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Full Research Article

Ameliorative Effects of Helix aspersa Extract against Liver Injury Induced by High Methionine Intake in Rats

S
Souad Lamda1,*
0000-0002-3189-5375
D
Dalila Naimi1
0000-0003-3304-4766
1École Nationale Supérieure de Biotechnologie Taoufik Khaznadar (ENSB), Ali Mendjeli, University City E66 BP 25100, Constantine, Algeria.

ABSTRACT

Background: Elevated circulating homocysteine (Hcy) has been proposed to be associated with nonalcoholic fatty liver disease. Hcy is produced as an intermediate during the metabolism of methionine, an essential amino acid obtained from dietary sources. The objective of this study was to investigate the effect of high methionine-intake on the rat liver and to explore the efficacy of Helix aspersa crude extract against the induced liver injury. 

Methods: For this purpose, twenty-four rats were divided into four groups: Control, Met (L-Methionine), HAH (Helix aspersa homogenate) and Met-HAH. Then, biochemical parameters were determined in serum, while liver supernatants were used for determination of total protein and malondialdehyde (MDA) levels. Liver injuries were assessed according to Ishak scoring.

Result: High methionine intake induced moderate hyperhomocysteinemia (HHcy), hyperlipidemia, moderate hepatitis, raised significantly liver markers, CRP (C-Reactive Protein) and lipid peroxidation compared to the control group. The combined treatment Met-HAH induced a significant decrease of Hcy, lipid profile, MDA, inflammation and liver markers and attenuated liver injuries. 

INTRODUCTION

The liver is a vital organ with a crucial role in the metabolism of organic molecules and the regulation of various physiological functions (Almazroo et al., 2016; Maurya et al., 2025). Hepatitis is an inflammatory condition of the hepatic parenchyma, resulting in elevated liver function markers. One of the most common causes of liver disease worldwide is nonalcoholic fatty liver disease (NAFLD) which is associated with obesity and steatosis (Adams et al., 2005; Gulati et al., 2018). In general, hepatitis is classified as acute or chronic based on the duration of the inflammation and damage to the hepatic parenchyma. Chronic hepatitis should be graded according to the severity of inflammation and hepatocellular injury (Cacciola et al., 2017; Elangovan et al., 2022; Unsaldi and Kalinbacak, 2025). The two key scoring systems used to determine the severity of the disease (mild, moderate or severe) are staging and Hepatitis Activity Index (HAI) grading (Ishak et al., 1995). Moreover, biochemical markers of liver dysfunction, including serum alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP), total bilirubin (TB) and albumin (Alb) are highly sensitive indicators of the occurred abnormalities. These tests aid in determining the extent of hepatic injury (Hoekstra et al., 2013; Singh et al., 2021; Lala et al., 2022). Healthy dietary patterns, intake of unsaturated fats and certain nutritional supplements (omega-3 fatty acids, probiotics, vitamin E and milk thistle) help protect against NAFLD and contribute to liver function (Miller, 2020; Zhang et al., 2021; Służały et al., 2024).
       
A recent study has demonstrated that high-methionine-intake may increase the risk of developing NAFLD (Yang et al., 2022). Homocysteine is a sulfhydryl-containing amino acid mainly produced by metabolism of methionine and catabolized in the liver. It can alter intracellular lipid metabolism and potentially promote hepatic fat accumulation. However, the underlying mechanism remains unclear and only a limited number of studies have explored the association between homocysteine and NAFLD prevalence (Dai et al., 2016; Yang et al., 2022).
       
Natural products are gaining great attention due to their biological effects, with specific interest in marine and land snails (Zizioli et al., 2022; Aouji et al., 2023). Gastropod mollusks have been used in medicine since antiquity. Crude snails’ extracts are considered therapeutic substances (Bonnemain, 2005; Conte, 2016).
       
Therefore, this study was conducted to investigate the possible involvement of HHcy in the induction of non-alcoholic fatty liver disease by correlating HAI scores with biochemical parameters. Besides, it aimed to assess the effects of Helix aspersa crude extract on liver damage induced by high-methionine-intake in rats.

MATERIALS AND METHODS

Drugs and chemicals
 
L-Methionine 98% was purchased from Acros Organics (Belgium), kinetic kits (ALT, AST and ALP) and colorimetric kits (Alb and TB) were obtained from Spinreact (Spain). All other chemicals were purchased from Sigma chemical company (St. Louis, MO, USA).
 
