Sodium Houttuyfonate Ameliorates β-amyloid1-42-Induced Memory Impairment and Neuroinflammation through Inhibiting the NLRP3/GSDMD Pathway in Alzheimer's Disease

Objective Our research is designed to explore the function of sodium houttuyfonate (SH) on Alzheimer's disease (AD) and its potential molecular mechanisms. Methods In our study, the Morris water maze (MWM) test was used to assess the role of SH on spatial learning and memory deficiency in amyloid-β peptide (Aβ)1-42-induced AD mice. We explored the functions of SH on proinflammatory cytokines, neuron apoptosis, and damage in vivo and in vitro by using an enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), flow cytometry, western blot, and Nissl staining. Moreover, the effect of SH on oxidative stress in vivo and in vitro was also detected. To explore the underlying molecular mechanisms of SH on AD, the expressions of proteins and mRNA involved in the NOD-like receptor pyrin domain containing-3/gasdermin D (NLRP3/GSDMD) pathway were determined using western blot, immunofluorescence staining, and qRT-PCR. Results Our data demonstrated that SH ameliorated spatial learning and memory deficiency in Aβ1-42-induced AD mice. Moreover, SH significantly improved hippocampal neuron damage and inhibited oxidative stress, neuroinflammation, and neuron apoptosis in Aβ1-42-induced AD mice and PC12 cells. The results also revealed that SH protected Aβ1-42-induced AD through inhibiting the NLRP3/GSDMD pathway. Conclusion The present study demonstrated that SH could ameliorate Aβ1-42-induced memory impairment neuroinflammation and pyroptosis through inhibiting the NLRP3/GSDMD pathway in AD, suggesting that SH may be a potential candidate for AD treatment.


Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disease, which is mainly characterized by progressive cognitive impairment and irreversible memory loss [1,2]. Recently, numerous studies have shown that the excessive production and deposition of amyloid-β peptides (Aβ) as senile plaques can initiate the process of neurodegeneration in AD [3,4]. There is a newly diagnosed AD case every 3 sec all over the world [5]. However, current treatment and drug could not effectively terminate or reverse the progression of AD. Therefore, it is urgent to search new therapeutic methods and medicines for the treatment of AD.
Houttuynia cordata is a commonly used traditional Chinese herb and possesses a variety of pharmacological activities, including anti-inflammatory activity, antivirus activity, and antibacterial activity [6]. Decanoyl acetaldehyde (houttuynin) is one of the main extracts from Houttuynia cordata. At present, sodium houttuyfonate (C 12 H 23 O 5 SNa, SH) was synthesized by decanoyl acetaldehyde and sodium bisulfite as a result of the instability of decanoyl acetaldehyde [7]. A study by Huh et al. [8] has reported that Houttuynia cordata could significantly improve cognitive deficits in AD. However, the effect of SH on AD has not been reported.
NOD-like receptors (NLRs) are a kind of cytoplasmic sensors or receptors that play an important role in the process of neuroinflammation [9]. NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome, a member of the NLR family, is a multiprotein complex consisting of the core protein NLRP3 and regulatory molecules procaspase-1 and apoptosis-associated speck-like protein (ASC). The activated NLRP3 inflammasome causes the cleavage of procaspase-1 into active caspase-1, which can induce the secretion of proinflammatory cytokines [10,11] and cleaves gasdermin D (GSDMD) into N-terminal poreforming domain (GSDMD-N) and the C-terminal repressor domain (GSDMD-C) [12]. Subsequently, GSDMD-N interacts with the cell membrane for forming pores. Meanwhile, proinflammatory cytokines flow out through the pores and then cause inflammatory responses causing pyroptosis [12,13]. Pyroptosis is an inflammatory form of programmed cell death that is associated with inflammasome activation [14]. Recently, increasing evidence has confirmed that the inhibition of pyroptosis can significantly prevent the progression of AD [15]. In addition, neuroinflammation is reported to be an important factor in the pathological procession of neurodegenerative disease, especially AD [16]. In this study, we detected the function of SH on AD and further investigated whether the mechanism is related to the NLRP3/GSDMD pathway.

Preparation of Aβ Oligomer.
A soluble Aβ oligomer was prepared as described by Xiang et al. [17]. Aβ 1-42 was dissolved in sterile H 2 O at a concentration of 1 mM, evaporated with high-purity N 2 blowing at 37°C for 30 min, and then incubated for 48 h at room temperature to form the Aβ oligomer. Subsequently, the stock solution of Aβ 1-42 was diluted in sterile phosphate-buffered saline (PBS) to the desired concentrations immediately before use.

