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. 2025 Apr 21;48(6):3959–3974. doi: 10.1007/s10753-025-02304-6

CLICs Inhibitor IAA94 Alleviates Inflammation and Injury in Septic Liver by Preventing Pyroptosis in Macrophages

Jing Liu 1,2,#, Jingwen Hu 1,2,#, Xulei Yao 1,2, Mengting Xu 1,2, Aini Yuan 1,2, Jianan Guo 1,2, Cui Wang 1,2, Yifei Le 1,2, Xingyu Yuan 1,2,✉, Dezhao Lu 1,2,✉
PMCID: PMC12722281  PMID: 40259192

Abstract

Macrophage pyroptosis represents a pivotal mechanism underlying acute liver injury during sepsis. Chloride intracellular channel proteins (CLICs) have been linked to inflammatory reflexes, with IAA94 serving as an inhibitor of channel formation characteristic of CLICs. In a mouse model, IAA94 demonstrated efficacy in reducing pro-inflammatory cytokines in liver tissues, decreasing macrophage in the liver, inhibiting the development of the pro-fibrosis phenotype, and alleviating tissue injury. Additionally, IAA94 exhibited inhibitory effects on the activation of NLRP3 inflammasome, leading to the suppression of pyroptosis in J774A.1 cells and the liver. Additionally, IAA94 was observed to impede the interaction between NEK7 and NLRP3. Furthermore, it was observed that the conditioned medium of pyroptotic macrophages treated with IAA94 induced an attenuated inflammatory response in hepatocytes in comparison to that induced by the conditioned medium of pyroptotic macrophages. However, NLRP3 overexpression impeded the beneficial effects of IAA94. In conclusion, IAA94 has the capacity to impede NLRP3 inflammasome formation-mediated pyroptosis by blocking CLICs-mediated chloride efflux and the inhibition of NEK7-NLRP3 interactions, thereby establishing CLICs as a promising therapeutic target against liver inflammation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10753-025-02304-6.

Keywords: IAA94, CLICs, Sepsis, Liver, Macrophages, NLRP3

Introduction

Sepsis is a systemic inflammatory response syndrome (SIRS) caused by infection that can result in multiorgan damage, which can in turn lead to multiorgan dysfunction and pose a significant threat to patient survival [1]. It is a major cause of mortality in intensive care unit (ICU) patients [2]. The liver plays a pivotal role in regulating metabolism and maintaining the stability of the internal environment [3]. Due to its distinctive physiological function and inflammatory response, the liver is among the most susceptible organs in patients with sepsis. Acute liver injury can manifest at any stage of sepsis, with the potential to be triggered in the early stages of the disease [4]. The mortality rate associated with sepsis-induced acute liver injury can reach as high as 38.2% to 68.0% [5]. A deeper comprehension of the underlying mechanisms of acute liver injury during sepsis could prove invaluable in the development of more effective therapeutic strategies.

The liver has the largest population of macrophages in the body, and macrophages are a principal cell type involved in the development of the liver's antimicrobial defences [6]. In response to pathogen-associated molecular patterns, liver macrophages are activated and subsequently release substantial quantities of inflammatory cytokines and chemokines into the body [7]. The excessive production of inflammatory factors induces an inflammatory response and damage to hepatic sinusoidal endothelial cells and hepatocytes, which in turn leads to the further production of danger-associated molecular patterns that reactivate the inflammatory response of macrophages [8, 9]. This process recruits additional circulating immune cells and promotes the infiltration of bone marrow-derived monocytes into the liver, where they differentiate into macrophages [10]. This results in a significant expansion of the hepatic macrophage pool and facilitates the aggregation and migration of infiltrating macrophages in the damaged liver [11]. These initial immune responses by macrophages frequently result in hepatic tissue injury and an intensification of the inflammatory response in the damaged liver [12]. Furthermore, the excessive immune and inflammatory response, in conjunction with liver injury, serve to exacerbate one another, ultimately leading to the development of liver dysfunction or even failure [13]. Macrophage pyroptosis has been identified as a pivotal pathogenic factor in sepsis-associated acute liver injury [14]. Classic pyroptosis is an inflammatory cell death mechanism triggered by the activation of the NLRP3 inflammasome and subsequent pore formation by GSDMD-N-terminal (GSDMD-NT) on the cell membrane [15, 16]. During sepsis, pyroptosis of macrophages induces inflammatory infiltration in surrounding tissues, leading to tissue damage and thereby exacerbating liver injury [17].