Preparation of HA crude extract
 
The used snails were housed in plastic cages and fed fresh lettuce leaves for two weeks under laboratory conditions, followed by a two-week fasting period. The snail homogenate was prepared according to the method of El Ouar et al. (2017) with modification. To remove mucus, the snails were washed five times in 10% NaCl solution. The shells were then removed and the entire soft body usually consumed, was used. Fresh snail homogenate was prepared just before use as a treatment for the animals. The specimens were weighed and homogenized in ice-cold phosphate buffer-saline (PBS).
 
Experimental design
 
The current study was conducted from May until November 2024 in the Bioengineering lab at ENSB (École Nationale supérieure de Biotechnologie) in Constantine, Algeria. Male albino Wistar rats aged of 10 weeks old (n=24) and weighing (200-250g) were obtained from Pasteur Institute (Algiers, Algeria). The animals were housed in standard cages under a controlled room temperature and a normal dark-light cycles. After two weeks of acclimation, the rats were divided into four groups (n=6 per group). The experimental design is outlined in Fig 1. Treatments were administered through gavage. In the Met group, HHcy was induced by high-methionine-intake (1g/kg/day) (Bhandari et al., 2011). HAH treated groups received HAH at a dose of 8g/kg/day (Lamda et al., 2017). All groups were fed the same diet as the control (standard animal food) with ad libitum access to water. All animal procedures were conducted in strict accordance with the ARRIVE Guidelines 2.0 for reporting in vivo experiments. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Fig 1: Scheme of experimental design (partially created with https://app.biorender.com/ , 2024).


       
At the end of the experiment, the rats were fasted overnight. All the animals were sacrificed and blood samples were collected into dry tubes. Serum was obtained by centrifugation (3500 rpm/10 min) and stored at -80°C until the biochemical analysis. The liver of each rat was excised and washed in PBS, dried and weighed. Liver specimens were then divided into three parts: the first part for total protein estimation, the second part for Malondialdehyde (MDA) evaluation and the third for histopathological investigation. Each liver sample was weighed and homogenized using a tissue homogenizer in ice-cold lysis buffer containing 2 0mM PBS, pH (7.4). The tissue homogenate was centrifuged at 10,000 rpm for 20 minutes, at 4°C, then the obtained supernatant was aliquoted into Eppendorf tubes and preserved at -80°C until use.
 
Measurement of serum biochemical parameters
 
The biomarkers were spectrophotometrically evaluated using kinetic kits (Spin react, Spain) according to the manufacturer’s instructions based on the method described earlier for ALT (Ref: SP41274), AST (Ref: 1001161), ALP (Ref: TK41233). Colorimetric kits (Spin react, Spain) were used for Alb (Ref: SP1001020) and TB (Ref: 1001041). Serum lipid levels were determined using enzymatic colorimetric methods with commercial kit (Spin react, Spain) for TC (Ref: 1001092) and triglycerides (Ref: 1001312). Homocysteine levels were determined using high performance liquid chromatography with fluorescence detection according to Minniti et al., (1998). CRP levels were measured using a Rat C-Reactive Protein ELISA Kit, catalog number ERCRP, (Invitrogen, USA). Levels of serum and liver MDA were determined according to the method of Ohkawa et al., (1979), previously described and supernatant protein content was estimated using Bradford assay (Bradford, 1976).
 
Liver histopathology and histological scoring
 
A small cross section of liver tissue was fixed in 10% formaldehyde then embedded in paraffin and sectioned into 5ìm slices for histological analysis. Tissues were stained with hematoxylin and eosin (H and E) and observed by histopathologist at 10× and 40× magnification under light microscope. Images were captured using an Optec Optical Technology® electronic camera. Liver specimens were examined semi-quantitatively based on an observational scoring system according to Ishak et al., (1995) and Goodman, (2007) assessing liver injury severity through the HAI.
 
Statistical analysis
 
Data were expressed as Means ± Standard deviation (SD). Statistical analyses and graph presentations were performed using one-way analysis of variance (ANOVA) via Graph Pad Prism 10 for Windows followed by Newman-keuls post hoc test. A P-value <0.05 was considered statistically significant.

