Indian Journal of Animal Research

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Protective effect of Bacopa monnieri against Methotrexate induced Hepatotoxicity in Wistar rats

Srinivas Tummala1, Anilkumar Banothu1,*, B. Kumar1, M. Lakshman2, G. Ambica3, Y. Ravikumar2
1Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science, PV Narsimha Rao Telangana Veterinary University, Rajendranagar, Hyderabad-500 030, Telangana, India.
2Department of Veterinary Pathology, College of Veterinary Science, Rajendranagar, Hyderabad-500 030, Telangana, India.
3Department of Veterinary Medicine, College of Veterinary Science, Rajendranagar, Hyderabad-500 030, Telangana, India.

Background: Methotrexate (MTX) is an anticancer drug and its clinical efficacy is limited to the toxicity associated with the liver. In this study, we examine the potential role of aqueous leaf extracts of Bacopa monnieri to counter methotrexate-induced hepatotoxicity. 

Methods: This study was undertaken by considering 36 numbers. Wistar male rats were randomly segregated into six groups, with six rats per group. Groups 1 and 2 were kept as normal controls and methotrexate was treated on the 9th day. While groups 3 and 4 received an aqueous extract of the leaf of B. monnieri at two varied doses of 150 and 300 mg/kg, respectively, as a treatment, Whereas groups 5 and 6 kept B. monnieri perse and the standard hepatoprotective drug silymarin @ 200 mg/kg and experiment was conducted for 14 days period. After the experimental procedure, AST, ALT, TNF-á and IL-10 were measured and histopathology of the liver was done.

Result: The present study revealed significant alterations in AST, ALT, TNF-alfa and IL-10 in methotrexate-treated rats (group 2) when compared to group 1 and treatment groups 4 and 5 revealed significant improvement and these results were validated through imaging in the histopathology of the liver. We conclude that Bacopa monnieri can efficiently reduce methotrexate-induced liver inflammation and can be used in the management of hepatotoxicity in the liver.

