TRPA1 Expressed by Hepatocytes and Liver Macrophages Does Not Mediate Inflammatory Infiltration and Steatosis in a Mouse Model of Chronic Alcohol-Induced Liver Injury

Highlights

Using RNAscope ISH, we prove Trpa1 mRNA expression in hepatocytes and liver macrophages. We confirm that acetaldehyde is capable of activating human TRPA1. Steatosis and inflammatory infiltration resulting from chronic alcohol exposure occur through a TRPA1-independent mechanism.

What are the main findings?

• Trpa1 mRNA is expressed in mouse hepatocytes and liver macrophages.
• Acetaldehyde is able to activate human TRPA1.
• Alcohol-induced liver inflammatory infiltration and steatosis are mediated through a TRPA1-independent mechanism in mice.

What are the implications of the main findings?

• The occurrence of Trpa1 in hepatocytes and liver macrophages suggested the role of this ion channel in inflammatory, fibrotic, and degenerative processes in the liver.
• The ability of acetaldehyde to activate human TRPA1 suggests that the ion channel might act as a mediator of alcohol-induced liver damage.
• Although steatosis and inflammatory infiltration resulting from chronic alcohol exposure are thought to occur through a TRPA1-independent mechanism, we hypothesize that the ion channel plays a role in alcohol-induced liver damage, particularly in late-stage fibrotic changes.

Abstract

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel, and its activator, the alcohol breakdown product acetaldehyde, plays a key role in the pathomechanism of alcoholic liver disease (ALD). We hypothesized that TRPA1 is expressed in the liver, can be activated by alcohol breakdown products, and plays a role in ALD. We aimed (1) to confirm the presence of TRPA1 in liver samples from C57BL6/J mice by RNAscope in situ hybridization combined with immunostaining, (2) to prove that alcohol breakdown products may activate human TRPA1 by calcium-imaging, and (3) to investigate the role of TRPA1 in a chronic continuous 20% alcohol drinking model involving Trpa1 gene-deficient (KO) mice. The liver enzyme levels were evaluated; moreover, the steatosis, portal and interface inflammatory infiltrations were assessed in PAS–hematoxylin-stained sections. We detected Trpa1 expression in both hepatocytes and liver macrophages. We observed elevated liver enzyme levels in wild-type mice. Significant inflammatory infiltration and steatosis developed in both WT and KO mice in response to alcohol; however, no significant differences were found between the genotypes. We conclude that Trpa1 is expressed in hepatocytes and liver macrophages; however, the chronic alcohol-induced steatosis and inflammatory infiltration develop through a TRPA1-independent mechanism.

1. Introduction

Excessive alcohol consumption is the main cause of liver disease in Western countries. Pathological alcohol consumption is responsible for 3 million deaths worldwide each year, accounting for 5.3% of all deaths, and has been proven to play a causal role in more than 200 diseases. In working-age young people (20–39 years), alcohol is responsible for 13.5% of deaths [1].

The liver is the primary site of alcohol breakdown and is therefore particularly sensitive to alcohol consumption. Intermediates, as well as by-products, of alcohol metabolism damage the liver through several mechanisms, such as inhibiting lipid metabolism, increasing inflammatory processes, and triggering fibrosis, which can ultimately lead to alcoholic liver disease (ALD) [2]. ALD is the most common chronic liver disease worldwide. ALD can develop from alcoholic fatty liver (AFL) and progress to alcoholic steatohepatitis (ASH), characterized by inflammation of the liver. Chronic ASH can eventually lead to fibrosis, cirrhosis, and, in some cases, even hepatocellular carcinoma (HCC). The pathogenesis of ALD involves the fatty degeneration of the liver (hepatic steatosis), oxidative stress, toxicity caused by acetaldehyde, a harmful metabolite of alcohol, and inflammation triggered by cytokines and chemokines [3].

Alcohol is detoxified mainly in the liver through a series of oxidative metabolic processes consisting of three main steps. First, a reversible oxidation of ethanol to toxic acetaldehyde takes place. In the second step, the acetaldehyde is irreversibly converted into acetate. The third and final step is the breakdown of acetate into water and carbon dioxide. The first, oxidative metabolic step is catalyzed by alcohol dehydrogenase (ADH), cytochrome P450 family 2 subfamily E member 1 (CYP2E1), and catalase. ADH is the main oxidizing enzyme, which binds to alcohol with high affinity and converts it into acetaldehyde in the cytoplasm. CYP2E1 can be induced by chronic alcohol consumption and breaks down ethanol in peroxisomes. Through the third pathway, catalase oxidizes ethanol in microsomes.

The conversion of acetaldehyde to acetate is primarily carried out by mitochondrial aldehyde dehydrogenase (ALDH). Acetaldehyde can also be metabolized by CYP2E1 in an NADPH-dependent manner. The reaction product, acetate, is unstable and decomposes spontaneously into water and carbon dioxide. If these oxidative pathways become overloaded, acetaldehyde accumulates and exerts its toxic effects. Acetaldehyde is the most important toxic metabolite and the main mediator of the fibrogenic and mutagenic effects of alcohol in the liver. It promotes the formation of adducts, causing the functional damage of key proteins, including enzymes, and DNA damage that results ultimately in mutagenesis [4].