RESULTS AND DISCUSSION

In the current study, abnormal liver function was observed after four weeks of methionine intake. Liver enzymes were significantly raised (p<0.0001) and total bilirubin was elevated (Table 1). Similar results were reported by Woo et al., (2006) and Derouiche et al., (2024). The results (Table 1) exhibited intermediate HHcy, hyperlipidemia and raised oxidative stress and inflammation, in methionine-treated rats compared to the control group. Similar results were found by Bhandari et al. (2011), Yang et al., (2022) and Derouiche et al. (2024). The Met group displayed a substantial increase in liver weight (Fold=1.31) and liver MDA (Fold=1.55) compared to the control group (Table 1). Similar results were reported by Woo et al., (2006) and Derouiche et al., (2024). The histopathological analysis of the Met group revealed marked liver injury, including interface hepatitis, parenchymal injury and inflammation (Fig 2). The hepatitis activity index (HAI = 10±1.1) was significantly increased (p<0.0001) compared to the control group. Histopathological investigation, confirmed biochemical results, demonstrating liver injury and moderate hepatitis. Liver alterations in methionine-treated rats closely resemble those found in NASH, lobular inflammation, apoptosis and varying degrees of hepatic fibrosis. Derouiche et al., (2024), reported less marked alterations with ballooned cells in the liver tissue of high-methionine-treated rats (1g/kg) for 21 days.

Table 1: Changes in liver markers, lipid profile, lipid peroxidation and inflammation markers before and after treatments in different groups.



Fig 2: Histopathological analysis.


       
It is well documented that HHcy induces oxidative stress through an autoxidation reaction, producing reactive oxygen species (ROS) and decreasing the expression of antioxidants (Jalal et al., 2023). Oxidative stress can trigger endoplasmic reticulum (ER) stress which activates transcription factors that upregulate genes implicated in lipid production (Skovierová et al., 2016). Wu et al., (2011) demonstrated that in HHcy rats, both liver and serum cholesterol and triglyceride levels are elevated. In addition, cholesterol synthesis and accumulation in mitochondria lead to mitochondrial dysfunction and its sensitization to oxidative stress and inflammatory cytokines (Nuño-Lámbarri et al., 2016). Li et al., (2021) demonstrated that homocysteine can stimulate production of CRP in vitro and in-vivo in high-methionine-diet-induced HHcy rat model. Recently, new observations suggest that increased intracellular Hcy levels boost ER stress response genes, leads to ROS generation, apoptosis promotor activation and NF-κB activation, resulting in oxidative stress and inflammation (Jalal et al., 2023).
       
The HAH group showed no significant differences in biochemical parameters compared to the control group (Table 1) and microscopic analysis of liver slides from the control and HAH groups revealed normal lobular organization (Fig 2).
       
HAH treatment remarkably ameliorated inflammation and reduced liver markers, lipid profile, oxidative stress, HHcy and CRP levels induced by high- methionine-intake in rats of Met group. These findings suggest that HAH positively influences lipid metabolism and exerts anti-oxidative and anti-inflammatory effects in the liver. In the Met-HAH group, relative liver weight and liver MDA levels showed a notable reduction, with liver MDA exhibiting a significant decrease (Table 1). Examination of HandE-stained slides from the Met-HAH group showed that HAH treatment significantly ameliorated the necro-inflammatory changes induced by methionine (Fig 2). Met-HAH rats exhibited a significant decrease in Hepatitis activity index (Fold=0.58) when compared to the Met group rats.
       
Bioactive natural products show promising effects against NAFLD, primarily by reducing hepatic fat accumulation, inflammation and fibrosis (Datta et al., 2023; Służały et al., 2024).
       
