Exposure to mercuric chloride (HgCl2) causes liver damage by disrupting the hepatic architecture, increasing oxidative stress, and altering cytokeratin integrity, resulting in hepatocyte fragility and impaired function. The present study evaluated the effects of Senecio biafrae leaf ethanol extract (SBLEE) on HgCl2-induced liver injury and cytoskeletal integrity.
Materials and methodsForty-two adult Wistar rats were divided into seven groups. The control group was given distilled water, whereas the other groups were given 4 mg/kg of mercuric chloride, orally, for 21 days, followed by either immediate sacrifice or a recovery period, and treatment with silymarin or various doses of SBLEE (300, 400, and 600 mg/kg) for 21 days.
ResultsThe histologic, immunohistochemical, and biochemical studies showed that HgCl2 significantly disrupted the hepatic architecture and cytokeratin networks, increased oxidative stress, and altered liver enzyme levels. Treatment with SBLEE restored cytokeratin integrity, reduced oxidative stress, and normalized the liver biomarkers, with results comparable to those of the silymarin and control groups.
ConclusionOur findings suggest that Senecio biafrae leaves have cytoprotective properties that help stabilize the hepatic cytoskeleton and preserve liver function after toxic injury.
La exposición al cloruro de mercurio (HgCl₂) causa daño hepático al alterar la arquitectura hepática, aumentar el estrés oxidativo y alterar la integridad de la citoqueratina, lo que provoca fragilidad y deterioro funcional de los hepatocitos. Este estudio evaluó los efectos del extracto etanólico de hojas de Senecio biafrae (SBLEE, por sus siglas en inglés) contra la lesión hepática inducida por HgCl₂ y la integridad del citoesqueleto.
Materiales y métodosCuarenta y dos ratas Wistar adultas se dividieron en siete grupos. Mientras que el grupo control recibió agua destilada, a los demás se les administraron 4 mg/kg de cloruro de mercurio por vía oral durante 21 días, seguido de sacrificio inmediato o de un período de recuperación y tratamiento con silimarina o dosis variables de SBLEE (300, 400 y 600 mg/kg) durante 21 días.
ResultadosLas evaluaciones histológicas, inmunohistoquímicas y bioquímicas mostraron que el HgCl₂ alteró significativamente la arquitectura hepática y las redes de citoqueratina, aumentó el estrés oxidativo y alteró los niveles de enzimas hepáticas.
Sin embargo, el tratamiento con SBLEE restauró la integridad de la citoqueratina, redujo el daño oxidativo y normalizó los biomarcadores hepáticos, con resultados comparables a los de los grupos de silimarina y control.
ConclusiónEstos hallazgos sugieren que las hojas de Senecio biafrae poseen propiedades citoprotectoras que ayudan a estabilizar el citoesqueleto hepático y a preservar la función hepática tras una lesión tóxica.
Mercury exposure remains a significant public health concern due to its widespread environmental distribution and bioaccumulative properties.1,2 Mercury exists in 3 predominant forms: elemental mercury (Hg), organic mercury (methyl mercury), and inorganic mercury (mercuric chloride [HgCl2]).3 HgCl2 is a well-known inorganic mercury compound that manufacturers have indiscriminately added to preservatives, laxatives, and even whitening cosmetics for a long time.4 Exposure to HgCl2 occurs through industrial effluents, contaminated water, and dietary sources, leading to accumulation in the liver.5,6 The liver is one of the primary organs that HgCl2 targets, and is essential for processes of absorption, transformation, metabolism, detoxification, and protein synthesis, making it highly susceptible to toxic insults.7,8 The liver forms a multicellular system, where its structural organization is maintained by a network of hepatocytes, sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells, all embedded within an extracellular matrix (ECM) that provides mechanical support and biochemical signaling.9,10 The ECM consists of a fine meshwork of reticulin fibers (primarily type III collagen), proteoglycans, and glycoproteins, which facilitate hepatocyte adhesion, communication, and regeneration.11,12 Intermediate filaments are key structural components of the cytoskeleton that provide mechanical strength and maintain cellular integrity in hepatic tissue.13,14 Cytokeratins are the most diverse group of intermediate filament proteins.15 Within the hepatocytes, cytokeratin filaments, particularly CK8 and CK18, form an essential component of the intermediate filament network, ensuring cellular stability and resistance to mechanical and toxic stress.1 These filaments interact with other cytoskeletal elements, including actin and microtubules, to maintain hepatocyte shape, facilitate intracellular trafficking,14 and regulate cell signaling pathways involved in apoptosis and regeneration.16 Cytokeratin filaments (CK8 and CK18) also play a protective role against oxidative stress by modulating the cellular response to reactive oxygen species and preventing organelle damage.17
Despite advances in cytoprotective and hepatoprotective therapies, phytotherapeutics remain a promising alternative due to their safety and efficacy.18Senecio biafrae leaves, commonly known as “African spinach”, are nutrient-rich edible vegetables widely consumed in West Africa, notably Nigeria, Benin, Ghana, etc. The leaves are known for their medicinal and therapeutic properties. They are rich in polyphenols, flavonoids, alkaloids, and essential vitamins, which contribute to their antioxidant, anti-inflammatory, and cytoprotective effects.19 However, the role of Senecio biafrae leaves in preserving cytokeratin integrity during hepatotoxicity remains unexplored. Therefore, understanding the crosslink between cytokeratin integrity and ECM stability is crucial in developing therapeutic strategies aimed at preserving hepatic architecture and attenuating liver damage caused by toxic insults, such as HgCl2.
