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Total Triterpenes of Wolfiporia cocos (Schwein.) Ryvarden & Gilb Exerts Antidepressant-Like Effects in a Chronic Unpredictable Mild Stress Rat Model and Regulates the Levels of Neurotransmitters, HPA Axis and NLRP3 Pathway

Purpose: Wolfiporia cocos is frequently used in traditional Chinese medicine to treat depression. However, antidepressant-like effects of the main active ingredients of Wolfiporia cocos, total triterpenes of Wolfiporia cocos (TTWC), are not well studied. This study aimed to investigate those effects and explore their specific mechanisms of action in depth. Methods: Chemical components of TTWC were analyzed using LC-MS. Depression-like behavior in rats were induced by chronic unpredictable mild stress (CUMS). The suppressive effects of TTWC (60, 120, 240 mg/kg) against CUMS-induced depression-like behavior were evaluated using the forced swimming test (FST), open field test (OFT) and sucrose preference test (SPT). Levels of 5-hydroxytryptamine (5-HT), glutamate (GLU), corticotropin-releasing hormone (CRH), interleukin-1 beta (IL-1beta), interleukin-18 (IL-18), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha) in different groups were determined by ELISA. Western blotting (WB) was used to detect the expression of NLRP3, ASC, pro-caspase-1, caspase-1, pro-IL-1beta, IL-1beta, pro-IL-18, and IL-18 in the prefrontal cortex. Additionally, the mRNA levels of NLRP3, ASC, caspase-1, IL-1beta and IL-18 were detected by RT-PCR. Results: A total of 69 lanostane-type triterpene acids of TTWC were identified. The results showed that TTWC exhibited an antidepressant-like effect in CUMS rats, reversed the decreased sugar preference in the SPT, reduction of immobility time in the FST, reduced the rest time, increased the total moving distance in the OFT. TTWC increased 5-HT levels and decreased GLU levels in the hippocampus. Moreover, TTWC decreased CRH levels in serum, indicating the regulation of over-activation of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, reduced serum levels of IL-1beta, IL-18, IL-6, and TNF-alpha. The WB results implied that TTWC inhibited the expression of NLRP3, ASC, caspase-1, IL-1beta, and IL-18 in the prefrontal cortex and enhanced the expression of pro-caspase-1, pro-IL-1beta, and pro-IL-18. Although most of the results were not significant, PCR results showed that TTWC inhibited the expression of NLRP3, ASC, caspase-1, IL-1beta, and IL-18 in the prefrontal cortex. Conclusion: TTWC treatment exerted an antidepressant-like effect and regulates neurotransmitters, HPA axis and NLRP3 signaling pathway. These results indicated the potential of TTWC in preventing the development of depression.


