Document Type : Original Article
1 Veterinary Pathology Division, Department of Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia
2 Veterinary Anatomy Division, Department of Veterinary Medicine, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia
Physical activity in general improves metabolic processes by stimulating muscles to contract actively, improving blood circulation, and supplying oxygen. On the other hand, it will create stressful conditions, which will increase cell damage and inflammatory responses in the liver. This study aimed to determine the effect of forest bee honey supplementation on the expression of HIF-1α, SOD, and TNF-α in rats that underwent the forced swimming test (FST) as a physical stress model. The physical activity conducted is at risk of disrupting to several organs due to the stress it causes in addition to the obtained health benefits. A total of 24 adult female rats were divided into four groups: Control (C) with FST only, (T1) FST and honey 2 g/day, (T2) FST and honey 4 g/day, (T3) FST and honey 6 g/day. A forced swimming test was conducted for five minutes per day for 14 days. The collected liver organs were histopathologically prepared by immunohistochemical staining for HIF-1α, SOD, and TNF-α proteins. The results showed that hepatic HIF-1α and TNF-α expression decreased in the honey-supplemented group, while hepatic SOD expression increased, although all three showed insignificant differences from each other. This study concludes that honey supplementation is incapable of increasing the expression of SOD as well as reducing the expression of HIF-1α and TNF-α in the liver of rats modelled by physical stress.
Physical activity in general has a positive effect on metabolic processes, stimulating muscles to actively contract, improving blood circulation, and supplying oxygen . On the other hand, physical activity will also induce stressful conditions, which will subsequently increase cell damage and inflammatory responses in the liver [2, 3]. The production of the cortisol hormone marks the occurrence of stressful conditions in the body . The production of cortisol in the body suppresses the production of brain-derived neurotrophic factor (BDNF), which protects against malondialdehyde (MDA) toxicity as a marker of oxidative stress caused by reactive oxygen species (ROS) . Physical activity could trigger an increase in malondialdehyde (MDA) levels and decrease Superoxide Dismutase (SOD) levels in the body [6, 7]. The body naturally has an antioxidant system that aims to prevent damage caused by ROS, including the enzymes Superoxide Dismutase (SOD), Glutathione Peroxidase (GPx), and Catalase . ROS in sufficient levels is beneficial in the cellular immune response, but when produced in excessive amounts beyond the capacity of antioxidant enzymes, it will cause oxidative stress conditions that are detrimental to the cell membrane .
Oxidative stress will trigger the activation of Hypoxia Inducible Factor (HIF-1α) through the MAPK pathway . In physiological conditions, activation of HIF-1α plays an important role in wound healing because it triggers angiogenesis. On the other hand, HIF-1α production is often associated with the pathogenesis of various hepatic disorders, such as fibrosis, hepatitis, and even cancer [11, 12]. Hepatic damage can be induced by an acute inflammatory response due to physical stress characterized by an increase in Tumor Necrosis Factor-α (TNF-α) . TNF-α production in the liver is carried out by Kupffer cells, which are involved in inflammation and hepatocyte apoptosis through caspase activation . Just like HIF-1α, TNF-α production in the liver is related to the pathogenesis of chronic hepatic inflammation that leads to fibrosis.
Stress due to physical activity is usually not realized by the individual and is often inevitable because it is a consequence of the work undertaken, such as in the case of sports athletes [14, 15].
Physical activity done with a sufficient portion is beneficial in the metabolic process of the individual while if done with high intensity will cause an increase in cortisol production and oxidative stress [16, 17].
To compensate for the decrease in endogenous antioxidants due to physical stress, exogenous antioxidants derived from food or beverages are needed.
Antioxidants are substances that can protect biological components from harm caused by chemical processes involving free radicals. They work by breaking down chains or stabilizing molecules . Thus, the best approach to alleviating oxidative stress is to reduce free radicals or optimize the body’s defences by multiplying antioxidants. Furthermore, antioxidants protect the tissue from oxidative damage .
Indonesia is rich in biodiversity and has many natural resources that can be beneficial to treat various illnesses .
