Antioxidant effect of ethanolic extract of Pleurotus Ostreatus on 4-hydroxy-2-nonenal (HNE) and glutathione (GSH) level in lung rats exposed to cigarette smoke

Introduction

Cigarette smoke is a mixture of more than 4,700 chemical components (nicotine, tar, benzene, carbon monoxide, etc.), reactive oxygen species (ROS), and a large source of free radicals that are divided into the gas phase and tar phase.^1–3 Free radicals can cause oxidative stress, and cause damage to important biomolecular (lipid, sugar, protein, DNA), membrane dysfunction, protein modifications, DNA damage, and enzyme inactivation. The effects of cigarette smoke on the body are influenced by many variables, including the dose and type of tobacco used, the route of administration, duration or duration of exposure, and other factors that may be present during stimulation. For inactive smokers and passive smokers, cigarette smoke causes a rapid dissolution of toxins in the mouth or respiratory epithelium systematically.^2,4–7 Acute and chronic exposure to cigarette smoke can result in tissue damage that can increase lipid peroxidation products and degradation products of extracellular matrix proteins, such as malondialdehyde (MDA), 8-isoprostane, hydrogen peroxide, and 4-Hydroxy-2-nonenal (HNE).^2,8,9

HNE is one of the cytotoxic lipid peroxidation end-product lipids from ω6 polyunsaturated fatty acids. Linoleic acid and arachidonic acid are potential precursors of HNE. 4-Hydroxy-2-nonenal can react with proteins and form HNE-adduct proteins with histidine, cysteine, and lysine. This complex can change the function of these proteins.^6,8 Cigarette smoke can increase lipid peroxidation and HNE formation, which then activates mitogen-activated protein kinase (MAPK) and the release of TGFβ1 and induction of γGCS. This eventually causes inflammation because there is a redox imbalance.^6,8

Oxidative stress can be inhibited by the body's defense system through antioxidants which can eliminate free radicals and minimize damage. Various enzymatic and non-enzymatic systems play a role in this process.^10,11 Antioxidants can be defined as chemical molecules containing monohydroxy/polyhydroxy phenols and work by inhibiting lipid peroxidation.^12–14 The appropriate antioxidants, both endogenous and exogenous antioxidants are important in the overall defense mechanism.^9,15 Glutathione (GSH) is the primary antioxidant because it produces in the body and is important for the first defense mechanism to free radicals. It is also a secondary antioxidant with N-acetylcysteine (NAC) and flavonoids that act as radical scavenging to prevent the chain reaction of free radicals, and play a role in various transcription factors, and detoxications. Cigarette smoke can make ineffectively of endogenous antioxidants and the body needs to supply exogenous antioxidants to maintain the defense.^9,15,16 Several studies have shown that smokers have lower levels of vitamins E, C, β carotene, and GSH in plasma compared to nonsmokers.^2,10,17 Glutathione is abundant in the body and is found in Epithelial lining fluid (ELF) in high concentrations and serve to protect various oxidants that enter inhalation. GSH concentrations in ELF are 10–100x higher than the levels in plasma. Exposure to chronic cigarette smoke can reduce GSH levels in ELF, plasma, and lungs after the GSH adaptive response period is exceeded.^18–20

Previous studies have shown that supplementation with vitamins C and E is expected to reduce the harmful effects of cigarettes. The effectiveness of most antioxidants is generally seen in proportion to the number of hydroxyls (OH) groups present in the aromatic ring so that natural substances appear to have better antioxidant activity than synthetic antioxidants.^10,16,21,22 One exogenous antioxidant that has been found in foods that have strong antioxidant effects is a P. ostreatus.^7,12,23

