Review: Progress of the NLRP3 inflammasome in tumours and perspectives for cholangiocarcinoma

Abstract

The NLRP3 inflammasome is a multi-protein complex that mediates intense inflammatory responses. Its activation and function are influenced by various factors, including the recognition of pathogen-associated molecular patterns and damage-associated molecular patterns, cellular stress responses, and metabolic disorders. Recent research has extensively studied the role of the NLRP3 inflammasome in tumors. Findings indicate that tumor cells and macrophages can regulate the activation of the NLRP3 inflammasome, promoting tumor development and progression. Conversely, the NLRP3 inflammasome can also exhibit anti-tumor effects through immune cells, such as dendritic cells. This has led to the development of treatment strategies, creating a comprehensive treatment system that includes sensitizers for radiotherapy and chemotherapy, inhibitors of the NLRP3 inflammasome pathway, and direct targeting of the NLRP3 inflammasome.Cholangiocarcinoma is a highly invasive and heterogeneous malignant tumor. Due to its non-specific symptoms, patients often present with advanced stages of the disease, and the mortality rate continues to rise annually. A deeper exploration of the mechanisms underlying cholangiocarcinoma’s occurrence and development is essential for improving diagnosis and treatment strategies. The application of multi-omics analysis in cholangiocarcinoma research lays a foundation for understanding its pathogenesis and potential treatments. This article systematically reviews the latest advancements in NLRP3 inflammasome research, including its regulatory mechanisms, role in promoting tumor development, and effects in anti-tumor immunotherapy. Additionally, by summarizing the mechanisms involved in cholangiocarcinoma, we hypothesize that the NLRP3 inflammasome may play a significant role in the occurrence and development of cholangiocarcinoma. Therefore, we conduct a multi-angle analysis of the potential relationship between the two and propose a hypothesis model. The goal of this article is to explore the role of the NLRP3 inflammasome in tumors and its potential relationship with cholangiocarcinoma, offering new insights for research on the link between cholangiocarcinoma-related inflammation and the disease itself.

Introduction

More than a century ago, Rudolf Virchow observed white blood cells in tumour tissue and first proposed a link between inflammation and cancer [1]. This landmark observation not only marked the beginning of research into the relationship between inflammation and cancer, but also provided important inspiration for subsequent scientific studies. Inflammation, as a defensive response of the body when stimulated by pathogens, tissue damage, etc., plays a crucial role in maintaining or altering the homeostasis of the body [2]. The core of the inflammatory response lies in its initial response to injury and infection and its role in tissue repair and restoration of homeostasis. However, when the inflammatory response persists without abating, it not only alters the internal environment of the body, but also provides key cellular and molecular conditions for the onset and development of cancer [3]. This relationship between inflammation and cancer forms a vicious circle: due to the persistence of the disease state and the difficulty of recovery, the body’s inflammatory response is continuously activated, which further aggravates the genetic instability of cancer cells and provides a favourable inflammatory microenvironment for cancer development, ultimately promoting the development of cancer [4]。.

In 2002, Martinon et al. first proposed the concept of the inflammasome [5]. Since then, the structure and function of inflammasomes have garnered significant attention from researchers [6–8]. However, most studies have primarily focused on the role of inflammasomes in inflammatory diseases [9–11]. It was not until 2009 that Ghiringhelli et al. first elucidated the crucial link between the NLRP3 inflammasome and the innate and adaptive immune responses to dead tumor cells [12]. This groundbreaking study highlighted the potential for more complex interconnections between inflammasomes and tumors. As research has progressed, the relationship between inflammasomes and various tumors has been explored more comprehensively. The NLRP3 inflammasome, a member of the inflammasome family [13], is a multi-protein complex that can detect various damage signals and activate downstream inflammatory responses, including the maturation and secretion of IL-1β [14]. The activation of the NLRP3 inflammasome is influenced by a range of factors, including the recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), as well as cellular stress responses and metabolic disorders [15]. These elements work together to create a complex network that regulates both the inflammatory response and cancer development. Understanding the intrinsic relationship between the NLRP3 inflammasome and tumor occurrence and progression is crucial for advancing prevention and treatment strategies for cancer.

Cholangiocarcinoma (CCA) can be categorized based on its anatomical location into three types: intrahepatic Cholangiocarcinoma (iCCA), perihilar Cholangiocarcinoma (pCCA), and distal Cholangiocarcinoma (dCCA). Typically, pCCA and dCCA are considered forms of extrahepatic Cholangiocarcinoma (eCCA) [16]. Although cholangiocarcinoma is less common than hepatocellular carcinoma, it often presents at an advanced stage due to its non-specific symptoms, leading to a rising mortality rate. The 5-year survival rate for CCA is only between 7% and 20% [17]. Known risk factors for CCA include cholangitis, choledochal cysts, choledochal stones, bile stasis, viral hepatitis, and liver cirrhosis [18–20]. Many of these factors can lead to bile stasis and inflammatory responses in the bile duct epithelium, indicating that inflammation may play a significant role in the onset and progression of CCA [21].

This article summarizes the latest research on the NLRP3 inflammasome, exploring its regulatory mechanisms, its role in promoting tumor development, and its implications for anti-tumor immunotherapy. By examining the effects of factors such as the nuclear factor-kappa B (NF-κB) pathway, phosphatase and tensin homolog (PTEN) deficiency, cholangitis, and bile acid stasis on CCA progression, we propose that the NLRP3 inflammasome may be crucial in the development of cholangiocarcinoma. Hence, we conducted an in-depth analysis of the relationship between these factors and suggested a hypothesis model. This article aims to investigate the role of the NLRP3 inflammasome in tumors and its potential connection to cholangiocarcinoma, offering new insights into the transition from cholangial inflammation to cholangiocarcinoma.

Recent advances in the assembly, activation, and regulation of the NLRP3 inflammasome

The structure of the NLRP3 inflammasome has been detailed in several excellent reviews. Thus, we will briefly describe its structure and summarize the latest advancements in its assembly, activation, and regulation.