The present study aimed to elucidate the cytoprotective potential of the Senecio biafrae leaf ethanol extract (SBLEE) against HgCl2-induced hepatic cytokeratin disruption and functional impairment in adult Wistar rats.
Materials and methodsType of studyA controlled animal experimental study was conducted, utilizing laboratory animals (Wistar rats). Liver injury was experimentally induced by HgCl2 administration and treated with SBLEE, to assess the ameliorative effects.
Chemicals and drugsHgCl2 (50 g), a product of British Drug Houses Limited (Poole, England), was supplied in its white crystalline form. Silymarin (Silybon-70®; 70 mg tablets) was purchased from Micro Labs Limited (India). All antibodies used were acquired from Novocastra Laboratories Limited (Newcastle upon Tyne, United Kingdom). Diethyl ether (≥99.8%) (500 mL), a colorless and volatile solvent, was obtained and used as an anesthetic agent. The product was manufactured by British Drug Houses Chemicals (Poole, England).
Plant extractionFresh Senecio biafrae leaves were obtained from the Fia market, Iyanfoworogi, via Ile-Ife, and were authenticated by a taxonomist at the Department of Botany, Obafemi Awolowo University, Ile-Ife. A voucher specimen (reference number IFE/18215) was deposited at the herbarium for reference purposes. The fresh Senecio biafrae leaves were air-dried, weighed, and pulverized. The pulverized leaves were extracted 3 times with 80% ethanol and continuously stirred with a magnetic stirrer at room temperature for 24 h. The extract was filtered using Whatman number 1 filter paper, and the filtrate was concentrated in vacuo, using a vacuum rotary evaporator (Buchi), and later freeze-dried in a lyophilizer. The ethanol extract was stored in a desiccator before phytochemical evaluation.
Phytochemical analysisComprehensive screening was carried out to check for the presence or absence of secondary metabolites in the extract. Tests were carried out to identify alkaloids, flavonoids, tannins, saponins, phenols, terpenoids, steroids, glycosides, anthraquinones, cyanogenic glycosides, and coumarins, among others. All tests were performed as previously described.18
Animal care and managementA total of 42 adult Wistar rats weighing between 130 g and 150 g were used for this study. The rats were bred at the Animal Holding Facility of the Department of Anatomy and Cell Biology, Obafemi Awolowo University, Ile-Ife. The rats were housed in plastic cages, kept under standard laboratory conditions of temperature, humidity, and light, fed standard laboratory rat chow (Ace feed, Osogbo, Osun State, Nigeria), and given free access to clean water. Ethical clearance was obtained from the Health Research and Ethics Committee (HREC) of the Institute of Public Health, Obafemi Awolowo University, Ile-Ife (IPH/OAU/12/2543). The rats received humane care according to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.20
Experimental designForty-two adult Wistar rats were randomly assigned to 7 groups (n = 6 per group).
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Group A: control group; rats received normal saline orally for 42 days
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Group B: exclusively HgCl2-exposed group; rats received 4 mg/kg HgCl2 daily for 21 days and were sacrificed 24 h after administering the last dose
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Group C: withdrawal group; rats received 4 mg/kg HgCl2 daily for 21 days and were observed for the subsequent 3 weeks before sacrifice
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Group D: silymarin-treated group; rats received 4 mg/kg HgCl2 daily for 21 days and were subsequently treated with 2 mg/kg silymarin, every 12 h, for 21 days (as reported by Awoniran and Adeyemi21
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Group E: low-dose test group; rats received 4 mg/kg HgCl2 daily for 21 days and were subsequently administered 300 mg/kg of SBLEE daily for 21 days
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Group F: mid-dose test group; rats received 4 mg/kg HgCl2 daily for 21 days and were subsequently administered 400 mg/kg of SBLEE daily for 21 days
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Group G: high-dose test group; rats received 4 mg/kg HgCl2 daily for 21 days and were subsequently administered 600 mg/kg of SBLEE daily for 21 days
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HgCl2, SBLEE, and silymarin dissolved in distilled water were administered orally for 21 days using a suitable oral cannula.