Chloride intracellular channel proteins (CLICs) represent a recently identified class of chloride channels [18]. They play a role in cellular processes such as oxidative stress, inflammation, signalling, migration, and proliferation. Additionally, they have been linked to a range of complex and fatal diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Among the aforementioned proteins, chloride channel protein 1 (CLIC1) is a 241-amino acid protein that belongs to the glutathione-S-transferases (GSTs) superfamily. It is highly conserved and widely distributed in mammalian tissues and subcellular structures. CLIC1 is dimorphic, exhibiting both a soluble and an intact membrane form, and represents a rare member of the'metamorphic proteins'. In response to oxidative conditions and pH changes, CLIC1 forms oligomers from the soluble monomeric form and aggregates at the cell membrane to form ion channels. In addition to chloride, CLIC1 facilitates the transport of anions, including iodide (I-) and bromide (Br-), which can alter the cellular microenvironment and regulate protein-nucleic acid and protein-protein interactions, thereby influencing the inflammatory response. Recent studies have revealed that CLIC1-mediated chloride efflux and ASC oligomerization are indispensable for NLRP3 inflammasome activation. However, the specific mechanism remains to be fully clarified.

IAA94 is an intracellular chloride channel protein blocker that inhibits CLIC1-dependent chloride permeability with an IC50 of 8.6 μM [19]. IAA- 94 has a high affinity for CLIC1-mediated chloride channels and has been employed to modulate CLIC1 chloride channel function and to investigate channel kinetics and function [20]. Previous studies have demonstrated that IAA94 plays a pivotal role in the prevention of cardiovascular disease, cancer, and viral infections [21–23]. In light of these observations, we sought to ascertain the role of IAA94 in macrophage pyroptosis and the mitigation of acute liver injury during sepsis. The present study demonstrated that IAA94 inhibits macrophage pyroptosis, reduces liver inflammation, and mitigates liver injury in septic mice. IAA94 inhibits NEK7 binding to NLRP3, impeding NLRP3 inflammasome formation and macrophage pyroptosis, which reduces the level of inflammation in liver cells in the macrophage-hepatocyte co-culture system. These findings suggest that IAA94 may have an important therapeutic role in the treatment of acute liver injury in septic mice.

Results

IAA94 Inhibits LPS-Induced Liver Injury

To ascertain the function of IAA94 in LPS-induced liver injury, mice were administered intraperitoneal injections of LPS, which imitates the effects of sepsis, and were subsequently given intraperitoneal injections of either IAA94 or a control solvent prior to the LPS injection (Fig. 1A). Initially, it was observed that the expression of CLIC1 dimers, a form of CLIC1 ion channel formation, was elevated in the livers of mice that had been treated with LPS. The channel-forming inhibitor of CLIC1, IAA94, was observed to inhibit the LPS-induced increase in CLIC1 dimer expression in the liver (Supplementary Fig. 1). Plasma alanine aminotransferase (ALT) and aminotransferase (AST) levels in mice were measured 6 h after performing LPS injections. Treatment with IAA94 resulted in a significant reduction in LPS-induced high plasma ALT and AST levels (Fig. 1B and C). To substantiate the impact of IAA94 on LPS-induced liver injury, the histopathological alterations in the livers of IAA94-treated mice were evaluated through H&E staining. The results of the histological examination of the livers of mice subjected to LPS treatment revealed the presence of lymphocytes and neutrophils, as well as evidence of cytolysis, tissue damage, and disorganised hepatocyte arrangement. However, the degree of recovery of liver inflammatory cell infiltration and tissue damage differed following IAA94 treatment (Fig. 1D). Macrophages are the primary drivers of the hepatic inflammatory response. To further elucidate the impact of IAA94 on the hepatic inflammatory response to liver injury, we conducted an immunofluorescence examination of the macrophage marker F4/80 in liver tissue. The results demonstrated that IAA94 significantly reduced the LPS-induced F4/80-positive macrophages in the liver (Fig. 1E).

Fig. 1.

Fig. 1

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IAA94 inhibits LPS-induced liver injury. A Study design protocol describing the experimental conditions of several groups of animals. Plasma levels of ALT (B) and AST (C) in mice. D HE staining of liver tissue sections. The arrows represent: areas of inflammatory cell infiltration. E F4/80 immunofluorescence results of mouse liver tissue sections. Experimental data values are the mean ± SEM of 6 replicates. *p < 0.05, **p < 0.01

