Imidazole Alkaloids Epiisopilosine and Epiisopiloturine Attenuate Acetaminophen‐Induced Liver Toxicity in Mice via Autophagy Modulation and Anti‐Inflammatory Effects
Ana Patricia de Oliveira, Gabriella Pacheco, André Luis Fernandes Lopes, Andreza Ketly da Silva Araujo, Letícia de Sousa Chaves, Simone de Araújo, Erick Bryan de Sousa Lima, Even Herlany Pereira Alves, Celso Martins Queiroz‐Junior, Mauro Martins Teixeira
TL;DR
Two natural compounds from Jaborandi plant reduce liver damage in mice caused by acetaminophen toxicity through autophagy and anti-inflammatory effects.
Contribution
The study demonstrates that epiisopilosine and epiisopiloturine protect the liver via autophagy modulation and inflammation suppression.
Findings
Epiisopilosine and epiisopiloturine significantly reduce APAP-induced liver toxicity in mice.
The compounds' protective effects are partially mediated through autophagy enhancement.
Inflammation and oxidative stress markers are reduced by these alkaloids.
Abstract
There is increasing interest in natural metabolites, such as alkaloids, due to their potential in treating liver diseases, including acetaminophen (APAP)‐induced hepatotoxicity. Alkaloids are known to modulate autophagy, a mechanism associated with liver protection. Epiisopilosine (EPIIS) and epiisopiloturine (EPI), imidazole alkaloids derived from Pilocarpus microphyllus (Jaborandi), exhibit anti‐inflammatory and hepatic immunomodulatory effects. Therefore, this study aimed to compare the hepatoprotective and autophagy‐modulating effects of EPI and EPIIS in a murine model of APAP‐induced hepatotoxicity. In the experimental design, male BALB/c mice received APAP (750 mg/kg, i.p.) to induce hepatotoxicity, followed by phosphate‐buffered saline (PBS), N‐acetylcysteine (NAC‐318 mg/kg, i.p.), or alkaloids (0.3, 1, or 3 mg/kg, i.p.) 30 min later. To assess the involvement of autophagy,…
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Figure 7| Complex (Ligand‐Protein) | ΔGbind
| Ki | Amino acids that interact through hydrogen bonds | Amino acids that perform hydrophobic interaction |
|---|---|---|---|---|
| Epiisopilosine‐2R3Z (1° active site) | −6.24 | 26.85 µM | Ile24 | Lys46, Arg22, Ala23, Met21, Ala32, Ser33, Gly25 and Pro31 |
| Epiisopilosine‐2R3Z (2° active site) | −6.17 | 29.87 µM | Ile24 and Gly25 | Lys46, Arg22, Ala32, Ala23, Met21, Pro31 and Ser33 |
| Epiisopilosine‐2R3Z (3° active site) | −6.22 | 27.75 µM | Ile24 and Gly25 | Lys46, Arg22, Ala32, Ala23, Met21, Pro31, Ser33 and Ile30 |
| Epiisopilosine‐2R3Z (4° active site) | −6.22 | 27.78 µM | Ile24 | Ala23, Arg22, Met21, Ala32, Ser33, Pro31, Ile30, Gly25 and Lys46 |
| Epiisopilosine‐2R3Z (5° active site) | −5.11 | 181.08 µM | Cys53 | Ile14, His13, Asp15, Glu57, Glu50, Gln51 and Arg52 |
| Epiisopilosine‐2R3Z (6° active site) | −6.17 | 29.91 µM | Arg5 and Ser35 | Thr6, Val7, Arg8 and Leu34 |
| Epiisopilosine‐2R3Z (7° active site) | −6.69 | 12.52 µM | Gln51, Pro2, Ile1, Leu3, Ile12, Cys11 and Cys53 | Asn10 |
| Epiisopiloturine‐2R3Z (1° active site) | −6.67 | 12.82 µM | Met21, Lys46 and Ala32 | Ala23, Arg22, Ser33, Gly25, Pro31, Ile30 and Ile24 |
| Epiisopiloturine‐2R3Z (2° active site) | −6.66 | 13.13 µM | Ala32, Lys46 and Met21 | Ile30, Ser33, Gly25, Pro31, Arg22, Ala23 and Il24 |
| Epiisopiloturine‐2R3Z (3° active site) | −6.67 | 12.82 µM | Ala32, Lys46 and Met21 | Pro31, Ile30, Gly25, Ser33, Ile24, Ala23 and Arg22 |
| Epiisopiloturine‐2R3Z (4° active site) | −6.3 | 24.1 µM | Ala32, Lys46, Ala23 and Ile24 | Arg22, Leu34, Ser33, Pro31, Ile30 and Gly25 |
| Epiisopiloturine‐2R3Z (5° active site) | −5.71 | 65.14 µM | Thr6, Ser35 and Arg5 | Thr6, Val7, Arg8 and Leu34 |
| Epiisopiloturine‐2R3Z (6° active site) | −5.71 | 65.14 µM | Thr6, Ser35 and Arg5 | Thr6, Val7, Arg8 and Leu34 |
| Epiisopiloturine‐2R3Z (7° active site) | −6.5 | 17.21 µM | His13, Ile12 and Ile1 | Arg5, Leu3, Gln51, Ile42, Cys53 and Asn10 |
- —Conselho Nacional de Desenvolvimento CientÍfico e TecnolÓgico10.13039/501100003593
- —CoordenaÇÃo de AperfeiÇoamento de Pessoal de NÍvel Superior10.13039/501100002322
- —Conselho Nacional de Desenvolvimento Científico e Tecnológico
- —Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
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Taxonomy
TopicsDrug-Induced Hepatotoxicity and Protection · Berberine and alkaloids research · Plant-based Medicinal Research
Introduction
1
Drug‐induced liver injury (DILI) has increasingly been recognized as a significant clinical concern over the past decade, with its incidence rising steadily since 2010 and reaching its highest levels between 2010 and 2020 (15.14 per 100,000 person‐years) [1]. Among the agents implicated in liver injury, acetaminophen (APAP, paracetamol) stands out as a major contributor, accounting on average for 55.7% of severe acute liver injury cases and approximately 7.3% of DILI cases [2]. This medication is one of the most widely used agents for pain relief and fever reduction worldwide [1, 3]. Owing to its wide availability, it is one of the leading agents involved in drug overdose cases, which can lead to acute liver damage, liver failure, and fatal outcomes [4, 5, 6]. This toxicity results from the accumulation of a reactive APAP metabolite, N‐acetyl‐p‐benzoquinone imine (NAPQI), in the liver [7, 8]. N‐acetylcysteine (NAC) is used as an antidote in cases of APAP overdose to prevent exacerbated liver injury and reduce mortality [9, 10]. However, some patients present hepatotoxicity even with the NAC administration [11, 12, 13].