Methotrexate (MTX) is a folate antimetabolite and derivative of aminopterin used as a cytotoxic anticancer agent in malignancies discovered during 1940, it acts by interfering with DNA biosynthesis, repair and cellular replication (Pawankalyan et al., 2022). Methotrexate was first employed as a treatment for acute leukemia in children back in 1948 and later on, it was also introduced as a therapy for psoriasis and rheumatoid arthritis (RA). The drug received FDA (Food and Drug Administration) approval for RA treatment in 1988 and for psoriasis treatment in 1972, becoming one of the most commonly used disease-modifying antirheumatic drugs globally (Tenti et al., 2023). The precise mechanism of MTX-induced hepatotoxicity is unknown at this time. Reactive oxygen species (ROS) metabolites have recently been discovered to play a key role in the hepatotoxicity of a variety of xenobiotics and medications. There is evidence that MTX can reduce oxygen uptake in isolated mitochondria and inhibit oxidative phosphorylation in the mitochondria, it can also inhibit the enzymes 2-oxoglutarate, isocitrate, malate and pyruvate dehydrogenases in mitochondria as well (Ghoneum and El-Gerbed, 2021). The consequences of ROS formation lead to stimulating transcription factors such as nuclear factor-kappa B (NF-κB) which can trigger an increase in the expression of genes related to the synthesis of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and IL-6. Subsequently, these agents contribute to tissue harm and the initiation of apoptosis (Sayed et al., 2022). For the treatment of liver disorders plants have been used as excellent sources for the management of liver toxicity due to the presence of phytochemicals like flavonoids, phenols, terpenoids and steroids (Gopi et al., 2010; Priyanka et al., 2020).
For centuries, Bacopa monnieri has been utilized as a traditional ayurvedic medicine in India to treat various illnesses and conditions, particularly as a nerve tonic and cardiotonic (Adams et al., 2007; Jain, et al., 2016). The plant is known to contain triterpenoid saponins, alkaloids and flavonoids, which have been found to have antioxidant properties (Hou et al., 2002; Allawadhi et al., 2021). In fact, studies have shown that alcohol extracts of Bacopa monnieri are effective in acting as antioxidants, free radical scavengers and anti-lipid peroxidative agents (Bhattacharya, 2011). Moreover, previous research has indicated that B. monnieri can inhibit the formation of superoxide anions in a dose-dependent manner and can also mitigate nitric oxide toxicity in rat astrocytes (Chougle et al., 2021).
B. monnieri’s phytochemicals, such as the alkaloid brahmine, nicotine, herpestine, bacosides A and B, saponins A, B and C, triterpenoid saponins, stigmastanol, -sitosterol, betulinic acid, D-mannitol, stigmasterol, -alanine, aspartic acid, glutamic acid and serine and pseudojujubogenin, are believed to have hepatoprotective effects (Manvitha et al., 2019: Jeyasri et al., 2020).
All chemicals were of analytical grade and they are obtained from Qualigens Pvt. Ltd., Mumbai and SRL Pvt. Ltd., Mumbai, India.
Plant material and preparation of leaf extract
To obtain the plant material, fresh leaves of B. monnieri were gathered near to Hyderabad, India and authenticated by a Scientist at Agricultural College, Hyderabad, India. Approximately 40-45 days were required for air-drying the leaves after being washed twice with distilled water. The dried leaves were then ground into a fine powder using a mechanical blender. Aproximately measured 100 gram of powder taken and was boiled by blending in a 100 ml of distilled water and stirred  for 15 minutes on a hot plate to obtain extract. Subsequently the extract was filtered by susing Whatman No.1 filter paper and stored at low temperature (4°C) until further use.
Animals and experimental design
In this experimental study, 36 Wistar male rats weighing in between 180±10 g were acquired from Vyas labs located in Hyderabad and were assigned to 6 groups (n=6) for administering varied treatments scheduled. The rats were placed in polycarbonated cages by maintaining ambient temperature, including a temperature of 20-22°C and a 12-hour light and dark cycle. The bedding material consisted of sterilized, dried, clean, autoclaved rice husk, replaced every other day. The rats were provided a standard balanced diet and access to drinking water ad libitum for the entire experimental period. Throughout the experimental period, the rats were given a nutritionally balanced diet.
Additionally, they were allowed to Reverse osmosis drink water freely. This ensured that the rats remained healthy and hydrated throughout the experiment. The Institutional Animal Ethical Committee reviewed and approved all the procedures and protocols used in the study, ensuring that the research complied with ethical standards. (No.2/22/C.V.Sc., Hyd. IAEC- Rats/29.02.2020) and the experiment was conducted in the Lab Animal House, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science, Rajendranagar, Hyderabad, in August 2021 in compliance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Experimental design with group-wise treatment protocol
Group 1 received normal saline (PO) as a sham, while group 2 rats were administered Methotrexate @ 20 mg BW via the IP route as a single dose on the 9th day of the experiment. Similarly, groups 4 and 5 were given low dose (150 mg/kg BW) and high dose (300 mg/kg BW) via the PO route and MTX administration on the 9th day. However, groups 3 and 6 received Bacopa (300 mg/kg, PO) and silymarin as a standard (200 mg/kg BW, PO), respectively. The treatment schedule was instituted for 14 days.
Blood and serum analysis
On the 14th day of the experiment, blood was withdrawn from the retro orbital flexus of the rats using serum vacutainers, then centrifuged at 3000 RPM for 15 minutes and the serum was separated and stored at -80°C until further analysis of liver biomarkers such as AST and ALT (Erba kit procedure by following IFCG Method, Kinetic). Following the blood collection, the rats were euthanized using carbon dioxide exposure and their liver tissues were collected, homogenized and stored at -80°C for TNF-á and IL10 (As per the Elisa kit procedure procured from Thermo Fisher Scientific, Bangalore) liver homogenate estimation and some peace of liver collected in the formalin for the histopathology (Singh and Sulochana, 1997) to draw possible conclusions.
Statistical analysis
The collected data was analyzed using the Statistical Package for Social Sciences (SPSS) version 25.0 and one-way analysis of variance (ANOVA) was applied. Duncan’s multiple comparison tests were used to test for differences between means and significance was considered at P<0.05.
Liver serum biomarkers
Rats treated with Methotrexate (Group 2) showed a significant increase (P<0.05) in serum parameters such as AST and ALT levels. However, the rats treated with the extract of Bacopa monnieri in groups 4 and 5 exhibited protection from the detrimental effects of methotrexate on these parameters. These values are comparable to the silymarin treated group (6) (Table 1).