The NLRP3 inflammasome is a trimeric protein complex made up of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and Pro-caspase-1 [22]. NLRP3 serves as the sensor for the inflammasome and consists of three domains: the C-terminal leucine-rich repeat (LRR), the NACHT domain with ATPase activity, and the N-terminal PYRIN Domain (PYD) [23]. The LRR mediates protein-protein interactions and contains an inhibitory protein that helps maintain the stability of NLRP3 in its resting state [24]. The NACHT domain can bind nucleotides and exhibits ATPase activity, which, when activated, promotes the oligomerization of NLRP3 [24]. The PYD domain is deubiquitinated and subsequently connects with the ASC homologous domain [25]. ASC acts as a linker; its N-terminal contains a PYD domain that connects with NLRP3, while its C-terminal has a caspase recruitment domain (CARD), which serves as a platform for recruiting Pro-caspase-1 [26]. Pro-caspase-1 functions as the effector and can be activated to become caspase-1. Caspase-1 consists of the N-terminal CARD, a large catalytic subunit (p20), and a small catalytic subunit (p10) [27]. Once activated, caspase-1 promotes the release of inflammatory factors IL-1β and IL-18, cleaves and activates Gasdermin D (GSDMD), and mediates inflammation and cell pyroptosis [27].

Although NLRP3 is expressed in various cells, its expression level is relatively low in the resting state [28]. It requires a “triggering” step to upregulate NLRP3 expression, facilitating the formation of the NLRP3 inflammasome [28]. Research by Yu et al. thoroughly analyzed the mechanism of NLRP3 activation and inflammasome assembly [29]. In simple terms, the triggering step involves the upregulation of NLRP3 and promotes its oligomerization. Following ATP hydrolysis and oligomer reorganization, NLRP3 gets activated and forms an open octamer, which aids in its transport to the microtubule organizing center [29]. Subsequently, NIMA-related kinase 7 (NEK7) dissociates the NLRP3 oligomer into a NEK7/NLRP3 dimer complex that exists in equilibrium with the dimer and monomer forms. The open NEK7/NLRP3 complex promotes the assembly of the NLRP3 inflammasome. In contrast, the closed NEK7/NLRP3 complex can be passively activated by the open NEK7/NLRP3 and contribute to the assembly process of the NLRP3 inflammasome [29]. Recent studies revealed that after the activation of the NLRP3 inflammasome, the formation of GSDMD pores leads to the efflux of K⁺. This triggers JNK1 to phosphorylate Thr 190/191 of NEK7 [30]. The phosphorylation enhances the interaction between NEK7 and NLRP3, further assembling and activating the NLRP3 inflammasome through a positive feedback mechanism [30]. However, this interaction can also be disrupted. Under lipopolysaccharide (LPS) stimulation, elevated levels of the endogenous metabolite isocyanate can modify Lys-593 of NLRP3, disrupting the interaction with NEK7 and limiting the formation of the NLRP3 inflammasome [31]. In conclusion, it is evident that NEK7 plays a pivotal role in the formation of the NLRP3 inflammasome. This information is illustrated in Fig. 1.

Fig. 1.

The role of NLRP3 in the assembly and activation of inflammasomes. NLRP3 is upregulated and oligomerised to form an open octameric structure. NEK7 forms a NEK7/NLRP3 complex with NLRP3 in a dimer/monomer equilibrium state, promoting assembly of the NLRP3 inflammasome. The formation of GSDMD pores and the outward flow of K⁺ cause JNK1 to induce the phosphorylation of NEK7, which further promotes the interaction between NEK7 and NLRP3, and activates the NLRP3 inflammasome through a positive feedback mechanism.However, upon LPS stimulation, isocyanate modifies NLRP3 through carbamoylation, disrupting the interaction between NEK7 and NLRP3 and limiting the formation of the NLRP3 inflammasome

The function of the NLRP3 inflammasome is heavily influenced by its activators. The primary activators of NLRP3 disrupt the membrane contact sites between the endoplasmic reticulum(ER) and endosomes (EECS) and impair the trafficking of endosomes to the trans-Golgi network (ETT). This disruption leads to the accumulation of phosphatidylinositol 4-phosphate (PI4P) within the endosome [32]. NLRP3 associates with the trans-Golgi network (TGN) through electrostatic interactions with PI4P and reacts to changes in endosomal components in response to various activators [32–34]. Once activated, NLRP3 is transported to the microtubule-organizing center (MTOC) along microtubules, where it interacts with NEK7, ultimately forming the NLRP3 inflammasome [35]. Mitochondria play a crucial role in the activation of the NLRP3 inflammasome and the process of endogenous apoptosis, although the exact mechanisms and their correlations remain unclear. To explain this complexity, Saller et al. proposed the “two activation signals” model for NLRP3 activation, demonstrating that both mitochondrial signals and additional signals are required for NLRP3 activation; a single signal alone is insufficient. This model highlights the intricate nature of NLRP3 activation and explores how mitochondria may facilitate the transition from cell apoptosis to NLRP3 inflammasome activation [36]. Their research found that NLRP3 activators, specifically oxidative phosphorylation (OXPHOS) inhibitors, disrupt mitochondrial cristae structure and limit cytochrome c release, thereby inhibiting cell apoptosis [36]. While this effect does not activate NLRP3 on its own, when paired with other NLRP3 activators, such as imiquimod, the OXPHOS inhibitor can trigger NLRP3 activation [36]. This study enriches our understanding of NLRP3 activation and underscores the essential role of mitochondria in this process. Despite the variety of stimuli that can activate the NLRP3 inflammasome, identifying a unified upstream signal has proven challenging. Baik et al. reported a common upstream mechanism in NLRP3 inflammasome activation, whereby activators like ATP and Nigericin induce the dissociation of hexokinase 2 (HK2) from the voltage-dependent anion channel (VDAC) on the outer mitochondrial membrane [37]. This triggers the activation of inositol triphosphate receptors, leading to calcium release (Ca²⁺) from the endoplasmic reticulum [37]. Once Ca²⁺ enters the mitochondria, it promotes VDAC oligomerization, creating large pores in the outer mitochondrial membrane that allow the release of mitochondrial DNA (mtDNA). This mtDNA then binds to the VDAC oligomer, recruiting NLRP3 and activating the inflammasome signal [37]. This research provides a new perspective on the activation mechanisms of the NLRP3 inflammasome. Although the initiation and activation mechanisms of the NLRP3 inflammasome have been continuously refined, there is still a lack of comprehensive reviews that systematically address these processes and the regulatory mechanisms involved. This understanding is crucial for a thorough examination of the NLRP3 inflammasome.

