Ethyl acetate extracts of Bungeanum ameliorates cognitive decits by suppressing NLRP3 inammasome-dependent pyroptosis in aging mice

Background: The NLRP3-mediated pyroptosis is a key player in the development of the age-associated neurodegenerative disease. Zanthoxylum bungeanum Maxim (Rutaceae), a homologous of medicine and foodstuff, has previously been demonstrated the potential prevention of cognitive dysfunction in aging mice. However, it is still unknown which fraction is the material basis responsible for their therapeutic effects and whether Z. bungeanum could confer anti-cognitive decits activity via restraining NLRP3-mediated pyroptosis. Thus, in the current study, we explored the active fraction of Z. bungeanum against cognitive decits and its underlying mechanism. Methods: In the present study, the D-galactose-induced mouse model of aging was established to explore the effect of cognitive impairment of four fractions of Z. bungeanum, including petroleum ether (PE), methylene chloride (DCM), ethyl acetate (EA), and n-butanol (N-BAI). We next activated the NLRP3 inammasome in BV-2 microglia cells to investigate the mechanisms for the neuroprotective effect of the active fraction of Z. bungeanum. Results: We demonstrated that the mice treated with EA had signicantly alleviated the memory decit induced by D-galactose. Meanwhile, EA up-regulated the cortex NeuN protein level, improved the survival and morphology of hippocampal neurons. We further found that EA signicantly alleviated oxidative damage, inhibited activation of microglia, and suppressed NLRP3 inammasome activation and subsequent cleaved Caspase-1, IL-1β, IL-18, and GSDMD-N. Correspondingly, in vitro data showed that EA protected BV-2 cells against Lipopolysaccharide (LPS) and adenosine triphosphate (ATP) elicited NLRP3 inammasome activation and pyroptosis, evidenced by declined the protein levels of NLRP3, cleaved Caspase-1, IL-1β, IL-18, and GSDMD-N. These data indicated that EA inhibited NLRP3-mediated pyroptosis. Conclusions: Our work revealed that the


Preparation of extraction
Solvents of four different polarities solvents (petroleum ether, dichloromethane, ethyl acetate and nbutanol) were used to fractionate Z. bungeanum extracts. The dried Z. bungeanum (500 g) was soaked in 5000 mL 95% ethanol for 24h and then ltered it. We repeated this operation one more time, and transferred the ltrate to a ask for evaporation. After the liquid was concentrated to 400 ml, we used petroleum ether, dichloromethane, ethyl acetate and n-butanol were used for extraction twice in turn.
These ltrates were concentrated by rotary evaporator. Finally, each extract was collected in a screw tube and kept them in a vacuum desiccator for 3-5 days to completely remove each organic solvent. The extracts were away from light stored at 4 °C until use. We referred to the fraction with petroleum ether as PE, the fraction with dichloromethane, as DCM, the fraction with ethyl acetate, as EA, and the fraction with n-butanol, as N-BAI.

Animals
Male Kunming mice (8 weeks old) were purchased from the Dashuo Experimental Animal Co., Ltd and housed under suitable conditions of temperature (22 ± 2℃) and humidity (65 ± 5%) in the animal observation room of Chengdu University of Traditional Chinese Medicine with free access to water and food. All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the Institute of Material Medica Integration and Transformation for Brain Disorders ethics committee approved the animal protocols. Mice were randomly divided into seven groups (n=6 in each group): solvent plus saline (control group), solvent plus D-gal (aging group), 0.9 mg/kg Hydergine plus D-gal (HG group), PE plus D-gal (PE group), DCM plus D-gal (DCM group), EA plus D-gal (EA group), and N-BAI plus D-gal (N-BAI group). Drugs were dissolved in 0.5% CMC-Na for administration, and the dose of each extract was equal to 450 mg/Kg of raw material. After oral administration of the for 1 h, 500 mg/kg D-gal was subcutaneously injected once a day for 42 days. After that, all the animals were executed for further study.

