent resistance of Que against cerebral ischemia injury. It provides new therapeutic targets for inflammation in cerebral ischemia injury and offers new perspectives for the clinical application of Que.

In summary, Que improved post-ischemic neuronal functional damage by regulating the pyroptosis axis through the NLRP3 inflammasome. It helped maintain neuronal morphology, alleviating neuronal pyroptosis and inflammatory responses, ultimately playing a protective role against cerebral ischemia injury (Fig. 5). These findings establish a robust theoretical foundation for further clinical investigations into the potential therapeutic benefits of Que.

Funding

This work was supported by National Natural Science Foundation of China (File No.81673639 and No.82174066), Zhejiang Chinese Medical University Project (File No.2022TS001).

Author Contributions

Huiling Yuan and Da Shen designed research; Huiling Yuan, Yue Wang, Da Shen, Lisheng Chu, Tingting Sang and Lin Li performed the research; Huiling Yuan and Da Shen analyzed the data and wrote the paper; Lijun Ge acquired funding, and helped to revised the paper. All authors read and approved the final paper.

Competing Interests

The authors declare no competing interests.

Data Availability

These data were used under license for the current study, so these data are not publicly available. However, the data are available from the authors upon reasonable request and with permission from corresponding authors.

Ethics approval

All experimental procedures were conducted following the ethical guidelines of the Zhejiang Chinese Medical University Animal Experiment and Research Center [Ethical Approval Number: IACUC-20220725-06].

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The authors declare no competing interests.

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The Molecular Mechanism of Quercetin Improving Cerebral Ischemia Injury by Regulating NLRP3 Inflammasome Mediated Pyroptosis Axis

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Abstract

Background

Objective

Methods

Results

Conclusion

Figures

1. Introduction

2. Materials and Methods

2.1 Materials

2.2 Methods

2.2.1 PT Stroke Model Preparation and Animal Grouping

2.2.2 Cell Culture

2.2.3 OGD/R Model Preparation and Grouping

2.2.3 MTT Assay

2.2.4 ROS Assay

2.2.5 LDH Assay

2.2.6 Hoechst33342/PI Staining

2.2.7 ELISA Assay

2.2.8 Western Blotting

2.2.9 Behavioral Tests

2.2.10 HE Staining and Nissl Staining

2.2.11 Immunohistochemical Detection

2.2.12 Statistical Analysis

3. Results

3.1 Effect of Different Concentrations of Que on OGD/R Cell Viability

3.2 Que Inhibits OGD/R-Induced Microglial Cell Damage

3.3 Que Inhibited Pyroptosis Induced by OGD/R

3.4 Que Reduced the Expression of Pyroptotic Inflammatory Factors

3.5 Inhibition of NLRP3 Inflammasome Activation by Que Attenuates OGD/R-induced BV2 Cell Pyroptosis

3.6 Que Ameliorated Motor Dysfunction and Neuronal Cell Injury in PT Mice

3.7 Que Inhibited Inflammasome Activation to Alleviate Pyroptosis Induced by Cerebral Ischemia

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

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