The initiation and assembly of the NLRP3 inflammasome are tightly regulated by various post-translational modifications (PTMs), such as acetylation, phosphorylation, and palmitoylation [38–40]. Additionally, Jing et al. identified a novel reversible PTM known as UFMylation, mediated by the coupling of ubiquitin-fold modifier 1 (UFM1) with the protein [41]. This study revealed that during the initiation stage of NLRP3 activation, the UFM1 E3 ligase UFL1 facilitates UFMylation by interacting with the NACHT domain of NLRP3. UFMylation can counteract K63-linked ubiquitination of NLRP3 and subsequent autophagic degradation, thus maintaining the stability of NLRP3 [41].This suggests that precisely regulating NLRP3 activity through the interplay of UFMylation, ubiquitination, and other PTMs may be a significant area for future research. The content mentioned above is illustrated in Fig. 2.

Fig. 2.

The NLRP3 inflammasome’s regulatory mechanism. During the initiation stage, the NACHT domain of NLRP3 undergoes UFMization, which maintains the stability of NLRP3 by inhibiting its ubiquitination and autophagic degradation, while also activating the NLRP3 inflammasome through two separate signals. Additional signals: NLRP3 activators induce dissociation of HK2 and promote oligomerisation of VDAC and release of mtDNA through the ER pathway, thereby recruiting NLRP3 to VDAC. Meanwhile, NLRP3 is activated by PI4P and transported to the MTOC, where it interacts with NEK7 to form the NLRP3 inflammasome. Mitochondrial signals: NLRP3 activators inhibit the OXPHOS pathway, suppress apoptosis and act with additional signals to activate the NLRP3 inflammasome

In summary, the combination of these studies elucidates the molecular mechanisms involved in the composition and activation of the NLRP3 inflammasome, providing a valuable theoretical foundation for understanding the pathological mechanisms of diseases and guiding targeted treatments.

NLRP3 inflammasome and tumours

Recent studies have shown that the NLRP3 inflammasome is a double-edged sword that can both promote tumour development and exert anti-tumour protective effects. We will now explore the tumour-promoting and anti-tumour mechanisms of the NLRP3 inflammasome in tumours.

The tumourigenic mechanism of the NLRP3 inflammasome

Matusiak M was the first to demonstrate, through histological analysis, that the NLRP3 inflammasome aggregates in human cancers. NLRP3⁺ macrophages co-localize with neutrophils to activate the inflammasome in tumors, promoting tumor development [42]. Particles from lung cancer can induce the M2-type transformation of macrophages in the TME, leading to the release of the key pro-inflammatory cytokine IL-1β through the activation of the NLRP3 inflammasome, thereby accelerating lung cancer progression [43]. M1-type macrophages are an essential component of the multiple myeloma (MM) microenvironment. Studies have shown that M1-type macrophages uptake accumulated β2-microglobulin (β2m), which aggregates into amyloid fibers under acidic conditions. This process leads to lysosomal swelling and rupture, release of reactive oxygen species (ROS), and subsequent activation of the NLRP3 inflammasome, ultimately promoting the growth of MM [44]. In invasive ductal carcinoma of the breast, Unc-51 Like autophagy activating kinase 1 (ULK1) is depleted in the hypoxic microenvironment, weakening the mitochondrial autophagy function of breast cancer cells. Damaged mitochondria cannot clear effectively, leading to ROS accumulation and subsequent activation of the NLRP3 inflammasome. This process eventually results in osteoclast differentiation and maturation, contributing to bone metastasis of breast cancer [45]. Additionally, the expression of the NLRP3 inflammasome is positively correlated with the immunosuppressive state of patients with diffuse large B-cell lymphoma. Its activation can upregulate programmed death-ligand 1 (PD-L1) and reduce the proportion of cytotoxic T lymphocytes (CTLs), thereby contributing to a tumor-immunosuppressive microenvironment. Further in vivo studies have indicated that IL-18 is a major effector factor in inhibiting anti-leukemia immunity [46]. Research has shown that hyaluronic acid (HA) fragments released by iron-laden hepatocellular carcinoma cells can upregulate Pro-IL-1β and the NLRP3 inflammasome in M1-type macrophages via the NF-κB pathway [47]. In parallel, M1-type macrophages secrete oxidized phospholipids that activate the NLRP3 inflammasome and release IL-1β, triggering neutrophil-mediated vascular remodeling, which creates conditions favorable for the invasive growth and lung metastasis of liver cancer [47]. Moreover, the tumor microbiota in patients with liver cirrhosis and hepatocellular carcinoma contains a high abundance of Stenotrophomonas maltophilia, which induces the formation of NLRP3 inflammasomes in hepatic stellate cells (HSC) through the NF-κB pathway, promoting liver cancer progression [48]. In pancreatic ductal adenocarcinoma (PDAC), the TLR4-NLRP3 inflammasome pathway, driven by tumor cells, secretes IL-1β, which activates quiescent pancreatic stellate cells (PSC) and establishes an immunosuppressive TME mediated by M2-type macrophages and various immunosuppressive cells [49]. Yang D and colleagues conducted a single-cell RNA sequencing analysis to identify changes in the PDAC tumor immune microenvironment following TREM2 depletion. They found that TREM2 and LPS synergistically elevated the expression of the NF-κB/NLRP3/IL-1 pathway in M1-type macrophages in PDAC, thereby accelerating tumor progression [50]. These findings offer insights into the complex TME of PDAC. The mechanism of the NF-κB-NLRP3 inflammasome is illustrated in Fig. 5.

Fig. 5.

Activation pattern diagram of the classical NF-κB-NLRP3 inflammasome signaling pathway

Classical cancer genes also correlate highly with the activation of the NLRP3 inflammasome. One study found that the deficiency of the breast cancer susceptibility gene 1 (BRCA1) upregulated the expression of mitofusin 1/2 (MFN1/2), causing mitochondrial dynamics dysfunction. This deficiency also inhibited mitochondrial division, disrupted stress-induced mitochondrial autophagy, and triggered NLRP3 inflammasome activation, creating a tumor-related microenvironment that promotes tumor proliferation and metastasis [51]. The oncogenic KRAS mutation occurs in various types of leukemia, driving cancer progression through its classical RAS/MEK/ERK pathway and enhancing the activation of the pro-inflammatory RAC1/ROS/NLRP3/IL-1β axis, thus promoting leukemia development [52]. The Missiroli S team discovered a trimeric complex on the mitochondria-associated endoplasmic reticulum membrane (MAMs) of macrophages, where promyelocytic leukemia protein (PML) regulates the interaction between the purinergic P2 × 7 receptor (P2 × 7R) and NLRP3 [53]. However, the absence of PML enhances NLRP3 inflammasome activation in M2-type macrophages, accelerating tumor progression and worsening prognosis. This provides new insights into the PML-P2 × 7R-NLRP3 axis that regulates the TME in human carcinogenesis [53].