IAA94 Inhibits LPS-Induced Hepatic Inflammation and Pro-Fibrosis

Pro-inflammatory cytokines have been demonstrated to play a pivotal role in the progression of liver injury in sepsis [24]. The expression of early pro-inflammatory factors, namely TNF-α, MCP- 1 and IL- 6, was compared in the livers of different treatment groups after 6 h of LPS treatment. This was achieved through the use of IHC staining (Fig. 2A, B) and Western blotting (Fig. 2C). The expression levels of TNF-α, MCP- 1 and IL- 6 in the liver were markedly elevated in mice following LPS treatment. Conversely, the expression levels of TNF-α, MCP- 1 and IL- 6 were markedly diminished in the livers of mice that had been pretreated with IAA94 and then subjected to an intraperitoneal injection of LPS, in comparison with those that had been administered an LPS injection alone. And qPCR results demonstrated that LPS increased the expression levels of IL- 1β, IL- 6 and TNF-α in liver tissue, and treatment with IAA94 inhibited the upregulation of these inflammatory factors caused by LPS (Fig. 2D).

Fig. 2.

Fig. 2

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IAA94 inhibits LPS-induced hepatic inflammation. A, B Immunohistochemistry of inflammatory factors in mouse liver tissue. C Expression of proteins related to inflammatory factors in mouse liver tissue. D Expression of inflammatory genes in mouse liver tissue. Values are the mean ± SEM of 4 replicates. *p < 0.05, **p < 0.01

Fibrosis is a common histological feature of progressive acute liver injury [25]. Injured hepatocytes may undergo significant alterations and exhibit a secretory phenotype [26]. The release of pro-fibrotic cytokines by injured hepatocytes stimulates fibrosis in interstitial fibroblasts, resulting in the development of a secretory phenotype [27]. Sirius red and Masson staining showed that LPS increased the level of liver fibrosis, which could be alleviated by IAA94 treatment (Fig. 3A, B). The protein expression of fibrosis-associated factors, including TGF-β1, MMP9 and α-SMA, was markedly elevated in the livers of mice treated with LPS in comparison to controls (Fig. 3C). However, a notable reduction in the expression of TGF-β1, MMP9, and α-SMA was observed in the livers of mice that underwent LPS treatment following IAA94 preconditioning, in comparison to mice that underwent LPS treatment alone (Fig. 3C). These findings collectively indicate that IAA94 has the potential to attenuate LPS-induced hepatic inflammation, pro-fibrosis, and injury.

Fig. 3.

Fig. 3

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IAA94 inhibits LPS-induced hepatic pro-fibrosis. A, B Sirius red and Masson stain in mouse liver tissue. C Expression of fibrosis-related proteins in mouse liver tissue. Values are the mean ± SEM of 4 replicates. *p < 0.05, **p < 0.01

IAA94 Inhibits LPS-Induced Pathways Associated with Pyroptosis in Liver

During sepsis, damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) activate the assembly of the NLRP3 inflammasome, which promotes pyroptosis and amplifies the inflammatory response and tissue injury [15]. By regulating the activity of the NLRP3 inflammasome, it may play an important role in attenuating the inflammatory response and related pathological processes in acute liver injury in sepsis [28]. Therefore, inhibiting the activation of the NLRP3 inflammasome is potentially significant for LPS-induced liver injury. In order to evaluate the function of IAA94 in NLRP3 inflammasome and cellular pyroptosis in liver tissues, we conducted an examination of the activation of NLRP3 inflammatory vesicles and the activation of pyroptosis-associated proteins in liver tissues of LPS-treated mice. Figure 4B illustrates the outcomes of immunoblotting, which demonstrate that LPS markedly elevated the expression of Caspase- 1 p20, GSMSD-NT, IL- 1β, IL- 18 and NLRP3 proteins in mouse liver tissues. The administration of IAA94 resulted in a notable reduction in the expression of Caspase- 1 p20, GSMSD-NT, IL- 1β, IL- 18 and NLRP3 proteins in mouse liver tissues. Macrophage pyroptosis plays a significant role in the pathogenesis of hepatic inflammatory injury [12, 29]. To ascertain the impact of IAA94 on the modulation of macrophage pyroptosis in liver tissues, we investigated the expression of proteins associated with pyroptosis in liver tissue macrophages through immunofluorescence confocal analysis. Figure 4A illustrates the results of immunofluorescence, indicating that LPS significantly increased the expression of Caspase- 1 p20 and GSMSD-NT in mouse liver tissue macrophages. These findings suggest that pretreatment with IAA94 significantly reduced the expression of Caspase- 1 p20 and GSDMD-NT in liver tissue macrophages compared to mice receiving LPS alone. The aforementioned results indicate a notable reduction in pyroptosis following treatment with IAA94.

Fig. 4.

