Thymoquinone Protects Against EGCG/LPS Induced ...

International Journal of Pharmacology and Clinical Trials, ISSN:2051-8293, Vol.26, Issue.1

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Thymoquinone Protects Against EGCG/LPS Induced Hepatotoxicity in Mice Ibrahim G. Saleh National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA Department of Pharmacology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt

Zulfiqar Ali National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA

Floyd D. Wilson Mississippi Veterinary Research and Diagnostic Laboratory, College of Veterinary Medicine, Mississippi State University, Pearl, MS. 39208, USA

Farid M. Hamada Department of Pharmacology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt

Mohamed F. Abd-Ellah Department of Pharmacology, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt

Larry A. Walker National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA Department of Pharmacology, School of Pharmacy, University of Mississippi, University, MS 38677, USA

Ikhlas A. Khan National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA Department of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677, USA Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11557, Saudi Arabia

Mohammad K. Ashfaq National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, MS 38677, USA Corresponding Author E-mail: [email protected]

ABSTRACT Green tea (GT) is a widely used beverage and folk medicine. Epigallocatechin-3-gallate (EGCG), the main polyphenol of GT has been demonstrated to impact a number of biological targets. Our previous work has shown that the combination of LPS and EGCG in male mice causes hepatotoxicity, perhaps related to a prooxidant effect. Thymoquinone (TQ) is a prominent constituent of “black seed”, Nigella sativa, and is known for a number of biological activities, including potent antioxidant and anti-inflammatory activities. It has been extensively used in the Middle East as a protectant against hepatic and renal injuries. In the current study we treated mice with oral doses of TQ before and concurrently with EGCG/LPS. Thymoquinone ameliorated the rise in the plasma concentrations of ALT, ALP, TB, Amylase and the decrease of A/G ratio to normal after exposure to EGCG/LPS. It also blunted the weight loss. No histopathological lesions or mortality was observed. It can be concluded from this study that TQ showed a hepatoprotective effect in a murine model of EGCG/LPS

induced hepatotoxicity. This hepatoprotective effect may be related to the antioxidant properties of TQ, but this conclusion needs further evaluation.

Keywords - Green tea, Epigallocatechin-3-gallate, Lipopolysaccharide, Thymoquinone, Hepatotoxicity.

1. INTRODUCTION The potential health benefits associated with green tea consumption have been partially attributed to the antioxidant properties of tea polyphenols in particular (−)epigallocatechin-3-gallate (EGCG) the major component of GT[1]. The antioxidant capacity of GT and its polyphenols has been assessed by several methods. Using the Oxygen Radical Absorbance Capacity (ORAC) assay, it has been found that green tea has much higher antioxidant activity against peroxyl radicals than vegetables such as garlic, spinach and Brussels sprouts [2]. Several clinical trials have demonstrated that a single dose of tea improves plasma antioxidant capacity of healthy adults within 30 to 60 minutes after ingestion. A

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significant rise in plasma antioxidant capacity was detected after consumption of brewed GT or black tea [3]. In general, the rise in plasma antioxidant capacity peaks about one to two hours after tea ingestion and subsides shortly thereafter. Repeated consumption of tea and encapsulated tea extracts for one to four weeks has been demonstrated to decrease biomarkers of oxidative status. In a trial of 40 male smokers in China and 27 men and women (smokers and non-smokers) in the United States, oxidative DNA damage, lipid peroxidation and free radical generation were reduced after consuming ~6 cups a day of GT for seven days [4]. Plasma malondialdehyde, another indicator of lipid peroxidation, was reduced in 20 healthy women, 23 to 50 years of age, consuming a high linoleic acid diet and administered an encapsulated tea extract (equivalent to 10 cups a day of green tea) for four weeks [5].

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The seeds of N. sativa as well as their oil have been widely used for centuries in the treatment of various ailments throughout the world and is an important medicinal entity in the Indian traditional system of medicine [23]. Thymoquinone has been extensively studied for its biological activities and therapeutic potential and shown to possess wide spectrum of activities such as diuretic, antihypertensive, antidiabetic, anticancer and immunomodulatory, analgesic, antimicrobial, anthelmintic, analgesic, anti-inflammatory, spasmolytic, bronchodilator, gastroprotective, hepatoprotective, renal protective and antioxidant properties [reviewed in 24]. Thymoquinone is widely used in the treatment of various diseases like bronchitis, asthma, diarrhea, rheumatism and skin disorders. It is also used as liver tonic, digestive, antidiarrheal, appetite stimulant, lactagogue, to fight parasitic infections, and to support immune system [reviewed in 25].

