1 Introduction
Tourette Syndrome (TS) is a chronic neuropsychiatric disorder commonly seen in childhood. It is primarily marked by involuntary, repetitive, quick, and purposeless muscle or vocal tics, which can be accompanied by emotional disturbances, hyperactivity, attention deficits, compulsive actions or thoughts, along with other behavioral issues (Hongyan et al., 2019). Various tic disorders (TD), including provisional tic disorder (PTD) and chronic motor or vocal tic disorder (CTD), fall under this classification, with TS being the most severe form (Müller-Vahl et al., 2019; Liu et al., 2020). Presently, TS treatment is categorized into behavioral and pharmacological therapies. Among behavioral approaches, only Comprehensive Behavioral Intervention for Tics (CBIT) is specifically endorsed by the American Academy of Neurology as effective. Nonetheless, its high cost restricts its widespread application. Pharmacological treatments mainly include atypical antipsychotics like aripiprazole, tiapride, and risperidone. Of note, tiapride is more commonly prescribed for children and adolescents compared to adults, often with favorable outcomes. However, prolonged use of these medications can result in extrapyramidal side effects such as muscle rigidity, tremors, and involuntary movements, along with drowsiness, nausea, increased prolactin levels, and weight gain. Although deep brain stimulation is employed for treatment-resistant cases, its limited sample size, varied study designs, and high costs hinder its broad implementation (Roessner et al., 2022; Andrén et al., 2022; Johnson et al., 2023). Therefore, there is a critical need to discover a safe and effective therapeutic approach for TS.
A growing body of evidence indicates a strong link between inflammatory cytokines and the development of TS, leading to significant alterations in serum and striatum levels of cytokines such as interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNF-α) (Long et al., 2019). Neuroinflammation, which is triggered by inflammatory stimuli, activates microglia within the central nervous system (CNS) as a fundamental protective mechanism. However, sustained neuroinflammation can result in neuronal dysfunction and even cell death Cherry (Cherry et al., 2014). Furthermore, neuroinflammation plays a critical role in a range of CNS disorders, including TS, autism spectrum disorder (ASD), Alzheimer’s disease (AD), and Parkinson’s disease (PD) (Mirarchi et al., 2023). Thus, mitigating the inflammatory response induced by overactive microglia and preventing subsequent cellular damage, alongside regulating neuroinflammatory processes, might be effective strategies for managing neuroinflammation and CNS disorders (Bachiller et al., 2018; Lim et al., 2018; Fatoba et al., 2020).
Acorus tatarinowii schott (The plant name “A. tatarinowii” has been verified through Kew Medicinal Plant Names Services (MPNS) and http://www.worldfloraonline.org. The plant name corresponds with the latest revision of “World Flora Online”. First published in Oesterr. Bot. Z. 9: 101 (1859). This name is a synonym of Acorus verus), commonly known as Shi Chang Pu, a traditional Chinese medicinal botanical drug first recorded in the “Shennong Ben Cao Jing,” possesses pharmacological properties such as sedation, anticonvulsant, antioxidant, anti-neuroinflammation, neurotransmitter regulation, and anti-apoptosis (Balakrishnan et al., 2022; Kim et al., 2022; Li et al., 2022; Tao et al., 2022; Wang M. et al., 2023). It has been widely used in treating neurological diseases (Zhang et al., 2019). Relevant pharmacological studies indicate that A. tatarinowii contains various metabolites, including volatile oils, flavonoids, alkaloids, and organic acids, with volatile oils being the primary active metabolites (Ma et al., 2015). Common extraction methods for volatile oils from A. tatarinowii include steam distillation, microwave-assisted extraction, ultrasonic extraction, and supercritical fluid extraction (Aziz et al., 2018; Bai et al., 2022). Several studies have identified its main active metabolites as α-asarone, β-asarone, methyleugenol, and caryophyllene (Limón et al., 2009; Wang et al., 2014; Lam et al., 2016; Shi et al., 2021). Numerous studies have confirmed that these metabolites, as important neuroprotective agents, can penetrate the blood-brain barrier, maintain the balance of neurotransmitters in the brain by adjusting excitatory and inhibitory responses, reduce neuroinflammatory responses, and effectively inhibit microglia-mediated neuroinflammatory responses by suppressing the production of pro-inflammatory cytokines (Chellian et al., 2017; Lee et al., 2018; Zhang et al., 2019; Ullah et al., 2021). Consequently, they exhibit neuroprotective effects (Balakrishnan et al., 2022; Hongyan et al., 2019; Kim et al., 2022; Ning et al., 2024), indicating positive potential for the prevention and treatment of CNS diseases (Shi et al., 2021; Bai et al., 2022). Therefore, these findings suggest that VOA may be an effective candidate for treating TS. Tiapride is one of the ten most commonly used antipsychotic drugs in pediatrics, particularly as a first-line treatment for children and adolescents with Tourette syndrome (Chellian et al., 2017; Roessner et al., 2022). It is widely used as a first-line medication for treating TS in Asian countries (Liu et al., 2020). Tiapride primarily exerts its therapeutic effects by blocking dopamine D2 receptors in the brain, with significant action in the striatum, which helps reduce extrapyramidal side effects. However, it can also cause adverse reactions such as dizziness, fatigue, drowsiness, and central nervous system effects, as well as gastrointestinal discomfort like nausea and vomiting (Roessner et al., 2022; Johnson et al., 2023). Therefore, we selected Tiapride as a positive control drug to evaluate the therapeutic potential of VOA in TS patients.