Animals.
Male ICR mice (6 weeks old, 25-30 g) were obtained from Vital River Experimental Technology Company (Beijing, China). All mice had free access to water and food and were maintained under a temperature of 22 ± 1°C, 60% humidity, and a 12 h light/dark cycle (lights on from 7:00 a.m. to 7:00 p.m.). Before the experiments, the animals were allowed to acclimate for seven days. The experimental protocol of our study was performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the ethics committee of our hospital.  Injection and Drug Administration. Fifty mice were randomly divided into five treatment groups with ten animals in each group: Control group, Aβ 1-42 group, Aβ 1-42 +SH (25 mg/kg) group, Aβ 1-42 +SH (50 mg/kg) group, and Aβ 1-42 +SH (100 mg/kg) group. Each mouse was anesthetized with intraperitoneal injection of pentobarbital sodium (40 mg/kg). Then, 3 μl Aβ 1-42 (10 ng/μl) was stereotaxically injected into the hippocampus CA1 subregion in which the coordinates were as follows: -2.3 mm anterior/posterior, ±1.8 mm medial/lateral, and -1.75 mm dorsal/ventral from the bregma. In addition, the control group mice received the same volume of saline. From the second day after surgery, the mice in the Aβ 1-42 +SH (25 mg/kg), Aβ 1-42 +SH (50 mg/kg), and Aβ 1-42 +SH (100 mg/kg) groups were administered by oral administration with corresponding doses once a day, while mice in the Control and Aβ 1-42 groups were administered with the same volume of saline for 28 days.

Morris Water Maze (MWM)
Test. The MWM test was used to evaluate the spatial learning and memory abilities of the mice. Simply, following the given SH for 28 consecutive days, each animal was subjected to finding the submerged platform through a daily acquisition test (4 trials per day). And the maximum trial length was 120 s. If a mouse did not locate the platform within 120 s, it was then guided to the platform and placed on the platform for 30 s. In this case, its escape latency was still marked as 120 s. This procedure lasted for 6 days, with two training sessions each day and a 15 min interval between the sessions. On the sixth day, the animals were subjected to a probe trial session in which the platform was removed from the pool and mice were allowed to search for it for 120 s. The escape latency, the time spent in the target quadrant, and the frequency of mice crossing the virtual platform were recorded and analyzed by using a video tracking system.

Nissl
Staining. The brain tissues of the mice were removed and postfixed in 4% formaldehyde solution at room temperature for 24 h. Subsequently, the brain tissues were dehydrated in graded concentrations of ethanol and then embedded in paraffin and transversely cut into 5 μm thick sections. Then, the tissue sections were treated with conventional Nissl staining and visualized with an optical microscope (magnification, ×400). For AD cell model in vitro, PC12 cells (1 × 10 4 cells/well) were planted into 96-well plates and cultured for 24 h. The PC12 cells were pretreated with SH (15, 30, 60, 120, and 240 μg/ml) or an inhibitor of NLRP3 inflammasome (CY-09, 10 μM) for 2 h and then treated with Aβ 1-42 (5 μM) for 24 h at 37°C. 2.7. Cell Viability. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used to assess the cell viability. PC12 cells (1 × 10 5 cells/well) were planted into 96-well plates and cultured overnight. After treatment of SH or Aβ 1-42 , 20 μl of MTT solution (5 mg/ml, Beyotime, Nanjing, China) was added to each well and incubated at 37°C for about 4 h. After removal of the MTT solution, PC12 cells were treated with 200 μl dimethyl sulfoxide (Beyotime) to dissolve the formazan crystals. Finally, the absorbance at 570 nm was recorded by using a microplate reader (Bio-Rad, USA).  Mediators of Inflammation (2 × 10 5 cells/well) were planted into 6-well plates and cultured for 24 h. Following treatment of SH or Aβ 1-42 , PC12 cells were incubated with 10 nM fluorescent probe of 2′,7′ -dichlorofluorescein diacetate (DCFH-DA, Sigma, USA) for 30 min at 37°C in the dark. The fluorescence signal was observed under a fluorescence microscope. In addition, the levels of SOD and MDA in both PC12 cells and brain tissues were measured by using the corresponding SOD and MDA detection kits (Beyotime) according to the manufacturer's instructions.
2.9. Flow Cytometry. The apoptosis of PC12 cells was assessed by using an Annexin V-fluorescein isothiocyanate/propidium iodide (FITC/PI) apoptosis detection kit (KeyGen Biotech, Shanghai, China) according to the instructions of the manufacturer. In brief, after treatment of SH or Aβ 1-42 , PC12 cells were harvested and resuspended in a binding buffer. Afterwards, the cells were stained by Annexin V-FITC and PI in the dark for 20 min. Finally, the cell apoptosis was detected by flow cytometer.
2.10. Immunofluorescence Staining. PC12 cells were fixed in 4% paraformaldehyde solution at room temperature for 20 min and then blocked with 5% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature. After that, the slices were incubated with rabbit anti-GSDMD antibody (1 : 100, #20770-1-AP, Proteintech, USA) and rabbit anti-NLRP3 antibody (1 : 200, #ab214185, Abcam, USA) overnight at 4°C. Following washed with PBS, the slices were incubated with Cy3-labeled goat anti-rabbit secondary antibody (1 : 500, Beyotime) for 1 h at 37°C in the dark. The cells were stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, USA) for 10 min at room temperature. The fluorescence images were randomly scanned with a confocal laser scanning microscope (Nikon, Tokyo, Japan) by a single investigator who was blind to the sample identity.
2.14. Statistical Analysis. All data were expressed as mean ± standard deviation and were analyzed by the SPSS22.0 software and GraphPad Prism 8.0 Software. One-way ANOVA combined with Turkey's multiple comparison tests was used to compare data. P values less than 0.05 were considered statistically significant.