Hepatic injury induced by diverse agents, such as APAP, involves complex inflammatory signaling, particularly mediated by interferon‐γ (IFN‐γ) and the chemokine CXCL10 (IP‐10) [14, 15]. IFN‐γ promotes the activation of CXCL10 in hepatocytes and Kupffer cells, leading to leukocyte recruitment, amplification of inflammation, and suppression of autophagic flux in the liver [14, 15, 16, 17]. Inhibition of the IFN‐γ/CXCL10 axis is associated with reduced cytokine release, attenuation of oxidative stress, and restoration of autophagy during liver injury [17, 18]. Autophagy, an intracellular degradation process, is a key cytoprotective mechanism that contributes to the removal of damaged organelles and reduces hepatocyte necrosis [19, 20, 21]. Therefore, compounds capable of modulating inflammation and autophagy may be promising candidates for mitigating APAP‐induced liver damage.
Over time, plants have developed various mechanisms to increase their growth and defense strategies against environmental stress, including the production of alkaloids, which are heterocyclic organic compounds containing nitrogen in their structures [22, 23, 24]. More than 20 groups of alkaloid classes have been described, including pyrrolidines, pyrrolizidines, quinolizidines, pyridines, and imidazoles [25, 26]. Among these, imidazole alkaloids have gained prominence in applications, especially those extracted from jaborandi leaves (Pilocarpus microphyllus Stapf ex Holm.), a species native to Brazilian flora [27].
Several imidazole alkaloids have been identified in jaborandi leaves, among which pilocarpine is the most important. Pilocarpine is extracted on a large scale and used as an active ingredient in medicines for treating glaucoma and dry mouth [27, 28, 29]. Other imidazole alkaloids, such as epiisopilosine (EPIIS) and epiisopiloturine (EPI), can also be obtained from industrial residues generated by the pilocarpine extraction [30, 31]. EPIIS and EPI are imidazole alkaloids (C_16_H_18_N_2_O_3_) and structural isomers that differ in stereochemical configuration, which may influence their biological activity and molecular interactions. The chemical structures of the isomeric alkaloids EPIIS and EPI are shown in Figure 1. Previous studies have reported distinct anti‐inflammatory and antiparasitic profiles of these compounds, suggesting that subtle structural variations can alter target affinity and pharmacological response [32, 33]. Therefore, a comparative evaluation of both isomers may provide insights into the structure‐activity relationship and mechanisms underlying their hepatoprotective potential.
Chemical structures of epiisopilosine (EPIIS) (A) and epiisopiloturine (EPI) (B).
The scientific community has shown considerable interest in these metabolites because of their anti‐inflammatory activity, which diminishes the generation of reactive oxygen species (ROS), inhibits the degranulation of neutrophils in vitro and in vivo, inhibits pro‐inflammatory cytokines, and their anti‐parasitic activity against S. mansoni, which reduces the harmful effects of this infection on liver tissue [30, 31, 33, 34, 35, 36]. Despite the latest advances in understanding the biological activities of EPI and EPIIS, there remains a need to gain a deeper understanding of their targets and functions in liver tissue, which is constantly exposed to disorders through the metabolism of various substances.
Given that some natural alkaloids can modulate inflammatory pathways and enhance autophagy through phosphatidylinositol 3‐kinase PI3K inhibition or CXCL10 suppression, EPI and EPIIS emerge as promising candidates for further investigation in models of APAP‐induced hepatotoxicity [17, 37, 38, 39, 40]. Therefore, this study aimed to evaluate and compare the potential therapeutic effects of these alkaloids in animals with APAP‐overdose‐induced liver injury and to determine whether the mechanism of action of these alkaloids involves increased autophagy in liver tissue.
Materials and Methods
2
Alkaloids Purification
2.1
EPI and EPIIS were obtained by extracting industrial waste from Pilocarpus microphyllus leaves and isolated according to the protocol established by Véras et al. [30]. Anidro do Brasil Extracões S. A. (Piauí, Brazil) provided the alkaloids. The samples were purified using high‐performance chromatography (HPLC; SHIMADZU Prominence, Tokyo).
Drugs and Reagents
2.2
We purchased acetaminophen from Sigma‐Aldrich (St. Louis, MO, USA Louis, MO, USA) and diluted it in PBS (saline phosphate buffer), maintaining a pH of 7.2 (37°C). Anti‐LC3B and anti‐Ly6G antibodies were purchased from Cell Signaling Technology (USA) and Abcam (USA), respectively. Antibody kits were used for cytokines (IL‐10, IL‐1β, IL‐6, TNF, and IFN‐γ) and chemokine CXCL1 (R&D Systems, USA). The other chemical compounds used were purchased from standard analytical suppliers.
Animals
2.3
Male BALB/c mice (25–30 g) were used in this study and maintained at 22°C under a 12:12 h light/dark cycle with free access to water and food. This study was carried out in accordance with the internationally accepted animal use standards (Guide for Care and Use of Laboratory Animals of the National Institute of Health), and the project was approved by the Ethics Committee on Animal Experimentation of the Federal University of the Parnaíba Delta (UFDPar) under number 36/20.
Experimental Design of APAP‐Induced Liver Toxicity in Mice
2.4
A post‐treatment model was used to evaluate the effects of alkaloids on hepatotoxicity induced by APAP overdose, using the protocol of Ishida et al. [14], with some modifications. First, male Mus musculus mice (BALB/c) weighing 25–30 g were subjected to 10‐h fasting before injury induction. After this period, the animals were intraperitoneally administered 750 mg/kg of APAP. After 30 min, the animals received the following treatments: the positive control group received 5 mL/25 g of saline phosphate buffer (PBS); test groups received epiisopilosine or epiisopiloturine at doses of 0.3, 1, and 3 mg/kg i.p.; and a group received the standard drug NAC (318 mg/kg i.p.). In addition, one group was used as a negative control and received only PBS intraperitoneally.
After 10 h of APAP administration, the animals were anesthetized with xylazine and ketamine (10 mg/kg i.p. and 60 mg/kg i.p., respectively), and blood was collected to assess liver function markers in the serum of the animals. Subsequently, euthanasia was performed using an overdose of ketamine and xylazine (300 mg/kg and 30 mg/kg, respectively, via i.p). An abdominal incision was made to collect liver samples for biochemical assays, cytokine evaluation, and histopathological analysis.
