Thymus vulgaris extract modulates dexamethasone induced liver injury and restores the hepatic antioxidant redox system

Abstract

Background

The liver is the largest important organ and the site for essential biochemical reactions and detoxifying toxic substances in the human body. Long-term, high-dose dexamethasone administration can cause severe alterations in liver function. Therefore, Thymus vulgaris leave extract possess a modulatory role on dexamethasone-induced hepatotoxicity by attenuating antioxidant defense system.

By subcutaneous route, animals will receive three doses per week for 8 weeks of dexamethasone (0.1 mg/kg. b. wt.) concomitant with oral administration of thyme aqueous extract (500 mg/kg b.wt.).

Results

DXM treatment led to a marked increase in the liver function enzyme activities that are successfully ameliorated by thyme aqueous extract. Thyme natural antioxidants augmented the antioxidant defense system that overcomes oxidative stress caused by dexamethasone. Conversely, although dexamethasone-treated animals rose lipid peroxidation, thyme extract pretreatment did the reverse.

Conclusion

Hepatotoxicity and oxidative stress caused by dexamethasone might improve by thyme natural antioxidants.

Background

The liver is the main site for powerful metabolism and elimination of chemicals, foreign substances, drugs, and toxic mixtures. Damage in the hepatic tissue accompanied by elevated tissue malonaldialdehyde, cellular necrosis, and reduced glutathione levels was diminished. Also, serum levels of cholesterol, bilirubin, transaminases, triglycerides, and alkaline phosphatase are raised in hepatic disease [4, 42]. Suprarenal medullary gland secretes glucocorticosteroids (GCs) or steroid hormones that have immunomodulation and regulating metabolism actions [7]. The ingestion of dexamethasone overdose wastes its effect as a long-acting anti-inflammatory synthetic steroid [29]. Synthetic and natural glucocorticoids are considered as a leader of the anti-inflammatory and immunosuppressive treatments. Acute and chronic inflammations as multiple sclerosis, eczema, and rheumatoid arthritis are widely treated with glucocorticoids. Furthermore, organ transplant, immunosuppressive regimes, and leukemia are also using glucocorticoids. Long-term use of oral glucocorticoids may lead to metabolic disease, osteoporosis, and cardiovascular disorder ([46] [44];). Long-term treatment of dexamethasone had several side effects, for example, initiation of the free radicals which might play a role in oxidative stress, skeletal muscle atrophy, and insulin resistance, although it is effective in immunosuppression and anti-inflammatory properties [15, 47]. Dexamethasone induced hepatic injury through excessive formation of free radical which caused oxidative stress [23].

Dietary natural antioxidant compounds have pay attention to its ameliorative effects, which are found in fruits, vegetables, and seeds. Also in traditional and alternative medicine, dietary antioxidant is considered as a treatment [24]. Thymus species are rich sources of secondary metabolites that play key roles in delaying the development of diseases such as cancer and chronic inflammation. Metabolites have notable radical scavenging and antimicrobial properties as the phenolic monoterpenoids, thymol, and carvacrol [43]. Also, flavonoids such as luteolin and apigenin derivatives and phenolic acids such as rosmarinic acids, cinnamic, and carnosic give an important contribution to the antioxidant capacity of thyme extracts [13]. In several experimental models of liver injury, several species of plant, for example, thyme, contain other metabolites, i.e., chemotypes (chemical phenotypes), cause hepatoprotective effects. Liver injury in rats induced by carbon tetrachloride (CCl4) could ameliorate by thyme extract and essential oil [1, 5]. The defensive effects of thyme essential oil and watery extract are found, versus paracetamol, cause hepatic damage. Plant secondary metabolites are natural products that were examined to be a recent source of therapeutic and pharmaceutical agents which characterized definitive plants. The entire body is affected by the liver, which is considered as the center of biochemical transformations. Particularly, the liver is susceptible to sudden increases in reactive oxygen species (ROS) levels plus further prooxidants as the center of detoxification and synthesis of a number of essential substances [17, 21]. The central role that controlled ROS concentrations is played by an enzyme (superoxide dismutase), which synthesizes hydrogen peroxide plus degraded by glutathione-dependent peroxidase or catalase into oxygen and water, although ROS (reactive oxygen species) concentrations are controlled by specialized metabolic pathways [38, 49]. Glutathione S-transferase (GST) supplemented this first line of antioxidant defense, which conjugates reactive electrophilic compounds with glutathione and initiates their detoxification [16]. Regarding the increasing oxidative stress and antioxidant defense of herbs, the current research is conducted to assess the protective role of thyme watery extract on DXM-enhanced hepatotoxicity in albino rats.