INTRODUCTION
Depression, which is characterized by depressive mood or loss of anhedonia, is one of the most common and costly psychiatric disorders. The global prevalence of depression is high; over 264 million people of all ages worldwide suffer from depression, leading approximately 80 thousand patients to commit suicide (www.who.int). Rising competition in modern society has led to a sharp surge in the likelihood of individuals to suffer from depression in recent years . Current treatments for depression mainly focus on the monoaminergic system, and the commonly used antidepressants are selective serotonin reuptake inhibitors, or norepinephrine reuptake inhibitors. Unfortunately, studies have shown that one-third of patients do not respond to the initial treatment, and almost half of them have a secondary response, often producing adverse reactions such as sexual dysfunction, nausea, tremor, and insomnia (Li et al., 2020a;Zhuo et al., 2020). To combat depression, the identification of natural medicines with better curative effects and increased safety is urgently needed.
Wolfiporia cocos (Schwein.) Ryvarden & Gilb is the dried sclerotium of the polyporaceae fungus Wolfiporia cocos, which often grows as a parasite on pine roots . Wolfiporia cocos, as a medicinal material and food, has existed for thousands of years in Chinese culture. At present, the secondary response of many antidepressants is inevitable, which on the other hand aggravates the disease of patients. Wolfiporia cocos, has the advantages of high safety and large yield, and its development as a therapeutic alternative may reduce the economic burden and physical torture of patients. In modern pharmacological research, it is widely used as a constituent of many formulae (including ding-zhi-xiao-wan, xiao-yao-san), wherein it plays a significant role in the treatment of neurodegenerative diseases (He et al., 2020;Liu et al., 2021). The main active components of Wolfiporia cocos are triterpenoids and polysaccharides (including watersoluble polysaccharides and alkali-soluble polysaccharides). Water-soluble polysaccharides exhibit a potent antidepressantlike effect by regulating monoaminergic neurotransmission and inactivation of inflammation (Huang et al., 2020). Similarly, alkali-soluble polysaccharides have exhibited their antidepressant-like effects by regulating neurotransmitters and alleviating neuroinflammation . Indeed, petroleum ether extract of Wolfiporia cocos has been confirmed to improve depression-like behavior by acting on gut microbiota and metabolites in chronic unpredictable mild stress (CUMS) rats (Gao et al., 2020). However, to date, most of the reported triterpenes of Wolfiporia cocos have been triterpene acids. Our previous study found only a tiny amount of Wolfiporia cocos triterpenes in petroleum ether extraction. Therefore, the possibility of the total triterpenes of Wolfiporia cocos (TTWC) exerting antidepressant-like effects is still unknown, and hence worth investigating.
Accumulating evidence shows that brain inflammation may be a critical factor in developing depression (Hodes et al., 2015). The NLRP3 inflammasome pathway, a key target in depression, mediates the production of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and interleukin-18 (IL-18) (Xu et al., 2016). Indeed, proinflammatory cytokines, including IL-1beta, interleukin-6 (IL-6), and tumor necrosis factoralpha (TNF-alpha), have been implicated in the etiology of depression, contributing to cellular damage, impaired neuronal plasticity, and neurotransmission in the prefrontal cortex and hippocampus (Felger and Lotrich 2013). Corroborating this observation, higher concentrations of proinflammatory cytokines, such as IL-6 and TNF-alpha, are observed more often in patients with depression than in healthy subjects (Dowlati et al., 2010).
TTWC, as a natural anti-inflammatory substance (Jeong et al., 2014;Lee et al., 2017), may have the potential to be developed into new antidepressant drugs. In our previous study, we have revealed that TTWC can regulate levels of 5hydroxytryptamine (5-HT), dopamine (DA), and other neurotransmitters in the hippocampus of sleep-deprived rats in the hippocampus, inhibit the hyperfunction of the hypothalamic-pituitary-adrenal (HPA) axis, and relieve anxiety symptoms. The present study is the first to describe the antidepressant-like effects of TTWC in a rat CUMS model. CUMS is a well-established procedure that induces depressionlike behavior in rodents (Willner 2017). Accordingly, the sucrose preference test (SPT), forced swimming test (FST), and open field test (OFT) were performed in CUMS-induced rats to analyze antidepressant-like effects of TTWC after administration. The possible impact of TTWC on depression was studied from the perspective of regulation of neurotransmitter levels, the HPA axis, and neuroinflammation. The technical strategy used in this study is shown in Figure 1.

Animals
All experimental procedures were approved by the Animal Ethics Committee of the Hubei University of TCM. All efforts were made to minimize suffering and the number of animals used to produce reliable data. Male Sprague Dawley rats (180-220 g) were supplied by Sanxia University. (Animal license No: SCXK (E) 2017-0012). The animals and protocols for this study were approved by the Ethics Committee of the Animal Experiment Center of Hubei University of Traditional Chinese Medicine (No: HUCMS202011001). Rats were housed under standard conditions to adapt to the environment for 7 days before study initiation and had free access to food and water. The standard conditions for temperature (23 ± 2°C) and light (12:12-h light/ dark cycle, lights on 8:00-20:00) were considered.

Chronic Unpredictable Mild Stress Procedure
The CUMS procedure was conducted as previously described, with minor modifications (Willner 1997). Rats in the unstressed group were housed together with free access to water and food. The water was removed for 24 h before the SPT. CUMS rats were housed singly and subjected to the following stressors for four continuous weeks: 45°cage tilt (12 h), water and food deprivation (24 h), cold swimming at 4°C (5 min), damp bedding (24 h), cage rotation (20 min), tail clip (2 min), and electrical stimulation (2 min). The stressors were randomly scheduled once daily during CUMS procedure and every stressor presented four times within 4 weeks. The same stressor could not repeat on two consecutive days to ensure the unpredictability and avoid the adaptation by rats.