Inhibiting the assembly and activation of the NLRP3 inflammatory body also exacerbates tumor progression. It was found that overexpression of Snail in lung cancer cells can induce epithelial-mesenchymal transition (EMT) in cancer cells, and then deliver miR-21 through exosomes to tumor-associated macrophages, promoting the M2-like polarization of macrophages [54, 55]. Mechanistically, miR-21 promotes NLRP3 phosphorylation and lysine-63 ubiquitination, reduces the interaction between NLRP3 and ASC, thereby inhibiting the assembly of the NLRP3 inflammatory body and weakening the anti-tumor immunity induced by chemotherapy [54]. In the hypoxic microenvironment of endometrial cancer, the highly expressed estrogen-related receptorα(ERRα) can bind to the NLRP3 promoter, thereby inhibiting the Caspase-1/GSDMD pathway, increasing the anti-apoptotic ability of cancer cells, and generating cisplatin resistance [56]. The soluble resistance-related calcium-binding protein (Sorcin) is significantly upregulated in HCC, and its high expression may compete with the NLRP3 inflammasome for binding to Ca²⁺, thereby inhibiting the activation of the NLRP3 inflammasome and promoting the progression of liver cancer [57]. The above content can be seen in Fig. 3; Table 1.

Fig. 3.

Mechanism diagram of how the NLRP3 inflammasome promotes tumor development

Table 1.

The NLRP3 inflammasome promotes tumor progression

Type of cancer	Mechanism	Effect on tumor	References
Colon Cancer	Activate the NLRP3 inflammasome in tumors	promoting effect	[42]
Lung Cancer	Activate the NLRP3 inflammasome of M2 type macrophages	promoting effect	[43]
MM	β2m activates the NLRP3 inflammasome	promoting effect	[44]
Breast Cancer	Disruption of the balance between mitochondrial autophagy and NLRP3 inflammasome	promoting effect	[45, 51]
Lymphoma	The NLRP3 inflammasome upregulates the expression of PD-L1	promoting effect	[46]
Liver Cancer	Activate the NF-ΚB-NLRP3 inflammasome pathway	promoting effect	[47, 48]
Pancreatic Cancer	Activate the TLR4-NLRP3 inflammasome pathway	promoting effect	[49]
Pancreatic Cancer	Depletion of TREM2 promotes the NLRP3 inflammasome	promoting effect	[50]
Leukemia	Activate the RAC1/ROS/NLRP3/IL-1β axis	promoting effect	[52]
Lung Cancer Melanoma	The PML-P2 × 7R-NLRP3 axis has been disrupted.	promoting effect	[53]
HNSCC Breast Cancer	miR-21 promotes the phosphorylation of NLRP3 and the lysine-63 ubiquitination process.	promoting effect	[54]
Endometrial Carcinoma	Inhibit the Caspase-1/GSDMD pathway	promoting effect	[56]
Liver Cancer	Inhibit the activation of the NLRP3 inflammasome	promoting effect	[57]
Colorectal Cancer	Hexokinase 2 inhibits the activation of NLRP3 inflammasome	promoting effect	[58]
Colorectal Cancer	5-Hydroxytryptamine enhances the activation of NLRP 3 inflammasome	promoting effect	[59]
OSCC	IL-6 regulates the JAK 2/STAT 3/Sox 4/NLRP3 axis	promoting effect	[60]
Breast Cancer Melanoma	The S1PR1/C3/PPIL1/NLRP3 pathway	promoting effect	[61]
Breast Cancer	Activation of NLRP3 in astrocytes	promoting effect	[62]
Colorectal Cancer	Alu RNA activates the NLRP3 inflammasome	promoting effect	[63]

Note:To enhance readers’ understanding of the role of the NLRP3 inflammasome in tumor development, this table summarizes not only the literature discussed in the main text but also includes additional studies that were not covered in that section

In conclusion, we believe that the role of the NLRP3 inflammasome in tumor progression is indispensable, and its specific effect depends on the tumor type, microenvironment status, and molecular interaction network. Targeted regulation of the NLRP3 inflammasome is expected to become a new strategy for cancer treatment, but it needs to be intervened in combination with specific pathological backgrounds.

The anti-tumour mechanism of the NLRP3 inflammasome

The activation of the NLRP3 inflammasome plays a crucial role in regulating the anti-tumor immune response. Research has shown that tumor-associated macrophages can upregulate CD146 during short-term stimulation, which inhibits the expression of the transmembrane protein 176B (TMEM176B). This process subsequently promotes the activation of the NLRP3 inflammasome, thereby enhancing anti-tumor immunity [64]. Additionally, radiation can induce the activation of the NLRP3 inflammasome, leading to increased production of IL-1 by macrophages, which in turn activates dendritic cells (DC) [65]. Activated DCs result in a more robust T cell immune response, contributing to a powerful anti-tumor effect [65]. Therefore, the anti-tumor immune pathway mediated by macrophages also appears to be dependent, in part, on DC. Zhivaki D et al. discovered that DC hyperactivators can excessively activate DCs through the NLRP3-Caspase-1-IL-1β/GSDMD pathway. This hyperactivation promotes their migration to the lymph nodes, stimulates the activation and differentiation of CD4⁺ T cells, and helps eliminate tumors in aged mice [66]. Furthermore, Dixon KO et al. highlighted the significant role of T cell immunoglobulin and mucin-containing molecule 3 (TIM-3) in DC function [67]. They found that the conditional absence of TIM-3 on DCs leads to the accumulation of ROS, activates the NLRP3 inflammasome, and promotes anti-tumor immunity [67]. This indicates that the activation of the NLRP3 inflammasome in DC is critical for an effective anti-tumor immune response. Mast cells (MC) also play a pivotal role as sentinels in the regulation of immune responses and inflammation. Single-cell transcriptomic studies have demonstrated that MCs possess pro-inflammatory properties regulated by the activation of the NLRP3 inflammasome within these cells. Pro-inflammatory MCs upregulate the expression of NKG2D and IFN-γ in MAIT cells through cytokine activation mechanisms, thus enhancing the innate anti-tumor activity of MAIT cells [68]. Moreover, regulating ASC can enhance the anti-tumor effects of the NLRP3 inflammasome. NADPH oxidase, the primary ROS-producing enzyme during inflammation [69], has its NCF4 subunit phosphorylated and distributed in a dot-like manner from the NADPH complex to the perinuclear region. This mediates ASC oligomerization, with NCF4 acting as a ROS sensor that balances ROS production and NLRP3 inflammasome activation, thereby contributing to the anti-tumor response of the NLRP3 inflammasome [70]. Studies have shown that NCF4 deficiency disrupts the inflammatory macrophage-IL-18-IFN-γ axis in the early stages of colorectal cancer in mice, resulting in more severe colorectal cancer [70]. The above-mentioned content can be found in Fig. 4; Table 2.

