Hepatoprotective effect of Thymus vulgaris extract on sodium nitrite-induced changes in oxidative stress, antioxidant and inflammatory marker expression

The herb thyme (Thymus vulgaris) has multiple therapeutic uses. In this study, we explored how T. vulgaris leaf extract protects liver cells against sodium nitrite-(NaNO2) induced oxidative stress. Mice were divided into four groups; each group received one of the following treatments orally: saline; T. vulgaris extract alone; NaNO2 alone; or T. vulgaris extract + NaNO2. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), reduced glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), IL-1β, IL-6, TNF-α, and total proteins were measured in serum using standard methods. TNF-α, hemooxygenase-1 (HO-1), thioredoxin, SOD, and GSH synthase, all of which are linked to oxidative stress, were measured using quantitative real-time PCR (qRT-PCR). In mice treated with T. vulgaris extract, the effect of NaNO2 on ALT and AST levels and total proteins was reduced, and its effect on antioxidant levels was reversed. Normally, NaNO2 causes hepatocyte congestion and severe hepatic central vein congestion. Tissues in the mice treated with T. vulgaris were restored to normal conditions. Our results demonstrate that NaNO2-induced hepatic injury is significantly reduced by pretreatment with T. vulgaris extract, which protects against hepatic oxidative stress and its associated genes at the biochemical, molecular, and cellular levels.

www.nature.com/scientificreports/ prepared using Bouin's solution; blood samples were collected and the extracted serum stored at -20 °C for later use.
Biochemical assessments. Serum ALT and AST levels were measured using a colorimetric spectrophotometer following the procedures in the user's manual. MDA levels were assessed using the technique developed by Ohkawa et al. 32 . SOD was measured using the method of Beutler et al. 33 , reduced GSH using the method of Nishikimi et al. 34 , and total protein levels were determined using the method of Lowry et al. 35 . These colorimetric methods were performed using a Bio-Rad Smart Tech Spectrophotometer according to the procedures in the product inserts provided with each kit. The reduced glutathione (GSH) and oxidized glutathione (GSSG) kits were purchased from Sigma-Aldrich Chemical (St. Louis, MO, USA). All chemicals used were of the highest purity and purchased from commercial suppliers in Saudi Arabia and Egypt.
Cytokine measurements. Several different kits for measuring parameters in mice, including mouse IL-1β (cat. No. E-EL-M0037), IL-6 (E-EL-M0044), and TNF-α (E-EL-M0049) were purchased from Elabscience (Houston, TX, USA). Serum cytokine levels were determined using Sandwich-ELISA kits in accordance with the product inserts and reference tables available at the Clinical Laboratory Sciences Department, Turabah University College, Taif University.
Quantitative real-time PCR (qRT-PCR) and gene expression. Total liver RNA was extracted and determined to be pure at 260/280 nm. Reverse transcription was performed using the Quanti-Tect reverse transcription kit with 1 µg total RNA, producing single-stranded complementary DNA (cDNA) using a two-step qRT-PCR reaction with a random primer hexamer. Amplification of cDNA was performed using SYBR Green master mix (Thermo Scientific, Waltham, MA, USA). Table 1 lists the primers used in thermal cycle qRT-PCR analysis, using the 2 -ΔΔCt method. β-actin was used as a 'house-keeping' (internal standard) gene, against which cDNA was normalized. Reactions were run on and analyzed using a 7500 FAST Real-Time PCR Detection System (Applied Biosystems, Foster City, CA, USA). qRT-PCR conditions were: 95 °C for 10 min (1st denaturation stage) and 40 cycles of 95 °C for 15 s (2nd denaturation stage) followed by 60 °C for 1 min (annealing and extension stage). Changes in intensity and expression of the specific genes listed in Table 1 under Analysis were determined using comparative cycle threshold (CT) values.
Histological examinations. After decapitation, typical tissue specimens from the livers of all animals were harvested according to a standardized necropsy protocol 36 and immediately fixed in Bouin's solution for 24 h. The fixed specimens were thoroughly washed under running tap water, dehydrated in an ascending series  www.nature.com/scientificreports/ of ethanol concentrations (70%, 80%, 90%, and 100%; 2 h each), cleared in two changes of xylene (1 h each), impregnated in two changes of soft paraffin (2 h each), embedded in paraffin blocks, cut into Sects. 4 μm thick, and routinely stained with hematoxylin and eosin following standard processing and staining protocols 37 . The stained sections were examined microscopically and any histopathological alterations observed were recorded. This was followed by multiparametric quantitative lesion scoring for the hepatic parenchyma in all groups following the protocol described by Khalil et al. 38 with a few modifications. For each animal, five randomly selected, non-duplicated microscopic profiles (40 × objective) were captured using an AmScope digital camera attached to an Olympus light microscope. These images were analyzed to allow calculation of the ratio of the area fractions of the hepatic central veins, portal blood vessels, sinusoids, necrosis, hemorrhage, fatty change, and leukocytic aggregations to the total areas using the image analysis software ImageJ (version 1.51v; Research Services Branch, NIH, Bethesda, MD, USA). The proportion of hepatic cells that exhibited pyknosis, single-cell necrosis, and vacuolar and hydropic degeneration with respect to the total number of hepatocytes per image was calculated subjectively. The frequency with which other lesions occurred (Kupffer cell hyperplasia, leukocytic infiltration, cholangiocyte hyperplasia and/or necrosis, and cholestasis) was determined by counting the number of lesions per image. Eventually, the results were expressed as percentages (mean ± SEM).