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable, non-selective cation channel. TRPA1 is a chemical nociceptor sensitive to various exogenous or endogenous harmful stimuli, such as irritating compounds, mechanical stimuli, reactive chemical substances, and signals associated with tissue damage and pain [5,6]. Various chemical and mechanical stresses affecting the membrane are converted into intracellular biochemical signaling via specific proteins, which can result in physiological/pathological responses [7,8]. During inflammatory processes, activated immune cells release various mediators that sensitize TRPA1 channels. Increased calcium influx indirectly regulates cytokine production through the modulation of intracellular pathways [9], including the production of proinflammatory cytokines, such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) [10,11,12,13,14]. TRPA1 also regulates local tissue inflammation and the progression of fibrosis, which is why it is currently the subject of numerous research studies [15]. TRPA1 also plays an important mediating role in the immune responses of inflammatory diseases [16,17]. It can be activated directly or indirectly by inflammatory signaling molecules or by heat and oxidants present in the inflammatory microenvironment. In addition, it participates in the activation of transcription factors and protein kinases in certain pro- and anti-inflammatory signaling cascades and is associated with neurogenic inflammation, osteoarthritis, allergic dermatitis, asthma, inflammatory bowel disease, migraine, cancer pain, gout, and other diseases, making it a promising target for drug development [18,19,20,21,22].

Based on our preliminary studies and data from the literature, we know that TRPA1 is also expressed in the liver [23,24,25]. Nevertheless, its exact localization was unclear, and due to the lack of specific antibodies, the results of immunohistochemical studies were questionable.

Endogenous agonists of TRPA1 include mediators released during inflammatory and degenerative processes (e.g., products of lipid peroxidation, free radicals, and fatty acids). Upon activation, calcium influx occurs, which influences intracellular processes and plays an important role in modulating degeneration, inflammation, or apoptosis [26]. According to the literature, activators of the TRPA1 ion channel include ethanol [27] and its breakdown products acetaldehyde [9,28,29] and acetate [30], which we hypothesize to directly affect liver cells and influence toxic liver damage mediated by TRPA1.

We hypothesize that the TRPA1 ion channel is expressed in hepatocytes of the liver and can be activated by ethanol and its degradation products; moreover, it plays a role in the pathomechanism of liver damage caused by chronic alcohol consumption. To prove our hypotheses, we set out three main aims: (a) to examine the expression of the TRPA1 ion channel in mouse liver tissue using RNAscope in situ hybridization (ISH) combined with immunofluorescent labeling, (b) to prove the functional activity of the human TRPA1 ion channel with alcohol and its breakdown products (acetaldehyde and acetate) using in vitro calcium imaging techniques on a Chinese hamster ovary (CHO) cell line overexpressing human TRPA1, and (c) to investigate the role of the TRPA1 ion channel in a mouse model of chronic alcohol exposure for 3 months involving wild-type (WT) and Trpa1-knockout (KO) mice, by evaluating blood biomarkers of liver injury and histomorphological changes characteristic for ALD.

2. Materials and Methods

2.1. Experimental Design

In our first experiment, we used liver samples from intact C57BL6/J mice. We examined Trpa1 mRNA expression by RNAscope ISH combined with ionized calcium-binding adaptor molecule 1 (IBA1) and CD68 immunostaining to identify liver macrophages; moreover, arginase-1 immunolabeling was used to detect hepatocytes.

In our second experiment, we used calcium-imaging techniques to examine whether alcohol and its metabolites (acetaldehyde and acetate) are capable of activating the TRPA1 ion channel in a TRPA1-overexpressing CHO cell line (CHO-K1, Cat. No.: ECACC#85051005; European Collection of Authenticated Cell Culture, Middlesex, United Kingdom).

In our third experiment, we examined the livers of Trpa1 KO and WT mice in a chronic alcohol drinking mouse model. We determined plasma liver enzyme (aspartate aminotransferase (AST) and alanine aminotransferase (ALT)) levels. We performed a morphometrical analysis of mouse liver sections upon periodic acid Schiff reaction (PAS) staining. Based on the evaluation of interface inflammatory infiltration, portal inflammatory infiltration and steatosis, we developed a scoring system to obtain a dataset allowing for reliable statistical assessment.

2.2. Animals

Trpa1 mRNA expression was detected in C57BL6/J mice obtained from Jackson Laboratories. The original Trpa1 WT and KO mouse breeding pairs used for the alcohol models were provided by Prof. P. Geppetti (University of Florence, Italy). In this model, Trpa1 is not completely deleted at the genomic level; instead, a targeted disruption was introduced that removes a critical exon encoding an essential region of the TRPA1 ion channel. As a result, the channel protein is nonfunctional, and TRPA1-mediated currents are completely abolished. This genetic design does not eliminate transcription from the Trpa1 locus. Consequently, Trpa1 mRNA can still be detected in KO tissue. For this reason, the detection of Trpa1 transcripts by PCR or RNAscope ISH does not indicate the presence of a functional TRPA1 channel in this model. The absence of TRPA1 protein function in the KO line has been conclusively demonstrated by electrophysiological tools, indicating the lack of TRPA1-dependent currents, and by a pharmacological approach utilizing TRPA1 agonists with obvious behavioral outcomes. Thus, the loss of channel function—rather than complete absence of Trpa1 mRNA—is the defining and biologically relevant feature of this knockout model [31]. Mice were bred in the animal facility of the Preclinical Research Center of the University of Pécs.