Fig. 4.

Mechanism diagram of NLRP3 inflammasome’s role in preventing tumor development

Table 2.

Anti-tumor mechanisms of the NLRP3 inflammasome

Type of cancer	Mechanism	Effect on tumor	References
Liver Cancer	Inhibit the expression of TMEM176B and activate the NLRP3 inflammasome	suppressor effect	[64]
Colon Cancer Melanoma	Radiation-induced activation of the NLRP3 inflammasome	suppressor effect	[65]
Colon Cancer Melanoma	Activate the NLRP3-IL-1β pathway of DCs	suppressor effect	[66]
Lung cancer Colon cancer	TIM-3 deletion activates the NLRP3 inflammasome	suppressor effect	[67]
Lung Cancer	MC activates the NLRP3 inflammasome	suppressor effect	[68]
Colorectal Cancer	NCF4 regulates ASC	suppressor effect	[70]

In summary, it is evident that the activation of the NLRP3 inflammasome in various immune cells can lead to significant anti-tumor effects. Additionally, regulating the structural composition of the NLRP3 inflammasome to influence its function is an important aspect of its anti-tumor mechanism. Combining NLRP3 inflammasome regulation with other tumor treatments can help in designing more effective tumor treatment strategies targeting NLRP3.

The potential of the NLRP3 inflammasome as a therapy in cancer treatment

The NLRP3 inflammasome plays a significant role in enhancing the sensitivity of tumors to radiotherapy and chemotherapy. R-spondin 3 (RSPO3), which is closely related to the Wnt/β-catenin pathway, is crucial in regulating radioresistance in non-small cell lung cancer (NSCLC) [71]. Research indicates that RSPO3 expression is downregulated in radioresistant NSCLC cells. However, overexpressing RSPO3 can activate the NLRP3 inflammasome through the β-catenin-NF-κB pathway, leading to pyroptosis and subsequently increasing tumor sensitivity to radiotherapy [72]. Typically, chemotherapy is combined with immune checkpoint inhibitors to improve the immune response to treatment. The endoplasmic reticulum stress sensor IRE1αserves as a key immune checkpoint that can limit the effectiveness of chemotherapy in breast cancer by preventing the accumulation of hazardous signals and the pyroptosis of cancer cells induced by taxanes [73]. Inhibiting IRE1α can enhance the immunogenicity of taxanes, allowing them to induce considerable dsRNA accumulation and activate the NLRP3-GSDMD pyroptosis signaling pathway. This activation helps recruit and activate antigen-presenting cells and initiate antigen-specific T cell responses [73]. It can be seen that the pro-apoptotic property of the NLRP3 inflammasome plays a significant role in inhibiting tumor progression. Temozolomide (TMZ), commonly used to treat glioblastoma, can induce the secretion of high mobility group box 1 protein (HMGB1) in rats. HMGB1 binds to the receptor for advanced glycation end products (RAGE) on macrophages, enhancing M1-like polarization through the NF-κB-NLRP3 inflammasome pathway. This results in a pro-inflammatory anti-tumor effect and increases the sensitivity of tumor cells to TMZ [74]. The mechanism of NF-κB-NLRP3 inflammasome is shown in Fig. 5.

Inhibitors of the NLRP3 inflammasome pathway exhibit promising anti-tumor effects. For instance, Sirtuin 7 (SIRT7) inhibits the assembly of the NLRP3 inflammasome and downregulates the activated ERK1/2 signaling pathway, thereby reversing acquired resistance to sorafenib [75]. In one case of KRAS-mutated chronic myelomonocytic leukemia (CMML), treatment with the IL-1 receptor inhibitor anakinra inhibited NLRP3 inflammasome activation, leading to a decrease in single-cell count and allowing the patient to successfully receive hematopoietic stem cell transplantation [76]. This highlights the potential clinical application of NLRP3 inflammasome inhibitors and IL-1 blockers. Interestingly, combining NLRP3 receptor inhibitors with anti-PD-L1 therapy has shown an antagonistic effect during lymphoma treatment [46]. The clinical application of NLRP3 inflammasome pathway inhibitors still encounters several challenges. However, it is clear that as our understanding of the role of the NLRP3 inflammasome in tumors deepens, these inhibitors could emerge as a significant approach for the clinical treatment of tumors.

Immune checkpoint blockade therapy (ICB) has long garnered significant attention; however, it is often associated with drug resistance [77]. Liu et al. demonstrated that 2-guanidinobenzimidazole (2GBI) can directly target NLRP3 and induce the activation of the NLRP3 inflammasome, leading to a synergistic enhancement of anti-tumor immunity when combined with ICB [77]. In recent years, the use of nanomaterials that target the NLRP3 inflammasome has also significantly improved the efficacy of ICB, as discussed in references [78, 79]. This indicates that directly targeting NLRP3 represents a viable strategy for inducing anti-tumor immunity and augmenting the effectiveness of ICB. The information presented above is summarized in Table 3.

Table 3.