Statistical analysis.
Seven mice per group were selected and one-way ANOVA and Dunnett's post hoc descriptive tests were performed using SPSS software version 22 (SPSS, IBM, Chicago, IL, USA) for Windows. The data are presented as mean ± standard error of mean; statistical significance is p < 0.05, indicated by letters with different symbols**.
Ethical Statement. All experimental procedures were carried out under National Institutes of Health Guidelines for the care and use of laboratory animals, and all procedures designed to minimize the suffering of animals were followed.

Results
Analysis and detection of T. vulgaris content. Analysis (using gallic acid as a standard curve) revealed that ethanolic T. vulgaris extract contained rosmarinic acid (38.23 mg/g dry weight thyme extract), luteolin-7-Oglucoside (17.3 mg/g), and caffeic acid (1.96 mg/g). These results are shown in Table 2. Concentrations of phenol and flavonoids were 221.65 and 115 mg/g, respectively, using rutin as a standard calibration curve (Table 1). Table 3 shows the major compounds contained in T. vulgaris extract. Ten major compounds were detected in T. vulgaris extract following GC-MS analysis; the molecular weight (MW), retention time (RT), and peak area vary among these compounds. The hydroxyl (OH), carbonyl (CO), acetyl (COCH 3 ), and nitro (NO 2 ) functional groups are also shown in Table 3.

Serum hepatic biomarkers.
We observed elevated ALT and AST levels and lower total proteins in the NaNO 2 -intoxicated groups (Groups 3 and 4), which indicated liver damage and dysfunction. In the group pretreated with T.vulgaris extract (Group 4), which received T. vulgaris extract for 14 days and then NaNO 2 for 24 h, we observed less pronounced changes in ALT and AST levels and total proteins (Table 4). Pretreatment with T. vulgaris extract normalized and returned ALT and AST levels and total proteins to their relative control levels. www.nature.com/scientificreports/ against NaNO 2 -induced hepatic toxicity ( Table 5). The effect of T. vulgaris extract on the glutathione system was evaluated by determining the ratio of GSH to oxidized glutathione (GSSG) in serum from NaNO 2 -intoxicated mice ( Table 5). The T. vulgaris extract had a positive effect on the glutathione status in NaNO 2 -injected mice by causing a concomitant increase in GSH levels and decrease in GSSG levels, which restored the GSH/GSSG ratio to control levels. The profound decrease in the GSH/GSSG ratio induced by NaNO 2 was normalized by T. vulgaris treatment for 15 days.   www.nature.com/scientificreports/ Mitigated impact of T. vulgaris extract on inflammatory cytokines. To examine the destructive impact of NaNo 2 on the immune state of mice, we measured the levels of pro-inflammatory cytokines. NaNO 2 injection increased serum levels of IL-1β, IL-6, and TNF-α in the NaNO 2 -intoxicated mice (Groups 3 and 4). Pretreatment of NaNO 2 -intoxicated mice with T. vulgaris (Group 4) mitigated these effects (Table 6), which eventually returned to normal levels. T. vulgaris extract normalized all altered pro-inflammatory cytokines.

Impact of T. vulgaris
Ameliorative effect of T. vulgaris extract on quantitative expression of liver genes. Nitrite toxicity induced liver damage and hepatic dysfunction as shown by the increases in hemeoxygenase-1 (HO-1), an oxidative stress biomarker, and TNF-α, an inflammatory cytokine ( Fig. 2A,B). Gene expression for TNF-α   www.nature.com/scientificreports/ mRNA was downregulated in the group pretreated with T. vulgaris extract ( Fig. 2A). Conversely, the HO-1 gene was upregulated in the T. vulgaris-treated group (Fig. 2B). The ameliorative effects of T. vulgaris extract on both TNF-α and HO-1 expression and its potential to restore and recover gene alterations caused by NaNO 2 intoxication are shown in Fig. 2A,B.