Table 1: The activity of AST, ALT, TNF á and IL-10 in varied treatment groups of rats.

Pro-inflammatory and anti-inflammatory activity
Group 2 showed considerable (P<0.05) elevated levels in the TNF-α and IL-10 (pg/mg) in liver homogenate compared to group 1. On the other hand, groups 4 and 5, which were treated with the B. monnieri, showed a magnificent (P<0.05) decrease in TNF-α and IL-10 concentration when compared to group 2. Notably, these values in groups 4 and 5 revealed similar to those of group 6, which was treated with the standard silymarin. The results are depicted in Table 1.
Histopathology of liver
The tissue sections of the liver of group 2 showed moderate to severe congestion of the central vein, degenerated hepatocytes with the presence of Kupffer cells and mild fibrous tissue proliferation in the perivascular area with disruption of the portal triad and dilated sinusoids (Fig 2 and 2a). In contrast, treated groups 4, 5 and 6 showed mild to moderate congestion of the central vein, dilated sinusoidal space. Mild degeneration of the hepatocytes in group 4 (Fig 4 and 4a), mild degeneration, mild proliferation of Kupffer cells and mild dilation of the periportal area in group 5 (Fig 5 and 5a), Normal radiating appearance of hepatic cords with mild dilation of central vein in group 6 (Fig 6 and6a), however, groups 1 and 3 revealed normal architecture (Fig 1, 1a, and 3,3a).

Fig 1: Group 1: Photomicrograph of liver demonstrating the liver’s normal architecture with the appearance of radiating pattern hepatic cords (arrow). H and E´10.


Fig 1a: In Group 1, a photomicrograph of the liver was obtained, which revealed the normal architecture of the liver with hepatic cords appearing in a radiating pattern (Indicated by an arrow). H and E´40.


Fig 2: Group 2: A photomicrograph of the liver displayed moderate congestion in the central vein (Indicated by an arrow), as well as mild dilation of sinusoidal spaces (Indicated by a chevron) H and E´10.


Fig 2a: In Group 2, a photomicrograph of the liver revealed moderate congestion in the central vein (Indicated by an arrow), mild dilation of sinusoidal spaces (Indicated by a chevron) and mild proliferation of Kupffer cells (Indicated by a down arrow head). H and E´40.


Fig 3: Group 3: A photomicrograph of the liver demonstrated the normal architecture of the liver with the appearance of hepatic cords in a radiating pattern: H and E´10.


Fig 3a: In Group 3, a photomicrograph of the liver was obtained, which exhibited the normal architecture of the liver with hepatic cords appearing in a radiating pattern. H and E´40.


Fig 4: In Group 4, a photomicrograph of the liver revealed the dilation and mild congestion of the central vein (Indicated by an arrow) and irregular hepatic cords with dilatation of sinusoidal space (Indicated by a chevron). H and E´10.


Fig 4a: Group 4: A photomicrograph of the liver displayed the dilation and mild congestion of the central vein (Indicated by an arrow) along with irregular hepatic cords exhibiting dilatation of sinusoidal spaces (Indicated by a chevron). H and E´40.


Fig 5: In Group 5, a photomicrograph of the liver demonstrated congestion of the central vein (Indicated by an arrow) and mild dilation of sinusoidal spaces (Indicated by a down arrow). H and Ex10.


Fig 5a: Group 5: A photomicrograph of the liver displayed congestion of the central vein (Indicated by an arrow), mild proliferation of Kupffer cells (Indicated by a chevron) and mild dilation of sinusoidal spaces (Indicated by a down arrow): H and E´40.


Fig 6: In Group 6, a photomicrograph of the liver depicted congestion of the central vein (Indicated by a down arrow) and mild dilation of sinusoidal spaces (Indicated by an arrow). H and E´10.