Therapeutic progress of NLRP3 inflammasome in tumor treatment

Therapy methods	Activation or inhibition of the NLRP3 inflammasome	Type of cancer	References
RSPO3 upregulation	Activation	Lung Cancer	[72]
IRE1a inhibitor	Activation	Breast Cancer	[73]
TMZ	Activation	Glioblastoma	[74]
SIRT7 inhibitor	Inhibition	Liver Cancer	[75]
IL-1R inhibitor	Inhibition	CMML	[76]
Targeting NLRP3	Activation	Lung Cancer Melanoma	[77]
Metal-ion-chelating phenylalanine nanostructures	Activation	Breast Cancer Colorectal Cancer	[78]
MDSC-targeting gold nanoparticles	Inhibition	Colon Cancer	[79]
tBSA/Cas9-PAR 2	Inhibition	Breast Cancer	[80]
Bacopa monnieri	Inhibition	OSCC	[81]
Shuanghua decoction	Activation	Liver Cancer	[82]
Inflammasome-Activating Nanovaccine	Activation	Melanoma Liver Cancer	[83]
Hydroxyapatite nanoparticles	Activation	Colorectal Cancer	[84]
schottky heterojunction	Activation	OSCC	[85]
Novel Aryl Sulfonamide Derivatives	Inhibition	Melanoma	[86]
Golgi Apparatus-Targeted Photodynamic Therapy	Activation	Lung Cancer	[87]

Therefore, whether used as an adjunct to radiotherapy and chemotherapy or as an independent anti-tumor approach, the NLRP3 inflammasome demonstrates beneficial effects. The development of tumor-targeted treatments and drugs that focus on the NLRP3 inflammasome holds significant theoretical and practical importance.

Analysis of the potential relationship between cholangiocarcinoma and the NLRP3 inflammasome

Progress in multimodal omics research on the microenvironment of cholangiocarcinoma

In recent years, multi-omics analysis of the TME has emerged as a prominent area of research, particularly in cholangiocarcinoma. To better understand the heterogeneity of T cells in different subtypes of biliary tract cancer (BTC), Nie et al. conducted a systematic comparison of the transcriptomic characteristics of tumor-infiltrating T cells across three BTC subtypes: iCCA, eCCA, and gallbladder cancer. This analysis was performed at both the single-cell and spatial levels [88]. The study revealed significant differences in the distribution of T cell clusters among the various subtypes. eCCA was characterized by T cell exhaustion, increased CXCL13 expression in CD^4 + helper T cells and CD8⁺ CXCL13⁺ exhausted T cells, more mature tertiary lymphoid structures, and fewer desert-like phenotypes. In contrast, iCCA exhibited a rich population of activated regulatory T cells, while gallbladder cancer was marked by significant infiltration of Th17 cells [88]. Building on 900 transcriptomic data points for iCCA, Martin-Serrano et al. developed a new classification system by selecting relevant TME components. They categorized iCCA into inflammatory categories (immune classical and inflammatory stroma) and non-inflammatory categories (desert-like, hepatic stem cell-like, and others). This classification offers a more comprehensive understanding of the iCCA microenvironment and lays a theoretical foundation for targeted treatments [89]. Most iCCA patients are diagnosed at an advanced stage, presenting challenges for diagnosis and treatment [90]. To gain a deeper understanding of the iCCA TME, Hong et al. employed spatial multi-omics to analyze the TME of iCCA patients, identifying five distinct subtypes with unique spatial and molecular characteristics [91]. They observed a cellular neighborhood rich in M2-type macrophages and T cells, and through single-cell RNA sequencing (scRNA-seq), they conducted a cell communication analysis. This analysis revealed the immunosuppressive effects of M2-type macrophages on anti-tumor CD8⁺ T cells [91]. Furthermore, M2 macrophages can promote dedifferentiation and proliferation of malignant iCCA cells, while malignant cancer cells can induce an immunosuppressive state in macrophages, creating a dedifferentiation-immunosuppressive cycle between these cells [92]. Interestingly, cells in the proliferative phase may be more sensitive to chemotherapy due to their active division, while differentiated cancer cells may exhibit chemotherapy resistance. The differences between these two cell states require further experimental verification, underscoring the importance of precise classification and research on malignant tumor cells [92]. Additionally, multi-omics techniques have shown significant advantages in analyzing the metabolic microenvironment [93],tumor front microstructure [94],genomic features, and methylation changes in cholangiocarcinoma [95, 96]. The application of multi-omics analysis in this field has enhanced our understanding of the TME in cholangiocarcinoma, creating favorable conditions for exploring the pathological mechanisms underlying the disease.

NF-κB pathway

NF-κB is a critical regulatory factor in inflammatory responses and plays a significant role in the development and progression of tumors [97].Recently, the role of the NF-κB pathway in CCA has garnered considerable attention. Guan C et al. confirmed the importance of the circular RNA (circRNA) circPCSK6 and its peptide derivative circPCSK6-167aa in iCCA [98]. They found that circPCSK6-167aa interferes with the ubiquitination of IκBα, prevents its degradation, and leads to the accumulation of non-ubiquitinated IκBα in the cell. This process inhibits the nuclear translocation of NF-κB and its transcriptional activity, thereby suppressing the proliferation, migration, and invasion of tumor cells in vivo [98]. Lewinska M et al. discovered that lysyl oxidase (LOX) derived from cancer-associated fibroblasts can disrupt the dimerization of cREL-NF-κB, promoting the development of CCA [99]. Moreover, TNF receptor-associated factor 3 (Traf3) has been identified as the most significantly mutated gene with functional deficiencies in the mouse iCCA model [100]. Traf3 inhibits the activation of NF-κB-Inducing Kinase (NIK), which promotes the activation of the non-classical NF-κB pathway, leading to significant growth of iCCA. In humans, low Traf3 or high NIK expression is closely linked to poor prognosis [100]. Additionally, the NF-κB pathway is involved in the metabolic reprogramming of iCCA [101]. These recent studies not only highlight the diversity and complexity of NF-κB functions but also underscore the critical role of NF-κB-mediated inflammation in the development of CCA.

Current research indicates that the classical NF-κB-NLRP3 inflammasome pathway operates as follows: Various PAMPs and DAMPs bind to toll-like receptors (TLRs), activating the NF-κB pathway, which in turn promotes the transcription of NLRP3, Pro-IL-1β, and Pro-IL-18 [102, 103]. After the assembly of the NLRP3 inflammasome, several activators [104] lead to the conversion of Pro-caspase-1 to caspase-1. This activation promotes the cleavage of GSDMD and the generation of IL-1β and IL-18. Following GSDMD cleavage, N-GSDMD oligomerizes to form pores in the plasma membrane, mediating cell apoptosis and facilitating the release of mature IL-1β and IL-18, thus triggering an inflammatory response [105, 106].The above-mentioned content can be seen in Fig. 5. Zhang T et al. found that CARD9 is significantly upregulated in CCA tissues and activates both the NF-κB and NLRP3 inflammasome pathways, leading to the excessive release of various inflammatory factors. This, in turn, activates the Hedgehog signaling pathway and accelerates the progression of CCA [107]. While this study confirmed the expression of NF-κB and NLRP3 inflammasomes in CCA for the first time, it did not fully elucidate the deeper relationship between NF-κB and NLRP3 inflammasomes in CCA. However, given the established importance of NF-κB in the development of CCA, we can confidently affirm the validity of the NF-κB-NLRP3 inflammasome signaling pathway. Future research may focus on exploring the function and regulatory mechanisms of the NLRP3 inflammasome during the progression of CCA, as this could reveal critical insights into the underlying pathological mechanisms of the disease.