P. ostreatus are nutritious, high in fiber, and contain many vitamins and minerals.^10,24–26 This oyster mushroom has strong antioxidant activity due to secondary metabolite compounds contained in it. Components in white oyster mushrooms that are believed to have antioxidant effects include vitamin C, beta-carotene, selenium, ergothioneine, and phenolic components for the primary component. Phenolic antioxidant activity is mainly due to its ability as a hydrogen donor reducing agent and oxygen quencher singlet and has a potential metal chelation effect.^7,12,27 Phenolic antioxidant derivatives will also induce GSH enzyme synthesis in human alveolar epithelial cells.^12,21,24 P. ostreatus as a whole when compared with winter and shitake mushrooms have antioxidant activity, reducing power, scavenging abilities, and higher total phenol content.^7,10,23,28 Previous studies have shown that P. ostreatus extract has a protective effect on the liver, kidneys, and brain.^7,29 Other studies have shown that ethanol extract of white oyster mushrooms has the effect of preventing an increase in MDA¹¹ levels and a decrease in lung density in rats induced by cigarette smoke.^16,24,28,30 In this study, we assess the antioxidant effect of P. ostreatus, their influence in preventing a decrease in GSH levels and preventing an increase in HNE levels, and their correlation in Wistar rat lung cells that are exposed to cigarette smoke.

Methods

Research tools and materials

The equipment used in this study were: a smoking pump/tobacco smoke discharger (designed for this research, more information below), HNE Elisa Kit Assay (Abbexa) 83, Reduced Glutathione (GSH) Assay Kit (Colorimetric) (K464-100) 84, tissue homogenizer, multichannel micropipette reservoirs; Multiscan GO Reader (Thermo Scientific 1510).

A tobacco smoke discharger (Figure 1) is a device that functions as a means of sucking cigarette smoke and flowing it into the test room (smoking room) with a certain flow speed. This tool utilizes electric power which is then converted into mechanical energy so as to produce a pressure difference sufficient for the suction and flow of cigarette smoke. The use of this tool is for research purposes because this tool helps to simulate a test room containing cigarette smoke with a certain intensity and its impact on test animals is investigated. The advantage of using this tool is the achievement of steady and measurable smoke flow stability so as to produce valid research data.

Figure 1. Tobacco smoke discharger.

Results

On macroscopic examination of the lungs, it was seen that the rat lung tissue was pink and did not show any significant color differences in each group. With a smooth lung surface, there were no lumps or nodules visible or palpable.

Table 1 shows that the group with the highest average HNE value was group 2 with HNE levels of 47.70 ± 5.35 pg/mol, while the group with the lowest HNE was group 1 with HNE 40 values, 40 ± 1.93 pg/mol.⁴⁹ HNE levels in group 3 and group 4 were higher than group 1, but lower than group 2. Group 3 had a lower HNE value than group 4.

The HNE data analyzed by K-Wallis test and value of P=0.03, which means that there is a significant difference in the mean HNE between the test groups. In this study, it may be concluded that cigarette smoke caused a significant increase in HNE levels compared to normal controls and ethanolic extract of P. ostreatus signicantly reduced the level.

The data analyzed with ANOVA test and the resut P-value for GSH was 0.00 (p≤ 0.05). There is a significant difference in the mean GSH between groups, with Bartlett's P-value greater than 0.05 so that the post-hoc Bonferroni test can be used seeing the difference in mean GSH between groups. Significant mean differences were found in almost all groups, except for differences between groups 3 and 4 (Table 1). It’s means that this study, cigarette smoke was shown to significantly reduce GSH compared to normal controls, and ethanol extract of P. ostreatus significantly increased GSH levels in the lung of rats exposed to cigarette smoke. The effect of the ethanolic extract of Pleurotus ostreatus in experimental animals was as good as the effect of NAC. The GSH and HNE parameters were then tested for correlation using Spearman’s rank analysis. The results show the value of P = 0.78 and rs = 0.06 so it can be concluded that there is no correlation between GSH and HNE.