Twenty-four hours after the final HgCl2 administration, the rats were euthanized under di-ethyl ether anesthesia, and a midline incision on the anterior body wall was made. Blood samples were collected with a 2 mL syringe by cardiac puncture, stored in 5 mL plain bottles, and left for 20 min at room temperature to clot for the biochemical assessment of liver marker enzymes. The whole liver was excised and weighed on a top-loading analytical balance. The median lobes of each liver were dissected, and all samples were taken from a carefully dissected median lobe to maintain uniformity, given that previous reports have shown that the different liver lobes respond to hepatotoxic agents distinctly.22Fig. 1 shows the lobes of an adult Wistar rat.
Rat body weight
Before euthanasia, the absolute body weights of the animals were measured using a top-loading weighing balance at the beginning (initial weight) and at the end (final weight) of the experiment. The weight change percentage was expressed as the difference between the final and initial body weights divided by the initial weight, multiplied by 100. Means were compared between the groups, using the Rahardjo et al. method,23 as described by Adeyemi and Awoniran.24
Histological processingFor optimal staining, the median lobes of the rats were fixed in 10% neutral buffered formalin (NBF) and totally immersed for 48 h. The tissues were trimmed to about 4−6 mm thick and processed via the paraffin wax embedding method of Drury and Wallington (1980),25 for light microscopy examination. Sections measuring 5 μm in thickness were produced on a rotary microtome (Laboid: LBM – RM2). They were stained with hematoxylin and eosin (H&E) and Gridley’s silver stain to visualize the general histoarchitecture and reticulin fibers, respectively.
Immunohistochemical studiesFor the immunohistochemical study, the processed liver tissue sections were stained with pan-cytokeratin antibody, according to the manufacturer’s instructions for light microscopy examination, and were utilized to visualize the filamentous cytoskeleton. Paraffin sections of the liver were cut at a thickness of 3 μm, on positively charged slides. Sections were deparaffinized with xylene and rehydrated through descending ethanol concentrations (100% 2 ×, 95%, 70% for 2 min) and then rinsed in distilled water for 5 min. Antigen retrieval was performed by boiling the slides in citrate buffer (pH 6.0) for 15 min, cooling them down for 20 min at room temperature, and rinsing them in phosphate-buffered saline (PBS). Peroxidase blocking was carried out on the sections by simply covering them with 3% hydrogen peroxide (H2O2) for 15 min. Sections were washed with PBS, and protein blocking was performed using avidin for 15 min. Sections were washed with PBS, and endogenous biotin in the tissue was blocked, using biotin for 15 min. Sections were incubated with pan-cytokeratin antibody for 60 min and biotinylated secondary antibody for 15 min. Horseradish peroxidase and 3,3’-diaminobenzidine (DAB) were used to visualize the antigen-antibody reaction in the tissues. Cells with specific brown colors in the cytoplasm, depending on the antigenic sites, were considered positive.
Biochemical proceduresLiver function marker assessmentThe serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured to assess hepatoxicity, using enzyme colorimetric assay kits and standard procedures.
Biochemical quantification of oxidative stress markersGlutathione (GSH) concentration and catalase (CAT) and superoxide dismutase (SOD) activities in the liver tissue were estimated. The frozen piece of the median lobe was weighed and homogenized with potassium phosphate (pH of 7.0 to 7.4), using an electric tissue homogenizer. The homogenates were centrifuged at 4,000 rpm for 10 min, and a clear supernatant was obtained.