PTEN

PTEN is a tumor suppressor gene that frequently undergoes mutations in human cancers [108]. Research indicates that PTEN dephosphorylates the Ser211 site of transcription factor EB (TFEB) through its protein phosphatase activity, thus enhancing the biosynthesis and acidification of lysosomes [109]. Conversely, PTEN deficiency leads to increased exosome secretion in tumor cells, which promotes the proliferation and invasion of CCA [109]. Additionally, circular RNA (cZNF215) competes with peroxiredoxin 1 (PRDX1) to inhibit its function. This competition prevents PRDX1 from binding to PTEN, resulting in the inactivation of the oxidative-induced PTEN/AKT pathway and further advancing the progression and metastasis of iCCA [110]. PTEN also plays a regulatory role in tumors by modulating the inflammatory response. Yang et al. created a novel mouse model of cholangitis that progresses to eCCA [111]. This model induces cholangitis by conditionally knocking out PTEN in cells, leading gradually to dysplasia and eventually to eCCA. This phenotypic change effectively replicates the essential histopathological features of the liver’s cholangitis-cholecystocarcinoma continuum [111]. Furthermore, Huang et al. discovered that the anti-tumor immune response and the tumor-suppressive effect induced by chemotherapy depend on the expression of PTEN in myeloid cells [112]. Specifically, PTEN interacts with NLRP3, dephosphorylating its Tyr32 site, which enhances the binding of NLRP3 to ASC, promoting the assembly of the inflammasome, production of IL-1β, and the infiltration and activation of T cells in the TME [112]. Notably, this study showed that adding IL-1β can reverse chemotherapy resistance caused by PTEN deficiency in myeloid cells, suggesting that PTEN expression in these cells may serve as a biomarker for predicting chemotherapy efficacy. Additionally, targeting the NLRP3-IL-1β axis might provide a strategic solution for chemotherapy resistance in patients with PTEN-deficient tumors [112]. This research not only uncovers new functions of PTEN in anti-tumor immunity and inflammation but also preliminarily explores the mechanisms by which PTEN deficiency contributes to the development of CCA, paving the way for precise treatments for PTEN-deficient CCA.

Primary sclerosing cholangitis (PSC)

PSC is a liver disease characterized by idiopathic bile duct inflammation and fibrosis, leading to narrowing and damage of the bile ducts [113]. PSC is a malignant disease affecting the pre-biliary tract. Chronic biliary inflammation in PSC can lead to cancer development through various pathways, including regulating cell proliferation, inducing DNA damage, and altering the extracellular matrix [114]. CCA is one of the most common and serious types of cancer faced by patients with PSC, and it is often found in the Pcca subtype [115]. According to statistics, patients with PSC have a risk of developing CCA that is 400 times higher than that of the general population [116]. The cumulative incidence of CCA over a 30-year period can reach as high as 20%, with nearly one-third of CCA cases diagnosed within the first year of a PSC diagnosis [114]. Researchers have identified a potential role for the NLRP3 inflammasome in the progression from PSC to CCA. In both mice and human PSC cases, the expression of TLRs in reactive biliary epithelial cells is increased. Continuous stimulation by LPS and DNA fragments leads to over-activation of the NLRP3 inflammasome, which in turn increases the secretion of IL-18 and promotes chronic inflammatory responses. Although the activation of the NLRP3 inflammasome does not appear to affect the proliferation of biliary epithelial cells, it does have a significant impact on their barrier function, leading to a marked reduction in the expression of the E-cadherin gene. This downregulation of E-cadherin contributes to periductal fibrosis, perivenous inflammation, and an increased susceptibility to liver and bile tumors [117, 118]. However, Matsushita et al. reached a contrasting conclusion, finding that low expression of the NLRP3 inflammasome in biliary epithelial cells in PSC patients is significantly associated with the development of CCA [119]. The discrepancies between these studies may be attributed to differences in sample size and the high heterogeneity of cholangiocarcinoma. Therefore, further research is needed to explore the causal relationship between NLRP3 and the transformation process from PSC to CCA.

Bile acid

CCA, a malignant tumor that originates in the bile duct, is closely associated with the accumulation of bile acids [120]. Additionally, the activation of the atypical NF-κB axis, specifically the LTβ/NIK/RelB pathway, by bile acids contributes to the progression of cholangiocarcinoma [121]. Numerous studies have indicated that bile acids act as DAMPs, significantly upregulating the mRNA levels of NLRP3 and IL-1β. This process induces prolonged Ca²⁺ influx, which, in conjunction with ATP, synergistically activates the NLRP3 inflammasome in macrophages [122–124]. Thus, the potential connection between accumulated bile acids and the NF-κB-NLRP3 inflammatory pathway, as well as their role in promoting the transformation of inflammation into CCA, is a subject that warrants further investigation.

The possible role of NLRP3 inflammasome in the occurrence and development of cholangiocarcinoma

In the previous section, we summarized the “double-edged sword” role of the NLRP3 inflammasome in tumor progression. We discussed the risk factors that contribute to CCA and the various mechanisms by which inflammatory factors, such as NF-κB, are involved in the progression of CCA. Additionally, we provided an in-depth analysis of the CCA microenvironment through multi-omics approaches.

Bile acid accumulation and PSC promote the development of CCA via the NLRP3 inflammasome. In biliary epithelial cells affected by PSC, the expression of TLRs is increased. Under continuous stimulation from LPS and DNA fragments, the NLRP3 inflammasome is overly activated, resulting in heightened secretion of IL-1β and IL-18, which creates a chronic inflammatory environment. The accumulated bile acids act as typical DAMPs and continuously trigger the activation of the NLRP3 inflammasome in biliary epithelial cells through the NF-κB pathway(Figure 5). This activation significantly decreases the expression of the E-cadherin gene and increases the susceptibility of biliary epithelial cells to carcinogenesis. The chronic inflammatory environment is conducive to mutations in the PTEN gene within these cells, inducing their transformation into cholangiocarcinoma cells. Moreover, persistent activation of the NLRP3 inflammasome within cholangiocarcinoma cells supports their own activation and proliferation.