Methods

Animal preparation

In the current investigation, 120–150 g (female) albino rats were used. The rats were obtained from Ophthalmology Research Center animal house in Giza, Egypt, according to the [12]. Two weeks ahead the beginning of the study, the animals were housed under examination to prevent any intercurrent infection. Rats were exposed to natural conditions (daily light-dark cycles and kept at room temperature). Clean water always existed and they were fed ad libitum.

Chemicals

By Sigma–Tec (Merck, Darmstadt, Germany, permission), DXM (Fortecortin® 8 mg—mono ampoule) is manufactured in Egypt—S. A. E by medicinal manufactures.

Herbal preparation

For medicinal plants (Cairo, Egypt), T. vulgaris L. (Lamiaceae) leaves were bought from Sekem Co. An ecologist identified T. vulgaris leaves and a labeled sample was present in Beni-Suef University, Faculty of Science, Botany Department, Egypt.

Water extract of Thymus vulgaris

Aerial material of dried thyme [40] weighing 30 grams was infused in distilled water (60 mL) for a day. By using filter paper, sample was filtered and the filtrate was kept at – 20 °C just for 3 days.

Drug administration

In the current finding, 0.1 mg/kg b.wt. of DXM dosage was used. Feng et al. [18] reported that this dose, previously, increased in the frequency of hepatotoxicity and ovarian toxicity in mammalian systems. Rats were injected three times per week with 0.1 mg/kg b.wt. of DXM (adjusted in sterile water proceeding to use) subcutaneously concomitant with 500 mg/kg b.wt. of TAE (thyme aqueous extract) for 8 weeks [40].

Experimental design

Thirty animals were distributed to:

• 1. G1 (Group 1) normal or negative control: daily for 8 weeks, animals were provided with distilled water.

• 2. G2 (Group 2) toxic group: animals were injected by subcutaneous route three times a week for 8 weeks with 0.1 mg/kg b. wt. of DXM.

• 3. G3 (Group 3) thyme- and DXM-treated group: rats were injected subcutaneously three times per week with 0.1 mg/kg b.wt. of DXM concomitant with oral administration of 500 mg/kg b. wt. of TAE for 8 weeks [40].

Sampling

Rats were sacrificed (in the morning) by cervical decapitation and a 5-mL blood sample was collected in a centrifuge tube from the jugular vein of each animal under light ether anesthesia [9]. At room temperature for 45 min, the blood sample was left to clot. For various physiological and biochemical analyses at 3000 rpm, the clear sera were separated for 15 min at 30 °C by centrifugation and kept frozen at – 20 °C.

Biochemical analyses

The serum samples were used to estimate serum alkaline phosphatase (ALP) activity kinetically [11, 36] method was used to determine LDH (lactate dehydrogenase) using Stanbio Laboratories reagent kit purchased from Texas, USA. Biodiagnostic kits were used to estimate ALT plus AST (alanine aminotransferase and aspartate aminotransferase) activities [35].

Hepatic oxidative stress and antioxidant enzymes analyses

The liver was removed rapidly after dissection and homogenized 0.5 g in 5-mL normal saline solution (0.9% NaCl “10% w/v”) by Teflon homogenizer. Liver clear supernatants used to measure the activity of catalase (CAT) were assayed following the method of Aebi [3]. Beutler et al. [10] method was used to measure glutathione (GSH), Ohkawa et al. [32] used to estimate lipid peroxidation (LP), and glutathione S-transferase (GST) and glutathione peroxidase (GSP) activities were estimated by the method of Habig et al. [22] and Paglia and Valentine [34] respectively. Glutathione reductase (GSR) activity was done using Goldberg and Spooner [20] method.

Histopathological study

Liver samples from each group washing with normal saline were fixed in 10% buffered formalin, inserted in paraffin wax, dehydrated and exposed to H& E (hematoxylin and eosin), then sectioned into 5-μm thickness according to the method of Banchroft et al. [8] to compare their morphologies.

Statistical analysis

Data are expressed as mean ± SEM for post hoc analysis, and one-way variance analysis was used to establish statistical differences followed by Tukey-Kramer methods. Data done by ANOVA (one-way analysis of variance) were used, were presented as mean ± SEM, and were analyzed. When P < 0.05, statistically significant data was considered by using GraphPad Prism 5 software for statistical analysis (San Diego, CA, USA).