Behavioral evaluation
The protocol followed the sequence of passive avoidance test and Morris water maze test. All tests were performed in conditions of dim light and low noise. The passive avoidance test was performed as previously described [18]. The test was performed daily for 2 consecutive days, and the rst day was the training trial, and the second day was the test trial. Mice were individually placed into the apparatus and permitted free exploration for 5 min. The mice were individually placed in a lit room and allowed free access to the corresponding dark room when a trap door was opened. The latency time taken to pass through the trap door was recorded as " Escape latency ", and the number of times the mice passed through the trap door was counted as " The number of errors ". If the mouse did not pass through the trap door within 300 s, a latency of 300 s was recorded.
The Morris water maze test was conducted based on earlier methodology [18]. In the orientation navigation experiment, the mice were placed in the opposite quadrant of the platform and allowed to swim until it found the hidden platform within 60 s. The time of nding the platform was recorded as" Escape latency ". If the mice could not nd the platform within 60 s, it was gently guided to the platform and kept there 10 s, and the was recorded as 60 s. The continuous period of the hidden platform experiment was 5 days. Next the spatial probe trial was performed by removing the platform on the sixth day. The swimming time in target quadrant and the number of crossing the platform were recorded in the spatial probe trial.

Hematoxylin and eosin staining
For hematoxylin and eosin (HE) staining, four mice brain samples in each group were collected and post xed in 4% paraformaldehyde. The hemisphere of each mouse was embedded in para n and cut into 4 μm slices, dewaxed, which were then stained successively with hematoxylin and eosin to observe lesions in the hippocampus. And only large intact cells with clearly stained cytoplasm and a distinct nucleus, usually with evident nucleoli, were counted as healthy neurons [19].

Measurement of the activity of SOD and the content of MDA
The activity of SOD and the content of MDA were determined strictly according to the kit instructions.

Tunel staining
The para n blocks were cut into 4 μm slices, dewaxed, repaired with proteinase K working solution, disrupted cytomembrane, incubated with TdT and FITC-12-dUTP for 1 h. After rinsing with phosphatebuffered solution (PBS), the nuclei were stained with DAPI, then anti-uorescence quenching agent was added. Then the samples were observed immediately under uorescence microscope after sealed with neutral balsam on slides. Pay attention to avoid light in the experiment.
2.9 Cell culture and Cell viability measurements BV-2 microglial cells were purchased from Chinese Academy of Sciences (shanghai, China). Cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM) with 10% fetal bovine serum and 1% streptomycin/penicillin, maintained at 37°C under 5% CO2. Cells in the exponential growth phase were used for all experiments. Cells were plated into 96-well plates at a density of 1×10 4 or plated into 6-well plates at 1×10 5 densities. After culturing for 24h, drugs were rst added for 1h, then 100 ng/ml LPS was added for 12 h, and nally, ATP (2.5 mM) was added for 3h. After the cells were treated as described above, 10μl of MTT solution was added to each well. Finally, after the plates were incubated in the incubator for 4h, remove the supernatant, add dimethyl sulfoxide, incubate in a shaker, and absorbance was measured at 490 nm. Cell experiments were independently performed at least three times.

Sample preparation and Mass spectrometry analysis
EA fraction was extracted with methanol solution by ultrasonic extraction, and ltered through a 0.22 μm micropore lm to yield the sample solution for HPLC-MS/MS. The sample was analyzed by an Agilent Technologies 1290 LC and an Agilent Technologies 6410 triple quadrupole mass spectrometer (Agilent Technologies, CA, USA). And the chromatographic separation was performed on a poroshell 120 EC-C18 column (100 × 4.6 mm, 2.7 μm, Agilent Technologies, CA, USA) at 29°C. The mobile phase consisted of H2O (containing 0.05% formic acid, A) and acetonitrile (B) in the gradient elution program: 0 ⟶ 3 min, 5% ⟶ 18% B; 3 ⟶ 10 min, 18% B; 10⟶ 20 min, 18% ⟶ 63% B. The ow rate was 0.3 mL/min, and the sample injection volume was 10 μL. Mass spectrometric scan was obtained by electrospray ionization (ESI) in positive-ion mode with a scanning interval m/z 100-1300. The main parameters for MS were set as follows: gas temperature, 300 °C; gas ow, 11L·min -1 ; nebulizer, 35 psig; capillary voltage, 4000 V; and atomizer pressure 15 psi (1 psi = 6.895 Kpa).

Statistical analysis
All dates are represented as the mean ± standard error of the mean (SEM). The escape latency in the Morris water maze test navigation phase was analyzed by two-way analysis of variance (ANOVA). For other data, date with a normal distribution was analyzed by one-way ANOVA. Student's test was performed for comparisons between two groups. And Bonferroni post hoc analysis was conducted, and the statistical signi cance was tested at P < 0.05.