Evaluation of Autophagy‐Associated Protective Mechanisms
2.5
Another protocol was performed after the first analysis to assess the implications of autophagy on the mechanism of alkaloid protection. First, the animals were treated with 80 mg/kg hydroxychloroquine (HCQ), an autophagy inhibitor, based on a study by Jackson, Hansen, and Gustafson [41]. After 2 h, the rats were administered 750 mg/kg acetaminophen. These animals were treated with the best dose of epiisopilosine and epiisopiloturine (1 mg/kg) or PBS 2 h after acetaminophen administration. After 10 h, the animals were euthanized as described above, and samples were collected to evaluate the markers of liver function, histopathological analysis, and biochemical dosages of oxidative stress.
Evaluation of Hepatic Function
2.6
The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assessed using standard enzymatic colorimetric methods with Labtest kits (Minas Gerais, Brazil) and a spectrophotometer (V‐5000 METASH, Microprecision, California, USA).
Histopathological Analysis of Hepatic Injury
2.7
Liver fragments were collected and fixed in 10% formalin for 24 h. The biological material was dehydrated using increasing concentrations of ethyl alcohol, processed, and embedded in paraffin. After these procedures, sections were obtained using a microtome, transferred onto slides, and stained with hematoxylin and eosin (H&E). Experienced researchers evaluated the slides using a microscope (Motic, Carl Zeiss, Gottingen, Germany) and a digital PowerShot A620 camera (Canon, Tokyo, Japan) to obtain microphotographs and to assess histopathological scores.
For histological analysis, we used the method described by Latchoumycandane et al. [42]. The severity of liver damage was divided into the following scores: 0‐without lesion; 1‐minimal lesion (only a few affected hepatocytes); 2, mild lesion (centrilobular necrosis in some lobes, one to two rings of necrotic cells); 3, moderate lesion (centrilobular necrosis in most lobules, two to three rings of necrotic cells); 4, marked lesion (centrilobular necrosis in all lobules, three to four rings of necrotic cells); and 5, severe injury (confluent panlobular necrosis, five rings of necrotic cells and hemorrhage). Afterward, sections were evaluated in a blinded manner by an experienced pathologist (D.F.P. Vasconcelos).
Ly6G Immunohistochemistry of Hepatic Tissue
2.8
Immunostaining of neutrophils in the liver tissues of animals treated with or without EPIIS and EPI against APAP‐induced injury was performed to estimate neutrophil migration to the liver in animals treated with a sublethal dose of APAP. Histological blocks were obtained from histopathological assessment samples, cut at 4 µm, and prepared into slides. The samples were incubated overnight with an anti‐Ly6G antibody (1:2000; EPR22909‐135; ab238132; Abcam, USA). Subsequently, the samples were incubated with a biotinylated secondary antibody, and color was revealed using 3,3′‐diaminobenzidine (DAB; Sigma‐Aldrich, USA).
In addition, the slides were stained with hematoxylin, assembled, and evaluated using a Motic microscope (Carl Zeiss, Gottingen, Germany) and adjusted to a digital camera (PowerShot A620, Canon, Tokyo, Japan). Cells labeled with Ly6G were counted in 15 fields. The results were expressed as Ly6G/mm^2^.
Malondialdehyde (MDA) Levels in Hepatic Tissue
2.9
According to Uchiyama and Mihara [43], MDA was used to measure tissue damage. Liver samples were homogenized in a 1.15% KCl solution using a T10 Basic Ultra Turrax (Ika Werk GmbH & Co. KG, Staufen, Germany). The absorbance was measured at 520 and 535 nm using a UV‐vis spectrophotometer (Shimadzu, Kyoto, Japan). The results are presented as nmol/g tissue MDA.
Reduced Glutathione (GSH) Levels in Hepatic Tissue
2.10
The methodology described by Sedlak and Lindsay [44] was used, with modifications, to measure the endogenous antioxidant GSH. For this purpose, liver samples were homogenized with Ultra‐Turrax in 0.02 M ethylenediaminetetraacetic acid (EDTA) to prepare a homogenate (10%). The obtained solution was evaluated at 412 nm using a visible spectrophotometer (V‐5000 METASH). The results are expressed as mg of GSH/g protein.
Superoxide Dismutase (SOD) Activity in Hepatic Tissue
2.11
The enzymatic activity of SOD was also evaluated, indirectly estimated by its ability to inhibit the photochemical reduction of the Griess reagent, following the procedures of Das, Samanta, and Chayne [45]. The liver samples were homogenized in phosphate buffer 0.05 M (pH 7.4), and the solution was centrifuged for 15 min (3,000 rpm, 4°C). The supernatant was removed to determine the total protein (Labtest kit). The results were measured using an ELISA reader (Polaris microplate reader, Celer Biotecnologia, Minas Gerais, Brazil) at 550 nm. The results are presented as SOD units per μg of protein.
Nitrate/Nitrite Levels in Hepatic Tissue
2.12
The peroxynitrite levels in liver tissue were measured indirectly using the nitrite metabolite and the Griess reagent, according to Green et al. [46]. The absorbance was measured at 550 nm. The results are presented as the concentrations of nitrite and nitrate‐Nox (μM).
Myeloperoxidase (MPO) Activity in Hepatic Tissue
2.13
The activity of MPO, present in neutrophils, was measured in the liver tissue of mice following the protocol described by Bradley et al. [47]. Liver samples were macerated using an Ultra‐Turrax in a cold potassium phosphate (pH 6.0) solution with 0.5% hexadecyl trimethyl ammonium bromide. After this procedure, the liver homogenates were centrifuged for 25 min (4000 rpm, 4°C). The supernatants of the samples were then added to a reaction solution (o‐dianisidine and H_2_O_2_ at 1%). The changes in the absorbance of the samples were evaluated at zero and 1 min at 450 nm. The results are presented as MPO units per gram of protein.
Cytokine Levels
2.14
The cytokines IL‐6, IL‐10, TNF, IFN‐γ, and CXCL1 were assessed. For this purpose, liver fragments were homogenized in PBS with a protease inhibitor (100 µM phenylmethylsulfonyl fluoride; 100 µM benzethonium chloride; 10 mM EDTA; 20 KI aprotinin A) and Tween 20 (0.05%) at a ratio of 0.1 g/ml. Then, the liver homogenates were centrifuged for 10 min (10,000 rpm, 4°C).
Liver tissue samples were analyzed at a 1:5 dilution for cytokines and a 1:10 dilution for CXCL1 in PBS with 0.1% BSA (bovine serum albumin). Antibody kits (R&D Systems, USA) were used according to the manufacturer's protocol. The capture antibodies were diluted in PBS (pH 7.4) for cytokine measurements, and sensitization occurred for 18 h at 4°C. The reaction was then blocked using PBS with 1% BSA. Samples, standards, and 0.1% BSA were added to the wells and incubated for 18 h. After this period, the wells were washed, and the detection antibody was added for at least 2 h. The reaction was detected by incubation with streptavidin conjugated with peroxidase (HRP‐Streptavidin Pharmingem, 1:200) and developed with phenylenediamine‐dihydrochloride. 50 µL of H_2_SO_4_ was added 30 min later to stop the reaction. The measurements were performed using an ELISA reader (Status‐lab systems, Multiskan RC, Brazil) at a wavelength of 490 nm.