Results

Biochemical changes

Variations in various serum parameters associated with liver function are shown in Table 1 and Figs. 1, 2, 3, and 4. Regarding liver function enzymes interrelated, the dexamethasone-managed group showed important surge of ALP, AST, ALT, and LDH activities. Thyme aqueous extract management effectively enhanced theses elevated enzymes.

Table 1 The defensive role of thyme aqueous extract on serum alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities in normal and dexamethasone (DXM) treated rats

Fig. 1

The defensive role of thyme aqueous extract on serum alkaline phosphatase activity in normal and dexamethasone (DXM)-treated rats. Percentage changes (%) were calculated by comparing normal group with DXM-treated group and pre-treated DXM groups with DXM-treated group

Fig. 2

The defensive role of thyme aqueous extract on serum aspartate aminotransferase (AST) activity in normal and dexamethasone (DXM)-treated rats. Percentage changes (%) were calculated by comparing normal group with DXM-treated group and pre-treated DXM groups with DXM-treated group

Fig. 3

The defensive role of thyme aqueous extract on serum alanine aminotransferase (ALT) activity in normal and dexamethasone (DXM)-treated rats. Percentage changes (%) were calculated by comparing normal group with DXM-treated group and pre-treated DXM groups with DXM-treated group

Fig. 4

The defensive role of thyme aqueous extract on serum lactate dehydrogenase (LDH) activity in normal and dexamethasone (DXM)-treated rats. Percentage changes (%) were calculated by comparing normal group with DXM-treated group and pre-treated DXM groups with DXM-treated group

The liver antioxidant defense system is shown in Table 2 and Figs. 5, 6, 7, 8, 9 and 10. Animals treated with thyme caused a great elevation of the liver glutathione level that was markedly increased; the proportion was 89.02% as competed with DXM-treated animals (− 44.76), which revealed important reduction in reduced glutathione level paralleled to non-treated animals. Liver CAT, GSP, GST, and GSR activities demonstrated a marked significant rise in TAE-treated group that corresponds to DXM group which reduced significantly. Dissimilarity, liver LP was elevated significantly as a result of DXM management, although the TAE produced a significant reduction of the elevated value.

Table 2 The defensive role of thyme aqueous extract on liver lipid peroxidation, liver glutathione levels, liver glutathione S-transferase, catalase, glutathione peroxidase, and glutathione reductase activities in normal and dexamethasone (DXM)-treated rats

Fig. 5

The defensive role of thyme aqueous extract on liver lipid peroxidation level in normal and dexamethasone (DXM)-treated rats. Percentage changes (%) were calculated by comparing normal group with DXM-treated group and pre-treated DXM groups with DXM-treated group

Fig. 6

The defensive role of thyme aqueous extract on liver glutathione level in normal and dexamethasone (DXM)-treated rats. Percentage changes (%) were calculated by comparing normal group with DXM-treated group and pre-treated DXM groups with DXM-treated group

Fig. 7

The defensive role of thyme aqueous extract on liver glutathione S-transferase activity in normal and dexamethasone (DXM)-treated rats. Percentage changes (%) were calculated by comparing normal group with DXM-treated group and pre-treated DXM groups with DXM-treated group

Fig. 8

The defensive role of thyme aqueous extract on liver catalase activity in normal and dexamethasone (DXM)-treated rats. Percentage changes (%) were calculated by comparing normal group with DXM-treated group and pre-treated DXM groups with DXM-treated group

Fig. 9

The defensive role of thyme aqueous extract on liver glutathione peroxidase activity in normal and dexamethasone (DXM)-treated rats. Percentage changes (%) were calculated by comparing normal group with DXM-treated group and pre-treated DXM groups with DXM-treated group.

Fig. 10

The defensive role of thyme aqueous extract on liver glutathione reductase activity in normal and dexamethasone (DXM)-treated rats. Percentage changes (%) were calculated by comparing normal group with DXM-treated group and pre-treated DXM groups with DXM-treated group

Histopathological changes

Figure 11a shows the microscopic hepatic examination in normal control group without pathological alteration. Conversely, hepatic cell degeneration, vacuolation, and pyknotic nuclei were produced in DXM-treated rats (Fig. 11b), whereas thyme pretreated group (Fig. 11c) showed normal hepatocytes with very little vacuolation.