Glutathione assayGSH levels were quantified using the Ellman’s reagent (5’,5’-dithiobis-2-nitrobenzoic acid, DTNB) method. Tissue homogenate (100 μL) was added to 1 mL of phosphate buffer (pH 8.0) and 100 μL of DTNB (0.01 M). After incubation for 10 min at room temperature, absorbance was measured at 412 nm.26
Catalase assayCAT activity was measured based on the decomposition of hydrogen peroxide (H2O2). Tissue homogenate (100 μL) was added to 1 mL of phosphate buffer (pH 7.0) and 500 μL of H2O2 (0.02 M). The decrease in absorbance was monitored at 240 nm for 60 s.27
Superoxide dismutase assaySOD activity was measured using the nitroblue tetrazolium (NBT) reduction method. Briefly, 100 μL of homogenate was mixed with 1 mL of phosphate buffer (pH 7.4), 100 μL of NBT solution, and 100 μL of riboflavin. The mixture was incubated under light for 10 min, and absorbance was read at 560 nm.27
Photomicrography and image analysisLiver sections were examined under a LEICA research microscope (DM750) connected to a digital camera (LEICA ICC50), and permanent photomicrographs were taken. Scaling bars were merged onto each micrograph. Gridley silver-stained and pan-cytokeratin-stained micrographs were quantified for staining intensity by adopting the method described by Amber et al.,28 using computer running image analysis software (ImageJ® NIH, USA), according to the manufacturer’s specifications. The ImageJ® region of interest (ROI) manager tool for analyzing specific areas of the micrographs was employed. The mean gray values for the 3 ROI were obtained, and means were computed and analyzed.
Statistical analysisThe data obtained were analyzed using GraphPad Prism (version 9.3), and results were expressed as mean ± standard error of mean (SEM). The presence of significant differences among means of the groups was determined using the one-way analysis of variance (ANOVA) with the Tukey post hoc test. The alpha level was set at 0.05.
Ethical considerationsThe present study complies with the current regulations on bioethical research, and ethical clearance was obtained from the Health Research and Ethics Committee (HREC) of the Institute of Public Health, Obafemi Awolowo University, Ile-Ife (IPH/OAU/12/2543). This study does not involve any human participants. All experimental procedures were conducted in strict accordance with the principles for laboratory animal care and use. The rats received humane care, efforts were made to minimize the number of animals used and to reduce pain, distress, or discomfort throughout the study period. This study complies with the guidelines outlined in the ARRIVE 2.0 (Animal Research: Reporting of In Vivo Experiments) and follows the principles of the NIH Guide for the Care and Use of Laboratory Animals.20
ResultsBody weight resultThe body weight of rats was measured throughout the 42-day experimental period, as shown in Fig. 2. Before HgCl2 administration, there was an insignificant difference in the average weights of the rats across the groups. However, after the experimentally induced hepatotoxicity, body weight dropped significantly across the groups (Fig. 2). This persisted throughout the experimental period. Treatment with SBLEE and silymarin caused a significant increase in body weight that was inconsistent and fluctuated up to the end of the experiment (Fig. 2).
Histological observation
Hematoxylin and eosin were used to stain liver sections for visualizing the general histoarchitecture of the liver. As shown in Fig. 3, the control rats produced intact morphological features, some of which included polyhedral hepatocytes, with characteristically round euchromatic nuclei with prominent nucleoli and normal radiating sinusoids (Fig. 3A). The characteristic features of a healthy liver were also observed in most of the rats treated with mid and high doses of SBLEE and in the silymarin-treated rats (Fig. 3D, F, and G). However, HgCl2-induced confluent areas of necrosis with numerous cytoplasmic and nuclear necrotic morphological changes were observed in the group B and C rats (Fig. 3). In addition, groups B and C showed disfigured architecture, intranuclear vesiculations, vacuolations, hemorrhagic congestion in the central vein, and sinusoidal dilatation. Rats treated with low-dose SBLEE also presented some of those abnormal morphological features (Fig. 3E).
Representative light microscopy micrographs of liver sections with H&E staining. (A) Polyhedral hepatocytes with prominent central vein (cv), eccentrically placed rounded euchromatic nuclei with prominent nucleoli (green arrow), and endothelial cells lining the radially apparent sinusoids are shown. (B, C, and E) show features of cell degeneration, pyknosis (yellow arrow), hepatocellular vacuolation (green circle), hemorrhagic congestion in the central vein, sinusoidal dilatation (blue arrow), and areas of necrosis (red circle). (D, F, and G) show features similar to the controls, with rounded nuclei and prominent nucleoli (black arrow) and radially apparent sinusoids. Scaling bars – 50 μm.
Gridley’s silver stain was utilized to visualize reticulin fibers of the hepatic ECM, examined by light microscopy. Fig. 4A–G shows the light microscopy photomicrographs. The control rats had a delicate meshwork of reticulin fibers at the portal space and within the extravascular spaces of Disse. However, HgCl2 caused breaking of the perisinusoidal reticular meshwork into lumpy, thickened, small fragments, as observed in groups B and C, as well as in the low-dose SBLEE group (Fig. 4B, C, and E). Mid and high doses of SBLEE restored a close-to-normal perisinusoidal reticular meshwork and the supporting reticulin fiber around blood vessels that was superior to treatment with silymarin (Fig. 4D, F, and G).