LC3B Expression
2.15
Liver proteins were extracted to perform western blotting for LC3B, an autophagy marker. Liver tissue was macerated in RIPA buffer (100 mg/800 μL). The homogenates of each tissue were then sonicated for 1 min and placed on ice for 5 min (four times). Next, the specimens were centrifuged at 14,000 × g for 15 min at 4°C, and the supernatants were collected. This process was repeated three more times to remove the fat layer.
After the protein extraction methods, the samples were quantified and calculated to obtain 30 μg of proteins, which were applied to each well of the electrophoresis gel to standardize the amount of protein tested. Then, the liver samples were diluted in buffer and heated in a dry bath at 100°C for 15 min. Tris‐glycine gel SDS‐polyacrylamide was prepared for electrophoresis at 12.5% for protein fractionation. After polymerization, a running buffer (TBS 1x) was added to the electrophoresis tank. Each sample was placed in the 1‐mm wells of the gel system and in the molecular weight marker rainbow (5 μL). Electrophoresis was run at 100 V with free amperage for 2 h.
Proteins were transferred to a PVDF membrane (0.40 A, free voltage, for 2 h). After transfer, the gel was discarded for antibody incubation, and the membrane was blocked with 3% skim milk. The milk was then removed, and the membranes were rinsed three times with TBST and once with TBS. The primary antibody of the cocktail (1:1000‐ antibody LC3B) was added and incubated overnight with agitation. After incubation, the membranes were rewashed. Then, the secondary antibody (anti‐rabbit IgG, 1:2000; Cell Signaling Technology, United States) was added for 1 h with stirring. The membranes were washed once more and then developed with 1 mL of NBT‐BCIP (nitro‐blue tetrazolium chloride/5‐bromo‐4‐chloro‐3'‐indolyphosphate p‐toluidine salt), followed by washing with distilled water. The membranes were photographed, and protein expression was analyzed using ImageJ software version 2.1 by measuring the peak areas of the gel bands. Results are expressed as the LC3B II/actin ratio.
Molecular Docking
2.16
The 3D structure of the possible protein mouse IP‐10 was obtained from the Protein Data Bank (PDB) [48] with the code PDB ID: 2R3Z. All docking procedures utilized the Autodock 4.2 package [49, 50, 51]. Protein and ligands were prepared for docking simulations with AutoDock Tools (ADT) version 1.5.6 [52]. The receptor was considered rigid; each ligand was considered flexible. Gasteiger and Marsili [53] partial charges were calculated after the addition fall hydrogens. Non‐polar hydrogen atoms of the protein and ligand were subsequently merged. A cubic box of 60 × 60 × 60 points with a spacing of 0.35 Å between the grid points was generated for the hole protein target. The affinity grid centers were defined. The global search Lamarckian genetical algorithm (LGA) [54] and the local search (LS) pseudo‐Solis and Wets [55] methods were applied in the docking search. Each ligand was subjected to 100 independent runs of docking simulations. Other docking parameters were set as the default values. The resulting docked conformations were clustered into families according to the RMSD. For a more detailed analysis, the coordinates of the selected complexes were chosen by the criterion of the lowest docking conformation of the cluster with the lowest energy in combination with a visual inspection.
Statistical Analysis
2.17
The results are indicated as the Standard Error of the Mean (S.E.M.). The results were analyzed with GraphPad Software Inc. (GraphPad Prism 6, California, USA), which used an analysis of variance (ANOVA), followed by the post‐hoc Newman‐Keuls test when necessary. In addition, the non‐parametric Kruskal–Wallis test, followed by the Dunn test, was used for histopathological analysis. We established statistical significance at p < 0.05.
Results
3
EPIIS and EPI Attenuate Liver Damage and Reverse the Histopathological Findings of Hepatotoxicity Induced by APAP
3.1
To evaluate liver function, altered by administering toxic doses of APAP, the markers ALT and AST were analyzed in the serum of mice submitted to the treatments. In the group that received 750 mg/kg of APAP, there was a statistically significant increase in both ALT (240.20 ± 11.73 U/l) and AST (218.20 ± 5.33 U/l) compared to the negative control group that received only PBS (ALT‐116.00 ± 5.86 U/l; AST‐117.6 ± 5.54 U/l), as shown in Figure 2A,B.
*Evaluation of liver damage in APAP‐induced hepatotoxicity. (A) Alanine aminotransferase (ALT), (B) aspartate aminotransferase (AST), and (C) relative liver weight in negative control animals (PBS), animals that received acetaminophen (APAP, 750 mg/kg, i.p.), and those treated with the alkaloids EPIIS or EPI at doses of 0.3, 1, or 3 mg/kg, or with the standard drug N‐acetylcysteine (NAC), administered 30 min after APAP. Data were analyzed by one‐way ANOVA followed by Tukey's post hoc test. (D) Quantitative analysis of liver microscopic scores was performed using the non‐parametric Kruskal–Wallis test followed by Dunn's post hoc test. (E) Representative macroscopic images of livers from APAP‐treated animals showing visible necrotic areas (dotted squares) in the APAP + PBS group, and representative liver photomicrographs (H&E staining). Scale bar: 50 μm; magnification: 200×. Nec, microscopic necrosis area; He, hemorrhage area; Neut, neutrophilic infiltrate. Data are expressed as mean ± Standard error of the mean (S.E.M.), n = 6 mice per group. # p < 0.05 versus negative control group ‐ PBS; p < 0.05 versus positive control group APAP + PBS; α p < 0.05 between EPIIS doses; β p < 0.05 between EPI doses.
Treatment with EPIIS and EPI, 30 min after acetaminophen administration, may prevent liver damage, as indicated by the reversal of elevated ALT and AST levels across all tested doses. It was also observed that a dose of 1 mg/kg was the lowest dose with the most significant effect for both EPIIS and EPI (p < 0.05). Thus, this dose was used for the other experiments. The NAC control drug also significantly reduced liver injury markers in a statistically significant way (p < 0.05). In Figure 2C, the relative weight of the livers of these animals is shown, and it is observed that mice that received only APAP and PBS showed an increase in this parameter. Treatment with alkaloids and NAC reversed this finding (p < 0.05).
The sublethal dose of acetaminophen generated necrotic lesions in the animals' livers, which were not present in the animals treated with the tested alkaloids and NAC, as shown in Figure 2E. Histological images indicate regions of centrilobular necrosis and pycnotic nucleus cells with hemorrhagic zones (Figure 2E). The evaluated histopathological scores, classified as lesions ranging from mild to intense injury, increased, showing a statistical difference compared with the group that received only PBS (Figure 2D). In addition, evident hydropic degeneration was observed in some fields, and the presence of leukocytes in sinusoids.