Discussion

In this study, exposure to kretek cigarette smoke in rats for six weeks was expected to cause significant oxidative stress in rats.^4,36 Free radicals in cigarette smoke can cause membrane damage through lipid peroxidation, which will cause a cascade of further oxidative damage, increase cell membrane rigidity, osmotic fragility, decrease the life span of erythrocytes, and disrupt lipid fluidity.⁷

Lipid peroxidation will produce secondary metabolites from peroxidation products such as malondialdehyde (MDA) and HNE.⁷ Lipid peroxidase also can cause changes in phospholipase to arachidonic acid which can further increase HNE and induce the release of inflammatory mediators such as prostaglanding and leukotrienes. This can cause the respiratory tract to become more vulnerable to inflammation and develop cell proliferation changes and cause airway obstruction.^37,38

4-Hydroxy nonenal is a specific metabolite which is very toxic, reactive and has high diffusion ability. The concentration of HNE in cells can be used for the determination of signaling for apoptosis, differentiation, or proliferation.^8,39 In this study the high HNE in rats exposed to cigarette smoke indicates that lipid membrane damage has occurred and poses a risk for further damage and can initiate a heavier process. Increased 4-HNE adducts have a good correlation with pulmonary function measurement through FEV1. This reinforces the suspicion that 4-HNE has an important role in the pathogenesis of COPD.^40,41

The ethanolic extract of P. ostreatus at a dose of 250 mg/kg body weight could inhibit the increase in HNE levels in rats exposed to cigarette smoke exposure, it can be concluded that extract of P. ostreatus can inhibit pulmonary lipid peroxidation due to free radicals from cigarette smoke.

The mechanism of P. ostreatus ethanolic extract in inhibiting the increase in HNE can be caused by several mechanisms, mainly because the hydroxyl group in the ethanolic extract of P. ostreatus become an excellent hydrogen donor to prevent membrane damage due to free radicals. Another possibility is to increase the synthesis of endogenous antioxidants such as glutathione.

Previous in vitro studies, showed the potential antioxidant activity of the ethanolic extract of P. ostreatus. the ethanolic extract of P. ostreatus with a concentration of 10 mg/mL has an effectiveness of 56.20% in inhibiting lipid peroxidation (LPO) activity, while ascorbic acid as a standard can inhibit 67.15%. The 50% inhibitory concentration for lipid peroxidation activity for extracts and ascorbic acid was 8 and 6 mg/mL.^7,28 The water-soluble P. ostreatus polysaccharide extract on Caco-2 cells showed inhibition of lipid peroxidation with MDA markers with fluctuating results for three days, the first day showed a decrease in MDA, but the third day the effect of the inhibition disappeared. This is thought to be correlated with GSH levels whose results are the opposite of MDA levels, on the same day.⁴²

In vitro test of ethanolic extract P. ostreatus has a potent reducing power and this effect increases with increasing extract concentration. The reduction ability at 10 mg/mL is 1.367, which is better than BHT (1,192).²³ In previous studies, ethanol extract of P. ostreatus in vivo was proven to be able to prevent increased levels of malondiadehyde (MDA) in the liver, kidney and brain in mice given CCl4 as hepatotoxic.²³

Antioxidant agents can inhibit the process of lipid peroxidation.¹¹ One of the most abundant endogenous antioxidant in the body is GSH.^21,36 In this study rats exposed to cigarette smoke showed a significant reduction in GSH compared to normal rat. The imbalance of oxidants and antioxidants in the body will cause oxidative stress.^43,44

GSH levels in the body depend on a balance between the synthesis process and the metabolic process of GSH to be inactive. Synthesis of de novo GSH is influenced by yGLCL and GLS and is influenced by GPx and glutathione reductase (GR) in the process of reducing GSH to GSSG. The highest GSH synthesis is in the liver, which is the place of primary synthesis, so if GSH decrease in other organs there should be a fairly extreme decrease before the liver. So far the GSH:GSSH ratio is one of the best indicators for assessing oxidative stress conditions in cells.^18,45,46 The body's defense system or endogenous antioxidants are not fully able to fight free radicals in the body, so exogenous antioxidant supplements are needed.¹⁰

Exogenous antioxidants can be synthetic antioxidants or antioxidants derived from natural ingredients such as medicinal plants. The most commonly used synthetic antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate and tert-butylhydroquinone. However, the use of BHA and BHT is very limited because it is suspected to be carcinogenic and cause liver damage. It is still very necessary to develop antioxidants that are sourced from natural sources.⁷ P. ostreatus are medicinal plants that have high antioxidant potential and can be increased GSH levels in male rats exposed to cigarette smoke in this study. The effect given is significant, and the result is the same as the effect arising from the administration of GSH precursors, namely NAC.