Fig. 4

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IAA94 inhibits LPS-induced Pathways associated with pyroptosis in liver. A Co-localization of macrophages (F4/80-labeled, red), Caspase- 1 p20 (green) and GSDMD-NT (yellow) in liver tissue of mice after IAA94 treatment. B Pyroptosis-associated protein expression. Values are the mean ± SEM of 4 replicates. *p < 0.05, **p < 0.01

IAA94 Inhibits Activation of Pyroptosis-Associated Proteins in Mouse Macrophages

The impact of IAA94 on macrophage NLRP3 inflammasome and pyroptosis-associated proteins was investigated in an in vitro setting. Firstly, it was observed that the dimeric and oligomeric forms of CLIC1 were increased in the mouse macrophage cell line J774 A.1 following treatment with LPS for 4 h and with Nigericin (Nig) for 45 min. Conversely, the dimeric and oligomeric forms of CLIC1 could be reduced by the action of IAA94 (Fig. 5A). Additionally, a reduction in intracellular chloride ion concentration was observed. The decline in chloride ions in macrophages induced by LPS in combination with Nig was prevented by IAA94 (Fig. 5B). And overexpression of CLIC1 has been showed to increase expression of the NLRP3 protein (Supplementary Fig. 3). Furthermore, the data demonstrated a notable elevation in the protein expression levels of NLRP3, GSDMD-NT, Caspase- 1 p20, IL- 1β, and IL- 18 in J774 A.1 cells following treatment with LPS for 4 h and Nig for 45 min (Fig. 5D). The IAA94 treatment resulted in a notable reduction in the levels of NLRP3, GSDMD-NT, Caspase- 1 p20, IL- 1β, and IL- 18 in J774 A.1 cells when exposed to LPS and Nig administration (Fig. 5D). The assembly of NLRP3 inflammasomes was observed following the formation of oligomer-activated NLRP3-inflammasome complexes by NLRP3 and ASC. As demonstrated in Figure 5C, the expression of inflammasome oligomers was detected in DSS cross-linked-treated J774 A.1 cells using Triton X-100 non-solubilised components, as determined by immunoblotting (Fig. 5C). It was demonstrated that treatment with LPS for 4 h and Nig for 45 min resulted in a notable elevation in the expression of ASC and NLRP3 protein oligomers in J774 A.1 cells. Following treatment with LPS and Nig, IAA94 was observed to significantly reduce the expression levels of ASC and NLRP3 protein oligomers in J774 A.1 cells (Fig. 5C). It has been demonstrated that NEK7 is essential for the formation of NLRP3-mediated inflammasome [30]. To ascertain the regulatory function of IAA94 on the interaction of NEK7 with NLRP3, the results of immunoprecipitation demonstrated that LPS treatment for 4 h versus Nig treatment for 15 min resulted in elevated interactions between NLRP3 and NEK7, as well as between NLRP3 and ASC, in J774 A.1 mouse macrophages (Fig. 5E). Treatment with IAA94 resulted in a significant reduction in NLRP3-NEK7 and NLRP3-ASC interactions in J774 A.1 cells following LPS + Nig treatment (Fig. 5E). Overall, in vitro, IAA94 was observed to reduce NEK7-NLRP3 interactions, inhibit NLRP3 inflammasome activation and suppress pyroptosis.

Fig. 5.

Fig. 5

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IAA94 Inhibits Activation of Pyroptosis-Associated Proteins in J774 A.1 cells. A Immunoblotting to detect the effect of IAA94 on CLIC1 in LPS + Nig-induced J774 A.1 cells. B Detection of intracellular chloride concentration in LPS + Nig-induced J774 A.1 cells. C Immunoblotting to detect the effect of IAA94 on NLRP3 and ASC oligomers in LPS + Nig-induced J774 A.1 cells. D Immunoblotting to detect the effect of IAA94 on pyroptosis-associated proteins in LPS + Nig-induced J774 A.1 cells; Data are expressed as the Mean ± SD of 4 independent replicates; *p < 0.05, **p < 0.01. E Immunoprecipitation to detect the effect of IAA94 on NLRP3 inflammasome in J774 A.1 cells

In addition, primary peritoneal macrophages (PMs) were extracted from mice for the purpose of validation experiments. The results demonstrated that LPS + Nig activated the pyroptosis of PMs, a finding that was corroborated by the results of both immunoblotting and immunofluorescence (Fig. 6A-D). As anticipated, IAA94 treatment was effective in alleviating LPS + Nig-activated PMs pyroptosis (Fig. 6A-D).

Fig. 6.