Results
3.1 Effects of four different polarities fractions of Z. bungeanum on cognitive impairment in aging mice.
The Passive avoidance test was rst used to evaluate the effects of PE, DCM, EA, and N-BAI fractions on fear memory disorder in aging mice induced by D-gal ( Fig. 1A and 1B). There was no signi cant difference among the groups during the training for the escape latency and the number of errors. However, in the testing trial, compared with control mice, the escape latency obviously was shortened (P < 0.05), and the number of errors obviously was increased (P < 0.05) in aging mice. Interestingly, the changes in aging mice were alleviated after treatment with EA and N-BAI fractions of Z. bungeanum, as indicated by longer latency and fewer errors (P < 0.05).
Morris water maze test was also used to assess the learning and memory abilities of mice ( Fig. 1C − 1F). In the orientation navigation test, all groups exhibited a shorter escape latency with the increase in training days (P < 0.0001, F [4, 174] = 10.89). However, the escape latency of aging mice was signi cantly longer than the control mice (P < 0.01, F [1, 50] = 10.40), indicating that the learning and memory abilities were signi cantly impaired in aging mice. But EA (P = 0.0144, F [1, 50] = 4.242) and N-BAI (P = 0.0447, F [4, 49] = 3.460) treatments shortened the escape latency of aging mice. In the spatial search test, aging mice displayed fewer the number of passing the platform zone and residence time in the target quadrant than the control group (P < 0.01), suggesting that the spatial memory of aging mice was damaged, and these changes can be ameliorated by hydergine treatment. Meanwhile, EA and N-BAI treatment reversed distinctly the D-gal-induced decrease in the number of passing the platform and residence time in the target quadrant (P < 0.05). Collectively, these results manifested that EA and N-BAI ameliorated amnesia in aging mice, which may act as an inhibitor of cognitive impairment in aging mice.
3.2 Effects of EA and N-BAI fractions on pathological damage and number of surviving neurons in hippocampus and cortex in aging mice.
To examine whether pathological was altered in the hippocampus of aging mice compared with normal mice and whether EA and N-BAI could defend it, HE staining was employed to analyze the morphology and number of survival hippocampal CA1 and CA3 neurons in different groups (Fig. 1G). The administration of D-gal induced a signi cant pathological injury of neurons in the hippocampus, characterized by atrophy, loose arrangement, and decreased number of neurons (P < 0.01). However, these changes were markedly halted by EA and N-BAI treatment (P < 0.05) ( Fig. 1J and 1I). The immunohistochemistry for NeuN was used to label surviving neurons. The results revealed aging mice had a signi cantly reduced number of surviving neurons in the cortex (P < 0.01) (Fig. 1H and 1K). Consistent with HE staining results, the number of positive neurons increased signi cantly in the EA and N-BAI treatment group compared with the aging mice. However, the number of surviving neurons in the EA group was higher than that in the N-BAI group in HE staining and the immunohistochemistry of NeuN (P < 0.05), indicating that EA was the optimal fraction. The above studies showed that EA was a major activity part of Z. bungeanum extracts against age-related neural lesions.
3.3 Effects of EA fraction on oxidative damage and neuroin ammation in aging mice.
To understand the speci c mechanism of the protective effect of EA in aging mice, we measured antioxidant enzyme activity, lipid oxide content, the expression of Iba1 and CD11b. As illustrated in Figs. 2A and 2B, the activity of SOD in the brain tissue was remarkably suppressed and the level of MDA was increased after exposure to D-gal compared with the control group (P < 0.01). However, after EA treatment, the phenomenon had been changed, indicating that EA could improve the redox balance of brain tissue in mice. To detect changes in microglia in aging mice, we performed immunolabelling of microglial marker Iba1. As shown in Figs. 2C and 2E, D-gal signi cantly increased Iba1 levels in the hippocampus CA1 regions (P < 0.01), but which was attenuated by EA (P < 0.05). Meanwhile, the CD11b protein expression, a marker of microglial activation, was increased in D-gal-induced aging mice compared with the control group (P < 0.01). EA treatment also inhibited the expression of CD11b compared with aging mice (P < 0.05) (Fig. 2D). Microglia is the main immune cells in the central nervous system, our results suggested that EA can alleviate microglial in ammation of the brain tissue in aging mice.
3.4 Effects of EA fraction on the activation of NLRP3 in ammasome and the pyroptosis of GSDMDmediated in aging mice.
NLRP3 in ammasome and GSDMD-mediated pyroptosis play an important role in microglial in ammation response. To verify the effects of EA on the activation of NLRP3-in ammasome and the pyroptosis of GSDMD-mediated in aging mice, the associating protein expression was tested by western blotting (Fig. 3). As expected, the expression of NLRP3, cleaved caspase-1, and cleaved Gasdermin D (GSDMD-N) of brain tissue were signi cantly increased in aging mice (P < 0.01), inversely, those could remarkably down-regulated by EA (P < 0.05). The release of activated IL-18 and IL-1β was increased in aging mice compared with the control group (P < 0.01). Interestingly, the levels of these factors in aging mice were signi cantly depressed after EA treatment (P < 0.05). Besides, the dUTP nick-end labeling (Tunel) assay was used to detect and verify the pyroptosis. The results showed a noticeable increase in death of the hippocampus and cortex of aging mice compared to the normal mice, and EA exhibited signi cant suppression of Tunel positive staining (Fig. 3F). Our results demonstrated that EA could suppress the activation of NLRP3 in ammasome and the pyroptosis of GSDMD-mediated in aging mice.