SH Ameliorates Spatial Learning and Memory Deficiency
in Aβ 1-42 -Induced AD Mice. The MWM test was used to evaluate the function of SH on the spatial learning and memory in mice. As shown in Figure 1(a), the escape latencies of the Control group, Aβ 1-42 group, Aβ 1-42 +SH (25 mg/kg) group, Aβ 1-42 +SH (50 mg/kg) group, and Aβ 1-42 +SH (100 mg/kg) group were progressively decreased in successive trials. When compared with the Control group, the escape latency of the Aβ 1-42 group was significantly increased at 6 days (P < 0:01). SH (25, 50, and 100 mg/kg) dramatically decreased the escape latency at 6 days in a dose-dependent manner (P < 0:01). In addition, the results of Figures 1(b) and 1(c) showed that mouse swimming time in the target quadrant and the frequency of platform crossing in the Aβ 1-42 group were significantly decreased in comparison with those in the Control group (P < 0:01). The mice treated with SH (25, 50, and 100 mg/kg) spent more time in the target quadrant and increased the frequency of platform crossing relative to the mice in the Aβ 1-42 group. In the subsequently experiments, SH (50 mg/kg) was used. All the results suggested that SH could ameliorate spatial learning and memory deficiency in Aβ 1-42 -induced AD mice.

SH Attenuates Aβ 1-42 -Induced Hippocampal Alterations and Neuroinflammation in Mice.
We firstly assessed the effects of SH (50 mg/kg) on hippocampus neuron survival by using Nissl staining (Figure 2(a)). The shrinkage and loss of the neurons were found in mice of the Aβ 1-42 group relative to the Control group. When compared with the Aβ 1-42 group, the treatment of SH (50 mg/kg) dramatically restored the impairment of neuronal loss and shrinkage. As Figure 2(b) showed, the MDA level in Aβ 1-42 group brain tissues was significantly increased compared with that in the Control group (P < 0:01), while the SOD level was markedly decreased (P < 0:01). Treatment with SH (50 mg/kg) dramatically attenuated the role of Aβ 1-42 on MDA and SOD (P < 0:01). The ELISA assay (Figure 2(c)) was further used to detect the levels of TNF-α, IL-1β, IL-6, and IL-18 in brain tis-sues. The results showed that the levels of IL-6, IL-1β, IL-18, and TNF-α in the Aβ 1-42 group were higher than those in the Control group (P < 0:01). On the contrary, treating SH (50 mg/kg) significantly reduced the levels of IL-6, IL-1β, IL-18, and TNF-α compared with the Aβ 1-42 group (P < 0:01). In addition, the results of the western blot (Figure 2(d)) revealed that the expressions of caspase-3 and Bax in Aβ 1-42 group brain tissues were significantly increased compared with those in the Control group (P < 0:01), while the Bcl-2 expression was markedly decreased (P < 0:01). Treatment with SH (50 mg/kg) dramatically attenuated the effect of Aβ 1-42 on the expressions of Bcl-2, Bax, and caspase-3 (P < 0:01). All the results indicated that SH (50 mg/kg) could attenuate Aβ 1-42 -induced hippocampal alterations, oxidative stress, neuroinflammation, and neuronal apoptosis in mice.   Mediators of Inflammation

Discussion
AD is a specific form of dementia in elderly people that affects more than 35 million individuals all over the world [18]. By 2050, the number of AD patients will increase to 135.46 million [19]. Although some progress has been made in the aspects of understanding the pathophysiology of AD, there are no effective treatments, and only a few medicines are currently available to patients. Thus, there is an urgent need to search innovative therapeutic strategies and effective drugs for the benefit of AD patients. In the present study, we confirmed that SH could ameliorate Aβ 1-42 -induced memory impairment, neuroinflammation, and pyroptosis through inhibiting the NLRP3/GSDMD pathway in AD.  [20]. Huh et al. [8] have reported that Houttuynia cordata could significantly improve cognitive deficits of AD mice. Therefore, SH was selected in our study to investigate its effects on AD treatment. In the present study, MWM test results confirmed that SH significantly ameliorated spatial learning and memory deficiency in Aβ 1-42 -induced AD mice, suggesting that SH could improve cognitive deficits and memory loss in the progression of AD mice.