Treatment with EPIIS and EPI promoted the reversal of APAP‐induced damage at the microscopic level, with 50% of the treated animals showing no injury or a reduction in histopathological scores. The histopathological analysis revealed only minimal or mild lesions. Thus, there was a statistically significant difference in histopathological scores between the alkaloids EPIIS and EPI compared to the positive control group (p < 0.05; Figure 2D). The standard drug NAC also significantly reduced the damaging effects caused by the toxic agent (p < 0.05).
EPIIS and EPI Reverse the Rise in Oxidative Stress Markers in Liver Damage Caused by APAP
3.2
MDA, a lipid peroxidation product, is measured to evaluate oxidative stress. The administration of APAP (750 mg/kg) resulted in an increase in MDA concentration in the liver of the animals, as shown in Figure 3A (554.80 ± 57.17 nmol/g) compared with the group that received only PBS (p < 0.05). Treatment with the alkaloids EPIIS and EPI led to a significant reduction in MDA levels in the model of APAP‐induced liver injury (263.10 ± 25.47 nmol/g; 313.90 ± 42.70 nmol/g, respectively). The standard drug NAC also reduced lipid peroxidation (p < 0.05).
*Effect of alkaloids on oxidative stress biomarkers and antioxidant parameters. (A) Malondialdehyde (MDA), (B) nitrite, (C) reduced glutathione (GSH), and (D) superoxide dismutase (SOD) levels in liver tissue of healthy animals (PBS), animals that received acetaminophen (APAP, 750 mg/kg, i.p.), and animal treated with the alkaloids epiisopilosine (EPIIS, 1 mg/kg) or epiisopiloturine (EPI, 1 mg/kg), or with N‐acetylcysteine (NAC) administered 30 min after APAP. Data were analyzed by one‐way ANOVA followed by Tukey's post hoc test (n = 6 mice per group). # p < 0.001 versus negative control mice group (PBS); p < 0.05 versus positive control group (APAP + PBS).
Peroxynitrite, another oxidant, was indirectly evaluated by measuring Nox levels (Figure 3B). As expected, this indicator increased in animals that received acetaminophen (0.112 ± 0.01 µM) compared with the negative control group. Treatment with the alkaloids tested and NAC reduced Nox levels (p < 0.05), indicating a reversal of the oxidative stress generated.
In addition to these markers, GSH and SOD antioxidants were measured in liver tissue samples. The GSH concentrations are shown in Figure 3C, indicating that the basal levels of GSH in the liver of BALB/c mice were 3.40 ± 0.23 μg of GSH/mg of protein. A significant reduction in GSH was observed (0.11 ± 0.02 μg of GSH/mg of protein) with a sublethal dose of APAP. However, alkaloids and NAC treatment maintained baseline GSH levels (2.72 ± 0.19 μg GSH/mg of protein; 2.00 ± 0.25 μg GSH/mg of protein; 2.2 ± 0.36 μg GSH/mg of protein, respectively).
The SOD concentration was also evaluated, with healthy animals showing levels of 7.99 ± 0.98 U SOD/mg of protein. The administration of a sublethal dose of APAP led to a decrease in this antioxidant concentration in liver tissue (2.97 ± 0.46 U SOD/μg of protein) of the animals. Treatments with the alkaloids EPIIS, EPI, and NAC maintained baseline levels of SOD 30 min after APAP administration, as can be seen in Figure 3 (D) (6.93 ± 0.36 U SOD/μg protein; 5.74 ± 0.22 SOD/μg protein; 6.10 ± 0.99 SOD/μg protein, respectively).
EPIIS and EPI Reduce the Recruitment of Neutrophils and Proinflammatory Cytokine Levels in the Liver of APAP‐Induced Liver Injury
3.3
To evaluate the inflammation induced by the administration of 750 mg/kg of APAP via i.p., neutrophil recruitment to the liver was analyzed by assessing the presence of neutrophils in the liver tissue and the expression of CXCL1 chemokine, which is related to the recruitment of these leukocytes. Immunohistochemistry was performed for the labeled neutrophils (Ly6G+ cells), and the presence of these inflammatory cells was confirmed (Figure 4A,B). There was an increase in Ly6G levels in the livers of animals treated with APAP (19.97 ± 1.18 Ly6G + /mm^2^) compared to the negative control group (12.50 ± 1.74 Ly6G + /mm^2^). EPIIS and EPI 30 min after APAP administration significantly reversed this finding (p < 0.05).
*Parameters associated with inflammation in APAP‐induced liver injury. (A) Representative photomicrographs of immunohistochemistry showing Ly6G+ cells. (B) Quantification of Ly6G+ labeled cells in 15 microscopic fields. Myeloperoxidase (MPO) activity (C) and CXCL1 levels (D) in the liver of mice subjected to acetaminophen (APAP, 750 mg/kg)‐induced liver injury, with or without treatment with epiisopilosine (EPIIS, 1 mg/kg), epiisopiloturine (EPI, 1 mg/kg), or the standard drug N‐acetylcysteine (NAC) administered 30 min after APAP. Cytokine levels of (E) interferon‐γ (IFN‐γ), (F) tumor necrosis factor (TNF), (G) interleukin‐6 (IL‐6), and (H) interleukin‐10 (IL‐10) were quantified in liver tissue homogenates. Data were analyzed by one‐way ANOVA followed by Tukey's post hoc test. Results are expressed as mean ± S.E.M (n = 6 mice per group). # p < 0.0001 versus negative control group (PBS); p < 0.05 versus positive control (APAP + PBS).
As expected, there was an increase in MPO (p < 0.05), a protein present in the azurophilic granules of neutrophils, confirming the presence of these leukocytes (Figure 4C) in the liver. In addition, there was an increase in CXCL1 expression in liver tissue, indicating neutrophil recruitment (Figure 4D).
Alkaloids significantly reduced neutrophil recruitment because they promoted the reversal of the increase in MPO and CXCL1 levels in the liver compared to the positive control group, which did not receive therapeutic drugs (p < 0.05). The standard drug NAC reversed the increase of MPO in the tissue.
The proinflammatory cytokines TNF, IL‐6, and IFN‐γ were analyzed in the liver samples of the animals, and their levels were elevated in APAP‐induced liver injury, as shown in Figure 4E–G (p < 0.05). Treatment with EPIIS and EPI significantly reversed the increase in these cytokine levels. Additionally, levels of the anti‐inflammatory cytokine IL‐10 were measured (Figure 4H), revealing an increase in this protein with EPIIS and EPI (13201.00 ± 5798.00 pg/100 mg; 19617.00 ± 2754.00 pg/100 mg, respectively). However, only EPI caused a statistically significant increase compared to the positive control (6631.00 ± 2101.00 pg/100 mg; p < 0.05).