Effects of EA fraction on NLRP3 in ammasome and pyroptosis in LPS + ATP treated BV-2 cells.
To further understand the effects of EA fraction on NLRP3 in ammasome and pyroptosis, the experiment that LPS + ATP induced NLRP3 activation and GSDMD-mediated pyrolysis was performed in BV-2 cells.
As illustrated in Fig. 4A, a signi cant toxic effect was observed after LPS + ATP treatment, evidenced by reduced cell viability in the LPS + ATP treated group compared with the control group (P < 0.01). Nevertheless, pretreatment with various doses of EA (5, 10, 20 µg/ml) increased the cell viability of BV-2 cells, demonstrating EA protected BV-2 cells from LPS + ATP induced cell death (P < 0.05). Moreover, levels of NLRP3 in ammasome and pyroptosis-associated proteins were measured (Fig. 4B). In Western blot assessment, the expression of NLRP3, cleavage capase-1, and GSDMD-N, the release of activated IL-18 and IL-1β were increased after LPS + ATP treatment. Nevertheless, these alternations were partially reversed by EA. These ndings re ected that EA can inhibit the activation of NLRP3 in ammasome and GSDMD-mediated pyroptosis in LPS + ATP treated BV-2 cells.

The chemical composition of EA active fraction of Z. bungeanum extracts
To examine the potential material basis for the EA fraction of Z. bungeanum extracts. HPLC-MS/MS was used to analyze the chemical composition of the optimal EA fraction. As shown in Table 1, compounds were identi ed and grouped as follows: Quercetin-3β-D-glucoside (16.403%) was recognized as the main constituents, and other components including Hirsutrin (8.360%), Epicatechin (7.966%), Chlorogenic acid (6.065%), Quercetin (7.562%), Quercetin 7-rhamnoside (4.923%), etc. It is dominated by avonoids and acids natural compounds in EA fraction, of which avonoids are more than 70% and acids are about 20% of all compounds.  [21]. In the present study, we demonstrated rich-bio avonoids EA fraction is the optimal fraction of Z. bungeanum in inhibiting age-related cognitive decline. We furtherly found that EA fraction relieved oxidative damage, silenced the NLRP3 in ammasome, and restraining pyroptotic death in vivo and in vitro, which suggested the potential for Z. bungeanum in prevention and therapy for oxidative stress and neuroin ammation. Amusing, to the best of our knowledge, our work is the rst systemic report of the presence of pyroptosis of the brain in D-gal-induced aging mice, although the neuroin ammation pathway has been reported [22]. In addition, GSDMD-mediated pyroptotic signaling pathway regulated by Z. bungeanum is also rst reported here.
Aging is one of the most important risk factors for various neurodegenerative diseases. D-gal is a reducing sugar found in humans and foods [23]. Low doses of D-gal can excrete from the body, but at high levels, it can be converted into aldose and hydroperoxide under the catalysis of galactose oxidase, resulting in the generation of ROS, which may subsequently cause oxidative stress and in ammation [24]. Injection of D-gal into mice could induce changes that resembled accelerated brain aging, characterized by cognitive function impairment [22]. Nowadays, D-gal-induced brain aging is established to be bene cial for age-related neurodegenerative diseases study. In this experiment, the passive avoidance test and the Morris water maze test were used to evaluate the cognition of the D-gal-treated aging mice. Hydergine be widely used for treating patients with either dementia or 'age-related' cognitive symptoms [25], therefore, it was tested as the positive control in the present study. The work showed that the EA fraction of Z. bungeanum extracts could signi cantly improve cognitive dysfunction, relieve pathological injury in D-gal-induced aging mice. Consequently, EA is the optimal fraction of Z. bungeanum extracts to ameliorate cognitive de cits in aging mice. In addition, HPLC-MS/MS was used to analyze the composition of EA, the results showed that EA mainly contained chemical components of avonoids. Flavonoids are common ingredients of numerous plants, which have a broad range of pharmacological activities, including anti-aging, antioxidant, anti-in ammatory, protect liver, etc [26,27]. This also explained the EA fraction of Z. bungeanum can act as an age-related cognitive decline inhibitor.