Drug Administration and Treatment
Before the CUMS procedure, the body weight and sucrose preference of each rat were tested. Based on results, rats were divided into two groups (48 rats in the CUMS-exposed group and 8 rats in the unstressed group). We ensured that there was no significant difference in body weight or sucrose preference among all groups. All rats in the CUMS-exposed group were subjected to CUMS for 4 weeks. After modeling, the body weight and open field total moving distance of each rat were measured. According to the results, the CUMS-exposed group was then divided into five groups: model, FH (1.8 mg/kg), TTWC-L (low), TTWC-M (middle), and TTWC-H (high) dose groups (60, 120, 240 mg/kg), with eight rats in each group. The dose of FH was converted from the clinical dose of 20 mg/d (Zhou et al., 2021). The unstressed and vehicle treated CUMS groups were administered saline every day, while the other groups received intragastric administration of the corresponding drugs for 2 weeks. The experimental procedure is shown in Figure 2.

Depression-Like Behavior in Rats Was
Determined Using Sucrose Preference Test, Forced Swimming Test, and Open Field Test

Sucrose Preference Test
The SPT was performed as previously described (Daodee et al., 2019). Rats were first habituated to sucrose solution (1% w/v) for 24 h: two bottles of 1% sugar water were placed in each cage, and one bottle was replaced with pure water 24 h later. After deprivation of food and water for 12 h, each rat was given free access to two bottles of solution (100 ml of tap water and 100 ml of 2% sucrose). The position of bottles was random. The test was completed 3 h later, and the sucrose preference was calculated using the following formula: sucrose preference (%) = sucrose consumption/(water consumption + sucrose consumption).

Forced Swimming Test
The FST was performed as previously described (Shi et al., 2019). Rats were placed in a cuboid with a water temperature of 24 ± 1°C. The immobility time of rats was monitored for 6 minutes. Each rat was forced to swim for 2 min, and the total duration of immobility was recorded in the remaining 4 min. Rats were considered to be immobile when they maintained floating in water without escaping from the cylinder.

Open Field Test
The OFT was carried out with reference to a previous study (Hassan et al., 2020). The experiment was conducted in four equal-sized square open boxes (40 × 40 × 50 cm). The test was carried out in a sound proof room under bright ambient light. A camera was installed directly above the central area of the four boxes. The rats were individually placed into center area, and the video analysis system was immediately activated to capture video automatically. The measuring time was 5 min for each rat, and the rats were placed in the center of the field and adapted to the field environment for 2 min. The rats were then recorded for 3 min. Floor surfaces and walls of the area were thoroughly cleaned with an alcohol solution and dried between each rat test. The single-subject tracking mode of the system was used to measure the rest time (rest time) and total moving distance (total distance).

Tissue Preparation
After the last behavioral test, rats were sacrificed, and serum and brain tissue were collected. Blood was collected from the abdominal aorta, centrifuged (3,500 rpm) for 15 min at 4°C and the serum was stored at −80°C. The hippocampus and prefrontal cortex of 36 rats (six randomly selected from each group) were removed on ice and stored at −80°C after freezing with liquid nitrogen. The whole brains of 12 rats (two rats randomly selected from each group) were stored in 4% paraformaldehyde solution at 4°C for paraffin embedding.