Although The Volatile Oil of A. tatarinowii (VOA) has shown potential therapeutic effects in the treatment of neuroinflammation and CNS diseases, its regulatory effects on neuroinflammatory damage in a TS rat model have not been fully explored. In particular, whether VOA can alleviate neuroinflammatory damage in TS model rats by the p38 MAPK/NLRP3/STAT3 signaling pathways requires systematic experimental investigation. In our previous studies (Feng et al., 2024), we first evaluated the therapeutic effects of different doses of VOA on a TS rat model, and the results demonstrated that High-dose VOA exhibited significant anti-tic effects. Based on these findings, the current study further focuses on the High-dose VOA group to investigate its potential in repairing neuroinflammatory damage by examining its inhibitory effects on the p38 MAPK/NLRP3/STAT3 signaling pathways.
4 Discussion
As previously mentioned, TD are a group of disorders that can be classified into PTD, CTD and TS based on the duration and type of symptoms. TS falls within the spectrum of TD and represents the most severe form. TS and TD share common characteristics, including involuntary, sudden, rapid, and repetitive movements or vocalizations (Liu et al., 2020). While distinguishing between these disorders aids clinical diagnosis, it can be challenging in clinical practice to accurately differentiate between TS and TD in a short period of time. When patients present with severe symptoms and it is difficult to immediately distinguish between TS and TD, the primary goal of treatment is to alleviate symptoms and improve the patient’s quality of life. Medications that can treat both types of tics are more aligned with clinical needs. Additionally, TD and TS share many pathological similarities, including neurotransmitter imbalances (such as dopamine) and abnormalities in the cortico-basal ganglia-thalamic circuitry (Jindachomthong et al., 2022; Ricketts, 2023). Neuroinflammation plays a crucial role in the pathogenesis of both TS and TD, with excessive activation of inflammatory factors potentially affecting the function of neural networks in the basal ganglia, leading to or exacerbating tic symptoms (Almeida et al., 2015; Skarphedinsson et al., 2015; Ramkiran et al., 2019). Thus, many medications used in clinical practice can treat both conditions simultaneously. VOA mentioned in this study is similarly applicable to both tic disorders, and based on our previous research, we further explored the efficacy of VOA in treating TS. This study focused on the role of the p38 MAPK/NLRP3/STAT3 signaling pathway in TS and demonstrated that VOA can inhibit pro-inflammatory cytokines, reduce microglial activation, and regulate neuroinflammation, thereby showing its potential efficacy in treating TS.
In our previous research (Feng et al., 2024), we found that VOA has a significant therapeutic effect on TD, with the highest efficacy observed in the High-dose group (51.12 mg/kg). TS is the most common manifestation of TD. IDPN is a commonly used neurotoxin. The IDPN model was first utilized by Diamond et al. (1982) to develop the TD model and can significantly increase stereotypic and motor behavior scores in rats (Cherry et al., 2014; Diamond et al., 1982). The IDPN model is a standard animal model for TS, capable of comprehensively reproducing the behavioral characteristics of TS; it has been used in many TS experiments (Zhao et al., 2015; Liu et al., 2020; Wang N. et al., 2022; Yang et al., 2022) and activate microglia in the striatum of these rats (Zhao et al., 2020; Wang X. et al., 2023). Therefore, in this study, we used an IDPN-induced TS rat model to evaluate the potential therapeutic effect of VOA in alleviating neuroinflammation in TS rats by modulating the p38 MAPK signaling pathway.