Impact of T. vulgaris extract on quantitative expression of antioxidant genes in liver. qRT-
PCR results showed that NaNO 2 downregulated thioredoxin, SOD, and GSH synthase mRNA expression ( Fig. 3A-C), leading to oxidative stress in the liver. The T.vulgaris-treated group (Group 2) showed positive correlation in the antioxidant genes examined, as T. vulgaris upregulated thioredoxin, SOD, and GSH synthase mRNA expression, demonstrating that the extract has important antioxidative properties (Fig. 3). In the group pretreated with T. vulgaris extract for 14 days followed by NaNO 2 intoxication for 24 h (Group 4), the altered mRNA expression of the examined genes returned to within the normal range as a result of pretreatment.
Histopathological examination. Upon microscopic examination of the specimens taken from the control and T. vulgaris-treated animals, we observed normal hepatic histological architectures: roughly hexagonshaped hepatic lobules with sinusoids converging from the periphery to the central vein and portal canals present at approximately three of the six angles of the lobule. The hepatic parenchyma between the portal canals consisted of hepatocytes arranged in cell plates (Fig. 4A,B). Exposure to NaNO 2 induced a wide variety of hepatopathic histological alterations, including circulatory changes (congestion of the central and portal veins and www.nature.com/scientificreports/ sinusoids), hepatocyte degeneration and necrosis (pyknosis, cellular swelling associated with vacuolations due to vacuolar and hydropic degenerations or fatty changes, and single cell necrosis), and inflammatory changes (intralobular and/or leukocytic infiltration or aggregations) (Fig. 4C,D). Interestingly, the biliary system did not show any noticeable histological alterations. T. vulgaris showed marked hepatoprotective effects against the NaNO 2 -induced hepatopathy. Although the hepatic tissue sections from the NaNO 2 + T. vulgaris-treated animals were not completely histologically normal, we observed a significant reduction in the severity and frequency of the NaNO 2 -induced histological changes. The most frequent lesions in this group were vacuolar vascular congestion, sinusoidal dilatation, hepatocyte vacuolation, and tiny mononuclear cell aggregations (Fig. 4E,F). www.nature.com/scientificreports/ The overall hepatic lesion scoring changes induced by NaNO 2 and possible amelioration by thymus extract are shown in Table 7.