(A–G) Representative light microscopy micrographs of liver sections with Gridley’s silver impregnation staining, showing reticulin fibers forming a delicate meshwork in the space of Disse (red arrow) and portal areas (blue parenthesis) outlined in A, D, F, and G and accentuated in B and C with thickened, collapsed, lumpy, and fragmented reticulin fibers (red circle). A less thickened, collapsed reticulin meshwork is seen in E. Scaling bars – 50 μm.
Fig. 5A–G shows the light microscopy photomicrographs of liver sections immunostained with pan-cytokeratin. The group A, D, F, and G rats had diffuse, homogeneous cytoplasmic cytokeratin-positive immunoreactivity. The HgCl2-exposed group B rats had depletion and delocalization of the hepatic cytokeratin, as evidenced by the nearly negative stain reactivity (Fig. 5B). There was intracytoplasmic aggregation of cytokeratin in the group C rats and mild periductal cytokeratin positive immunoreactivity in the low-dose SBLEE group (Fig. 5C and E). The cytoplasmic cytokeratin was restored in a dose-dependent manner with mid and high doses of SBLEE, which was comparable to the results in the silymarin-treated group (Fig. 5D, E, and G).
(A-G) Representative light microscopy micrographs of liver sections with pan-cytokeratin immunoreactivity. A, D, F, and G show diffuse, homogeneous cytoplasmic cytokeratin positive immunoreactivity (brown color). B, C, E show mild periductal positive immunoreactivity. Scaling bars – 50 μm.
Fig. 6 shows the percentage of the area covered by reticulin fiber. This parameter was significantly reduced in the exclusively HgCl2-exposed group B rats (4.36 ± 0.34) and the group C rats (6.51 ± 0.34), when compared with the control group A rats (11.33 ± 0.49). However, when compared with the group B results, a high dose of SBLEE significantly increased the area percentage (9.77 ± 0.14) and was superior to treatment with silymarin (8.11 ± 0.19). There was a significant difference between the silymarin-treated group D (8.11 ± 0.19) and the high-dose SBLEE group G rats (9.77 ± 0.14). There was also a significant difference between the low-dose SBLEE group E rats (7.43 ± 0.16) and a non-significant difference in the mid-dose SBLEE group F rats (8.61 ± 0.33), when compared with the high- dose SBLEE group G rats (9.77 ± 0.14) (F-ratio = 52.60; p < 0.0001).
Percentage of the area covered by the reticulin fiber of Wistar rats exposed to HgCl2 toxicity. Each bar represents the mean ± SEM. The bars with the symbol (α) are significantly different from the group A rats and those with the symbol (β) are significantly different from the group B rats, using the one-way ANOVA and the Tukey test at p < 0.05.
Fig. 7 shows the percentage of the area covered by cytokeratin. HgCl2 significantly reduced and depleted cytokeratin immunoreactivity in the HgCl2-exposed group B rats (11.13 ± 0.05), when compared with control group A (64.57 ± 2.96). However, treatment with SBLEE significantly increased this parameter, especially in the mid and high-dose SBLEE groups (50.64 ± 4.14 and 51.94 ± 3.81, respectively), when compared with the exclusively HgCl2-exposed group B. This increase was similar to that obtained with the silymarin-treated group (33.39 ± 2.07). Cytokeratin immunoreactivity was significantly reduced in the group E and C rats (31.75 ± 0.58 and 26.88 ± 1.78, respectively) when compared with control group A (64.57 ± 2.968) (F-ratio = 47.38; p < 0.0001).
Percentage of the area of pan-cytokeratin immunoreactivity of Wistar rats exposed to HgCl2 toxicity. Each bar represents the mean ± SEM. The bar with the symbol (α) is significantly different from the group A rats, the group with the lowercase letter (a) is not significantly different from the group A rats, and the bar with the symbol (β) is significantly different from the group B rats, using the one-way ANOVA and the Tukey test at p < 0.05.
Fig. 8A–C shows the biochemical assessment of the ALT, AST, and ALP serum liver enzymes, revealing the following: HgCl2significantly increased the activities of serum liver enzymes (ALT, AST, and ALP), when compared with the control group (p < 0.05) (Fig. 8A–C). However, SBLEE caused a significant dose-dependent decrease in the serum level of ALT, AST, and ALP in the group E, F, and G rats, when compared with the exclusively HgCl2-exposed group B. Silymarin also significantly decreased the serum enzyme levels in group D, when compared with the HgCl2-exposed group B rats (ALT; F-ratio = 458.00, AST; 1061.00, ALP; 155.00; p < 0.0001) (Fig. 8A–C).
