Protective Effects of Melatonin on Hepatic Injury Due to Chronic Monosodium Glutamate Consumption in Adult Female Rats

Research Article

Int J Nutr Sci. 2023; 8(2): 1074.

Protective Effects of Melatonin on Hepatic Injury Due to Chronic Monosodium Glutamate Consumption in Adult Female Rats

Selin Akbulut1,2; Hilal Nisva Levent1,3; Sevil Arabaci Tamer4,5; Berrak Caglayan Yegen4; Serap Sirvanci1*

1Marmara University, School of Medicine, Department of Histology and Embryology, Istanbul, Turkey

2Sanliurfa Education and Research Hospital, Spermiogram Unit, Sanliurfa, Turkey

3Kartal Lutfi Kirdar Education and Research Hospital, In Vitro Fertilization Unit, Istanbul, Turkey

4Marmara University, School of Medicine, Department of Physiology, Istanbul, Turkey

5Sakarya University, School of Medicine, Department of Physiology, Sakarya, Turkey

*Corresponding author: Serap Sirvanci Marmara University, School of Medicine, Department of Histology and Embryology, Istanbul, Turkey. Email: ssirvanci@marmara.edu.tr

Received: May 31, 2023 Accepted: June 28, 2023 Published: July 05, 2023

Abstract

In the present study, it was aimed to investigate the effects of Monosodium Glutamate (MSG) consumption on rat liver and the possible antioxidant and anti-inflammatory effects of melatoninin protecting against MSG-induced hepatic damage. Thirty two adult female rats were randomly divided into 4 groups. For 30 days, MSG (2g/kg/day) or melatonin (4mg/kg/day) was given daily in drinking water or both melatonin and MSG were given simultaneously at the same doses, while normal drinking water was given to the control group. Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) levels were investigated in the serum of rats decapitated at the end of the experiment. In liver sections, Hematoxylin-Eosin (H-E), Sirius red, Periodic Acid Schiff (PAS) staining for histopathological damage examination, Nuclear Factor-kappa B (NF-κB), smooth muscle a-actin [alpha-Smooth Muscle Actin (a-SMA)], NADPH Oxidase (NOX-2), Transforming Growth Factor (TGF)-β1, SMAD2, SMAD7 immunohistochemistry were applied. At the end of the experiment, % body weight change in MSG group rats was high, and serum ALT and AST levels were increased compared to control group, while these levels weredecreased with melatonin administration. In addition, vacuolization in hepatocytes, dilatation in sinusoids, increase in Kupffer cells andinflammatory cell foci were detected in the MSG group. When melatonin wasadded to MSG, decreased hepatocyte glycogen content and SMAD7 immunoreactivity with increased a-SMA were observed along with improvements in NF-kB, NOX-2, TGF-β1, SMAD2 immunoreactivity. Our findings suggest that melatonin, with its antioxidant and anti-inflammatory effects, may be a potential agent to prevent liver damage that can be caused by MSG in the diet.

Keywords: Monosodium glutamate; Liver; Melatonin; Oxidative stress; Inflammation

Introduction

Today, with the advancement in food technology and change in dietary habits, production and consumption of processed, packaged, and ready-to-eat food are increased. Monosodium Glutamate (MSG) is one of the food additives, which is widely used as a flavor enhancer [1,2]. Although these substances have several benefits and functions in terms of food industry, their effects on human health are controversial. Following its absorption, MSG is metabolized in the liver and the liver is one of the most vulnerable organs to toxic insults [3]. In various animal models using MSG, it was reported that MSG is toxic to the liver, brain, thymus, ovary, tuba uterina, testes, kidneys, and hematopoietic system [4]. Toxic effects of MSG application was associated with increased lipid peroxidation, decreased glutathione levels, and decreased catalase and superoxide dismutase activities in the tissues [5]. Oral application of MSG at the doses which is thought suitable for humans, caused increase in liver oxidative stress markers [6]. Besides, it was also reported that its application resulted in obesity, diabetes, steatosis in hepatocytes, inflammation, fibrosis, neoplastic changes, nodular lesions and degeneration in biliary ducts [6,7]. In a study in which rat pups were fed with MSG for 1 year starting from neonatal period; obesity, increase in serum lipid profiles, deteriorated glucose tolerance and metabolic syndrome was observed [8]. Even MSG was given orally, increased insulin levels were observed within 3 minutes [9]. Another study using MSG application reported hyperinsulinemia, obesity, adipocyte dysfunction and related reproductive system disorder [10]. MSG was reported to cause toxic effects on the liver and costic effects on gastrointestinal tract epithelium [11].

Melatonin is a hormone which has a role in sleep cycle, regulation of circadian rhyhm, and regulation of many biological functions such as reproduction and immunity [12]. It has been reported in different studies that antioxidant and antiinflammatory effects of melatonin might be useful for ameliorating hepatotoxicity caused by toxic agents [13,14].

In the present study, it was aimed to investigate possible protective effects of melatonin on the liver injury caused by MSG ingestion, by morphological and biochemical methods.

Materials And Method

Animals

Three-month-old adult female Wistar albino rats were used in the present study. Animals were obtained from Marmara University, The Experimental Animal Implementation and Research Center. All experiments were done according to the National Guidelines on Animal Experimentation and were approved by the Marmara University Local Ethical Committee for Experimental Animals (43.2021.mar). The animals were housed in a 12-hr light/dark cycle and humidity controlled room.