Among species of land snail, HA is the most popular and extensively consumed in the North Mediterranean. Mollusk meat is highly nutritious because it contains high levels of proteins, essential amino acids, abundant vitamins and minerals and minimal fat (Cağıltay et al., 2011). Snail protein is particularly value of its excellent amino acid profile. In a study by Matusiewicz et al., (2018), the crude protein content in lyophilized foot tissues of HA was the highest (80.74%), while crud fat was the lowest (less than 4%). Bongiorno et al., (2024), described the biochemical composition of HA, highlighting that polyunsaturated fatty acids were predominant in the fatty acid profile which was primarily consisted of polyunsaturated fatty acids, followed by saturated fatty acids and monounsaturated fatty acids. Additionally, Matusiewicz et al., (2018), reported a high concentration of n-3 polyunsaturated fatty acid in the foot tissues of HA. Furthermore, previous studies, have demonstrated the presence of a high content of enzymatic and non-enzymatic antioxidants in the foot tissues of HA, such as glutathione reductase, selenium-dependent glutathione peroxidase, total glutathione peroxidase, glutathione S-transferase, superoxide dismutase and catalase and reduced glutathione. The highest antioxidant enzyme activities were those of glutathione reductase and glutathione peroxidase (Ramos-Vasconcelos and Hermes-Lima, 2003; Nowakowska et al., 2012).
       
Our results suggested that HA crude extract showed many biological properties and was remarkably effective in modulating oxidative stress and inflammation, possibly due to its composition of nutritional and bioactive compounds. 

CONCLUSION

To our knowledge, this is the first in vivo demonstration of the anti-inflammatory-antioxidant properties of HA snail crude extract against high-methionine-intake-induced liver injury. Our biochemical and histopathological analysis prove that HHcy induced by subchronic high-methionine-intake leads to intermediate hepatitis (NASH-like alterations), inflammation, oxidative stress and hyperlipidemia. Furthermore, our findings indicate that HAH ameliorated the deleterious effects of high-methionine-intake on liver tissue and biochemical parameters. These results suggest that HAH may exert regulatory influence over critical biological processes involved in liver disorders, including lipid metabolism, oxidative stress and inflammation. However, these results should be further supported by confirmation of the molecular mechanism(s) involved. Nutritional therapy is fundamental in NAFLD/NASH management, as no medically sanctioned therapies are currently available. The present study opens new perspectives in the search for new natural therapies against HHcy and associated diseases.

ACKNOWLEDGEMENT

The present study was supported by the Algerian Ministry of Higher Education and scientific research. The authors express their gratitude to Dr. Ahlem Chelghoum, for reviewing the entire article.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Algerian National Council of Ethics for Health Sciences under the authority of Ministry of Health.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Ameliorative Effects of Helix aspersa Extract against Liver Injury Induced by High Methionine Intake in Rats

    S
    Souad Lamda1,*
    0000-0002-3189-5375
    D
    Dalila Naimi1
    0000-0003-3304-4766
    1École Nationale Supérieure de Biotechnologie Taoufik Khaznadar (ENSB), Ali Mendjeli, University City E66 BP 25100, Constantine, Algeria.

    ABSTRACT

    Background: Elevated circulating homocysteine (Hcy) has been proposed to be associated with nonalcoholic fatty liver disease. Hcy is produced as an intermediate during the metabolism of methionine, an essential amino acid obtained from dietary sources. The objective of this study was to investigate the effect of high methionine-intake on the rat liver and to explore the efficacy of Helix aspersa crude extract against the induced liver injury. 

    Methods: For this purpose, twenty-four rats were divided into four groups: Control, Met (L-Methionine), HAH (Helix aspersa homogenate) and Met-HAH. Then, biochemical parameters were determined in serum, while liver supernatants were used for determination of total protein and malondialdehyde (MDA) levels. Liver injuries were assessed according to Ishak scoring.

    Result: High methionine intake induced moderate hyperhomocysteinemia (HHcy), hyperlipidemia, moderate hepatitis, raised significantly liver markers, CRP (C-Reactive Protein) and lipid peroxidation compared to the control group. The combined treatment Met-HAH induced a significant decrease of Hcy, lipid profile, MDA, inflammation and liver markers and attenuated liver injuries. 