Fig. 6

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IAA94 Inhibits Activation of Pyroptosis-Associated Proteins in primary peritoneal macrophages of mice. A Immunoblotting to detect the effect of IAA94 on CLIC1 in LPS + Nig-induced PMs; Data are expressed as the Mean ± SD of 4 independent replicates; *p < 0.05, **p < 0.01. B-D Immunofluorescence detection of NLRP3, ASC, Caspase- 1 p20 and GSDMD-NT expression in PMs

IAA94 Inhibits Inflammation Activation in a Coculture System of J774 A.1 and AML- 12 Cells

In order to ascertain the impact exerted by cytokines secreted by macrophages on the inflammatory response of hepatocytes, serum-starved AML- 12 cells were treated with conditioned cultures of J774 A.1 cells treated with IAA94 (or DMSO) for a period of 24 h. Following this, the aforementioned treated AML- 12 cells were subjected to qPCR. J774 A.1 mouse macrophages were pretreated with IAA94, followed by treatment with LPS for 4 h and Nig for 60 min. Following the treatment of AML- 12 cells with a conditioned medium derived from macrophages, a significant increase in the expression of IL- 6, MCP- 1 and TNF-α was observed (Fig. 7A). Nevertheless, the treatment of AML- 12 cells with macrophage-conditioned medium after the pre-treatment of IAA94 resulted in a notable reduction in the mRNA expression of IL- 6, MCP- 1 and TNF-α when compared with the control group, which was treated with LPS and Nig alone (Fig. 7A). To further elucidate the regulatory mechanisms underlying IAA94-mediated alleviation of hepatocyte inflammation induced by pyroptotic macrophages, we overexpressed NLRP3 in J774 A.1 cells (Supplementary Fig. 3). The results indicated that the mRNA expression of IL- 6, MCP- 1 and TNF-α was decreased in hepatocytes treated with macrophage-conditioned medium following treatment with IAA94 (Fig. 7B). In contrast, the expression of these inflammatory factors mRNA was decreased in AML- 12 cells treated with macrophage-conditioned medium following the addition of NLRP3 overexpression, which reversed this phenomenon (Fig. 7B).

Fig. 7.

Fig. 7

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IAA94 Inhibits Inflammation Activation in a Coculture System of J774 A.1 and AML- 12 Cells. A The application of qPCR to assess the impact of macrophage-conditioned medium on the expression of inflammatory factors mRNA in hepatocytes. Data are expressed as the Mean ± SD of 4 independent replicates; *p < 0.05, **p < 0.01. B The application of qPCR to assess the impact of NLRP3-overexpressed macrophage-conditioned medium on the expression of inflammatory factors mRNA in hepatocytes. Data are expressed as the Mean ± SD of 4 independent replicates; *p < 0.05, **p < 0.01

Discussion

This study demonstrated the efficacy of IAA94, an intracellular chloride channel inhibitor, in the inhibition of the inflammatory process, fibrosis and liver injury in a murine model of LPS-induced sepsis-associated acute liver failure. The mechanism of action of IAA94 hinges on the inhibition of ion channel formation via the suppression of CLICs, the blocking of chloride efflux, and the hindrance of the reciprocal binding of NEK7-NLRP3. This, in turn, impedes the formation of NLRP3 inflammasome and subsequent pyroptotic cell death, thereby alleviating hepatocyte inflammation induced by macrophage pyroptosis (Fig. 8).

Fig. 8.

Fig. 8

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Anti-inflammation effect of IAA94 in liver tissue by inhibition of pyroptosis signaling pathways. IAA94, an intracellular chloride channel inhibitor, inhibits the inflammatory process, fibrosis and liver injury in a murine model of LPS-induced sepsis-associated acute liver failure. The mechanism of action of IAA94 hinges on the inhibition of ion channel formation via the suppression of CLICs, the blocking of chloride efflux, and the hindrance of the reciprocal binding of NEK7-NLRP3. This, in turn, impedes the formation of NLRP3 inflammasome and subsequent pyroptotic cell death, thereby alleviating hepatocyte inflammation induced by macrophage pyroptosis. DAMPs and PAMPs: damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs)

CLICs family-mediated chloride regulation is regarded as a pivotal mechanism in cellular immune regulation [31]. Inhibiting CLICs-mediated chloride efflux has been observed to alleviate peritoneal macrophage inflammation and suppress the expression levels of circulating inflammatory factors in septic mice [32]. Conversely, the inhibition of CLICs has been demonstrated to exert a beneficial influence on the progression of chronic inflammatory diseases, including atherosclerosis and pulmonary hypertension [33–35]. Nevertheless, their function in acute liver inflammation remains uncertain. This study demonstrates that an increase in the dimeric/multimeric form of CLIC1, one of the earliest identified proteins of the CLICs family and typically represented, was observed in both LPS-induced liver tissues and LPS-combined with Nig-treated macrophages. These suggest that CLIC1 ion channels are activated. Moreover, our study corroborates the notion that chloride ion efflux in response to inflammation is a consistent phenomenon at the cellular level, as evidenced by previous research in this domain [32, 36]. In this study, we employed IAA94, an inhibitor of CLICs, to elucidate the mechanism of CLICs in acute liver injury and to evaluate the potential of CLICs as therapeutic targets for inflammatory diseases.