Macrophages can be classified into two types: M1, which have an anti-tumor phenotype, and M2, which have a pro-tumor phenotype [125]. The activation of the NLRP3 inflammasome in macrophages within the CCA microenvironment is crucial for tumor development. In this microenvironment, PTEN within M1 macrophages is prone to mutation, which inhibits the interaction between PTEN and NLRP3. This inhibition impairs T-cell infiltration and activation mediated by the NLRP3-IL-1β pathway, ultimately preventing timely clearance of cancerous biliary epithelial cells and promoting CCA progression. Additionally, the CCA microenvironment encourages the transformation of macrophages from the M1 to the M2 phenotype. Unlike M1 macrophages, M2 macrophages can initiate vascular remodeling mediated by neutrophils through the NLRP3 inflammasome pathway, facilitating CCA proliferation and establishing an immunosuppressiveTME. However, the role of the NLRP3 inflammasome in the TME varies across different CCA subtypes. Within the iCCA microenvironment, M2 macrophages can release pro-inflammatory factors via the NLRP3-IL-1β pathway, further promoting iCCA cell proliferation and the activation of regulatory T cells. This contributes to the establishment of an immunosuppressive microenvironment that supports iCCA growth. In the eCCA microenvironment, activation of the NLRP3 inflammasome in M2 macrophages intensifies immunosuppressive effects on CD8⁺ T cells. Moreover, eCCA cells can upregulate PD-L1 through the NLRP3 inflammasome pathway, leading to a decrease in CTLs and fostering an immunosuppressive eCCA microenvironment characterized by T cell exhaustion.

We propose that the NLRP3 inflammasome may play a critical role in the processes of CCA occurrence and development based on these hypotheses. It is important to note that this hypothesis model is a reasoned speculation grounded in existing research and carries some ideal qualities. Further investigation by researchers is needed to verify the specific mechanisms involving the NLRP3 inflammasome in the development of CCA.

Conclusion and prospects

In this review, we have elaborated on the latest regulatory mechanisms of the NLRP3 inflammasome. Through a systematic analysis of recent research on the NLRP3 inflammasome in various TME states, we have revealed the complexity of its mechanisms and its “double-edged sword” effect on tumor development. Although the role of the NLRP3 inflammasome in CCA has not been extensively studied, preliminary evidence suggests relationships between the NLRP3 inflammasome and factors such as cholangitis, bile stasis, NF-κB, and PTEN. These factors are significant contributors to the development of CCA. Therefore, we believe there is a crucial connection between the NLRP3 inflammasome and the onset and progression of CCA. Further exploration of this relationship is important for understanding the transformation of cholangial inflammation into cholangiocarcinoma.

Abbreviations

PAMPs

pathogen-associated molecular patterns

DAMPs

damage-associated molecular patterns

CCA

Cholangiocarcinoma

iCCA

intrahepatic Cholangiocarcinoma

pCCA

perihilar Cholangiocarcinoma

dCCA

distal Cholangiocarcinoma

eCCA

extrahepatic Cholangiocarcinoma

PTEN

phosphatase and tensin homolog

ASC

apoptosis-associated speck-like protein containing a CARD

LRR

leucine-rich repeat

PYD

N-terminal PYRIN domain

CARD

caspase recruitment domain

GSDMD

gasdermin D

NEK7

NIMA-related kinase 7

JNK1

c-Jun N-terminal Kinase 1

LPS

lipopolysaccharide

EECS

endoplasmic reticulum-endosome membrane contact sites

ETT

endosome-to-trans-Golgi network trafficking

PI4P

phosphatidylinositol 4-phosphate

TGN

trans-Golgi network

MTOC

microtubule-organizing center

OXPHOS

oxidative phosphorylation

HK2

Hexokinase 2

VDAC

voltage-dependent anion channel

ER

endoplasmic reticulum

mtDNA

mitochondrial DNA

PTM

post-translational modification

UFM1

ubiquitin-fold modifier 1

MM

Multiple Myeloma

β2m

β2-Microglobulin

ROS

reactive oxygen species

ULK1

Unc-51 Like autophagy activating kinase 1

PD-L1

programmed death-ligand 1

CTL

Cytotoxic T Lymphocyte

HA

hyaluronic acid

S.maltophilia

Stenotrophomonas maltophilia

HSC

hepatic stellate cells

PSC

pancreatic stellate cell

TREM2

triggering receptor expressed on myeloid cells 2

BRCA1

The breast cancer susceptibility gene 1

MFN1/2

mitofusin1/2

MAMs

mitochondria-associated endoplasmic reticulum membrane

PML

promyelocytic leukemia protein

P2X7R

P2X7 receptor

EMT

epithelial-mesenchymal transition

ERRα

estrogen-related receptor α

Sorcin

Soluble resistance-related calcium-binding protein

TMEM176B

transmembrane protein 176B

DC

dendritic cells

TIM-3

T cell immunoglobulin and mucin-containing molecule 3

MC

mast cells

RSPO3

R-spondin 3

TMZ

Temozolomide

HMGB1

high mobility group box 1 protein

RAGE

receptor for advanced glycation end products

SIRT7

Sirtuin 7

CMML

chronic myelomonocytic leukemia

ICB

immune checkpoint blockade therapy

2GBI

2-guanidinobezimidazole

TME

tumor microenvironment

BTC

biliary tract cancer

circRNA

circular RNA

LOX

lysyl oxidase

Traf3

TNF receptor-associated factor 3

NIK

NF-κB-Inducing Kinase

TLRs

toll-like receptors

TFEB

transcription factor EB

PRDX1

peroxiredoxin 1

Authors’ contributions

ZQZ: Writing – original draft, review and editingYWX: Writing – original draft HZ: Writing – original draft BT: Writing – original draft QD: Writing – review and editing BZD: Writing – review and editing CZZ: Writing – review and editing, study concept or design All authors reviewed the manuscript.

Funding

The study was supported by National Natural Science Foundation of China (Grant No. 82370890), Taishan Scholars Program of Shandong Province (grant number 2019010668 and NO. tsqn202312382), Natural Science Foundation of Shandong Province (grant number ZR2021MH171, ZR2023MH243), Shandong Higher Education Young Science and Technology Support Program (grant number 2020KJL005 and 2023KJ224).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.