    INTRODUCTION

    The liver is a vital organ with a crucial role in the metabolism of organic molecules and the regulation of various physiological functions (Almazroo et al., 2016; Maurya et al., 2025). Hepatitis is an inflammatory condition of the hepatic parenchyma, resulting in elevated liver function markers. One of the most common causes of liver disease worldwide is nonalcoholic fatty liver disease (NAFLD) which is associated with obesity and steatosis (Adams et al., 2005; Gulati et al., 2018). In general, hepatitis is classified as acute or chronic based on the duration of the inflammation and damage to the hepatic parenchyma. Chronic hepatitis should be graded according to the severity of inflammation and hepatocellular injury (Cacciola et al., 2017; Elangovan et al., 2022; Unsaldi and Kalinbacak, 2025). The two key scoring systems used to determine the severity of the disease (mild, moderate or severe) are staging and Hepatitis Activity Index (HAI) grading (Ishak et al., 1995). Moreover, biochemical markers of liver dysfunction, including serum alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline phosphatase (ALP), total bilirubin (TB) and albumin (Alb) are highly sensitive indicators of the occurred abnormalities. These tests aid in determining the extent of hepatic injury (Hoekstra et al., 2013; Singh et al., 2021; Lala et al., 2022). Healthy dietary patterns, intake of unsaturated fats and certain nutritional supplements (omega-3 fatty acids, probiotics, vitamin E and milk thistle) help protect against NAFLD and contribute to liver function (Miller, 2020; Zhang et al., 2021; Służały et al., 2024).
           
    A recent study has demonstrated that high-methionine-intake may increase the risk of developing NAFLD (Yang et al., 2022). Homocysteine is a sulfhydryl-containing amino acid mainly produced by metabolism of methionine and catabolized in the liver. It can alter intracellular lipid metabolism and potentially promote hepatic fat accumulation. However, the underlying mechanism remains unclear and only a limited number of studies have explored the association between homocysteine and NAFLD prevalence (Dai et al., 2016; Yang et al., 2022).
           
    Natural products are gaining great attention due to their biological effects, with specific interest in marine and land snails (Zizioli et al., 2022; Aouji et al., 2023). Gastropod mollusks have been used in medicine since antiquity. Crude snails’ extracts are considered therapeutic substances (Bonnemain, 2005; Conte, 2016).
           
    Therefore, this study was conducted to investigate the possible involvement of HHcy in the induction of non-alcoholic fatty liver disease by correlating HAI scores with biochemical parameters. Besides, it aimed to assess the effects of Helix aspersa crude extract on liver damage induced by high-methionine-intake in rats.

    MATERIALS AND METHODS

    Drugs and chemicals
     
    L-Methionine 98% was purchased from Acros Organics (Belgium), kinetic kits (ALT, AST and ALP) and colorimetric kits (Alb and TB) were obtained from Spinreact (Spain). All other chemicals were purchased from Sigma chemical company (St. Louis, MO, USA).
     
    Preparation of HA crude extract
     
    The used snails were housed in plastic cages and fed fresh lettuce leaves for two weeks under laboratory conditions, followed by a two-week fasting period. The snail homogenate was prepared according to the method of El Ouar et al. (2017) with modification. To remove mucus, the snails were washed five times in 10% NaCl solution. The shells were then removed and the entire soft body usually consumed, was used. Fresh snail homogenate was prepared just before use as a treatment for the animals. The specimens were weighed and homogenized in ice-cold phosphate buffer-saline (PBS).
     
    Experimental design
     
    The current study was conducted from May until November 2024 in the Bioengineering lab at ENSB (École Nationale supérieure de Biotechnologie) in Constantine, Algeria. Male albino Wistar rats aged of 10 weeks old (n=24) and weighing (200-250g) were obtained from Pasteur Institute (Algiers, Algeria). The animals were housed in standard cages under a controlled room temperature and a normal dark-light cycles. After two weeks of acclimation, the rats were divided into four groups (n=6 per group). The experimental design is outlined in Fig 1. Treatments were administered through gavage. In the Met group, HHcy was induced by high-methionine-intake (1g/kg/day) (Bhandari et al., 2011). HAH treated groups received HAH at a dose of 8g/kg/day (Lamda et al., 2017). All groups were fed the same diet as the control (standard animal food) with ad libitum access to water. All animal procedures were conducted in strict accordance with the ARRIVE Guidelines 2.0 for reporting in vivo experiments. All efforts were made to minimize animal suffering and to reduce the number of animals used.

    Fig 1: Scheme of experimental design (partially created with https://app.biorender.com/ , 2024).


           
    At the end of the experiment, the rats were fasted overnight. All the animals were sacrificed and blood samples were collected into dry tubes. Serum was obtained by centrifugation (3500 rpm/10 min) and stored at -80°C until the biochemical analysis. The liver of each rat was excised and washed in PBS, dried and weighed. Liver specimens were then divided into three parts: the first part for total protein estimation, the second part for Malondialdehyde (MDA) evaluation and the third for histopathological investigation. Each liver sample was weighed and homogenized using a tissue homogenizer in ice-cold lysis buffer containing 2 0mM PBS, pH (7.4). The tissue homogenate was centrifuged at 10,000 rpm for 20 minutes, at 4°C, then the obtained supernatant was aliquoted into Eppendorf tubes and preserved at -80°C until use.
     