Tiapride, a selective D2 receptor (dopamine receptor) antagonist, has been used for years in the treatment of TS. Clinical and experimental studies have demonstrated that Tiapride has significant anti-tic effects (Chen, 2019; Pringsheim et al., 2019; Fekete et al., 2021). Acorus tatarinowii is a traditional medicinal botanical drug with a long history of use, known for its ability to restore vitality in the brain, nervous system, and cardiomyocytes, as well as its neuroprotective effects. It is widely used in the treatment of central nervous system, cardiovascular, and cerebrovascular diseases, and also exhibits antibacterial and antioxidant properties (Bai et al., 2022). The anti-inflammatory effects of essential oil metabolites in VOA have been demonstrated in both in vitro and in vivo studies against various stimulus-induced inflammatory responses (Zhong et al., 2020; Kim et al., 2022). Among these metabolites, 3-Methyl-pyrrolo [2,3-b]pyrazine exhibits various biological activities, including antibacterial, anti-inflammatory, antiviral, antifungal, antioxidant, antitumor, and kinase inhibition effects (Dehnavi, 2021). In a 90-day subchronic study with oral administration of α-Irone in 15 male and 15 female rats, no evidence of adverse toxic effects was observed upon necropsy (Oser et al., 1965). A dose of 0.4 g/kg of 3-Methyl-pyrrolo [2,3-b]pyrazine has been identified as safe in preliminary toxicity screenings (Stoner et al., 1973).1-Phenylcyclohexylamine shows potent anticonvulsant properties and is capable of inducing cumulative neuroplastic changes and alterations in brainwave patterns (Abiero et al., 2021; Thurkauf et al., 1990). β-asarone demonstrates multiple pharmacological activities, including antioxidant, anti-inflammatory, anti-apoptotic, anticancer, and neuroprotective effects (Meng et al., 2021; Balakrishnan et al., 2022). A subacute in vivo toxicity study indicated that oral administration of β-asarone (100 mg/kg for five consecutive days) led to reduced body weight and food consumption without causing mortality in rats prior to withdrawal (Balakrishnan et al., 2022). Citral, a monoterpene, has been extensively studied for its neuroprotective effects (Shamsi et al., 2024). It also exhibits antipyretic, antitumor, antibacterial, and anti-inflammatory activities (Dudai et al., 2005; Chaouki et al., 2009; Bachiega and Sforcin, 2011; Yang et al., 2013; Emílio-Silva et al., 2017.), inhibiting oxidative activity and enhancing brain-derived neurotrophic factor expression while reducing COX-2 and NF-κB expression and suppressing microglial activation (Katsukawa et al., 2010; Yang et al., 2013; Gonçalves et al., 2020; Charret et al., 2021; Habib et al., 2021). A 14-day toxicity assessment in male mice confirmed Citral’s safety, supporting its potential as a promising, innovative, and safe molecule for treating immune-inflammatory conditions and pain states (Gonçalves et al., 2020). Geraniol, an acyclic monoterpene derived from plants, has demonstrated anti-inflammatory, anticancer, antioxidant, and antibacterial effects in various in vivo and in vitro models (Rajendran et al., 2025). Studies suggest that Geraniol holds promise as a potential anti-inflammatory and cancer chemopreventive agent (Ben Ammar, 2023), exerting anti-inflammatory and immunomodulatory effects by reducing the expression of IL-6, IL-8, IL-1β, COX-2, NF-κB, and TNF-α (Wang et al., 2016; Ye et al., 2019; Pandur et al., 2024). Additionally, it reduces the expression of MAPKs (such as p38 MAPK), inhibiting microglial cells and neuroinflammation (Pandur et al., 2024; Rajendran et al., 2025). Trans-Calamenene, a metabolite contributing to the aroma and bioactivity of essential oils, exhibits antibacterial properties and may be considered as a candidate for natural antimicrobial agents (Gooré, 2017). Cis-Calamenene, the cis isomer of Calamenene, influences the maturation and function of human monocyte-derived dendritic cells, playing a role in immunomodulation by driving Th1 polarization, which is crucial for cellular immune responses, particularly in anti-infection and antitumor immunity (Takei et al., 2006).