Groups were as follows:

Control group (n=8): Animals were given normal drinking water for 1 month.

Melatoningroup (n=8):4 mg/kg/daymelatonin was added to drinking water for 1 month.

MSG group(n=8):2 g/kg/day MSG was added to drinking water for 1 month [15].

MSG+melatonin group (n=8):2 g/kg/day MSG and 4 mg/kg/daymelatonin were added to drinking water for 1 month.

Chemicals

In the present study, MSG with =98.0% purity was used (L-glutamic acid monosodium salt monohydrate, Sigma-Aldrich, USA, 49621). Average water consumption of each rat in one day was calculated as approximately 10-12ml/100g. MSG was daily prepared and given in drinking water as 2g/100ml according to the animal’s body weight. Thus, each rat was given MSG 2g/kg/day.

Melatonin was used as =98.0% purity level (Sigma-Aldrich, USA, M5250). It was given in drinking water as 4mg/100ml.

Experiment Protocol

Weight of the animals was measured at the beginning and the end of the study. The percentage of weight loss or weight gain of each animal was calculated [(weight change/first weight)x100]. Lee index was used for evaluation of obesity [16]. Values >310 were accepted as obesity.

At the end of the experiments, rats were deeply anesthetized with ketamine (100mg/kg) and xylazine hydrochloride (10mg/kg), intracardiac blood samples were obtained and then they were decapitated.

Biochemical Evaluation

Blood samples obtaned from decapitated animals were centrifuged at 3000rpm for 10 min. Serum and liver tisues were kept at -20°C until biochemical evaluations were done. Alanine Aminotransferase (ALT) and aspartate transaminase levels in serum were detected by using commercial kits with an autoanalyzer (Human/300) in Vetlab Veterinary Diagnostics Laboratory.

Light Microscopic Preparation and Histological Scoring

Liver tissues were fixed in 10% neutral buffered formalin. After dehydration in ascending series of ethanol, tissues were cleared in xylene. Tissues were embedded in paraffin after incubating in liquid paraffin at 60°C overnight. Four-micron-thick sections were stained with Hematoxylin and Eosin (H&E) for histopathological evaluation and with Sirius red for collagen density. PAS reaction was applied for evaluating glycogen content in hepatocytes. Sections were examined under an Olympus DP72 camera attached Olympus BX51 photomicroscope (Tokyo, Japan).

Five randomly selected areas in H&E stained sections under X20 objective were examined. Vacuolization in hepatocytes, vasocongestion, sinusoidal dilatation and Kupffer cell infiltration were evaluated. Histopathological findings were scored as follows: 0, no pathological finding; 1, mild; 2, moderate; 3, severe pathological findings [17]. Five areas in the sections stained with Sirius red were examined under X40 objective and evaluated by using Image J program; and percentage of stained areas were calculated. Glycogen content in sections stained with PAS was scored as 0, no staining; 1, mild; 2, moderate; 3, heavy staining [18].

Immunohistochemistry

Liver sections were immunostained for a-SMA, NF-κB, NOX-2, TGF-β1, Smad2, and Smad7. Four-micron-thick sections were incubated in 37°C and then deparaffinized in xylene. Sections were incubated in pure ethanol, 96% ethanol, and in 3% H2O2 in methanol for blockage of endogenous peroxidase activity. For antigen retrieval, sections were exposed to citrate tampon in microwave and incubated in blocking solution. Sections were incubated in primary antibodies (a-SMA, Abcam, ab7817, 1:200; NF-κB, Santa Cruz, sc-109, 1:100; NOX-2, Bioss, bs-3889R, 1:200, TGF-β1, Santa Cruz, sc-52893, 1:100; Smad2, Santa Cruz, sc-101153, 1:200; Smad7, Santa Cruz, sc-101152, 1:200) at 4°C. Then, biotinylated secondary antibody (SHP125, ScyTek Laboratories, Inc. USA) and Horse-Radish Peroxidase (HRP) solution were applied. After applying DAB chromogen (DAB Chromogen/Substrate Bulk Pack, ACK500, ScyTek Laboratories, Inc. USA), sections were counterstained with Mayer’s hematoxylin and mounted with Entellan. For negative controls, PBS was used instead of primary antibodies. Randomly selected five areas were photographed under X40 objective and percentage of NF-κB,a-SMA, NOX-2, TGF-β1, Smad2 and Smad7 positive areas were evaluated by using Image-J program.

Statistical Analysis

Data were analyzed by using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) program and presented as mean±S.E.M. One-Way-ANOVA and Tukey multiple comparison tests were used. p<0.05 was considered as significant.

Results

Weight Follow-upand Calculation of Lee Index

Weight gain in the animals in MSG group was increased compared to the control group (p<0.001). Weight gain in MSG+melatonin (p<0.01) and melatonin (p<0.0001) groups were significantly decreased compared to MSG group (Figure 1a). Lee index score was increased in MSG group compared to the control group (p<0.0001), but decreased in melatonin (p <0.0001) and MSG+melatonin (p<0.01) groups as compared to the MSG group (Figure 1b).