Honey is one of the natural ingredients that can be easily obtained and contains ascorbic acid, carotenoids, phenolic acids, flavonoids, and simple sugars . Antioxidants contained in honey act as free radical scavengers under oxidative stress conditions and were proven to increase the production of GPx, SOD, and CAT as endogenous antioxidants in rats and improve the histological structure of the liver [22, 23]. Many animal models of stress and depression have been made using the forced swimming test along with observations on the liver, such as oxidative damage , MDA levels , and glycogen levels  of the liver . This study attempted to determine whether the administration of wild bee honey supplements increased the expression of endogenous antioxidants, especially SOD, and decreased the expression of HIF-1α and TNF-α proteins in the livers of rats modelled by physical stress.
Results and Discussion
The mean protein expression of HIF-1α, TNFα, and SOD in the whole population showed the results, as indicated in Table 1 and Figure 1.
The forced swimming test as a physical stress model accompanied by honey supplementation was proven to reduce the expression of HIF-1α and TNF-α in the liver but based on statistical analysis, it was considered insignificant (p>0.05) as can be observed in Table 1 and Figure 1. The highest expression of HIF-1α and TNF-α was obtained in group C, which only underwent swimming activities without honey supplementation. Based on Table 1 and Figure 1, it can be further observed that between groups of forced swimming test treatment with honey supplementation, hepatic SOD expression gradually increased along with the increase in honey dose, with the least amount in group C, which did not get any honey. Although the values varied, the differences were not significant (p>0.05) (Table 1). The expression of HIF-1α, TNFα, and SOD presented in immunohistochemical staining can be further observed in Figures 2, 3, and 4.
Based on the immunohistochemical overview of the liver depicted in Figure 2, it can be observed that there is an expression of HIF-1α in the hepatic parenchyma of rats modelled by physical stress, even though there is no significant difference in expression between groups. ROS that arises due to stress will stimulate MAP/ERK Kinase (MEK) to phosphorylate p300 so that there is an increase in the transcription of the HIF-1α molecule in the liver even under normoxia . While in Figure 3, it is appeared that TNF-α expression is very weak to almost zero in the liver. TNF-α production in the liver is carried out by Kupffer cells, which play a role in inflammation and hepatocyte apoptosis through caspase activation . Apoptosis begins with a decrease in BCl-2 activity on the mitochondrial membrane so that membrane permeability changes. These changes cause the release of cytochrome C into the cytosol, which will activate Apaf-1. This activation is followed by a caspase cascade on pro-caspase 9 to process caspase 3, which will damage DNA . From Figure 4, it can be observed that the expression of SOD in the group with 6 g/day honey supplementation appears to be stronger in colour change when compared to other groups, although in statistical analysis the difference was considered insignificant.
The FST procedure used in this study was shown to be associated with HIF-1α production in the liver. This result is in line with other studies that revealed that physical stress of swimming can increase the ROS production, which will induce the activation of HIF-1α in the liver . In addition, the FST procedure proved to be positively correlated with hepatic TNF-α production, following other studies that prove that physical stress can increase TNF-α expression in the liver . The antioxidant content of honey is known to suppress cell damage due to physical stress and ROS [29, 30]. Furthermore, honey has also been shown to increase the production of endogenous hepatic antioxidants SOD as well as GPx, GSH, and catalase [22, 23].
However, honey efficacy was not significantly proven in this study. This may be due to the concentration of ROS being too high, so the dose level used in this study was insufficient to compensate for the cell damage that occurred even though there was an increase in SOD expression. Other studies have shown that excessive consumption of honey can distort the hepatic sinusoid circuit and necrosis of hepatocytes . Similar results also revealed that honey consumption can cause haematological changes as well as damage to hepatic and renal cells . This damage is thought to be due to the accumulation of heavy metals such as Pb and Cd in honey from the environment where the honey is taken. The accumulation of these metals then initiates higher ROS . Therefore, the length of time honey is given and the amount of dose given should be of utmost concern so that honey consumed provides benefits to the body and not the other way around. The dose level of honey and the duration of the study in this study are not sufficient to prove that honey supplementation can prevent general hepatic damage in rats modelled under physical stress. However, the observed parameters in this study do not fully represent the hepatic quality of an individual; other supporting data such as AST, ALT, and GGT profiles are needed for higher validity.