Nevertheless, the potential toxicity of these metabolites must be acknowledged. The primary toxic metabolites of A. tatarinowii are the asarones found in its volatile oil, which have been shown to exhibit cardiotoxic, hepatotoxic, reproductive toxic, and carcinogenic effects (Jaiswal et al., 2015; Cartus and Schrenk, 2016; Bai et al., 2022). Specifically, studies indicate that doses of β-asarone above 500 mg/kg can result in acute mortality in mice, with toxicity increasing in a dose-dependent manner. Therefore, the use of essential oil must be strictly controlled within a safe dosage range. Precise regulation of the dosage, administration time, syndrome differentiation, and target system of the essential oil is crucial to prevent potential harm to humans. In this study, the therapeutic dose was based on clinical standards, calculated according to a 25 kg child’s body weight, with a clinical dose of 8g of A. tatarinowii, and was then converted to a corresponding dose for rats to ensure the safety and efficacy of the experimental research. Therefore, in this study, we used Tiapride as a positive control drug to compare its efficacy with that of VOA. In the motor and stereotyped behavior experiments, we found that both Tiapride and VOA significantly reduced the behavioral scores in TS rats.
Neuroinflammation refers to the inflammatory response within the central nervous system (CNS), characterized by elevated levels of pro-inflammatory cytokines, microglial activation, infiltration of peripheral leukocytes, and neuronal tissue damage (Estes and McAllister, 2014). In this process, microglia, the primary immune cells within the CNS, become activated in response to pathological stimuli and release large amounts of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. These cytokines not only further activate microglia but also attract more peripheral immune cells to infiltrate the CNS, exacerbating neuroinflammation (Chen et al., 2019). For instance, in Parkinson’s disease (PD), neuroinflammation is considered one of the key factors driving neurodegeneration. Persistent microglial activation and excessive release of pro-inflammatory cytokines lead to the progressive loss of dopaminergic neurons, ultimately resulting in the onset and progression of motor symptoms. Microglia are key regulators of neuroinflammation, playing a significant role in the onset and progression of neurological diseases. Overactivated microglia are considered markers and drivers of neuroinflammation-related diseases (Whitney et al., 2009; Balakrishnan et al., 2022). Postmortem analyses of TS patients have shown increased numbers of CD45+ microglia in the striatum of the basal ganglia, with these cells exhibiting morphologies consistent with neurotoxic activation (Lennington et al., 2016). A recent PET study also demonstrated increased activation of microglia in the brains of TS patients (Kumar et al., 2015). In this study, we established a TS rat model using the neurotoxin IDPN. Through this model, we observed significantly elevated levels of pro-inflammatory cytokines, including TNF-α and IL-6, in the striatum of Model group rats, indicating a pronounced inflammatory response. Additionally, we detected significantly increased expression levels of microglial activation markers CD11 b and COX-2, further confirming the presence of an active microglial state and enhanced neuroinflammatory response in the TS rat model.
Fully activated microglia exhibit neurotoxic characteristics, specifically the M1 phenotype, producing pro-inflammatory mediators such as TNF-α, IL-1β, and IL-6. TNF-α and IL-6 are major stimulators of the inflammatory response in the CNS (Erta et al., 2012; Almolda et al., 2014; Recasens et al., 2021), possessing immunomodulatory and inflammation-regulating properties that can increase blood-brain barrier (BBB) permeability and promote neuronal death (Fujihara et al., 2020; Li et al., 2022). As a prototype immunoregulatory cytokine, IL-10 can control and modulate the production of pro-inflammatory cytokines both in vitro and in vivo (Ouyang et al., 2011; Leech et al., 2017), playing a crucial role in the interaction between the immune system and the nervous system (Long et al., 2019; Liu et al., 2021). It has been shown to induce the M2 phenotype of microglia or promote the conversion from M1 to M2, thereby effectively suppressing the production of inflammatory cytokines (Zhou et al., 2019; Cunha et al., 2022) and facilitating tissue remodeling and repair (Hoogland et al., 2015; Moehle and West, 2015; Tang and Le, 2016). We treated the Model group rats with VOA, Tiapride, and SB203580, respectively, and found that the levels of pro-inflammatory cytokines in the striatum of TS rats were significantly reduced after treatment, accompanied by decreased expression of the microglial marker CD11b, a reduction in the M1 marker COX-2, and an increase in the M2 marker CD163, indicating a transition and repair of neuroinflammation. Among these, VOA showed the most pronounced therapeutic effect, suggesting that VOA can effectively repair neuroinflammation.