(A–C) Effects of SBLEE on serum liver enzyme activities of Wistar rats exposed to HgCl2 toxicity. (A) Alanine aminotransferase (ALT) activity, (B) Aspartate aminotransferase (AST) activity, and (C) Alkaline phosphatase (ALP) activity. Each bar represents the mean ± SEM. Bars with the different symbols (α, β) and lowercase letters (c, d, x) differ significantly (p < 0.05). (a) = not significantly different from the control group A, (b) = significantly different from the HgCl2-exposed group B; (x) = significantly different from group D, (c) = significantly different from the mid-dose SBLEE-treated group F, and (d) = significantly different from the low-dose SBLEE-treated group E. Statistical comparison was performed using the one-way ANOVA followed by the Tukey’s post hoc test at p < 0.05.
Fig. 9A–C shows the assessment of the GSH, CAT, and SOD oxidative stress biomarkers, in adult Wistar rat livers, revealing that HgCl2 significantly decreased (p < 0.05) GSH concentrations and CAT and SOD activities in the group B and C rats, when compared with control group A. However, SBLEE caused a significant dose-dependent increase in GSH concentration and CAT and SOD activities in groups E, F, and G, respectively, when compared with the group B rats. Silymarin also significantly increased the oxidative biomarker activities when compared with the group B rats (GSH F-ratio = 656.90; CAT 37.10; SOD 484.00; p < 0.0001) (Fig. 9A–C).
(A–C) Effects of SBLEE on liver activity of antioxidant enzyme activities of Wistar rats exposed to HgCl2 toxicity. (A) Reduced glutathione (GSH) concentration, (B) catalase (CAT) activity, and (C) superoxide dismutase (SOD) activity. Each bar represents the mean ± SEM. Bars with the different symbols (α, β) and lowercase letters (c, d, x) differ significantly (p < 0.05). (a) = Not significantly different from the control group A, (b) = Significantly different from the HgCl2-exposed group (B); (x) = Significantly different from group D, (c) = Significantly different from the mid-dose SBLEE-treated group F; (d) = Significantly different from the low-dose SBLEE-treated group (E). Statistical comparison was performed using one-way ANOVA followed by the Tukey’s post hoc test at p < 0.05.
The protective effects of SBLEE against HgCl2-induced hepatic injury can be attributed to its multifaceted mechanisms of action.29 The results of the present study highlight the cytoprotective efficacy of SBLEE against HgCl2-induced hepatic injury and cytoskeletal disruption. The hepatocyte cytoskeleton is primarily composed of intermediate filaments, particularly cytokeratins (CK8 and CK18), which maintain cell shape, resist mechanical stress, and support intracellular transport.30,31 Their disruption, often marked by cytokeratin aggregation or loss, is a hallmark of liver injury and a diagnostic indicator of hepatocellular damage. This structural compromise is often accompanied by ECM disorganization, resulting in impaired hepatic architecture, sinusoidal collapse, and functional deterioration.32,33
In the present study, HgCl2 exposure led to a low-to-negative expression of pan-cytokeratin immunoreactivity in the exclusively HgCl2-exposed group and mild immunoreactivity in the withdrawal and low-dose SBLEE groups. This decreased expression of immunoreactivity is a result of HgCl2 exposure, which induces oxidative stress, leading to increased production of ROS that cause protein oxidation and lipid peroxidation.34 This oxidative environment results in the phosphorylation and misfolding of cytokeratins, promoting their aggregation and eventual degradation. The disruption of CK8 and CK18 destabilizes hepatocyte architecture, causing loss of cell polarity, impaired bile canaliculi formation, and increased cellular fragility.33,35 Cytokeratins provide mechanical support to epithelial cells, including hepatocytes, and their disruption can lead to increased cellular susceptibility to injury, which may result in cell lysis and necrosis.36 Furthermore, cytokeratin degradation sensitizes hepatocytes to apoptosis.33,36 The disassembly of the cytokeratin network is associated with the activation of apoptotic pathways, particularly those mediated by Caspase 3, resulting in increased hepatocyte death.37 Cytokeratins also act as a scaffold for various signaling pathways that regulate apoptosis, proliferation, and inflammation, further exacerbating liver damage.36,38 Conversely, groups treated with SBLEE, particularly at higher doses, exhibited strong cytokeratin expression, comparable to that of the control and silymarin-treated groups. This preservation of cytokeratin integrity suggests that SBLEE not only protects hepatocytes from oxidative damage but also helps stabilize and maintain the structural integrity of their cytoskeletal framework, reducing cellular injury and promoting recovery from HgCl2-induced damage. These findings align with the previous study by Gao et al.,39 which demonstrated that natural antioxidants, such as those found in plant extracts, play a crucial role in mitigating mercury-induced liver damage by preserving the structural integrity of liver cells and facilitating tissue regeneration.