Animals were housed in the animal facility of the Department of Pharmacology and Pharmacotherapy at the University of Pécs in standard polycarbonate cages (365 mm × 207 mm × 140 mm, Sealsave IVC cage, Cat. No: 1248L, Tecniplast, Buggugiate, Italy) under controlled temperature and humidity and a 12 h light–dark cycle. Mice were fed ad libitum with standard rodent chow. Control mice had unlimited access to tap water at all times. In contrast, the alcohol-treated group was not provided with tap water at all, but their only source of fluid was 20% ethanol solution. All procedures were performed in accordance with the animal welfare license issued by the Animal Welfare Committee and the Scientific Ethics Council for Animal Experiments of the University of Pécs (BA02/2000-25/2021; issue date: 2 September 2021) in accordance with the 1986 guidelines of the European Commission and Act XXVIII of 1998 on the protection and welfare of animals. Microbiological monitoring was performed annually (FELASA “S”). The authors complied with the ARRIVE guidelines.

2.3. Calcium-Imaging

The effects of ethanol, acetaldehyde, and acetic acid at 10 mM concentrations were tested on CHO cells expressing human TRPA1 receptors. The volatile quality of acetaldehyde made the pipetting of concentrated solutions impossible, so the highly concentrated solutions were measured by mass and always stored in completely filled tubes to prevent evaporation. The cells were plated on ibidi 96-well square plates (#89626) at a density of around 80% confluency on the day of measurement. For each experiment, at least 6 wells were used on two different days. The Fura-2 AM dye was purchased from Invitrogen (#F1221, Carlsbad, CA, USA) and dissolved in DMSO at a 1 µg/µL concentration. In each well, the cells were stained in 200 µL DMEM medium containing 0.5 µg Fura-2 dye for 30–45 min. Then, the dye was removed, the cells were washed with 200 µL extracellular solution (ECS), and finally, 200 µL ECS was added to each well. Fluorescence (335 nm and 380 nm excitation, 520 nm emission) was measured on a ClarioStar Plus plate reader machine (BMG Labtech, Offenburg, Germany) in plate mode once every minute. First, a baseline was established by four measurement points. Then, the different compounds were added by the machine in 100 µL volumes of ECS to reach the final concentration of 10 mM in the well, and the fluorescence was measured for 25 min. Finally, as a positive control to test the reaction of the cells, mustard oil was injected in 50 µL ECS to reach a 100 µM final concentration in the well, and the reaction was monitored for an additional 2 min. The Fura-2 340/380 ratio was calculated from the raw data by the Mars analysis software (Version V5.02 R5) of the plate reader. The fluorescent signals were measured in each well once per minute (1/min sampling rate) with the exception of the 4th and 29th minutes when the injection of the examined compound and the control mustard oil occurred, respectively. In single-point statistical analysis, the peak signal between 5 and 28 min of each curve was compared to the signal at 3 min just before the compound was added. For area under the curve analysis, first, a baseline for each curve was established by averaging the first four measurements (at 0–3 min time points), then the difference between the baseline and each data point between 5 and 28 min was added together to obtain the area under the curve, for the whole time frame that the compound took effect. No significant differences were observed in the calculated baseline Fura-2 ratios between TRPA1-expressing and control TRPA1-negative CHO cells. Statistical analysis of all the differences was done by the Welch t-test (Graphpad online version).

2.4. Mouse Model of Chronic Alcohol Consumption

Briefly, 12-week-old male mice were handled for two weeks. The animals were randomly assigned to four experimental groups: half of the animals were assigned to the control group, and the other half to the alcohol-treated group, which was further divided into subgroups according to genotype: WT and Trpa1 KO mice (n = 10–12/group). No animals were excluded from the study, and no mortality or humane endpoint-related exclusions occurred during the experimental period. Members of the control group consumed tap water ad libitum, while mice in the alcohol groups were subjected to chronic alcohol consumption for 3 months, during which they were allowed to drink only a 20% alcohol solution dissolved in tap water as their sole source of fluid. In the week prior to the start of the experiment, the mice were habituated to the alcoholic solution. We gradually increased its concentration from 5% to 20%, increasing by 5% every two days [32,33,34]. At the end of the experiment, the mice were transcardially perfused, and liver samples were collected.

2.5. Perfusion and Sample Collection

Deep anesthesia was induced with intraperitoneal urethane (2.4 g/kg). Loss of pain reflexes was verified by tail pinch. After anesthesia, body weight was measured, and tail samples were collected for later genotype verification. The chest cavity was quickly opened; 500 µL blood was collected by a left ventricular puncture into a pre-chilled syringe pre-filled with 50 µL 7.5 m/m% EDTA (Sigma, Zwijndrecht, The Netherlands) solution. The blood samples were filled into pre-chilled tubes and centrifuged at 3000 g for 5 min. Supernatant plasma samples were collected and stored at −20 °C for liver enzyme measurements.

After blood sampling, mice were transcardially perfused with 50 mL of ice-cold 0.1 M phosphate-buffered saline (PBS) (pH: 7.4), then 150 mL of 4% paraformaldehyde (PFA, Sigma) solution dissolved in Millonig buffer (pH 7.4). Liver tissue samples were collected after perfusion, in all cases from the left lobe, and then post-fixed in PFA solution at 4 °C for 72 h. The samples were then embedded in paraffin and sectioned at 5 µm by a microtome (Thermo Fisher Scientific, Waltham, MA, USA). Sections were mounted on Superfrost Ultra Plus slides (Thermo Fisher Scientific) and subjected to PAS–hematoxylin (PAS-H) staining or RNAscope combined with immunofluorescence.