Discussion
Our study confirmed that NaNO 2 intoxication increases the levels of ALT and AST in serum, decreases total proteins, affects cytokine levels and gene expression (leading to an imbalance in antioxidant mechanisms), and causes histopathological changes, resulting in severe oxidative stress in liver tissues. However, pretreatment with T. vulgaris extract prevented or reversed these effects, confirming the antioxidant effect of T. vulgaris against NaNO 2 -induced hepatic dysfunction. The elevated levels of ALT and AST, decreased total serum proteins, and the histopathological alterations that we observed, are all indicators of toxicity and also well-known quantitative measures of liver activity 39 . While functional liver damage can be indicated by high serum levels of AST, a more liver-specific indicator is the enzyme ALT. ALT catalyzes the conversion of alanine into pyruvate and glutamate, making serum levels of ALT a more reliable indicator of liver damage 40 . Our findings demonstrated that the membrane structure and integrity of the liver cells, which would otherwise have been destroyed by NaNO 2 , remained undamaged and uncongested, providing strong evidence for the protective effect of T. vulgaris extract also described previously 17,41 .
Sharma et al. 42 noted that hepatoprotective plants are characterized by significant levels of polyphenols and flavonoids, major compounds also found in T. vulgaris. Flavonoids promote the expression of enzymes involved in the production of glutamylcysteine synthetase and thioredoxin, leading to increased intracellular GSH levels 43,44 . The antioxidant assays used in this study, which targeted five different types of ROS molecules (MDA, SOD, GSH, GSSG, and GSH/GSSG), confirmed the strong antioxidant effect of the T. vulgaris extract. Our findings are in agreement with those of previous studies that reported the high phenolic content and strong antioxidative effect of T. vulgaris extract 17 . Inhibition and decreased function of the antioxidant system causes the accumulation of H 2 O 2 and hepatic cell decomposition 45 . Treatment with T. vulgaris extract significantly reversed these decreases in antioxidant enzyme activity.
Our study showed that NaNO 2 intoxication reduced antioxidant activity and increased both oxidative stress and lipid peroxidation in agreement with previous reports 11,30 . Antioxidant defenses are affected by the expression of genes such as thioredoxin, SOD, and GSH 46 , and oxidative stress is regulated by several other downstream genes, including HO-1 47 . Hyun 48 showed that many plant extracts play a critical role in increasing antioxidant activity and reducing ROS. Overexpression of a particular hemoxygenase-1 reported here in thymus control group and downregulation in the NaNO 2 -treated groups, confirmed the potential of T. vulgaris extract. HO-1 is normally involved in the regulation of mitochondrial biogenesis, neurogenesis, and angiogenesis 48 , causing an increase in protein expression 49 . As such, HO-1 plays an important role in mechanisms of oxidative stress and inflammation 50 . By controlling IL-1β activation 51 , HO-1 regulated the expression of inflammasome-associated genes 52 . The antioxidant mechanism is regulated by downstream genes that control oxidative stress, such as Nrf2 and HO-1 47,53 . Our findings confirmed that HO-1 expression plays a key role in the regulation of hepatic oxidative stress regulated by T. vulgaris extract. To explore the potential mechanism underlying the hepatoprotective effect of T. vulgaris extract, we examined the role of HO-1 signaling. Most reports have indicated that HO-1 plays a role in the activation and expression of some antioxidant and anti-inflammatory cytokine genes 54,55 . All were coincided with our reported findings.
Our study confirmed that T. vulgaris extract regulated the expression of pro-inflammatory cytokines, such as IL-1β, thereby ameliorating the NaNO 2 -induced inflammatory response. The inflammatory response causes increased production of IL-1β, IL-6, and TNF-α cytokines 56 , which play a leading role in sepsis and fever. NaNO 2 intoxication has the same effect 57 , yet our study found that cytokine levels in mice pretreated with T. vulgaris www.nature.com/scientificreports/ extract returned to normal. Therefore, T. vulgaris extract prevented NaNO 2 -induced hepatic toxicity by ameliorating disrupted serum levels of IL-1β, IL-6, and TNF-α. Reactive oxygen species (ROS) play a vital role in maintaining various human physiological processes 58 , but at high levels, they can be cytotoxic and cause liver damage 59 . Therefore, mechanisms exist to maintain appropriate ROS levels in the liver and other organs, thereby preventing oxidative stress and promoting redox homeostasis 59 . Our study found that while NaNO 2 intoxication induced lipid peroxidation, pretreatment with T. vulgaris reversed these effects. In part, this was due to the recovery of depleted GSH, which plays a significant defensive role against oxidative stress 60 .
Hydroperoxides are converted into alcohols and water in a process catalyzed by thioredoxin, a cytosolic protein 61 . Thioredoxin has several antioxidant functions, including scavenging for free radicals, removing hydrogen peroxide, protecting against oxidative stress, as well as broader functions in genetic transcription, DNA and protein repair, immunostimulant roles, apoptosis, and cell proliferation. As a thiol-specific antioxidant, thioredoxin shares many of the same functions as glutathione systems and the two are understood to act in tandem in the management of oxidative stress 62 . In our study, NaNO 2 downregulated thioredoxin and GSH synthase expression, but these returned to normal in mice pretreated with T. vulgaris extract.
In addition to biochemical alterations, the hepatotoxic effect of NaNO 2 has been further implicated in a wide variety of degenerative, circulatory, and inflammatory alterations. The etiopathogenesis of these hepatopathic morphological alterations are likely to be multifactorial and mediated by NaNO 2 -induced oxidative damage and inflammation due to the formation of ROS and lipid peroxidation. The latter is associated with lysosomal and mitochondrial membrane rupture and subsequent release of digestive proteases 63 and activation of the apoptosisrelated Bax and caspase-3 genes 64 .
Treatment with NaNO 2 caused hepatic dysfunction, oxidative stress, and alteration in the levels of inflammatory cytokines. Pretreatment with T. vulgaris extract alleviated these responses to toxicity and restored the levels and mRNA expression of the proteins and genes. The collective summary for the protective impact of T. vulgaris on NaNO 2 -induced liver dysfunction are clearly shown in Fig. 5. www.nature.com/scientificreports/

Conclusion
T. vulgaris extract exerted a protective effect against oxidative stress in liver tissues caused by NaNO 2 intoxication. The effect was observed in different antioxidant systems and through the regulation of pro-inflammatory cytokines. In short, the common herb thyme (T. vulgaris) can be used in the prevention and treatment of hepatic oxidative stress.

Data availability
Data are available upon request. www.nature.com/scientificreports/