The decrease in CD11 b and COX-2 levels in the striatum of TS rats further demonstrated VOA’s anti-inflammatory effects. CD11b, a microglia-specific surface marker protein, is a marker of pro-inflammatory polarization and is associated with the activation of M1 (pro-inflammatory) microglia. Increased expression of CD11 b correlates positively with the degree of microglial activation in various central nervous system diseases (Roy et al., 2008; Zhou et al., 2019; Liu et al., 2022). COX-2, a typical marker of pro-inflammatory cytokines, is an enzyme involved in the synthesis of pro-inflammatory prostaglandins. In the early stages of neuroinflammation, activated microglia mediate neuroinflammation and activate pro-inflammatory signaling pathways through COX-2 release (Aïd and Bosetti, 2011; López and Ballaz, 2020). Conversely, VOA treatment led to elevated levels of CD163, a hemoglobin-haptoglobin (HbHp) complex receptor, and a microglia-specific anti-inflammatory marker with strong antioxidant and anti-inflammatory properties. CD163 is a HbHp complex receptor and a microglia-specific anti-inflammatory marker with strong antioxidant and anti-inflammatory properties, playing a role in resolving inflammation and promoting tissue repair (Van Gorp et al., 2010; Pey et al., 2014; Sternberg et al., 2019; Han et al., 2022; Sharma et al., 2023). Consistent with these findings, we observed that VOA significantly downregulated CD11 b and COX-2 levels and increased CD163 levels in TS rats, indicating a downregulation of M1 pro-inflammatory mediators and an upregulation of M2 anti-inflammatory mediators, facilitating a reparative effect in the brains of TS rats. Notably, the increase in IL-10 further promoted the elevation of CD163 levels, both contributing to inflammation repair, adjusting the activation state of microglia, and alleviating neuroinflammation in TS rats.
Inflammation is characterized by membrane perforation mediated by the gasdermin protein family and sustained release of pro-inflammatory cytokines (Liu X. et al., 2016; Xu et al., 2018; Oh et al., 2020; De Schutter et al., 2021). Studies have shown that TNF-α can activate the NLRP3 inflammasome and caspase-1 (Yao et al., 2022). Activation of NLRP3 and caspase-1 in neurons leads to neuroinflammation (Hua et al., 2022). The NLRP3 inflammasome is a cytoplasmic protein complex composed of NLRP3, ASC, and Caspase-1 (Zhang et al., 2021), and its activation can lead to the proteolytic activation of Caspase-1 and increased secretion of pro-inflammatory cytokines IL-1β and IL-18, serving as a central signaling pathway for neuroinflammation (Kanthasamy et al., 2019; Weis et al., 2021). The NLRP3 inflammasome is one of the most widely studied and well-characterized inflammasomes in microglia. (De Rivero Vaccari et al., 2014; Han et al., 2021). NLRP3 activation is a crucial marker of M1 polarization and a key factor in inhibiting neuroinflammation (Han et al., 2023; Du et al., 2024), playing a critical role in microglial overactivation (Zhou et al., 2016; Dempsey et al., 2017; Mao et al., 2017). Its activation is closely related to neuronal damage in various neurological diseases (Wang Z. et al., 2019; Wang et al., 2022 M.). In this study, VOA intervention significantly reduced the levels of inflammatory cytokines such as TNF-α and IL-6 in TS rats, modulating the activation state of microglia and effectively regulating the neuroinflammatory state. The detection results showed that the decrease in the expression levels of NLRP3, a key marker of M1 polarization, and its downstream factors caspase-1 and GSDMD/GSDMD-NT proteins, also indicated that VOA could effectively reduce neuroinflammation in TS rats, serving as an effective regulation of M1 polarization.