2.6. Plasma Liver Enzyme Measurements

Plasma AST and ALT levels were measured from blood samples collected before the perfusion, using an automated routine enzymatic colorimetric assay. The assay was performed at an accredited laboratory (Department of Laboratory Medicine, Medical School, University of Pécs, Hungary) utilizing the Cobas Pro c503 analyzer (Roche Diagnostics GmbH, Mannheim, Germany).

2.7. PAS–Hematoxylin Staining

The slides were treated with xylene three times for 5 min, then incubated in a series of alcohol solutions of gradually decreasing concentration (100%, 96%, and 70%), and finally rinsed with distilled water. The samples were then held in 0.5% periodic acid for 5–10 min and washed again with distilled water. The samples were then treated with Schiff’s reagent (BioGnost, Zagreb, Croatia) for 15 min, rinsed with tap water for 5 min to remove excess dye, and finally stained with hematoxylin to visualize the cell nuclei. The sections were then placed in xylene for 3 min twice and covered with a water-free mounting medium (DePeX, Fluka Chemie AG, Buchs, Switzerland).

2.8. RNAscope ISH Combined with Immunostaining

RNAscope ISH technique was used to detect Trpa1 mRNA expression in paraffin-embedded liver sections [35]. The mounted sections were dried at room temperature for 3 h and then incubated at 60 °C for 60 min. We applied the protocol suggested in the RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD, Hayward, CA, USA) manual for pretreatment, probe hybridization, signal amplification, and channel development. Trpa1 mRNA was detected by a mouse-specific Trpa1 probe (ACD, Cat. No. 400211) and visualized by cyanine 3 (Cy3) dye at a dilution of 1:750.

As technical controls, we used mouse 3-plex positive control probes (ACD; Cat. No.: 320881) and 3-plex negative control probes (ACD; Cat. No.: 320871). The 3-plex positive control probes gave a well-detectable signal in the liver, while the negative control probes did not give any recognizable fluorescence.

Immunofluorescence labeling was performed after completing the RNAscope ISH protocol. The samples were incubated with rabbit IBA1 (1:1000; Cat. No: 019-19741; FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany; RIDD: AB_839504) and mouse CD68 (1:200; Cat. No: M087601; Agilent, Santa Clara, CA, USA; RIDD: AB_2074844) primary antibodies for 24 h at 24 °C to identify macrophages. Another set of samples was incubated with rabbit arginase-1 (1:50; Cat. No: ACI3058; Biocare Medical, Pacheco, CA, USA) primary antibody to identify the hepatocytes. After two 15 min washes with PBS, the samples were incubated with Alexa 488-conjugated donkey anti-rabbit (Cat. No: 711-545-152; Jackson Immunoresearch, Ely, UK) or Alexa 647-conjugated donkey anti-mouse (Cat. No: 715-605-150; Jackson Immunoresearch) or Alexa 647-conjugated donkey anti-rabbit (Cat. No: 711-607-003; Jackson Immunoresearch) secondary antibodies, respectively, at (1:500) for 3 h. After 2 × 15 min washes with PBS, the sections were treated with 4′,6-diamidino-2-phenylindole (DAPI) for the visualization of cell nuclei. After washes, the slides were covered with ProLong Gold Antifade (Thermo Fisher Scientific, Cat. No. P10144) medium and stored at −20 °C until digitization.

2.9. Microscopy and Morphometry

A Nikon Microphot FXA microscope was used to examine PAS-H-stained sections, which were digitized using a Spot RT color digital camera (Nikon, Tokyo, Japan). During sampling, the left hepatic lobe was consistently removed from all experimental animals and its entire cross-section was systematically scanned with a light microscope, and inflammatory foci were counted across the complete cross-sectional area. We developed a scoring system to evaluate the liver damage, which allowed for the reliable, unbiased assessment of the severity of lesions resulting from chronic alcohol exposure. Three parameters were evaluated in the scoring system: the number of interface and portal inflammatory infiltrates and the percentage of steatosis in the entire cross-section surface area. Portal and interface inflammatory infiltrates were evaluated separately, and inflammatory foci were counted per entire cross-section. Interface inflammation was considered a marker of more severe liver injury, as inflammatory cells breach the limiting plate and come into direct contact with hepatocytes, which may lead to hepatocellular damage, bridging necrosis, and subsequent fibrotic changes. The degree of inflammatory infiltration was scored in increments of five foci: 0–5 foci = 0 points, 6–10 = 1 point, 11–15 = 2 points, 16–20 = 3 points, 21–25 = 4 points and 26–30 inflammatory foci corresponded to 5 points. Steatosis was quantified as the percentage of the total cross-sectional area affected and scored in 20% increments: absence of steatosis = 0 points, 0–20% = 1 point, 21–40% = 2 points, 41–60% = 3 points, 61–80% = 4 points and 81–100% = 5 points (

Table 1. Histological scoring system used for liver injury assessment.

Inflammatory Infiltration Score		Steatosis Score
Score	Number of Inflammatory Foci	Score	Percentage of Hepatocytes Affected
0	0–5	0	None
1	6–10	1	0–20%
2	11–15	2	21–40%
3	16–20	3	41–60%
4	21–25	4	61–80%
5	26–30	5	81–100%

). The histological assessment was performed by an expert histopathologist who was not aware of the identity of the preparations. This blinded assessment was preferred to avoid human bias.