Fig. 11

a A transverse section of a liver of normal control (distilled water) rat showing a central vein, hepatic cords, and sinusoids in between, × 400. b A transverse section of a liver of dexamethasone group showing degenerative changes (d), vacuolation (V) all over the hepatocytes and pyknotic nuclei (↑), × 400. c A transverse section of a liver of dexamethasone and thyme group showing normal central vein (CV), hepatocytes with very little cell vacuolation, × 400

Discussion

In the existing results, dexamethasone induced liver injury by increasing AST, ALT, ALP, and LDH activities. However, TAE management effectively enhanced the elevated enzyme activities of the above-mentioned parameters. These investigations are in agreement with other studies [23, 42] which declared that any harm in the liver can weaken its functions and cause numerous implications on human health as it is the chief site of intense metabolism and excretion and plays an essential task in excretion plus detoxification of several exogenous beside endogenous components, and hepatic damage resulted from the distortion of these metabolic functions. Corticosteroid therapy associated with liver injuries leads to elevated liver function enzymes. Hydroxyl radicals plus superoxide anions enhanced inflammatory cell infiltration in the portal area, accompanied by liver injury, may be attributed to DXM which induces cell membrane oxidative damage leading to fatty liver change [26].

The histopathological alterations induced by DXM in the existing study agree with Safaei et al. [37] who revealed that dexamethasone caused necrosis, infiltration in the inflamed tissue, and severe hepatocyte erosion. Dexamethasone declines hepatocyte proliferation activity and, accordingly, their regeneration capacity [31]. High doses of glucocorticoids, especially dexamethasone, suppress the expression of hepatocyte growth factor due to declined proliferation capacity. The liver cell morphological alterations were associated with sinusoid plus central vein dilatation [27]. Dexamethasone elevated the osmotic pressure, disturbing electrolyte balance and affects the sodium-potassium pump in the liver cells due to cell vacuolization and blebbing [2].

Regarding the oxidative stress, the existing findings agreed with [24] who have indicated that the major cause of liver injury induced by dexamethasone is oxidative stress that is produced from the extreme formation of free radicals. Elevated amounts of dexamethasone markedly reduced the actions of whole antioxidant capacity in addition to superoxide dismutase, resulting in oxidative stress by increasing the volumes of peroxide hydrogen plus malondialdehyde [30]. Herbs may have a variety of phytochemicals that differ from phenolic complexes for antioxidant and bitter complexes which stimulate the digestive system beside several other pharmacological characteristics including antifungal and antibacterial effects [14].

Oxidative stress returns an inequality among the capabilities of the biological systems to fix the damage or to quickly detoxify the reactive intermediates and the reactive oxygen species’ systemic expression. The formation of free radical plus peroxides from redox state disturbance of the cell leads to damage of all cell components including breaking the DNA strand and base damage [6]. ROS generated indirectly and mostly from base damage, for example hydroxyl radical and superoxide radical “hydrogen peroxide.” Proteins, lipids, and DNA are the main cellular components affected through oxidative stress. Oxidative stress has significant role in numerous pathogenesis of degenerative diseases like cancer, diabetes, cardiovascular disorders, or neurodegenerative diseases [41]. So, balance between oxidant and antioxidant particles produced the complex task of oxidant progression in cells. Several enzymes performed protective actions against ROS, for example, glutathione peroxidase, superoxide dismutase (SOD), and catalase, in addition to non-enzymatic components as glutathione, vitamin E tocopherol, beta-carotene, and ascorbate. Reduction in the capacity of this antioxidant system raises inactivated ROS level, enzymatic or non-enzymatic reactions activated ROS formation, and ROS generation altered transition potential and mitochondrial permeability [28, 45]. Pro-apoptotic factors (cytochrome C) were initiated according to these variations. Moreover, oxidative stress may induce changes (reversible and irreversible) in sensitive proteins that are concomitant frequently with neurodegenerative disturbances [33, 48]. Lipid hydroperoxides resulted from cholesterol glycolipids, cholesterol esters, phospholipids, and unsaturated fatty acids. The natural antioxidants are necessary to repair the oxidative damage as the internal defense system cannot offer complete protection versus the over-all oxidative stress that humans are exposed to. Plants are source of several phytochemicals with in vitro antioxidant activity—a reason to be promising anti-oxidative stress agents [25]. Greater production of free radicals, in particular ROS, might be caused by extreme dexamethasone amounts. Free radicals increase mitochondrial permeability, mitochondrial dysfunction, and apoptosis in cells and decreased cellular energy production [19, 39].

Conclusion

Overall, DEX-induced hepatotoxic effect appeared either by liver enzyme activity elevation or by liver homogenate antioxidant activity reduction in addition to liver histological perturbations. Although, thyme extract restored these effects, applying thyme as a medicinal plant requires formerly attention, and additional studies about thyme assess safety as well as benefits.