As a principal organ of the body in terms of its external resistance and metabolic regulation, liver tissue exerts an essential influence on the advent of acute liver injury in the context of sepsis [13]. Previous studies have demonstrated that crosstalk between macrophage inflammation and hepatocyte facilitates the promotion of inflammatory responses and injury in liver tissue [10, 37, 38]. In the context of normal liver tissue, resident immune cells have been observed to play a role in the normal metabolic processes of the liver [6]. Nevertheless, it has been demonstrated that patients with diseases such as sepsis experience an increased influx of monocytes into liver tissue and abnormal activation of previously resident macrophages, which subsequently differentiate to generate pro-inflammatory macrophages [39]. The resulting inflammatory response, characterised by the production of pro-inflammatory cytokines, leads to tissue damage and structural disruption of the liver [40]. Additionally, the inflammatory process may propagate to other metabolic organs, ultimately leading to a systemic inflammatory response [41]. Therefore, elucidating the mechanisms underlying liver tissue inflammation is of paramount importance. Furthermore, it is essential to develop therapeutic interventions that target liver tissue macrophages to prevent and control the inflammatory cascade. The data from this study indicate that IAA94 may be an effective inhibitor of inflammatory processes in a mouse model. Furthermore, it seems to facilitate partial recovery of liver tissue damage. Prolonged inflammation is observed to be associated with alterations in tissue remodelling in a number of pathological conditions [42]. Prolonged inflammatory stress can lead to the development of fibrosis, characterised by ECM components within and around the affected tissue [43]. It is widely accepted that the process of pro-fibrosis is initiated by an inflammatory response and progresses to tissue damage [44]. Previous studies have demonstrated that pharmacological CLIC1 inhibition is associated with a decline in liver fibrosis [45]. The findings indicate that IAA94 is an effective inhibitor of the aberrant activation of pro-fibrotic proteins in mouse liver tissue induced by LPS.

The reduction of inflammation and extracellular matrix remodelling in liver tissue can be achieved by the inhibition of NLRP3 inflammasome and subsequent cellular pyroptosis [28]. Of particular significance is the role of macrophage pyroptosis, particularly at the initial stages [12, 29]. NEK7 is the smallest member of the mammalian NIMA-associated kinase (NEK) family of serine/threonine kinases, which comprises 11 members in total (NEK1-NEK11) [46]. It is suggested that NEK7 plays a pivotal role in the NLRP3 inflammasome, regulating NLRP3 oligomerisation and inflammasome assembly [15, 30]. It is now widely accepted among the scientific community that an increase in intracellular K⁺ concentration can drive NLRP3 inflammasome assembly via NEK7 and promote pyroptotic cell death [47]. As detailed in our data, IAA94 was observed to inhibit the interaction of NEK7 with NLRP3, thereby impeding the formation of the NLRP3 inflammasome and cellular pyroptosis. These findings align with those reported in previous studies. Furthermore, pretreatment with IAA94 was observed to mitigate hepatocyte inflammation resulting from pyroptotic macrophages. It is proposed that CLICs-mediated chloride efflux is closely associated with the assembly of the NLRP3 inflammasome and the onset of cellular pyroptosis. The role of IAA94 in preventing cellular pyroptosis is also attributable to its ability to impede the assembly of NLRP3 inflammatory vesicles. We conducted an experiment in which we overexpressed NLRP3 in macrophages in vitro. The results demonstrated that IAA94's capacity to inhibit the formation of NLRP3 inflammatory vesicles and prevent hepatocyte inflammation induced by pyroptotic macrophages was partially reversed. These suggest that CLIC-mediated chloride efflux is involved in NEK7-NLRP3 interactions and NLRP3 inflammasome assembly. Hence, IAA94 exerts its inhibitory effect by targeting chloride channels in CLICs.