Sections labeled with RNAscope ISH and immunofluorescence staining were digitized using an Olympus FluoView 1000 confocal microscope (Olympus, Europe, Hamburg, Germany). Digital images were recorded with sequential scanning for each fluorophore to ensure the reliable detection of fluorescent signals and to avoid false positivity from overlapping emission spectra. During scanning, the confocal aperture was set to 80 µm, the optical layer thickness to 3.5 µm, and a 40× and 60× magnification objective and 1024 × 1024 pixel resolution were used. We selected the excitation and emission spectra for the fluorophores using the built-in parameters of the FluoView software (FV10-ASW; Version 0102, Olympus, Europe, Hamburg, Germany). DAPI was excited at 405 nm, Alexa 488 at 488 nm, Cy3 at 550 nm and Alexa 647 at 647 nm by laser beams. The samples were recorded on all three channels at the appropriate wavelengths, and the resulting images were digitally merged. The following three virtual colors were assigned to the fluorophores: DAPI—blue, Alexa 488—green, Cy3—red and Alexa 647—white.

2.10. Statistics

The data are expressed as the mean ± standard error of the mean (SEM) for each experimental group. The normality of the data sets was tested using the Shapiro–Wilk test [36], and the homogeneity of variances was tested using Bartlett’s chi-square test [37]. Statistical analysis was performed using Statistica 8.0 (StatSoft, Tulsa, OK, USA) software (alpha = 5%). The data were analyzed using two-way analysis of variance (ANOVA; variables: treatment and genotype), followed by Tukey’s post hoc test. Further post hoc evaluation was performed based on the first- or second-order effects obtained in the ANOVA tests. Spearman’s rank test was also applied to search for correlations. Datasets obtained in the calcium-imaging were evaluated by Welch’s t-test.

3. Results

3.1. The Trpa1 mRNA Is Expressed in Mouse Hepatocytes and Liver Macrophages

We confirmed that Trpa1 mRNA is expressed in the liver (Figure 1) using ultrasensitive RNAscope ISH combined with immunohistochemistry. Arginase-1 was used to identify the hepatocytes, which were Trpa1 positive (Figure 1A,A’). CD68, combined with IBA1 immunofluorescence staining, was used as a marker of macrophages. The CD68 and IBA1 staining was necessary because hepatocytes and liver macrophages cannot be unambiguously distinguished by assessing their nuclear morphology exclusively. Based on the localization of the fluorescent signals, transcripts can be found in liver macrophages (Figure 1B–D’) as well as in hepatocytes with large, round nuclei (Figure 1D’).

The distribution of Trpa1 mRNA signal dots appeared to be relatively diffuse. The Trpa1 was not enriched in either periportal or centrilobular areas; moreover, the expression did not exert any specific pattern of lobular distribution.

3.2. Acetaldehyde Is Able to Activate Human TRPA1

No significant differences were observed in the calculated baseline Fura-2 ratios between TRPA1-expressing and control TRPA1-negative CHO cells.

The results show that neither ethanol nor acetic acid was able to activate the human TRPA1 receptor at the 1–10 mM concentration range (Figure 2). Low acetaldehyde concentrations were similarly not effective, but 10 mM acetaldehyde showed a weak and slow activation, at least compared to the 100 µM mustard oil reaction. The peak of the reaction was reached 6 min after the exposure to the acetaldehyde and slowly decreased over time. The maximum reaction to 10 mM acetaldehyde at time point 10 min (relative to time point 3 min) was one-third compared to the control mustard oil reaction at time point 30 min (relative to time point 3 min), 0.0772 ± 0.0336 ratio change compared to 0.265 ± 0.116 ratio change (mean ± SD), respectively. The statistical analysis of the Fura-2 signal increase caused by 10 mM acetaldehyde showed a significant difference between the TRPA1 expressing and TRPA1 negative control cells at the 10 min time point compared to the corresponding 3 min time point (Welch’s t-test: mean ± SD for TRPA1-negative cells: 0.0275 ± 0.0122, n = 12; mean ± SD for TRPA1+ cells: 0.0772 ± 0.0336, n = 18; t = 5.7442, df = 22, SEM_delta = 0.009, 95%CI_delta = 0.0318–0.0677, p < 0.0001. Yuen–Welch test: 20% trimming, mean ± SD for TRPA1-negative cells: 0.0280 ± 0.0114, n = 10; mean ± SD for TRPA1+ cells: 0.0764 ± 0.0265, n = 14, t = 6.1011, df = 18, SEM_delta = 0.008, 95%CI_delta = 0.0318–0.0651, p < 0.0001).

Similarly, the comparison of the area under the curve between 5 and 28 min indicated a significant difference between TRPA1+ and control cells exposed to 10 mM acetaldehyde. (Welch’s t-test: mean ± SD for TRPA1-negative cells: 0.3552 ± 0.2762, n = 12; mean ± SD for TRPA1+ cells: 1.360 ± 0.4786, n = 18; t = 6.5575, df = 28, SEM_delta = 0.153, 95%CI_delta = 0.6909–1.3187, p < 0.0001. Yuen–Welch test: 20% trimming, mean ± SD for TRPA1-negative cells: 0.3562 ± 0.2189, n = 10; mean ± SD for TRPA1+ cells: 1.3283 ± 0.4439, n = 14; t = 6.3654, df = 22, SEM_delta = 0.153, 95%CI_delta = 0.6554–1.2889, p < 0.0001).