EPIIS and EPI Reduce Hepatotoxicity via Autophagy
3.4
Hydroxychloroquine Inhibits the Hepatoprotective Effect of EPIIS and EPI in APAP‐Induced Liver Injury
3.4.1
The sublethal dose of acetaminophen generated necrotic lesions in the livers of BALB/c mice, as shown in Figure 5A. The alkaloids EPIIS and EPI at a dose of 1 mg/Kg partially protect the liver from liver injury 2 h after APAP administration in BALB/c mice (Figure 5A–C) by promoting a reduction in serum ALT levels and AST in the serum of the animals compared to the injured group (p < 0.05). The increase in liver weight was also reduced by alkaloid treatment (p < 0.05; Figure 5D).
*Autophagy assessment of hepatoprotective effects. (A) Representative photographs showing visible necrotic areas (dotted squares) and liver photomicrographs from healthy animals (PBS), animals treated with acetaminophen (APAP, 750 mg/Kg i.p.), and animals treated with the alkaloids epiisopilosine (EPIIS) or epiisopiloturine (EPI) at a dose of 1 mg/kg administered 2 h after APAP. Additional groups include animals treated with hydroxychloroquine (HCQ) + APAP with or without subsequent treatment with EPIIS and EPI. Scale bar 50 μm, magnification 200×. Nec‐ area of microscopic necrosis; He‐ area of hemorrhage. (B) Analysis of ALT (alanine aminotransferase) and (C) AST (aspartate aminotransferase) levels in the serum of animals. (D) Relative liver weight. Parametric data were analyzed by one‐way ANOVA followed by Tukey's post hoc test. (E) Liver histopathology scores with quantitative analysis of liver microscopic scores. Non‐parametric data were analyzed using the Kruskal–Wallis test followed by Dunn's post hoc test. Results were expressed as mean ± S.E.M (n = 6 mice per group). # p < 0.05 versus negative control group (PBS); p < 0.05 versus positive control (APAP + PBS group); ßi p < 0.05 versus EPIIS group; ßT p < 0.05 versus EPI group.
To evaluate the possible involvement of autophagy in the beneficial effects of the studied alkaloids, we used HCQ, an autophagy inhibitor. However, the administration of HCQ 2 h before acetaminophen significantly inhibited the effects of the EPIIS and EPI alkaloids in a statistically significant way (p < 0.05).
In addition, Figure 5A shows the effects caused by the administration of HCQ before APAP at macroscopic levels, where it is noted that this drug also inhibited the protective effect of alkaloids. The administration of HCQ also contributed to centrilobular necrosis and bleeding in liver tissue (Figure 5A,E). However, this autophagy inhibitor inhibited the beneficial effects of EPIIS and EPI 2 h after APAP administration.
Hydroxychloroquine Inhibits the Anti‐Inflammatory and Antioxidant Effects of EPIIS and EPI, Which Increase LC3B II Expression in APAP‐Induced Hepatic Damage
3.4.2
These results are consistent with those previously presented because the use of HCQ 2 h before APAP administration inhibited the protective effects of the studied alkaloids. Thus, there was no reduction in lipid peroxidation (MDA) with the treatment of EPIIS (717.00 ± 72.65 nmol/g tissue) and EPI (802.30 ± 76.86 nmol/g tissue) 2 h after HCQ + APAP, which differed from what happened to animals that were treated with alkaloids and did not receive HCQ (p < 0.05, Figure 6A). The same occurred for nitrite (Figure 6B) because the reduction of this oxidative marker caused by the administration of EPIIS and EPI was inhibited with the use of HCQ in the model of hepatic injury induced by APAP.
*Effects of alkaloids on oxidative stress, antioxidant, and autophagy parameters. (A) Malondialdehyde (MDA), (B) nitrite levels, (C) reduced glutathione (GSH), (D) superoxide dismutase (SOD) activity, and (E) myeloperoxidase (MPO) activity in liver tissue from healthy animals (PBS), animals treated with acetaminophen (APAP; 750 mg/kg, i.p.), and animals treated with the alkaloids epiisopilosine (EPIIS; 1 mg/kg) or epiisopiloturine (EPI; 1 mg/kg) administered 2 h after APAP. Additional groups include animals treated with hydroxychloroquine (HCQ) + APAP, with or without subsequent treatment with EPIIS or EPI. Data are expressed as mean ± S.E.M (n = 6 mice per group). # p < 0.05 versus negative control group (PBS); *p < 0.05 versus positive control group (APAP + PBS); βᵢ p < 0.05 versus EPIIS group; βᵀ p < 0.05 versus EPI group. (F) LC3B‐II protein expression in liver tissue from animals treated with APAP (750 mg/kg, i.p.) followed by EPIIS or EPI (1 mg/kg, 2 h post‐APAP), or PBS. Data were analyzed using one‐way ANOVA followed by Tukey's post hoc test and are expressed as mean ± S.E.M. (n = 4 mice per group). p < 0.05 versus PBS group; #p < 0.05 versus APAP + PBS group.
The antioxidant tripeptide GSH and enzyme SOD were presented at baseline levels following treatment with EPIIS (2.95 ± 0.34 mg GSH/g protein; 9.02 ± 0.42 U SOD/µg of protein) and EPI (2.71 ± 0.26 mg GSH/g protein; 9.14 ± 0.74 U SOD/µg) after damage induction. However, this effect was drastically abolished with the use of the autophagy inhibitor (HCQ + APAP + EPIIIS‐ 0.32 ± 0.02 mg GSH/g protein and 5.22 ± 0.54 U SOD/µg of protein; HCQ + APAP + EPI‐ 0.31 ± 0.02 mg GSH/g protein and 5.60 ± 0.59 U SOD/µg of protein), Figure 6C,D.
In Figure 6E, it is noted that the alkaloids EPIIS and EPI were also able to reduce myeloperoxidase levels in the liver tissue after 2 h of APAP injury induction (1.52 ± 0.22 U/g protein; 1.52 ± 0.12 U/g protein, respectively) compared with the injured group (4.02 ± 0.23 U/g protein). In addition, alkaloids showed no protective effect on MPO levels following pre‐treatment with HCQ (p < 0.05).
To determine whether the alkaloids studied increase autophagy, the expression of the LC3B protein in the liver tissue of mice was analyzed. As shown in Figure 6F, there was a tendency to increase the expression of the marker in the tissue of BALB/c mice that received APAP (750 mg/kg i.p.). However, the result was not statistically significant (2.02 ± 0.37 LC3BII/actin). When the animals received APAP and were treated with EPIIS or EPI, the expression of the autophagic marker increased (2.49 ± 0.13 LC3BII/actin; 2.96 ± 0.26 LC3BII/actin, respectively) compared with the healthy group (1.29 ± 0.16 LC3BII/actin). However, only EPI showed a significant difference in the expression of the autophagic marker when compared to the group treated with PBS in APAP‐induced liver injury (p < 0.05).