Effect of SH on Aβ 1-42 -Induced Neuroinflammation In
Vivo and In Vitro. SH has been reported to have therapeutic effects on various diseases due to its anti-inflammatory activity and antibacterial activity [21][22][23][24]. Researchers have found that neuroinflammation plays a critical role in the progression of AD [25]. Singhal et al. have suggested that neuroin-flammation is induced by the increased generation of inflammatory factors [26]. Demirci et al. have reported that the levels of proinflammatory cytokines (IL-6, IL-1β, IL-18, and TNF-α) in AD patients were higher than those in healthy volunteers [27]. In this study, we found that Aβ 1-42 treatment significantly increased the levels of IL-6, IL-1β, IL-18, and TNF-α both in vivo and in vitro. SH significantly decreased the levels of IL-6, IL-1β, IL-18, and TNF-α in Aβ 1-42induced AD in vivo and in vitro. These data suggested that SH could prevent Aβ 1-42 -induced AD through inhibiting neuroinflammation.

Effect of SH on Aβ 1-42 -Induced Neuron Pyroptosis
In Vivo and In Vitro. Galimberti and Scarpini [28] have reported that neuroinflammation is closely associated with neuronal apoptosis in AD. Neuronal apoptosis is identified to play an important role in learning and memory deficiency in AD [29]. In the present study, the results of the western blot       [30]. Therefore, we focused on ROS and the origin of oxidative stress via determining the levels of oxidative stress markers (MDA and SOD). In our study, we found that Aβ 1-42 treatment remarkably increased the MDA level and decreased the ROS level in vivo and in vitro. SH treatment significantly inhibited the generation of ROS and MDA and promoted the SOD level in Aβ 1-42 -induced AD in vivo and in vitro. The data confirmed that SH could prevent Aβ 1-42induced AD through inhibiting oxidative stress.

4.5.
Effect of SH on the NLRP3/GSDMD Pathway in Aβ 1-42 -Induced PC12 Cells. Recently, more and more studies have indicated that inflammasomes are closely related to neurodegenerative diseases: activated NLRP3 was observed in AD [31]. It is reported that NLRP3 inflammasome plays important roles in Aβ-induced inflammation [32]. When NLRP3 inflammasome is activated, NLRP3 and ASC can cut procaspase-1 into active caspase-1 [33]. The active caspase-1 then induces a secondary cascade of events associated to the releases of proinflammatory cytokines such as IL-1β and IL-18 [10]. In addition, the active caspase-1 also cleaves GSDMD into GSDMD-N and GSDMD-C [12]. After that, inflammatory factors flow out from the cells through the pores caused by GSDMD-N, which further trigger an inflammatory response and cause pyroptosis [12]. Recently, increasing evidence has confirmed that the inhibition of pyroptosis can significantly prevent the progression of AD [15]. Our data showed that SH significantly decreased the expressions of NLRP3, ASC, caspase-1, and GSDMD-N in Aβ 1-42induced AD in vitro, suggesting that SH could inhibit the activation of the NLRP3/GSDMD pathway in AD. Then, to further verify whether SH could prevent AD through the NLRP3/GSDMD pathway, an inhibitor of NLRP3 inflammasome (CY-09) was used in our experiment. The results showed that the effects of SH on proinflammatory cytokines and cell apoptosis were enhanced by CY-09, indicating that SH could protect Aβ 1-42 -induced AD through inhibiting the NLRP3/GSDMD pathway.
The nosogenesis and progression of AD involve many factors. However, our present study was confined to exploring the mechanism of SH on the NLRP3/GSDMD pathway and oxidative stress. Whether SH can alleviate the symptoms of AD by other pathways remains to be further studied.

Conclusion
In summary, we demonstrated that SH could ameliorate Aβ 1-42 -induced memory impairment, neuroinflammation, and pyroptosis through inhibiting the NLRP3/GSDMD pathway in AD. The findings of our study reveal that SH could serve as a therapeutic approach for the treatment of AD. In the future, further study might be focused on other underlying mechanisms of SH on AD.

Data Availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Disclosure
Yuequan Zhao and YunPeng Tian are co-first authors.