3.3. Alcoholic Inflammatory Infiltration and Steatosis Mediated Through a TRPA1-Independent Mechanism in Mice

We examined the effects of chronic alcohol exposure over three months in the liver using WT and Trpa1 KO mice. According to ANOVA, neither genotype nor alcohol exposure had a significant main effect on liver enzyme levels; however, a strong interaction was found between the variables for both AST (p = 0.02) and ALT (p = 0.0009). There was no baseline difference between the two genotypes for AST, but alcohol exposure significantly increased the hepatic enzyme levels in WT mice, according to the post hoc comparison (p = 0.04), which was not observed in KO mice (Figure 3A). When assessing ALT, we measured a higher basal level in the absence of the functional Trpa1 gene (p = 0.004). Interestingly, ALT increased in WT mice upon alcohol exposure (p = 0.001), while it remained unchanged in KO animals, according to the post hoc tests (Figure 3B).

By the morphometric evaluation of PAS-H-stained liver samples, both alcohol treatment (p = 3.175 × 10⁻⁷) and genotype (p = 0.0245) had a significant main effect on portal inflammatory infiltration according to the two-way ANOVA, without interaction (p = 0.786). There was no statistically significant baseline difference between genotypes, based on the results of the post hoc comparison. At the same time, there was a significant increase in portal inflammatory infiltration in both the WT (p = 0.00066) and KO (p = 0.00063) genotypes (Figure 4A,B).

The examination of the interface inflammatory infiltration showed that only the alcohol treatment had a significant main effect (p = 0.0006) according to ANOVA, while genotypes did not (p = 0.373). There was no significant interaction between the factors (p = 0.521). According to post hoc tests, alcohol treatment significantly increased the interface inflammatory infiltration in WT mice compared to the control (p = 0.0123) (Figure 4A,C).

When evaluating the steatosis score using a two-way ANOVA, the genotype had no significant effect (p = 0.438); however, alcohol treatment did have a significant effect (p = 5.737 × 10⁻⁵), with no interaction (p = 0.438). Based on the post hoc comparisons, the steatosis score increased significantly in both the WT (p = 0.0452) and KO genotypes (p = 0.00432). However, no significant difference was observed between genotypes (Figure 4A,D).

Results of the correlation analysis using Spearman’s rank correlation revealed significant positive associations among all histological parameters, including steatosis, portal inflammatory infiltration, and interface inflammatory infiltration. In addition, a significant positive correlation was observed between the liver enzymes AST and ALT. However, no significant correlations were identified between any of the histological parameters and the liver enzyme levels.

Table 2. Results of correlation analyses. Significant positive correlations were observed among all of the histological parameters and among the plasma concentrations of liver enzymes. No significant correlations were detected between histological parameters and liver enzyme levels. AST: aspartate aminotransferase; ALT: alanine aminotransferase; r: Spearman’s rank correlation coefficient. Significant positive correlations are highlighted in bold.

	Steatosis	Portal Inflammatory Infiltration	Interface Inflammatory Infiltration	AST	ALT
Steatosis	×	r = 0.52 p = 3.5126 × 10−4	r = 0.45 p = 2.411 × 10−3	r = 0.19 p = 0.287	r = 0.06 p = 0.711
Portal inflammatory infiltration	r = 0.52 p = 3.5126 × 10−4	×	r = 0.71 p = 9.645 × 10−8	r = 0.26 p = 0.135	r = 0.26 p = 0.144
Interface inflammatory infiltration	r = 0.45 p = 2.411 × 10−3	r = 0.71 p = 9.645 × 10−8	×	r = 0.32 p = 0.07	r = 0.25 p = 0.156
AST	r = 0.19 p = 0.287	r = 0.26 p = 0.135	r = 0.32 p = 0.07	×	r = 0.63 p = 9.091 × 10−5
ALT	r = 0.06 p = 0.711	r = 0.26 p = 0.144	r = 0.25 p = 0.156	r = 0.63 p = 9.091 × 10−5	×

includes the correlation coefficients and p-values, where the significant positive correlations are highlighted in bold.

4. Discussion

We examined Trpa1 mRNA expression in mouse liver tissue. There are several reports in the literature on the expression of the TRPA1 ion channel in mouse [23,24,25] and human livers [38], but these data are contradictory. Several studies have shown that the reliability of widely used anti-TRPA1 antibodies is questionable [39,40,41]. Other research groups have used PCR-based assays for studies at the mRNA level. An important limitation of this method is that Trpa1 mRNA is detected in samples prepared from tissue homogenates, that lacks the morphological context. More particularly, they do not provide unambiguous evidence on the identity of hepatic cells that express the TRPA1 ion channel.

Here, we demonstrate Trpa1 mRNA expression in the liver by RNAscope ISH, an ultrasensitive and specific technique providing evident morphological information to localize the mRNA. Using multiplex labeling, some of the Trpa1-positive cells were identified as macrophages, while based on nuclear morphology and arginase-1 labeling, the other cells with Trpa1 mRNA content correspond to hepatocytes. We have to state that this morphological mRNA localization was not complemented with protein-level or functional validation. Nevertheless, inflammatory and toxic hepatic states recruit not only mobile immune cells but also the resident liver cells, i.e., hepatocytes and Kupffer cells [42,43,44]. Therefore, the observation that TRPA1 is expressed in these cells suggests that this ion channel may contribute to the regulation of inflammatory and toxic responses in the liver.