Hematoxylin and Eosin Staining
The whole brain was fixed with a 4% paraformaldehyde for more than 24 h, and the trimmed tissue was placed in the dehydration box. The dehydration box was placed in the dehydrator to dehydrate with a gradient of alcohol. Specimens were embedded in paraffin and sliced. Paraffin sections were baked, dewaxed, and hydrated. Sections were placed in distilled water, and a hematoxylin aqueous solution was added for 3 min. This step was followed by hydrochloride ethanol differentiation for 15 s, slightly washed with water, and placed in bluing buffer for 15 s. The specimen was then washed with running water and eosin-stained five 5 min, washed with running water, cleared, mounted, and the morphology and number of pyramidal cells and Nissl's bodies in the hippocampus and prefrontal cortex were observed under a 400× microscope.
Quantification of Brain-Derived Neurotrophic Factor, 5-HT, Dopamine, Glutamate, NE in Hippocampus, and Corticotropin-Releasing Hormone, Adrenocorticotropic Hormone, Corticosterone, Interleukin-1 Beta, Interleukin-18, Tumor Necrosis Factor-Alpha, Interleukin-6 in Serum by Enzyme-Linked Immunosorbent Assay Rat hippocampus were used to determine the levels of BDNF and other neurotransmitters associated with CUMS-induced depression-like behavior. Serum was used to evaluate the activity of the HPA axis and proinflammatory cytokines related to CUMS-induced depression-like behavior. Levels of BDNF, 5-HT, DA, GLU, and NE in the hippocampus and levels of CRH, ACTH, CORT, IL-1beta, IL-18, TNF-alpha, and IL-6 in the serum of rats were measured using ELISA kits. The experiment was performed strictly under the ELISA instructions, and the concentration was normalized according to the standard curve.    Detection of NLRP3, ASC, Pro-caspase-1, Caspase-1, Pro-Interleukin-1beta, Interleukin-1beta, Pro-Interleukin-18, and Interleukin-18 Content in Prefrontal Cortex by Western Blotting Total protein was prepared from the prefrontal cortex of behavioral test rats. Approximately 40 μg of protein for each sample was loaded on electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking with 5% skimmed milk powder at room temperature for 1 h, the membrane was incubated with primary antibody at 4°C overnight, rinsed three times with Tris-buffered saline/Tween 20 (TBST) for 5 min, and incubated with HRPconjugated secondary antibody for 30 min at 37°C. The membrane was rinsed four times with TBST for 5 min and then analyzed using a chemiluminescence imaging system. Relative expression of protein was detected by the ratio of the grayscale of target protein band/grayscale of beta-actin band. The mean intensity values were obtained from three independent experiments.
Detection of NLRP3, ASC, Caspase-1, Interleukin-1beta, and Interleukin-18 mRNA in Prefrontal Cortex by Quantitative Reverse Transcription-Polymerase Chain Reaction Quantitative Reverse Transcription-Polymerase Chain Reaction was performed according to the SYBR Green PCR kit instructions. The expression levels of ASC, caspase-1, IL-1beta, and IL-18 mRNA in the prefrontal cortex were determined. The sequences of the primers used for real-time PCR are listed in Table 1. The amplification procedure was as follows: 95°C for 30 s (95°C for 15 s, 60°C for 30 s) × 40. The data were analyzed using Nanodrop 2000 software, and the expression levels of ASC, caspase-1, IL-1beta, and IL-18 mRNA were determined.

Statistical Analysis
The experimental data were expressed as the mean ± standard deviations (SD). The experimental data were statistically analyzed and plotted using SPSS (version 25.0; SPSS Inc., Chicago, IL, United States) and GraphPad Prism 7 software (GraphPad Software Inc., San Diego, CA, United States). One-way analysis of variance (ANOVA) was used for comparison between groups, dunnett method was used for homogeneity of variance, Games-Howell method was used for heterogeneity of variance, where p < 0.05 indicated a significant difference, and p < 0.01 indicated a very significant difference.

Composition Analysis of Total Triterpenes Of Wolfiporia Cocos
Ultra-high performance liquid chromatography-quadropole timeof-flight mass spectrometry (UPLC-QTOF-MS/MS) determined components of TTWC in both positive and negative modes. Our results corroborated findings from previous literature and identified 69 chemical constituents in the negative ion mode (Feng et al., 2018;Zou et al., 2019). Extracted ion chromatograms of the 69 triterpenoids are shown in Figure 3.

Effects of Total Triterpenes Of Wolfiporia Cocos on Depression-like Behavior Induced by Chronic Unpredictable Mild Stress in Rats
The depression-like behavior in CUMS rats was assessed to examine antidepressant-like effects of TTWC using SPT, FST, and OFT.

Effects of Total Triterpenes of Wolfiporia Cocos on Sucrose Preference Test
According to the results, sucrose preference remained consistent across all rats before the modeling process, as shown in Figure 4A. In the SPT ( Figure 4B), sucrose preference among different groups significantly differed. Compared with that in the unstressed group (0.836 ± 0.091), CUMS rats showed significant decrease in the sucrose preference rate (0.574 ± 0.136, p < 0.01). After 14 days of administration, compared with that of the vehicle treated CUMS group, the sucrose preference rate of the other groups improved, and significant differences in the TTWC-L, TTWC-M, and TTWC-H groups (0.816 ± 0.094, p < 0.01; 0.817 ± 0.086, p < 0.01; 0.845 ± 0.116, p < 0.01) were observed.