p38 MAPK, the member of the MAPK family most closely associated with inflammation, is considered a central mediator of cellular signal transduction pathways and a key mediator of pro-inflammatory cytokine production (Cuenda and Rousseau, 2007; Bachstetter and Eldik, 2010; Meng et al., 2014; Li et al., 2018; Wang M. et al., 2022). It can mediate inflammatory responses and promote the activation of the NLRP3 inflammasome by inducing the production of inflammatory cytokines and chemokines (O’Neil et al., 2018; Wang S. et al., 2019; Hu et al., 2020), playing a crucial role in neurological diseases and CNS inflammatory responses (Liu et al., 2020; Liu et al., 2023). p38 MAPK is an important metabolite of autoimmune neuroinflammation (Motawi et al., 2023), acting as a significant mediator in signal transduction pathways involved in inflammation, cell cycle, cell death, cell differentiation, development, and tumorigenesis. Studies have shown that blocking the p38 MAPK signaling pathway attenuates the expression of pro-inflammatory cytokines and the NLRP3 inflammasome in inflamed mouse lung tissues. Pretreatment with SB203580 significantly reduced the expression of pro-inflammatory cytokines, IL-1β, TNF-α, and IL-6 by blocking the p38 MAPK signaling pathway (Li et al., 2018). p38 MAPK has anti-inflammatory functions through its negative regulation of the NLRP3 inflammasome, with a deficiency in p38 MAPK leading to upregulation of NLRP3 expression. Treatment with the p38 Inhibitor SB203580 can suppress the expression of NLRP3 inflammasome-related proteins (Liu et al., 2023). STAT3, a transcription factor, regulates cytokine-induced pro-inflammatory and anti-inflammatory responses, playing a key role in modulating microglia-induced inflammatory responses. Specifically, IL-6 can promote the induction of STAT3, leading to the expression of various genes involved in inflammation (Wu et al., 2019). Studies have shown that inflammatory factors like IL-1β and TNF-α can promote IL-6 secretion by upregulating NF-κB activity, thereby indirectly activating STAT3 (Liu F.-T. et al., 2016; Wu et al., 2019; Youssef et al., 2022). The p38 MAPK signaling pathway can regulate the downstream NF-κB pathway, mediating the gene expression of NLRP3 and related inflammatory factors (Huo et al., 2023). Treatment with the p38 MAPK Inhibitor SB203580 reduced levels of p-STAT3, TNF-α, and IL-10 after LPS stimulation. Inhibition of p38 MAPK can prevent STAT3 phosphorylation, indicating an interaction between the STAT3 and MAPK signaling pathways (Meng et al., 2014). As a key regulator of STAT3 phosphorylation, inhibiting p38 MAPK can reduce TNF-α secretion, weaken the inflammatory response, and completely block STAT3 phosphorylation, allowing the p38 MAPK-mediated STAT3 pathway to exert anti-inflammatory and antioxidant effects. Reduced STAT3 activity may also lead to decreased expression of downstream inflammatory mediators such as COX-2 and TNF-α, which were also found to be reduced in VOA-treated rats. Blocking the p38 MAPK signaling pathway resulted in decreased secretion of TNF-α by microglia, enhancing its protective effects. Therefore, inhibiting p38 MAPK phosphorylation can reduce NLRP3 protein expression, decrease TNF-α release, lower the expression of pro-inflammatory proteins COX-2 and CD11b, suppress IL-6 release, and increase levels of IL-10 and CD163, thereby inhibiting STAT3 activation and exerting inhibitory effects on microglia-induced neuroinflammation. Our findings are consistent with the above observations. Specifically, in the TS rat model, upregulation of the p38 MAPK signaling pathway was observed, followed by elevated levels of pro-inflammatory cytokines (IL-6 and TNF-α), activation of NLRP3, leading to the proteolytic activation of Caspase-1 protein, accompanied by increased expression of STAT3 protein. Correspondingly, under VOA treatment, levels of inflammatory cytokines (IL-6 and TNF-α) were reduced, expression of pro-inflammatory proteins COX-2 and CD11 b decreased, and levels of IL-10 and CD163 increased. This was accompanied by the attenuation of neuroinflammation and decreased expression of involved inflammatory signaling pathways p38 MAPK/NLRP3/STAT3.