Considering the different functional roles of hepatocytes and Kupffer cells, TRPA1 activation is likely to produce cell-type-specific effects in the liver. Hepatocytes, beyond their metabolic and detoxifying functions, actively participate in the control of liver inflammation. They respond to cellular and organelle stress by release of pro-inflammatory mediators [45]. In this context, TRPA1 activation in hepatocytes may primarily modulate metabolic stress responses, redox imbalance, and stress-induced inflammatory signaling.

In contrast, Kupffer cells are liver-resident macrophages that orchestrate innate immune responses and undergo functional polarization under pathological conditions [45,46]. Therefore, TRPA1 activation in Kupffer cells may influence macrophage activation states, cytokine production, and immune cell recruitment, amplifying inflammatory responses. These distinct, cell-specific, TRPA1-dependent signaling pathways could contribute differently to metabolic injury versus immune-driven inflammation during alcohol-associated liver disease.

Numerous studies have confirmed that the mouse TRPA1 ion channel can be activated by ethanol [27] and its breakdown products acetaldehyde [9,28,29] and acetate [30], which also lead to calcium influx. Due to its relevance to human translation, our research group investigated the ion channel in vitro using calcium-imaging techniques on a CHO cell line overexpressing human TRPA1. We found that the ion channel can be activated by acetaldehyde, which is known to be the main mediator of alcoholic liver damage [4,47,48]. These findings are consistent with our previous in silico studies, where we showed that acetaldehyde is the most potent activator of human TRPA1 among alcohol and its metabolites [49]. While acetaldehyde is capable of inducing moderate activation of the channel at supraphysiological concentrations in vitro, this activation is unlikely to be a major determinant in vivo. The activation observed in Figure 2 is likely pharmacological rather than physiological in the context of in vivo liver concentration. Since the in vitro studies were performed on CHO cells, their in vivo translation to humans is limited. We plan to conduct further studies on human liver cell lines to determine the acetaldehyde concentration required to activate the TRPA1 ion channel in human hepatocytes.

According to the results of our current in vivo study, ethanol and acetic acid are unable to activate the human TRPA1 ion channel.

We compared Trpa1 WT and KO mice in an in vivo model of chronic alcohol consumption. The animals had ad libitum access to a 20% ethanol solution (which was their exclusive source of fluid), while the control groups received tap water. The experimental protocol was based on previously described, well-accepted, and widely used methods [32,34]. The 3-month period of alcohol consumption corresponds to approximately 10–12 years of alcoholism, expressed in human years.

It is well-known that liver damage induced by heavy drinking causes a rise in hepatic enzyme levels [50,51], which was confirmed in our WT mice. Importantly, we observed that KO animals exerted increased baseline ALT levels, which did not show an additional rise under the influence of alcohol. The significantly higher baseline ALT levels in Trpa1 KO mice suggest the role of TRPA1 as a stress sensor that modulates hepatic metabolic regulation and oxidative stress signaling. TRPA1 activation attenuates mitochondrial dysfunction and lipid accumulation in models of diet-induced steatotic liver disease, while Trpa1 deficiency exacerbates these metabolic disturbances. This suggests an inherent role for TRPA1 in maintaining basal hepatic metabolic homeostasis [52]. Indeed, TRPA1, similarly to other related TRP channels, functions as a cellular danger and oxidative stress sensor involved in neuroimmune coordination and tissue protection, connecting energy balance and cellular stress responses to metabolic processes. Therefore, loss of TRPA1 may compromise these stress-adaptive protective pathways, potentially increasing basal hepatocellular stress and baseline ALT levels [53,54].

Liver samples were examined using PAS-H staining to assess morphological changes in portal- and interface-type inflammatory infiltration and steatosis. Inflammation and steatosis developed both in WT and KO mice following alcohol treatment, confirming the validity of the model [55,56,57,58]. Plasma transaminases and histological injury scores reflect overlapping but distinct aspects of liver damage. ALT and AST elevations primarily indicate altered hepatocellular membrane permeability and enzyme leakage, whereas increased histological damage scores emphasize structural changes such as steatosis, inflammation, and necrosis. Consequently, discrepancies between biochemical and histological measures are well recognized in both experimental models and human alcoholic liver disease [59,60,61].

In alcohol-related liver injury, increases in plasma ALT and AST levels may occur that might not be accompanied by proportional changes in conventional histologic scores, because, especially in the early stages of the disease or when the injury is focal, the damage remains predominantly metabolic yet without obvious morphological consequences. In line with this, previous studies have shown that transaminase elevations may reflect hepatocellular stress, mitochondrial dysfunction, or sublethal injury that is not fully captured by semi-quantitative histological scoring approaches. Indeed, correlations between plasma transaminase levels and severity of histological alterations are frequently modest or inconsistent in alcoholic liver disease. Several clinical and preclinical studies report weak associations between ALT/AST and histological signs of inflammation or necrosis, underscoring that transaminases are sensitive but not specific indicators of structural liver injury [62,63]. Consistently, we found no significant correlations between ALT/AST levels and histology scores of the individual animals, suggesting that the biochemical injury signals may reflect hepatocellular injury mechanisms not fully represented by the applied histologic scoring system. This highlights the importance of integrating biochemical and histologic readouts rather than interpreting them as independent or redundant measures [64].