The mechanism by which ethanolic extract of P. ostreatus enhances the synthesis of GSH is suspected to be through several mechanisms as follows: 1) increasing the expression of enzymes yGLCL and GLS which play an important role in the synthesis of glutathione in the body; 2) increasing cysteine residues in the body, which is one of the glutathione amino acids, this mechanism is almost the same as the mechanism of action of NAC; 3) P. ostreatus contain selenium which is considered as one of the precursors of glutathione with a mechanism that cannot be clearly explained.^7,24,26,45 All three of the above mechanisms as a whole cause ethanol extracts of P. ostreatus to increase levels of glutathione in the body.

In this study, the potency of the ethanolic extract of P. ostreatus at a dose of 250 mg/kg BW in preventing the decrease in GSH levels in rats exposed to cigarette smoke was as good as the potency of N-acetylcysteine (NAC). N-acetylcysteine is an antioxidant that contains thiol which is also a precursor of GSH. N-acetylcysteine increases GSH synthesis and maintains a balance of GSH levels in cells. The thiol group in NAC can reduce free radicals and has functioned as a chelating agent which is very useful in the case of heavy metal poisoning. N-acetyl cysteine can also penetrate cell membranes, so that its level are high intracellular.^34,47

The antioxidant potential of P. ostreatus is supported by the content of secondary metabolites contained therein. The results of phytochemical tests previously showed that white oyster mushrooms contain many alkaloids, steroids, flavonoids, tannins, saponins and phenolic components. Previous research also showed that P. ostreatus can contain about 100 different bioactive components. This fungal cell wall contains many non-starch polysaccharides, where β-glucans are the most attractive components, and contain phenolic components such as protocatechuic acid, gallic acid, homogentisic acid, rutin, mirisetin, chrysin, naringin, tocopherol like α-tocopherol, γ-tocopherol, ascorbic acid and β-carotenoids.^12,30

Other research also states that the ethanol extract of P. ostreatus contains many polyphenols or phenolic components in which there are many flavonoids such as routine, also contain β-carotenoids, vitamin A and C, and selenium. In this study, the results of testing of compound markers using HPLC showed that the ethanol extract of P. ostreatus used in the study contained rutin (flavonoids) and cinnamic acid (phenolic acid).^12,47

The ethanolic extract of P. ostreatus in vitro has antioxidant power that can trap hydroxyl radicals and superoxide, inhibit lipid peroxidation, reduce ferric ions, chelating ferrous ions and quenching 2,3-diazabicyclo[2,2,2]oct-2-ene (ene DBO). This extract also shows good antioxidant activity in vivo by reducing lipid peroxidation and increasing enzymatic and non-enzymatic antioxidant activity.^24,28

Other studies mention that water soluble crude polysaccharide from P. ostreatus shows the superior ability of free radical scavenging and NOS, due to the alleged role of the P. ostreatus β-glucan antioxidant potential. This means that P. ostreatus can be a herb that can be developed into a good natural antioxidant.⁴⁸ Some of the above studies are strongly related to the mechanism of aging, whereas studies to see the effect of ethanol extracts of white oyster mushrooms on endogenous antioxidant levels in rat models with COPD have not been examined. This is why this research can pave the way to develop the use of natural antioxidants in COPD cases. Provision of antioxidants and anti-inflammatory drugs alone in COPD patients can be used based on several conditions that need to be considered. Currently exogenous antioxidants are often used in COPD patients among mucolytic antioxidants such as erdostein, carbocystein and NAC.^34,46