In the following we explored the neuroprotective mechanisms by which EA fraction acts. Our results re ected that EA fraction could reverse the increase of lipid peroxides and the decrease of antioxidant enzyme activity induced by D-galactose. This is consistent with our previous researches [17]. The accumulation of excessive oxidative stress causes neuroin ammation in the brain, which subsequently impairs cellular activity and destabilizes neuronal homeostasis, ultimately leading to neuronal loss. Signi cantly, microglia are detectors of in ammation in the central nervous system. In healthy brains, microglia exist in a resting state. When exposed to pathological injury, microglia changed from a resting stage to activated phases. Some protein expression changes, such as CD11b and CD68, have been used as markers of microglia activation [28]. We monitored the effects of EA fraction on D-gal-induced morphological changes in Iba1-immunoreactive microglia and CD11b immunoblotting. Results showed EA can suppress oxidative damage and microglial activation in aging mice induced by D-gal.
In ammatory signals can induce the production of in ammasomes. The NLRP3 in ammasome is the mainly studied in ammasome, which is a critical factor to initiate the process of neurodegeneration [29]. Mounting evidence suggests that administration of D-gal shows aging neuroin ammation phenotypes, such as microglial activation, increases of IL-1β, IL-6, and TNF-α levels, and activation of NLRP3 in ammasomes [30]. The results are entirely consistent with our research. Previous studies also have shown that Z. bungeanum extract inhibited the expression of TNF-α, IL-1β, and IL-12, and the activation of NLRP3 in dextran sulfate sodium-induced murine experimental colitis [31,32]. Consistent with the results, our study demonstrated that the activation of NLRP3 in ammasome was inhibited by EA active fraction in the brain of D-gal-induced aging mice and BV-2 cells induced by LPS + ATP.
Cell death and in ammation are fundamental characteristics in the initiation and development of neurological dysfunction. GSDMD-mediated pyroptosis plays crucial roles in the pathogenesis of multiple neurological diseases and age-related degeneration [33][34][35]. Pyroptosis is a type of lytic programmed cell death, distinguished from apoptosis and necrosis [8,36]. When the body is subjected to noxious stimuli, in ammasomes formation through caspase-1-dependent and/or caspase-4/5/11-dependent pyroptosissignaling pathways activation may be triggered via intracellular and extracellular signaling pathways [37]. Next, gasdermin D (GSDMD), the pyroptosis executioner, as a substrate of both caspase-1 and caspase-11/4/5, releasing several in ammatory mediators such as IL-1β and IL-18 and leading to cell pyroptosis [8]. In this study, we found that neurons displayed characteristic features of pyroptosis in the brain of D-gal-induced aging mice, as evidenced by increased levels of caspase-1, IL-1β, IL-18, GSDMD-N, and Tunel-positive cells.
It is noteworthy that the speci c role of pyroptosis has not yet been fully elucidated in D-gal-induced aging mice. The activation of NLRP3 in ammasome and GSDMD-mediated pyroptosis induced by LPS + ATP in BV-2 cells [38]. The pro-caspase-1 can indirectly connect with the adaptor protein apoptosisassociated speck-like protein (ASC), which combines with a pattern recognition receptor to form a macromolecular complex, known as in ammasomes [39]. After the activation of in ammasomes, procaspase-1 is cleaved to form active caspase-1, which may cleave the GSDMD protein molecule to form active N-terminal kinase portions to cause cell membrane perforation [40]. And baicalin has been reported to have a wide range of anti-in ammatory activities, blocking the NLRP3-GSDMD signaling in vitro [41].
Therefore, baicalin was tested as the positive control in LPS + ATP induced BV-2 cells. The result of the experiment in vitro was in correspondence with that of the experiment in vivo, activation of NLRP3 in ammasome and GSDMD-mediated pyroptosis signaling were inhibited by EA fraction in BV-2 cells induced by LPS + ATP. These works proved that EA fraction suppressed microglial pyorptosis by inhibiting the activation of NLRP3 in ammasomes.