Effects on Hippocampus and Prefrontal Cortex Tissue Morphology
HE staining of pyramidal cells in the CA3 region of the rat hippocampus, as viewed under a microscope, showed normal morphology and a tight arrangement of the pyramidal cells in the unstressed group. In contrast, the vehicle treated CUMS group demonstrated loosely arranged pyramidal cells with missing cells and a fuzzy structure. Moreover, the number of Nissl bodies in the prefrontal cortex and hippocampus was reduced in the vehicle treated CUMS group. In TTWC treated groups, the pyramidal cell morphology was improved, and the number of pyramidal cells and Nissl's bodies were also increased to a certain extent. Figure 6 shows the effects on hippocampal and prefrontal cortex tissue morphology.

Effect of Total Triterpenes of Wolfiporia
Cocos on the Level of Interleukin-1beta, Interleukin-18, Interleukin-6, and Tumor Necrosis Factor-Alpha in Serum CUMS induced inflammation in different brain regions, with increased levels of cytokines including IL-1beta, IL-18, IL-6, and TNF-alpha in the serum (

DISCUSSION
CUMS, acknowledged as a putative animal model of depressivelike behavior and an imitation of life stress, is reliable and stable (Willner 1997;Mineur et al., 2006). Lack of pleasure is an essential manifestation of depression, and SPT is the gold standard for evaluating the degree of pleasure deficiency in depressed animals (Weiss 1997;Zhou et al., 2021). In this study, compared with rats in unstressed group, CUMS rats showed a lower degree of preference for sucrose after 4 weeks of CUMS treatment, which increased the sucrose preference index and demonstrated an antidepressant-like effect. The FST is commonly used to evaluate depression-like behavior in rodents, in which the immobility time reflects the degree of despair in rats (Porsolt et al., 1978). The immobility time in the vehicle treated CUMS group was significantly prolonged, which was reduced upon TTWC administration. The OFT was used to quantitatively evaluate rodents' spontaneous activity exploration behavior and state of depression. Rats' depressivelike behavior was assessed by their desire to explore the open field (Wang et al., 2014). We found that the desire to study the open field decreased in the vehicle treated CUMS group, the total moving distance decreased, and rest time was prolonged. However, these indices were reversed upon TTWC administration. Analysis of SPT, FST, and OFT results showed that rats in the vehicle treated CUMS group exhibited apparent  As shown in the previous studies, the hippocampus plays a vital role in the regulation of emotional learning and expression (Liu and Aghajanian 2008;Yao et al., 2020), while the prefrontal cortex is crucial in transforming emotional information into stress behavior and is involved in the neurological mechanisms of stress adaptation and pathology (Mcklveen et al., 2013), both of which are more easily affected in depression (Sheline et al., 1996;Luo et al., 2020). Among the findings of altered brain structure and function in depression, the most consistent finding is the reduced volume of the prefrontal cortex (PFC) and hippocampus (Savitz and Drevets 2009). In addition, studies have shown that the levels of neurotransmitter and inflammation in PFC and hippocampus are also consistent (Ashraf et al., 2019;Shen et al., 2019;Lama et al., 2021). Therefore, we observed the tissue morphology of PFC and hippocampus, and selected hippocampus and PFC tissues respectively to detect neurotransmitter levels and inflammation expression. It is generally recognized that depression is associated with an imbalance in the impairment of BDNF and certain neurotransmitters such as 5-HT, DA, GLU, and NE in the central nervous system (Tavakoli et al., 2018). BDNF is a potent regulator of long-term synaptic plasticity and mediates learning and memory. Low levels of BDNF signaling lead to hippocampal and cortical neural plasticity deficits, which are followed by neurodegenerative disorders, such as Alzheimer's disease and major depression (Lu et al., 2013). The monoamine hypothesis is the first neurobiochemical theory of depression, which holds that deficiency of NE, 5-HT, or DA in the synaptic space occurs in vivo. It is also the leading cause of depression (Mcewen et al., 2010). Glutamate is the primary excitatory neurotransmitter in the brain. Once glutamate is released, glutamate receptors are stimulated excessively, leading to