Fig 6a: In Group 6, the photomicrograph of the liver shows a central vein with congestion (Indicated by a down arrow) and mild dilation of sinusoidal spaces (Indicated by an arrow): H and E´40.

MTX is a chemotherapeutic agent with cytotoxic properties that finds broad application in the treatment of various forms of cancer. However, MTX often exerts severe adverse effects and causes hepatotoxicity, including progressive development of fibrosis and cirrhosis. The primary cause of MTX-related liver damage is toxic metabolites produced by the drug. The toxicity of MTX was demonstrated in our study, where the toxic control group (Group 2) showed a significant increase in AST and ALT levels compared to the normal control groups (Groups 1 and 3). This finding is consistent with a previous report (Helal and Said, 2020; Namratha et al., 2021) that showed marked liver injury in rats treated with MTX. Similarly other authors also deomonstrated that protective effect of resveratrol and vitamin-e in 5-flourouracil induced hepatotoxicity (Harish et al., 2021), Terminalia arjuna against Experimental Hepatotoxicity due to Cisplatin in Rats (Sneha et al., 2021) and postulated protective effects due to antioxidant properties of these plants. However, our study also found that the administration of B. monnieri, which has antioxidant properties, significantly reversed the changes in serum AST and ALT activities in the toxic control group. This indicates that B. monnieri has the potential to alleviate MTX-induced liver damage. Furthermore, the groups treated with different doses of B. monnieri showed significant improvement in hepatic biomarkers and the results were comparable to the group treated with the standard silymarin extract containing silybinin, silydianine and silychristine, which also attenuated the increased AST and ALT levels.
Concurrently with oxidative stress, inflammation is pivotal in MTX-induced hepatotoxicity’s pathogenesis (Fouad et al., 2020). The current data indicate that a single i.p. injection of MTX was able to increase level of TNF-α which might be due to excess generation of ROS in MTX-treated rats increase neutrophil infiltration and enhance transcription of proinflammatory cytokines including TNF-α (Kumar and Reddy et al., 2012; Taskin et al., 2021). In the present investigation, MTX administration significantly elevated the tissue TNF-α levels compared to normal control rats. Administration of B. monnieri and silymarin for 14 days before MTX decreased inflammatory cytokine levels. The anti-inflammatory effects of B. monnieri and silymarin could be mediated by suppressing NF-kB regulation including COX-2, LOX and TNF α (Sharma et al., 2020). The reactive metabolite 7-hydroxy methotrexate is formed as a result of methotrexate metabolism. This metabolite has been found to modify cellular macromolecules, generate intracellular oxidative stress and mediate cytokine responses. These events are considered critical components in the pathophysiology of hepatotoxicity. Furthermore, the intracellular stress pathways lead to the sensitization of proinflammatory cytokines and the inhibition of anti-inflammatory cytokines. In this work, animals treated with B. monnieri and silymarin significantly showed the elevated level of anti-inflammatory interleukin IL-10 in groups 4, 5 and 6 compared to MTX group, suggesting that B. monnieri and silymarin possessed immune modulating activity in liver and immune system. Further these results were substantiated by the change in histopathology of liver in the MTX treated group and appreciable improvement in Bacopa treated groups.
Based on the findings, B. monnieri treatment provided hepatoprotection similar to silymarin against MTX-induced hepatotoxicity, although the rats given a high dose of B. monnieri had better results due to the presence of phytoconstituents in the B. monnieri. Based on the findings and histological analysis, it can be determined that B. monnieri extract can prevent MTX-induced hepatotoxicity and that B. monnieri extract can be administered as a hepatoprotective agent to combat the drug’s harmful effects.
The study found that MTX resulted in liver injury by increasing inflammatory markers, liver biomarkers and causing structural changes under microscopic examination. However, administering aqueous leaf extract of B. monnieri to rats injected with MTX helped reduce inflammation and liver damage. This suggests that the protective action of B. monnieri against Methotrexate toxicity is due to the presence of phytochemicals. Overall, the study confirms the beneficial role of B. monnieri in protecting against MTX-induced liver toxicity.
The authors acknowledge the support provided by the College of Veterinary Science, PVNR TVU, Rajendranagar, Hyderabad for facilitating the research work.

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