HgCl2 is a potent environmental toxicant that induces significant metabolic and physiological alterations, leading to profound effects on body weight regulation.40,41 As observed in the present study, the early and consistent manifestation of HgCl2-induced hepatotoxicity was the significant drop in absolute body weight of the experimental rats. The weight loss was driven by the catabolic state induced by mercury toxicity, in which increased oxidative stress and inflammatory cytokine release stimulate proteolysis and lipolysis, resulting in reduced nutrient absorption, increased energy expenditure, and muscle atrophy, and ultimately causing significant body weight loss.42 The administration of SBLEE has shown promising potential in mitigating such adverse effects. In our study, rats treated with SBLEE, following HgCl2 exposure, exhibited significant improvements in body weight, compared with the untreated groups. However, recovery was inconsistent and fluctuated throughout the experimental period. This inconsistent weight gain might suggest that while both SBLEE and silymarin have therapeutic potential, their ability to restore body weight after severe liver toxicity may be limited or require a longer recovery period. The phytochemicals in Senecio biafrae leaves have been reported to enhance hepatic metabolic activity, facilitating better nutrient absorption and utilization.43 The improved metabolic state exhibited by SBLEE administration promotes protein synthesis and lipid metabolism, thereby increasing muscle mass and fat deposition, which contributes to weight gain, as was observed in our study. This recovery in body weight gain correlated with improved liver histoarchitecture, further supporting tissue repair and regeneration, enhancing the recovery of damaged hepatocytes and restoring liver function.44
The liver’s histoarchitecture is highly organized and essential for its diverse functions. Exposure to HgCl2 toxicity induces significant structural and functional damage to the liver through oxidative stress and direct cytotoxicity.45,46
In the present study, HgCl2 exhibited significant histoarchitectural distortions. Said distortions included widespread hepatocyte degeneration, hepatocyte vacuolation, central vein congestion, and sinusoidal dilatation. Hepatocellular necrosis, which is the death of liver cells, was marked by pyknosis, karyorrhexis, and karyolysis. Pyknosis is often considered a hallmark of irreversible cellular injury and indicates severe damage to the liver cells.47 Such findings confirm the hepatotoxic effects of HgCl2 and its capacity to induce significant structural alterations in the liver. In contrast, the liver sections in groups E, F, and G, respectively treated with SBLEE, demonstrated significant improvements. Hepatocyte morphology was largely preserved, with reduced evidence of degeneration and necrosis. Sinusoidal congestion and inflammatory cell infiltration were notably diminished, highlighting the anti-inflammatory properties of SBLEE. The dose-dependent cytoprotective and ameliorative effects of SBLEE observed in our study indicate that higher doses of the extract provide greater protection against oxidative damage, as confirmed by the improved liver architecture at the highest dose (600 mg/kg), which was comparable to the results of the silymarin-treated group. The control group A rats exhibited healthy liver histoarchitecture, indicating the safety of SBLEE at the administered dose. SBLEE helps maintain cellular integrity and prevents hepatocellular damage, characterized by intact cellular borders and organized sinusoidal alignment.