    Measurement of serum biochemical parameters
     
    The biomarkers were spectrophotometrically evaluated using kinetic kits (Spin react, Spain) according to the manufacturer’s instructions based on the method described earlier for ALT (Ref: SP41274), AST (Ref: 1001161), ALP (Ref: TK41233). Colorimetric kits (Spin react, Spain) were used for Alb (Ref: SP1001020) and TB (Ref: 1001041). Serum lipid levels were determined using enzymatic colorimetric methods with commercial kit (Spin react, Spain) for TC (Ref: 1001092) and triglycerides (Ref: 1001312). Homocysteine levels were determined using high performance liquid chromatography with fluorescence detection according to Minniti et al., (1998). CRP levels were measured using a Rat C-Reactive Protein ELISA Kit, catalog number ERCRP, (Invitrogen, USA). Levels of serum and liver MDA were determined according to the method of Ohkawa et al., (1979), previously described and supernatant protein content was estimated using Bradford assay (Bradford, 1976).
     
    Liver histopathology and histological scoring
     
    A small cross section of liver tissue was fixed in 10% formaldehyde then embedded in paraffin and sectioned into 5ìm slices for histological analysis. Tissues were stained with hematoxylin and eosin (H and E) and observed by histopathologist at 10× and 40× magnification under light microscope. Images were captured using an Optec Optical Technology® electronic camera. Liver specimens were examined semi-quantitatively based on an observational scoring system according to Ishak et al., (1995) and Goodman, (2007) assessing liver injury severity through the HAI.
     
    Statistical analysis
     
    Data were expressed as Means ± Standard deviation (SD). Statistical analyses and graph presentations were performed using one-way analysis of variance (ANOVA) via Graph Pad Prism 10 for Windows followed by Newman-keuls post hoc test. A P-value <0.05 was considered statistically significant.

    RESULTS AND DISCUSSION

    In the current study, abnormal liver function was observed after four weeks of methionine intake. Liver enzymes were significantly raised (p<0.0001) and total bilirubin was elevated (Table 1). Similar results were reported by Woo et al., (2006) and Derouiche et al., (2024). The results (Table 1) exhibited intermediate HHcy, hyperlipidemia and raised oxidative stress and inflammation, in methionine-treated rats compared to the control group. Similar results were found by Bhandari et al. (2011), Yang et al., (2022) and Derouiche et al. (2024). The Met group displayed a substantial increase in liver weight (Fold=1.31) and liver MDA (Fold=1.55) compared to the control group (Table 1). Similar results were reported by Woo et al., (2006) and Derouiche et al., (2024). The histopathological analysis of the Met group revealed marked liver injury, including interface hepatitis, parenchymal injury and inflammation (Fig 2). The hepatitis activity index (HAI = 10±1.1) was significantly increased (p<0.0001) compared to the control group. Histopathological investigation, confirmed biochemical results, demonstrating liver injury and moderate hepatitis. Liver alterations in methionine-treated rats closely resemble those found in NASH, lobular inflammation, apoptosis and varying degrees of hepatic fibrosis. Derouiche et al., (2024), reported less marked alterations with ballooned cells in the liver tissue of high-methionine-treated rats (1g/kg) for 21 days.

    Table 1: Changes in liver markers, lipid profile, lipid peroxidation and inflammation markers before and after treatments in different groups.



    Fig 2: Histopathological analysis.


           
    It is well documented that HHcy induces oxidative stress through an autoxidation reaction, producing reactive oxygen species (ROS) and decreasing the expression of antioxidants (Jalal et al., 2023). Oxidative stress can trigger endoplasmic reticulum (ER) stress which activates transcription factors that upregulate genes implicated in lipid production (Skovierová et al., 2016). Wu et al., (2011) demonstrated that in HHcy rats, both liver and serum cholesterol and triglyceride levels are elevated. In addition, cholesterol synthesis and accumulation in mitochondria lead to mitochondrial dysfunction and its sensitization to oxidative stress and inflammatory cytokines (Nuño-Lámbarri et al., 2016). Li et al., (2021) demonstrated that homocysteine can stimulate production of CRP in vitro and in-vivo in high-methionine-diet-induced HHcy rat model. Recently, new observations suggest that increased intracellular Hcy levels boost ER stress response genes, leads to ROS generation, apoptosis promotor activation and NF-κB activation, resulting in oxidative stress and inflammation (Jalal et al., 2023).
           