Conclusions
Collectively, rich-bio avonoids EA fraction was a potential candidate responsible for the elite therapeutic potential of age-related cognitive de cits, the mechanism of action is related to inhibiting oxidative stress, the activation of NLRP3 in ammasome, and GSDMD-mediated pyroptosis. A detailed illustration of the study is shown in Fig. 5. However, our study still has a few limitations. Firstly, the active components in the EA fraction have not been further identi ed. Besides, our experiments focus on the classical pathway of pyroptosis, and the non-classical pathway remains to be explored. Next, our team will carry out indepth research on these problems.

Availability of data and materials
The datasets and materials supporting the conclusions of this article are included within the article.

Competing interests
The authors declare that they have no con ict of interest.

Consent for publication
Not applicable.

Authors' Contributions
Meihuan Zhao performed the methodology, the practical work and drafted the manuscript; Yuan Dai validation and supervision; Ping Li completed western blotting analyses; Jie Wang completed HPLC-MS/MS analyses; Tengyun Ma did the data curation; Shijun Xu conceived, designed the study and critically reviewed, edited, and revised the paper contributed the conception and design of the study.
Ethics approval and consent to participate All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the Institute of Material Medica Integration and Transformation for Brain Disorders ethics committee approved the animal protocols. Figure 1 Screening the optimal part of four fraction of Z. bungeanum to improve cognitive function in D-galactose induced aging mice. The cognitive performance of mice in the passive avoidance test (A-B) and in the Morris water maze (C-F), as determined by the escape latency (A) and the number of errors (B) on the passive avoidance test of mice, the escape latency (C), representative traces of each group (D), swimming time in the target quadrant (E) and the number of passing the platform (F) in the Morris water maze test. Representative pictures of hematoxylin-eosin (HE) staining in hippocampus CA1 and CA3 regions (G), quantifcation of neuronal survival in the hippocampal CA1 region (I) and CA3 region (J).
Representative pictures of NeuN staining in cortex (H), and quantifcation of positive neurons in cortex (K).
Scale bars = 50 μm. All data in A-F are presented as mean value ± SEM for n=6. Data in G-K are presented as mean value ± SEM for n = 4, ##P< 0.01 versus the control group, *P < 0.05 versus the model group, &P < 0.05 is the comparison between EA and N-BAI. Comparison of escape latency was performed by twoway analysis of variance (ANOVA) in the Morris water maze during the ve consecutive days training trial days, Student's test was used for comparison between EA and N-BAI groups, others were performed by one-way analysis of variance (ANOVA).

Figure 2
Effects of EA fraction on oxidative damage and neuroin ammation in D-galactose induced aging mice.
Effects of EA on superoxide dismutase (SOD) activities (A), malondialdehyde (MDA) levels (B). Representative immunohistochemical images of Iba1 in the hippocampal CA1 regions of mice (C), quantifcation of Iba1 positive neurons (E). Western blot and quantitative analysis the protein expression of CD11b, GAPDH was used as loading control (D). Scale bars = 25 μm. All dates are presented as mean value ± SEM for n = 5, ##P< 0.01 versus the control group, *P < 0.05 versus the model group. All dates were performed by one-way analysis of variance (ANOVA).

Figure 3
Effects of EA fraction on NLRP3-dependent in ammasome activation and GSDMD-mediated pyroptotic in D-galactose induced aging mice. Western blotting was used to further examine the NLRP3, cleaved caspase-1, cleaved GasedrminD (GSDMD-N), cleaved IL-1β and cleaved IL-18 protein expression (A). And Western blot and quantitative analysis of NLRP3, cleaved caspase-1, GSDMD-N, cleaved IL-1β, cleaved IL-18 protein in brain tissues, GAPDH was used as loading control (B-F). pyroptotic cell death was estimated by TUNEL staining (G). All dates are presented as mean value ± SEM for n = 3, ##P< 0.01 versus the control group, *P < 0.05 versus the model group. All dates were performed by one-way analysis of variance (ANOVA). Figure 4