Reticulin fibers, primarily type III collagen, form a delicate meshwork around hepatocytes, and within the space of Disse, play a crucial role in maintaining hepatic microarchitecture by supporting sinusoidal structures and regulating the mechanical properties of the liver parenchyma.48,49 The preservation of reticulin fibers observed in the Gridley’s silver-stained sections in our study provides additional evidence of the role of SBLEE in maintaining liver architecture and extracellular matrix preservation. The extracellular matrix of the liver is composed of collagen, glycoproteins, proteoglycans, and fibrous proteins, including reticulin fibers.50 Disruption of the ECM, as seen in hepatotoxic conditions, such as HgCl2 exposure, leads to fibrosis, altered hepatic microcirculation, and impaired liver function.49,51 The progressive remodeling of the ECM in response to toxic injury compromises hepatocyte polarity, disturbs metabolic zonation, and promotes pathological conditions, such as cirrhosis and hepatocellular carcinoma.52,53 In the group exclusively exposed to HgCl2, reticulin fibers appeared disorganized, fragmented, and sparse, reflecting the disruption of the liver’s structural framework. The integrity of these fibers is crucial for sustaining hepatic microcirculation and structural stability, hepatic repair, and regeneration. Their degradation is a hallmark of severe hepatic injury.48 HgCl2 exposure leads to sinusoidal narrowing, increased hepatic stiffness, and impaired microcirculation, which restricts nutrient and oxygen delivery to hepatocytes, causing further cellular damage. The thickening of reticulin fibers observed in our study suggests that HgCl2 exposure started the early stages of liver fibrogenesis and hepatic necrosis, highlighting the severity of the toxicant’s impact on the liver, reflecting severe structural disorganization. This change aligns with the effect of cell death on interstitial substances of the intercellular space. Contrastingly, in the control group, the reticulin fibers ensure that the liver’s structural framework remains intact, enabling efficient bile flow and nutrient exchange between blood and hepatocytes.49,52 Interestingly, the administration of SBLEE and silymarin doses appeared to reverse some of these fibrotic changes, as evidenced by the preservation of reticulin fibers and the reduced thickening of the reticulin network, thereby enhancing restorative effects on the liver histoarchitecture and ECM integrity. This suggests that SBLEE not only protects against oxidative damage but may also inhibit the fibrogenic process by modulating the activity of hepatic stellate cells and downregulating transforming growth factor-beta (TGF-β) signaling, which reduces excessive collagen deposition and ECM remodeling.54,55 The ability of SBLEE to counteract fibrosis further underscores its potential as a cytoprotective and anti-inflammatory agent, enhancing its regenerative capacity by promoting hepatocyte proliferation and inhibiting apoptotic pathways.29 The preservation of the reticulin network supports hepatocyte anchorage and cellular communication, which facilitates tissue repair and regeneration.56
Serum liver enzymes (ALT, AST, and ALP) are widely regarded as indicators of hepatic function and integrity.57 Their elevated levels in the exclusively HgCl2-exposed group reflect hepatocyte membrane damage and compromised liver function.58 Elevation of ALT is often proportional to the extent of hepatocyte necrosis and is a key marker in assessing acute liver injury, as ALT is a cytosolic enzyme primarily found in the liver.59 The dramatic rise in ALT, AST, and ALP in groups B and C is consistent with the toxicological profile of HgCl2, which has been shown to cause extensive liver damage by triggering oxidative stress, disrupting cellular structures, and impairing normal liver function.24 The control group, in contrast, exhibited much lower ALT, AST, and ALP activities, which is indicative of healthy liver function. The administration of graded doses of SBLEE significantly reduced the enzyme levels in a dose-dependent manner, indicating its ability to protect hepatocytes and maintain membrane stability.
Similarly, the gradual improvement of GSH concentration and CAT and SOD activities in rat livers, through the administration of SBLEE at different doses, was highlighted in the present study. The ability of SBLEE to restore antioxidant enzyme activity suggests its role in mitigating oxidative stress, a key mechanism of HgCl2-induced hepatotoxicity, as evidenced by restored antioxidant enzyme activities in the SBLEE and silymarin-treated groups. The highest dose of SBLEE restored SOD levels close to those of the control group, further demonstrating the therapeutic potential of Senecio biafrae leaves in ameliorating oxidative stress induced by mercury exposure. The antioxidant activity of SBLEE may be attributed to its rich phytochemical composition, including flavonoids and phenolics, which are known to scavenge free radicals and enhance endogenous antioxidant defenses.60 For example, a previous study4 reported that not all antioxidant treatments are equally effective against heavy metal-induced oxidative stress, given that the bioavailability and mechanism of action of phytochemicals can vary. In contrast, our study indicated a consistent and potent effect of SBLEE in restoring GSH concentration and CAT and SOD activities, suggesting that Senecio biafrae leaves may have a broader and more effective antioxidant spectrum, compared with other plant extracts. These findings underscore the potential of SBLEE to counteract oxidative stress and its associated deleterious effects on liver tissue.
ConclusionThe cytoprotective potential of Senecio biafrae leaves is attributed to their ability to stabilize the hepatic cytoskeleton, thereby attenuating structural damage and preserving liver function. These findings position Senecio biafrae leaves as a promising natural cytoprotective agent, with cytoskeletal stabilization as a key mechanism underlying their cytoprotective and ameliorative effect.
Author contributionsProf. D.O. Adeyemi and A.O. Ibitoye conceived and designed the study; A.O. Ibitoye analyzed and interpreted the data; and D.O. Adeyemi supervised the research.
Consent for publicationBoth authors have read and approved all aspects of the manuscript for its publication.
Manuscript approvalThe manuscript has been read and approved for publication in the Revista de Gastroenterología de México by both authors.
The authors declare there is no conflict of interest.
The authors wish to thank M.S. Ige, of the Department of Anatomy and Cell Biology of the Obafemi Aolowo University, for the technical assistance in preparing the histological slides.