Cigarette smoke is known to reduce levels of total glutathione (GSH + GSSG) in the respiratory tract. Glutathione is widely known to be present in ELFs and airway epithelium. Glutathione plays an important role in cellular redox balance and is considered to be the most important antioxidant against inhalable reactive components. The mechanism underlying the total glutathione depletion is presumably because there is a part of the cigarette smoke phase which will react irreversibly with GSH to form a non-reduced GSH derivative (GSX), thereby causing GSH depletion. Under normal conditions, intracellular GSH will generally be stored in a reduced form, but the imbalance between GSH and oxidized gluthatione (GSSG) will change under conditions of oxidative stress, and the ratio between GSH and GSSG will illustrate how intracellular cells condition. In vitro and in vivo studies state that under oxidative stress conditions the free sulfhydryl (-SH) group of GSH will become oxidized, and exposure to the cigarette smoke phase in vitro and in vivo will generally reduce GSH but the amount of GSSG does not increase significantly. Another study concluded that cigarette smoke does not cause GSH oxidation to GSSG, but rather causes a reaction so that it forms glutathione-aldehyde derivates or non-reduced GSH derivatives (GSX), which in turn will reduce total GSH. This will cause chronic antioxidant deficiency. Chronic smokers will continue to be exposed to free radicals and with the decrease in GSH the negative effects of these free radicals will be even higher. This condition causes GSH synthesis which is essential for cell defense and lung protection. Depletion of GSH Intracellular depletion will significantly trigger oxidative stress and stimulate signal transduction pathways, cell proliferation, apoptosis and inflammation.⁴¹

4-Hydroxy nonenal is a toxic molecule and can further increase the level of ROS in cells through the mechanism of mitochondrial dysfunction 4-Hydroxy-2-nonenal, because mitochondria are organelles that are susceptible to HNE. This condition will further increase the inflammatory response that will occur.⁸ One of the mechanisms for detoxifying HNE from cells is by forming conjugations with GSH (glutathione-4-hydroxynonenal/GSHNE) to then be taken out of the cell. This conjugation process is catalyzed by glutathione-S-transferases (GSTs), which play an important role in regulating HNE levels in cells. There are two groups of proteins involved in transporting foreign species from within cells: namely ATP-binding cassette (ABC) transporters and non-ABC transporters. ABC transporters, including P-glycoprotein (Pgp) and multidrug resistance-associated protein (e.g., MRP1). Various xenobiotics and natural metabolites such as GSHNE will be transported past MRP1. Non-ABC transporters involved in transporting toxins, some drugs and antineoplastic agents and various glutathione conjugations are Ral-binding protein 1 (RalBP1), also known as RLIP76. This protein is abundant in various body tissues and is bound to cell membranes.³⁹

This reinforces the hypothesis that GSH will affect HNE levels in the network, because high GSH will cause HNE to decrease because it forms GSHNE. The data in this study also show that the levels of GSH and HNE for each group are contradictory, if the GSH rises then the HNE will decrease, but the statistical analysis states that the data obtained is not significant enough to reinforce that there is a correlation between GSH levels and HNE levels in the lung of rats exposed to cigarette smoke. Insignificant results between GSH and HNE correlations can also be due to the limited number of samples, so the results cannot be applied to the population.

One important factor that can break the cascade of cigarette smoke inflammatory processes early is antioxidants. Glutathione is an endogenous antioxidant found in many cells including lung cells and ELF. During the synthesis and use of glutathione in a balanced body, no further damage is expected, but high consumption of glutathione can cause levels to decrease in the body. In this condition exogenous antioxidant supplements are needed which can help the work of endogenous antioxidants such as GSH. P. ostreatus are expected to optimize the body's endogenous antioxidants. Ethanol extract of P. ostreatus is expected to increase the synthesis of GSH in the body and help the body's defense of antioxidants through the mechanism of hydrogen donors from its hydroxyl groups. This plant can be developed into a potential source of antioxidants as well as a potential anti-inflammatory.

Conclusions

Ethanol extract of P. ostreatus inhibits the increase in levels of HNE and increases level of GSH in the lungs of male Wistar strain rats exposed to cigarette smoke exposure. There is no correlation between GSH levels and HNE in male Wistar strain rats exposed to cigarette smoke. This study was carried out according to well prepared research methods, however there are some research limitations, such as exposure to cigarette smoke carried out per group in one smoking chamber so that there may be variations in getting exposure to cigarette smoke for each experimental animal in one group, and the procedure for measuring oxidative stress and antioxidant parameters was not carried out as soon as possible after lung tissue collection, it is possible to reduce the levels of the parameters examined, especially for the GSH parameter.

Data availability