swelling and apoptosis of nerve cells and neurological disorders (Castillo et al., 1996). Therefore, we determined the levels of BDNF and neurotransmitters (5-HT, DA, GLU, and NE) in the hippocampus of rats. Our study revealed that in the CUMS group, the levels of BDNF, 5-HT, DA, and NE were decreased, and GLU levels were increased considerably. Their levels were reversed after intervention with FH and TTWC. Furthermore, the hippocampal and prefrontal cortex were improved morphologically and functionally by FH and TTWC administration. This observation indicated that TTWC might exert an antidepressant-like effect by regulating the levels of BDNF and neurotransmitters. In addition, we also hypothesized that it might promote the recovery of the hippocampus and prefrontal cortex.
The HPA axis, as a significant part of the neuroendocrine system, controls reactions to stress and regulates emotion (Swaab et al., 2005). Long-term chronic stress can lead to dysfunction of the HPA axis and increased secretion of CRH, ACTH, and CORT, which is considered to be closely involved in the pathogenesis of human depression (Fischer et al., 2017). The function of the HPA axis often returns to normal when depression patients or depressive animals receive effective treatment or when patients display spontaneous remission (Raone et al., 2007). Therefore, concentrations of CRH, ACTH, and CORT in the serum were measured. CRH and ACTH levels were increased in CUMS rats, with CORT levels being elevated to some extent. The administration of TTWC decreased CRH, ACTH, and CORT levels in the serum of CUMS rats. These results suggest that the inhibition of hyperfunction of the HPA axis may be an essential mechanism of the antidepressant-like effect of TTWC.
Growing evidence has revealed that suppressing the activation of NLRP3 and reducing the release of inflammatory cytokines plays a vital role in normalizing hyperactivity of the HPA axis (Lu et al., 2014). In addition, a positive correlation between neuroinflammation and depression has been reported in many clinical studies (Miller and Raison 2016). Stress from society, work, or life tend to cause elevated levels of proinflammatory cytokines such as IL-1beta, IL-18, IL-6, and TNF-alpha in both the periphery and brain, leading to sickness behavior syndrome; after treatment with antidepressants, the elevated levels of proinflammatory cytokines in some depression patients return to normal. (Miller et al., 2009). In the present study, CUMS rats showed increased levels of IL-1beta, IL-18, IL-6, and TNF-alpha in the serum. Notably, TTWC treatment decreased levels of IL-1beta, IL-18, IL-6, and TNF-alpha in the serum, indicating that the antidepressant-like effect of TTWC may be related to the inhibition of neuroinflammation in the prefrontal cortex. To further explore the mechanism of TTWC action, we investigated in depth the NLRP3 inflammasome, which controls the maturation of IL-1beta and IL-18.
The NLRP3 inflammasome is the most widely characterized NLR inflammasome complex. Canonical NLRP3 inflammasome complex is an intracellular protein complex consisting of the sensor NLRP3, the adaptor ASC, and pro-caspase-1. According to the previous literature, upon sensing danger signals, NLRP3 monomers, via their leucine-rich repeat motif domains, may interact with the pyrin domain of ASC, which then recruits the cysteine protease pro-caspase-1 via a caspase recruitment domain (Jo et al., 2016). Oligomerization of pro-caspase-1 proteins induces their proteolytic cleavage into active caspase-1 (Yang et al., 1998). Active caspase-1 cleaves precursor cytokines pro-IL-1beta and pro-IL-18, generating biologically active cytokines IL-1beta and IL-18 and, under certain conditions, to induce proptosis, a form of programmed inflammatory cell death (Guo et al., 2015). IL-1beta is a critical pro-inflammatory cytokine that can produce signal amplification cascades involving multiple inflammatory factors, thus aggravating inflammatory responses (LaRock et al., 2016). According to these findings, our study showed that the expression of NLRP3, ASC, Caspase-1, IL-1beta, and IL-18 was significantly reduced compared to those of the vehicle treated CUMS group. Levels of pro-caspase-1, pro-IL-1beta, and pro-IL-18 increased considerably in the FH and TTWC groups, suggesting that the inhibition of proinflammatory cytokine release induced by TTWC depends on the inhibition of the NLRP3 signaling pathway.