In conclusion, IAA94 was observed to alleviate inflammatory, pro-fibrotic and tissue damage responses in mouse liver tissues resulting from lipopolysaccharide (LPS)-induced inflammation. This was achieved by preventing recruitment of macrophages, pro-inflammatory responses and the activation of the NLRP3 inflammasome. Furthermore, IAA94 was observed to inhibit NLRP3 inflammasome activation and cellular pyroptosis by blocking CLICs-mediated ion channel formation, preventing chloride ion efflux, and hindering the binding effect of NEK7 to NLRP3. In light of these findings, it may be posited that CLICs represent a novel drug research target for the modulation of inflammatory injury in the liver.

Materials and Methods

Animal Feeding, Grouping and Drug Administration

A straightforward random method was used to divide 18 male C57BL/6 mice into three groups, each comprising six mice: an initial acclimatisation period of one week, after which the mice were assigned to one of three groups: a control group (receiving saline), an LPS model group and an IAA94 treatment group. The male mouse is notable for its relatively stable behaviour, which renders it relatively impervious to hormonal changes in the body. Furthermore, it has been demonstrated to exhibit excellent repeatability, a quality that has contributed to its selection as a subject for further study. The mice in the control group were given saline on a single occasion, while the IAA94 treatment group received 50 mg/kg of IAA94 by gavage six hours before the LPS modelling treatment. In order to induce acute liver injury in mice, the animals in the aforementioned model group and IAA94 treatment group were injected intraperitoneally with LPS (10 mg/kg) after the administration of IAA94 for a period of 1 h. Subsequent to this, the mice were anaesthetised via intraperitoneal injection of pentobarbital at a dose of 40 mg/kg at 6 h following the injection of LPS, in order to obtain liver and blood samples.

Detection of AST and ALT Levels

The analysis of plasma levels of AST and ALT was conducted in accordance with the instructions provided by the manufacturer in order to assess liver function in mice.

Histological Analysis

The liver tissue was fixed in an ethanol gradient overnight, cleared with xylene, dipped in wax and embedded in paraffin wax, and subsequently cut into 4 µm slices using a microtome. Subsequently, the wax strips were partitioned and placed in a warm water bath maintained at 37 °C. The slices were then subjected to a baking process of five hours. Thereafter, a series of elutions with xylene, ethanol, and distilled water were performed, followed by staining of the slices with HE staining solution and sealing with neutral gum for observation of tissue damage. To examine the migration of macrophages within the hepatic tissue of mice, paraffin sections of liver tissue were labelled with the F4/80 antibody (#70076, Cell Signaling Technology) through immunofluorescence. Subsequently, the macrophages was observed under a fluorescence microscope.

Cell Culture

The mouse macrophage cell line J774 A.1 was procured from ATCC. The cells were grown in DMEM medium, supplemented with 10% FBS, and cultured in a humidified incubator at 37 °C, with 5% CO₂ atmosphere. The AML- 12 mouse hepatocyte cell line (CRL- 2254) was sourced from the American Type Culture Collection (ATCC). The cells were cultivated in DMEM/F12, supplemented with 10% foetal bovine serum (FBS, FS201 - 02, TransGen Biotech, China). The cells were cultured at 37 °C in a humidified incubator with 5% CO2, with the addition of 1 μM dexamethasone (DEX, D4902, Sigma) and insulin-transferrin-selenium (I3146, Sigma-Aldrich).

Co-Culture of Macrophages and Hepatocytes

The co-culture of macrophages and hepatocytes underwent a modification based on the results of previous studies [48, 49]. The J774 A.1 macrophages were treated with IAA94 for a period of four hours. Subsequently, the macrophages were stimulated with LPS (1 μg/mL) for a further four hours, followed by a one-hour incubation with 10 μM Nig. Following this, the macrophages were incubated in DMEM medium (serum-free, 0.5% fatty acid-free BSA) for a period of 24 h, after which the medium was harvested. The cells were then cultured in DMEM medium (serum-free, 0.2% fatty acid-free BSA) for 24 h. After this period, the medium was harvested and centrifuged at 400 × g for 10 min. The resulting supernatant was then collected and designated as macrophage-conditioned medium (CM). The aforementioned conditioned medium was then used to culture AML- 12 cells for a period of 24 h. The serum-starvation of AML12 cells is a two-fold process intended to control cell proliferation and to render them sensitive and unaffected by serum.