In Silico Interaction of EPIIS and EPI With Mouse CXCL10/IP‐10 Protein
3.5
The results concerning molecular docking of the EPIIS and EPI with the mouse CXCL10/IP‐10 protein are presented in Table 1. Optimal molecular affinity parameters were obtained from the interaction between the EPIIS ligand with the protein 2R3Z, on the seventh active site. The affinity was observed with a binding energy equal to −6.69 kcal.mol^−1^, and an inhibition constant of 12.52 µM (Table 1; Figure 7A). For EPIIS, interactions were observed with amino acids Gln51, Pro2, Ile1, Leu3, Ile12, Cys11, and Cys53 of the active site via a hydrogen bridge. Furthermore, the molecular affinity of EPI with 2R3Z, on the first and third active site (Table 1; Figure 7B), with binding energy equal to −6.67 kcal.mol^−1^, and an inhibition constant of 12.82 µM. Interaction with the amino acids Met21, Lys46, and Ala32 of the active site was observed.
3D molecular docking of epiisopilosine and epiisopiloturine with CXCL10 (PDB ID: 2R3Z). (A–B) Docking of epiisopilosine (red) with the 2R3Z protein (Chain A: coral; Chain B: pink; Chain C: light blue; Chain D: aquamarine) at the seventh active site, illustrating the ligand position within the binding pocket (A) and the corresponding hydrogen‐bond interactions (B). (C–D) Docking of epiisopiloturine (purple) with the same protein at the first and third active sites, showing the active binding pocket (C) and the respective hydrogen‐bond interactions (D).
Discussion
4
Acetaminophen, one of the most used antipyretic drugs on the planet, has been implicated in liver toxicity due to overdose, which is linked to fulminant liver necrosis and may lead to death [3, 6, 56]. However, currently available therapies are not entirely safe and effective. Therefore, there is a need for prospecting compounds extracted from plants that may be promising in toxicity caused by this antipyretic. In this context, the imidazole alkaloids EPIIS and EPI, isomers extracted from Jaborandi (Pilocarpus microphyllus) leaves, stand out because of their anti‐inflammatory and antioxidant activities and evidence of hepatic immunomodulation [31, 33, 34, 36]. In addition, in vivo studies have shown the absence of toxicity observed for these alkaloids demonstrated an acceptable safety profile for studies in animal models [31, 36]. These characteristics make these secondary metabolites promising candidates for preventing and treating liver injuries, such as those induced by acetaminophen.
In the present study, the therapeutic use of these alkaloids was evaluated in a mouse model of liver injury induced by a sublethal dose of acetaminophen. BALB/c mice euthanized 10 h after administration of 750 mg/kg APAP showed increased relative liver weight and elevated ALT and AST markers, which, in high concentrations in serum, indicate liver injury. Additionally, the collected livers showed necrotic spots visible to the naked eye, along with regions of centrilobular necrosis and hemorrhage in histopathological analysis.
Treatment with EPIIS and EPI, 30 min after APAP administration, significantly reversed these injury parameters, reducing tissue necrosis, hemorrhage, serum transaminases, and relative organ weight. These results support our hypothesis that these alkaloids exert protective effects on liver tissue. We hypothesize that EPIIS and EPI exert hepatoprotective effects since the reduction of liver granulomas caused by Schistosoma mansoni in animals treated with these alkaloids also suggests protective effects on the liver, not only schistosomicidal activity, as previously reported by Guimarães and collaborators [31, 36]. Furthermore, these results corroborate findings for other alkaloids, whose biological effects are mainly due to their anti‐inflammatory and antioxidant actions [57, 58].
Imidazole alkaloids are known for their antioxidant potential and ability to eliminate free radicals directly, with the imidazole group playing a key role in electron transfer [58, 59]. Consistent with these findings, EPI and EPIIS exhibited antioxidant effects in vivo, thereby reducing lipid peroxidation resulting from oxidative stress [34, 60]. In addition, Rocha et al. [33] demonstrated that these phytoactive compounds directly reduced ROS production in neutrophils.
This antioxidative effect of EPI and EPIIS may help reduce the damage caused by APAP overdose because in this type of intoxication there is an increase in reactive oxygen and nitrogen species, such as peroxynitrite, associated with lipid and protein damage in hepatocytes, leading to lipid peroxidation [61, 62, 63]. In the present study, MDA levels, a marker of lipid peroxidation, were elevated after APAP administration, along with an increase in Nox activity. The imidazole alkaloids significantly reduced these oxidative agents.
Moreover, the liver metabolism of APAP produces the reactive metabolite NAPQI, which binds to GSH and lowers its antioxidant activity [64, 65]. Corroborating this, we observed a significant reduction in GSH and SOD levels after paracetamol administration. Treatment with EPI and EPIIS reversed this reduction, maintaining antioxidant activity and preventing oxidative damage, as also reported for other alkaloids with hepatoprotective action [34, 66, 67, 68].
Since toxic doses of APAP cause inflammation in liver tissue, we evaluated parameters related to inflammatory cell migration. We observed an increase in the local inflammatory response, measured by the enzyme myeloperoxidase, a product of azurophilic granules in neutrophils, the main leukocytes involved in acute inflammation [69, 70]. As expected, APAP administration also led to an increased release of CXCL1, a key chemokine for neutrophil recruitment, and an increase in the neutrophil marker Ly6G in liver tissue [71].
These inflammatory cells are drawn to the liver due to the release of proinflammatory cytokines, mainly TNF‐α, which promotes leukocyte mobilization and neutrophil degranulation [72, 73]. This cytokine also regulates IL‐1β and IL‐6, which are associated with IFN‐γ, a protein directly involved in APAP pathogenesis [74, 75]. Accordingly, there was an increase in IFN‐γ, TNF, and IL‐6 in APAP‐treated mice, contributing to neutrophil migration to the liver.
The alkaloids studied here lowered the levels of these proinflammatory cytokines, which helped protect against liver damage. They also decreased levels of CXCL1, reducing leukocyte recruitment, as evidenced by the reduction of Ly6G‐labeled cells and MPO in liver tissue. These results corroborate previous studies demonstrating the anti‐inflammatory effects of these imidazole alkaloids in vivo and in vitro [33, 34, 60, 66].