    The HAH group showed no significant differences in biochemical parameters compared to the control group (Table 1) and microscopic analysis of liver slides from the control and HAH groups revealed normal lobular organization (Fig 2).
           
    HAH treatment remarkably ameliorated inflammation and reduced liver markers, lipid profile, oxidative stress, HHcy and CRP levels induced by high- methionine-intake in rats of Met group. These findings suggest that HAH positively influences lipid metabolism and exerts anti-oxidative and anti-inflammatory effects in the liver. In the Met-HAH group, relative liver weight and liver MDA levels showed a notable reduction, with liver MDA exhibiting a significant decrease (Table 1). Examination of HandE-stained slides from the Met-HAH group showed that HAH treatment significantly ameliorated the necro-inflammatory changes induced by methionine (Fig 2). Met-HAH rats exhibited a significant decrease in Hepatitis activity index (Fold=0.58) when compared to the Met group rats.
           
    Bioactive natural products show promising effects against NAFLD, primarily by reducing hepatic fat accumulation, inflammation and fibrosis (Datta et al., 2023; Służały et al., 2024).
           
    Among species of land snail, HA is the most popular and extensively consumed in the North Mediterranean. Mollusk meat is highly nutritious because it contains high levels of proteins, essential amino acids, abundant vitamins and minerals and minimal fat (Cağıltay et al., 2011). Snail protein is particularly value of its excellent amino acid profile. In a study by Matusiewicz et al., (2018), the crude protein content in lyophilized foot tissues of HA was the highest (80.74%), while crud fat was the lowest (less than 4%). Bongiorno et al., (2024), described the biochemical composition of HA, highlighting that polyunsaturated fatty acids were predominant in the fatty acid profile which was primarily consisted of polyunsaturated fatty acids, followed by saturated fatty acids and monounsaturated fatty acids. Additionally, Matusiewicz et al., (2018), reported a high concentration of n-3 polyunsaturated fatty acid in the foot tissues of HA. Furthermore, previous studies, have demonstrated the presence of a high content of enzymatic and non-enzymatic antioxidants in the foot tissues of HA, such as glutathione reductase, selenium-dependent glutathione peroxidase, total glutathione peroxidase, glutathione S-transferase, superoxide dismutase and catalase and reduced glutathione. The highest antioxidant enzyme activities were those of glutathione reductase and glutathione peroxidase (Ramos-Vasconcelos and Hermes-Lima, 2003; Nowakowska et al., 2012).
           
    Our results suggested that HA crude extract showed many biological properties and was remarkably effective in modulating oxidative stress and inflammation, possibly due to its composition of nutritional and bioactive compounds. 

    CONCLUSION

    To our knowledge, this is the first in vivo demonstration of the anti-inflammatory-antioxidant properties of HA snail crude extract against high-methionine-intake-induced liver injury. Our biochemical and histopathological analysis prove that HHcy induced by subchronic high-methionine-intake leads to intermediate hepatitis (NASH-like alterations), inflammation, oxidative stress and hyperlipidemia. Furthermore, our findings indicate that HAH ameliorated the deleterious effects of high-methionine-intake on liver tissue and biochemical parameters. These results suggest that HAH may exert regulatory influence over critical biological processes involved in liver disorders, including lipid metabolism, oxidative stress and inflammation. However, these results should be further supported by confirmation of the molecular mechanism(s) involved. Nutritional therapy is fundamental in NAFLD/NASH management, as no medically sanctioned therapies are currently available. The present study opens new perspectives in the search for new natural therapies against HHcy and associated diseases.

    ACKNOWLEDGEMENT

    The present study was supported by the Algerian Ministry of Higher Education and scientific research. The authors express their gratitude to Dr. Ahlem Chelghoum, for reviewing the entire article.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Algerian National Council of Ethics for Health Sciences under the authority of Ministry of Health.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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