Immunoblotting Assay

Initially, cell lysis buffer for Western and IP was incorporated into the samples, which subsequently underwent a 30-min incubation period on ice. Following this, the samples underwent a 20-min centrifugation process at 4 °C and 12,000 r/min. Subsequently, the supernatant was aspirated, and the protein concentration was determined. This was followed by denaturation, sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) separation, and transfer to a polyvinylidene fluoride (PVDF) membrane via transblotting. The proteins were subsequently transferred to the PVDF membrane, which was then closed with a protein-free closure solution for one hour at room temperature. The particular primary antibody was then added and left to incubate overnight at 4 °C under shaking conditions. The primary antibodies used are listed in Table S1. On the following day, the membrane was washed by TBST. Thereafter, the corresponding HRP-labelled secondary antibody working solution was added and incubated for one hour at room temperature. Subsequent to this, the membrane was washed, and the protein bands were chromatographed using ECL luminescent liquid. The bands were then detected in the imaging system using the ImageJ software, and a greyscale analysis of the bands was conducted.

Immunohistochemistry Assay

The obtained liver tissue sections were deparaffinised in xylene and gradient ethanol, then they were placed in buffer and kept for 2 h in order to obtain full exposure of the antigenic determinants. Subsequently, the tissue sections were immersed in a 3% hydrogen peroxide solution and incubated at room temperature for 10 min. They were then washed three times with PBS and incubated overnight at 4 °C with specific primary antibodies (Table S2). The following day, the sections were washed again and drops of HRP-labelled secondary antibodies corresponding to the species were incubated at room temperature for 30 min. They were then washed and the DAB colour was developed for 2 to 5 min until brownish-yellow particles were observed under a microscope. Finally, the reaction was terminated using deionised water. The reaction was terminated by the addition of deionized water. The sections were then lightly re-stained with a hematoxylin stain for a further 2 min before being washed again. They were subsequently dehydrated through a series of gradient alcohols, immersed in xylene, allowed to dry, sealed with neutral gum, and observed, recorded, and analysed under a bright-field microscope.

Extraction of Peritoneal Macrophages

In this experiment, male C57BL/6 mice were injected intraperitoneally with thioglycolate broth to induce peritoneal macrophages. After a period of three days, the mice were euthanised by cervical dislocation, and the body was disinfected with 75% ethanol and then fixed on an anatomical plate. The skin was then cut along the midline of the abdomen to expose the peritoneum, and 5 mL of pre-cooled sterile PBS (containing 1% penicillin–streptomycin) was injected intraperitoneally. After a gentle abdominal massage for 5 min, the wash solution was recovered. Centrifugation of the cell suspension at 1000 rpm for 5 min was followed by the discarding of the upper layer and the resuspension of the pellet in DMEM medium containing 10% FBS. The inoculation of the cell suspension into a culture dish was then performed, and the dish was placed into a 37 °C incubator containing 5% CO2.Subsequent to the attachment of the cells, the suspended cells were removed and the adherent macrophages were retained for the subsequent experiments.

Data Analysis

All data from animal experiments obtained in this study are shown as mean ± standard error of mean (SEM), while all data from cell experiments are shown as mean ± standard (SD). For all analyses and graphs were prepared by GraphPad Prism 8.0. Statistical significance was set at P < 0.05.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 2906 KB) (2.8MB, docx)

Acknowledgements

We appreciated the technical support from the Public Platform of Medical Research Center, Academy of Chinese Medical Science, Zhejiang Chinese Medical University. We also acknowledge the BioRender (www.biorender.com.), as Graphical Abstract, the schematic diagram of Figs. 1A and 8 were created with BioRender platform.

Author Contributions

Author Contributions: Conceptualization, D.L., Y.L. and XY.Y.; methodology, J.H., J.L. and X.Y.; validation, J.H., X.Y. and M.X.; investigation, J.H., J.L., A.Y. and J.Guo; resources, M.X. and C.W.; writing—original draft preparation, J.L. and J.H.; writing—review and editing, XY.Y. and D.L.; supervision, C.W. and D.L.; project administration, C.W. and D.L.; funding acquisition, XY.Y. and D.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Natural Science Foundation of China (82074057), the Opening Project of Key Laboratory of Integrative Chinese and Western Medicine for the Diagnosis and Treatment of Circulatory Diseases of Zhejiang Province (2 C32004,2 C32101) and “the Postgraduate Scientific Research Fund” of Zhejiang Chinese Medical University (2022YKJ23).

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Institutional Review Board Statement

The animal study protocol was approved by Medical Code and Ethics Committee of Zhejiang Chinese Medicine University (protocol code 20230619–19 and approved on 19 June 2023).

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Jing Liu and Jingwen Hu contributed equally to this work.

Contributor Information

Xingyu Yuan, Email: starmoveyxy@163.com.

Dezhao Lu, Email: ludezhao@126.com.

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Associated Data

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Supplementary Materials

Supplementary file1 (DOCX 2906 KB) (2.8MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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