IFN‐γ and its downstream chemokine CXCL10 (IP‐10) play a crucial role in amplifying the inflammatory response during APAP‐induced hepatotoxicity. IFN‐γ promotes CXCL10 expression in hepatocytes and Kupffer cells, leading to enhanced leukocyte recruitment and production of TNF‐α and IL‐6, further exacerbating hepatocellular injury [14, 15, 16, 17]. Moreover, CXCL10 has been reported to impair autophagic flux and autolysosome formation in hepatocytes, linking chronic inflammation to reduced cellular repair [17].
In the present study, in silico analysis demonstrated that both EPIIS and EPI interact moderately with CXCL10. However, experimental studies need to be conducted to prove whether this interaction interferes with this signaling axis. If this interaction is proven experimentally, it may provide a plausible molecular mechanism for the observed increase in autophagy and reduction in inflammatory mediators, as inhibition of the IFN‐γ–CXCL10 pathway has been associated with restoration of autophagy and attenuation of liver injury. Zhang et al. [17] demonstrated that CXCL10 impairs autophagosome‐lysosome fusion, leading to defective autophagic flux in hepatocytes. Therefore, the predicted interaction of EPI and EPIIS with CXCL10 may alleviate this impairment, contributing to the restoration of autophagy and hepatocellular homeostasis. In silico methods, such as molecular docking, have revolutionized early‐stage drug discovery by providing structural insights into protein‐ligand interactions. However, they possess intrinsic limitations that must be acknowledged. A primary constraint is the lack of cellular context; most docking simulations are performed in isolation, failing to account for the crowded intracellular environment, pH variations, and the presence of competing biomolecules that influence binding affinity in vivo (Ganesan et al. (2017). In silico polypharmacology: drug repurposing and mechanism‐of‐action predictions. Drug Discovery Today. Acessar via ScienceDirect.).
Once we confirmed that EPI and EPIIS reduce APAP‐induced hepatotoxicity, we investigated mechanisms related to their protective effect. Increased autophagy is associated with liver protection in APAP‐induced injury, promoting the removal of damaged mitochondria and toxic protein adducts [19, 21]. Pharmacologic induction of autophagy is therefore a promising strategy for managing APAP‐induced hepatic injury [20].
Given these findings, we aimed to evaluate whether EPI and EPIIS exert their protective effects through autophagy‐related modulation. Hydroxychloroquine, an autophagy inhibitor, was administered to confirm this involvement. The protective effects of both alkaloids were abolished by HCQ, indicating the possible involvement of autophagy to their hepatoprotective action. LC3B‐II expression increased in animals treated with EPIIS and EPI, indicating potential effects on autophagy‐related protein expression, which may facilitate the removal of components damaged by NAPQI.
It is well established in the literature that the hydroxychloroquine blocks autophagy at late stages [76]. However, HCQ has many non‐specific effects and is generally considered to have low hepatotoxic effect when used at standard doses but can precipitate marked transaminase elevations and even acute liver injury [77]. General studies document HCQ's ability to deacidify lysosomes and change lysosomal enzyme activity [78]. Likewise, HCQ exerts multiple immunomodulatory effects that are relatively non‐specific (i.e., they alter innate and adaptive immunity across conditions) [79, 80].
Although EPI and EPIIS are structural isomers, subtle stereochemical differences may underlie their distinct biological activities. EPI and EPIIS differ in the spatial orientation of the imidazole group, which can influence hydrogen bonding and docking interactions with biological targets [32, 81]. These structural differences may explain the stronger anti‐inflammatory and pro‐autophagic responses observed for EPI, evidenced by higher IL‐10 and LC3B‐II levels. The distinct binding modes of these alkaloids with CXCL10 in the in silico analysis further support that stereochemistry modulates interaction with proteins involved in inflammation and autophagy. Thus, while both metabolites display hepatoprotective potential, we suggest that EPI may possess a more favorable conformation for modulating key targets within the IFN‐γ–CXCL10–autophagy axis.
From a bioprospecting perspective, these findings reinforce Jaborandi as a promising natural source of bioactive imidazole alkaloids for developing hepatoprotective agents. The present study provides evidence that EPI and EPIIS are associated with modulation of hepatic autophagy‐related markers and attenuation of hepatic inflammation, in addition to exhibiting a predicted interaction with CXCL10. Together, these findings suggest that the hepatoprotective effects of these alkaloids may involve, at least in part, the regulation of oxidative stress and inflammatory responses, possibly through mechanisms related to autophagy modulation and the IFN‐γ–CXCL10 signaling axis. However, more experiments are needed to prove the involvement of this autophagic pathway.
Conclusion
5
In conclusion, this study provides the first experimental evidence that the imidazole alkaloids epiisopilosine and epiisopiloturine attenuate APAP‐induced hepatotoxicity and are associated with reductions in oxidative stress and inflammatory markers. The observed modulation of autophagy‐related parameters, together with the predicted interaction with CXCL10, suggests a potential involvement of autophagy‐related pathways and the IFN‐γ‐CXCL10 axis in their hepatoprotective effects. Although these findings support a mechanistic hypothesis linking these pathways to liver protection, further studies are required to confirm causal relationships. Given their natural origin and favorable toxicity profile, EPIIS and EPI emerge as promising lead compounds for the development of hepatoprotective agents.
Author Contributions
Ana Patricia de Oliveira: Project Administration (lead), conceptualization (supporting), investigation (equal), data curation, and writing – original draft preparation (lead). Gabriella Pacheco: Validation (lead) and investigation (equal). André Luis Fernandes Lopes: Methodology (lead), investigation (equal). Andreza Ketly da Silva Araujo: Methodology (equal), writing – review and editing (supporting). Letícia de Sousa Chaves: Methodology (supporting), investigation (equal). Simone de Araújo: Investigation (equal), data curation (supporting). Erick Bryan de Sousa Lima: Methodology and data curation (equal). Even Herlany Pereira Alves: Methodology (supporting), validation (equal). Celso Martins Queiroz‐Junior: Methodology and investigation (equal). Mauro Martins Teixeira: Resources (supporting). Vivian Vasconcelos Costa: Resources (supporting). Jéssica Maria Teles Souza: Validation (supporting), investigation (equal). Ayslan Batista Barros: Validation (supporting), investigation (equal). Paulo Sérgio de Araujo Sousa: Validation (supporting), formal analysis (supporting). Jefferson Almeida Rocha: Resources (supporting), formal analysis (lead). Ana Jérsia Araújo: Resources (supporting), methodology (supporting). José Delano Barreto Marinho Filho: Resources (supporting), methodology (supporting). Leiz Maria Costa Véras: Resources (supporting). Daniel Fernando Pereira Vasconcelos: Resources (supporting), validation (equal). Jand Venes Rolim Medeiros: Conceptualization, funding acquisition, resources and supervision (lead).
Conflicts of Interest
The authors declare no conflicts of interest.
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