Although inflammation and steatosis also developed in Trpa1 KO mice following chronic alcohol exposure, we found no significant difference in the degree of liver damage compared to the wild-type genotype in the absence of Trpa1.

This result is surprising in light of our original assumption that acetaldehyde, which is known to play a major role in alcoholic liver damage [3], contributes to the development of ALD through TRPA1 activation, among other pathways. Accordingly, we expected milder damage in the KO genotype. Therefore, our results suggest that TRPA1 may play a subordinate role in the process of alcohol-induced inflammation and steatosis in the liver, mediated probably by TRPA1-independent mechanisms.

Interestingly, TRPA1 also influences the regulation of tissue fibrosis. Therefore, TRPA1 is currently the focus of numerous studies related to heart, kidney, pancreas, intestine, bladder, corneal, synovial and dermal fibroses [15,25,65,66,67,68,69,70,71,72,73,74,75]. TRPA1 signaling has been linked to fibroblast activation, calcium-dependent signaling, chronic inflammatory responses, and extracellular matrix remodeling in these organs. These processes are commonly involved in fibrotic progression. Hepatic fibrogenesis shares key mechanistic features with these extrahepatic fibrotic conditions, including inflammation-driven stromal cell activation and sustained stress signaling. Based on our own findings and those reported in the literature, we speculate that TRPA1 may play a role in the development of fibrosis in the later stages of ALD. Notably, since the current 3-month ethanol feeding model primarily captures steatosis and inflammation rather than advanced fibrosis, the proposed role of TRPA1 should be considered preliminary and requires direct experimental validation. We plan to conduct further, longer-term experiments to investigate this in the future.

As to the limitations, we used developmental Trpa1 KO mice in our experiments. Therefore, it cannot be ruled out that compensatory mechanisms have developed as a result of gene deficiency, such as the upregulation of other TRP and/or calcium channels expressed in the liver. This ultimately may have increased calcium influx into the cells, thus increasing the extent of toxic liver damage even in the absence of TRPA1. Based on data from the literature, in the absence of TRPA1, potential candidates for compensatory changes include TRPV1, TRPV3, and TRPV4 channels known to be involved in hepatocellular injury and inflammatory signaling. Furthermore, TRPC1 and TRPC6 may regulate calcium homeostasis and hepatic stellate cell activation, while TRPM2 is a redox-sensitive channel implicated in oxidative stress-induced liver injury [76].

An additional limitation of the study is the exclusive use of male mice. This experimental design was chosen to minimize variability related to sex-dependent differences in alcohol consumption patterns and to avoid potential confounding effects of estrous cycle-associated hormonal fluctuations in female mice. Nevertheless, in the view that alcohol-related behaviors, metabolic regulation and underlying disease mechanisms may differ between sexes, moreover, liver enzyme levels may depend on the actual sex hormone levels [77] and related gynecological diseases [78]; the use of male mice only limits the generalizability of our findings [79,80].

5. Conclusions

Our research group has successfully proved the Trpa1 mRNA expression in the liver using RNAscope ISH on hepatocytes and liver macrophages; moreover, we have confirmed that acetaldehyde is capable of activating human TRPA1. Although the present findings suggest that steatosis and inflammatory infiltration resulting from chronic alcohol exposure are thought to occur mainly through a TRPA1-independent mechanism, the question of whether the ion channel plays a role in ALD-associated late-stage hepatic fibrosis requires future experimentation. Confirmation of TPRA1-mediated mechanisms in the background of late-stage toxic liver damage may define TRPA1 as a new potential drug target for mitigating the harmful effects of ALD.

6. Future Directions

Although the current study shows that TRPA1 is not essential for the initial histopathological and biochemical characteristics of chronic alcohol-induced liver injury, mounting evidence indicates a possible role for TRPA1 signaling in fibrotic processes in other organs, such as the lungs, kidneys, heart and skin [73,81]. In these systems, TRPA1 activation has been associated with fibroblast activation, extracellular matrix remodeling, and chronic inflammatory signaling. Based on these observations, we hypothesize that TRPA1 contributes to the later stages of alcohol-associated liver disease, especially the transition from inflammation to fibrosis.

The three-month, 20% ethanol drinking protocol used in the present study primarily models the early-to-mid stages of alcohol-associated liver disease. This paradigm is not optimized to induce advanced fibrotic remodeling. Therefore, to address the potential contribution of TRPA1 to hepatic fibrosis, experimental approaches that promote progressive fibrogenesis are required, such as extended-duration alcohol exposure or binge-on-chronic alcohol models.

Future studies will therefore directly assess TRPA1’s involvement in hepatic fibrogenesis using extended alcohol exposure protocols and fibrosis-specific endpoints. These studies will include histological fibrosis-specific staining (e.g., Sirius Red), a quantitative assessment of collagen deposition, an analysis of hepatic stellate cell activation markers (e.g., α-SMA), profibrotic signaling pathway evaluation (e.g., TGF-β) and pharmacological inhibition of TRPA1 signaling. These experiments are essential to determine whether TRPA1 is a context-dependent modulator of liver fibrosis and a viable therapeutic target in advanced stages of alcohol-related liver disease.

In addition, future studies incorporating female mice will be required to determine whether sex-specific differences in alcohol intake, hormonal modulation, and TRPA1-dependent signaling contribute to alcohol-induced liver injury and disease progression.