Materials and Methods
This study had been approved by the Ethics Commission of the Faculty of Veterinary Medicine, Universitas Airlangga (1.KEH.041.04.2022).
The experimental animals used were three-month-old female Wistar rats (Rattus norvegicus) with an average body weight of 200 grams. The total population of 24 rats was divided into four treatment groups using a complete randomization system so that each group contained six rats. Rats were acclimatized with adequate food and water for seven days. During the study, the rats were placed in cages measuring 53 × 30 × 17 cm with a base of wood chips and placed in a room with a temperature of 34 °C and 50% humidity. The forced swimming test is a method used to model animal stress and depression, according to Porsolt . Rats were exercised in a 50 cm diameter and 60 cm high barrel filled with water to approximately 2/3 of the height of the barrel for five minutes every day for 14 days.
There were four treatment groups: C with the forced swimming test (FST) alone; T1 with the FST and 2 g/day honey; T2 with the FST and 4 g/day honey; and T3 with the FST and 6 g/day honey. Honey was given by oral gavage. FST was performed for 14 days, and the rats were sacrificed on day 15. Rats were sacrificed with a combination of ketamine and xylazine injections. A laparotomy procedure was performed to collect the liver.
The organs were prepared for histological examination as follows : each animal's liver was fixed in 10% neutral buffered formalin. The fixed tissues were cleared with xylol after being dehydrated in a graded series of alcohols. The tissues were then infiltrated with molten paraffin at 56–60 ᵒC. From a solid block of tissue, serial sections of 3 µm thickness were cut, washed, and placed in object glass.
The tissues were deparaffinized and rehydrated. Then the tissue slides were incubated with hydrogen peroxide for 10-15 minutes, and blocking was performed for 5 minutes. Antibody incubations were conducted using primary antibodies (HIF-1α (1:200), TNFα (1:50), and SOD (1:200)) for 60 minutes and secondary antibodies for 30 minutes. Subsequently, the slides were incubated with streptavidin peroxidase for 10 minutes. Furthermore, the addition of DAB chromogen and substrate was carried out for 15 minutes. Likewise,counterstaining was carried out using Mayer's hemalum solution.
Hepatic immunohistochemical protein expressions were documented using a Nikon Eclipse Ci microscope at 200x magnification and interpreted using the IRS scoring system. Expression was considered positive if there was a brownish-yellow colour change due to antigen-antibody binding in hepatocytes. Observations were made in five fields of view, and the results were averaged. The data obtained were then statistically analysed by ANOVA using SPSS for Windows.
The conclusion that can be drawn from this study is that supplementation of Apis dorsata honey at the dose level given is unable to reduce the expression of HIF-1α and TNFα and increase the SOD expression in physical stress model rats. In future studies, it is suggested to use a more customized dose of honey and to determine the overall hepatic profile, other research parameters such as AST, ALT, and GGT are needed.
The authors express their gratitude to Agung Budianto Achmad, DVM, M.Sc., who supported the data analysis in this study.
No potential conflict of interest was reported by the authors.
This study was funded by Universitas Airlangga with Grant No. 251/UN3/2022.
All authors contributed to data analysis, drafting, and revising of the paper and agreed to be responsible for all the aspects of this work.
Mey Vanda Pusparina Sajida
HOW TO CITE THIS ARTICLE
Hani Plumeriastuti, Widjiati Widjiati, Mey Vanda Pusparina Sajida, Annise Proboningrat. Effect of Forest Bee Honey (Apis dorsata) Supplementation on Expression of HIF-1α, SOD, and TNF-α in Rats (Rattus norvegicus) Liver Exposed to Physical Stress. J. Med. Chem. Sci., 2023, 6(10) 2348-2356