The anticarcinogenic mechanisms of EGCG include inhibition of mitogen activation protein-kinases, the 2. AIM activation of activator protein-1, and cell transformation [6]. EGCG has also been shown to cause inhibition of In light of the cited literature TQ appears to have a growth factor receptor phosphorylation, such as epidermal protective effect on liver [26], especially in LPS induced growth factor receptor (EGFR), platelet-derived growth injuries [27, 28] where TQ was shown to inhibit LPSfactor receptor (PDGFR), and HER-2/neu [7]. It has also induced pro-inflammatory cytokines such as IL-5, IL-13, been reported to cause G0-G1 phase arrest [8], induce IL-1ß, TNFα, MMP-13, COX-2 and PGE-2. It was apoptosis [9], inhibit DNA methyltransferase activity [10], reported earlier that a subtoxic dose of LPS when and inhibit aberrant arachidonic acid metabolism [11]. It combined with a subtoxic dose of monocrotaline, caused was shown earlier that EGCG could cause auto-oxidation an increase in the blood level of leptin, interleukin-6 (ILin cancer cells. A significant amount of H2O2 was 6) [29]. Also, we know from our previous study that generated from EGCG in many cell culture systems [12]. EGCG in high doses behaves as a pro-oxidant as it showed The fact that the addition of catalase, which eliminates an over-expression of (OX.LDL, CXCL16) and of proH2O2, can prevent EGCG-induced apoptosis (completely inflammatory cytokines (TNFα, TGFß) [30]. In this study or partially) suggests that the H2O2 generated from EGCG we selected TQ as it possesses both anti-oxidant and antiauto-oxidation may be at least partially responsible for the inflammatory properties. Our aim was to determine if preinduction of apoptosis [13, 14]. At higher doses, the treatment of mice with TQ would protect against apoptotic effect of EGCG may affect normal cells such as hepatotoxicity in mice caused by multiple high IG doses of hepatocytes. In other words, EGCG may exert some of its EGCG and single IP dose of LPS. toxic effects via auto-oxidation or pro-oxidation leading to 3. MATERIALS AND METHODS apoptosis. Many animal studies and clinical case reports have implicated EGCG in hepatotoxicity [15]. Others and 3.1 Animals we have studied the effect of high doses of EGCG on - Male ND-4 mice were obtained from Harlan Lab mouse liver and the possible underlying conditions that (Indianapolis, IN, USA) at 5 weeks of age and 23 - 28 g might contribute to EGCG-induced hepatotoxicity [16, body weight, housed in micro isolator cages with corn cob 17]. bedding, on 12 h light/dark cycle, at 72°F (22 °C)and 35Oxidative stress occurs when pro-oxidants overweigh the 50% relative humidity. Mice were fed on (TekLad 57001) anti-oxidant mechanisms [18]. Liver is the major organ laboratory chow and water ad libitum. All animals were concerned with the detoxifying process. It possesses an fasted for at least 8 hours before any treatment. All animal enzymatic system to overcome the hazardous effect of the study protocols were approved by the Institutional Animal free radicals. This enzymatic system includes glutathione Care and Use Committee (IACUC), University of peroxidase (GSH-Px), glutathione s transferase (GST), Mississippi, USA. lactate dehydrogenase (LDH), superoxide dismutase (SOD) and catalase (CAT). When the exposure to a prooxidant exceeds the capacity of detoxification mechanisms, the first organ to be injured is liver. This can lead to steatotic changes in liver that can lead to hepatitis [19]. Many natural products possess anti-oxidant effect. Among these natural products, Nigella sativa with its main constituent, thymoquinone (TQ), possesses powerful antioxidant [20] and anti-inflammatory properties [21, 22].

3.2 Chemicals 3.2.1- Epigallocatechin - 3- gallate (EGCG) was purified to 97% from a hot water extraction of green tea leaves. The initial hot water extract was further purified by separating the catechin fraction with ethyl acetate. This was then subjected to chromatographic separation of EGCG from the catechin fraction in ethanol/water followed by crystallization or spray drying.



3.2.2-Thymoquinone (TQ): (99%) [2-Isopropyl- 5-methyl1,4-benzoquinone] obtained from Sigma-Aldrich (St Louis, MO, USA).

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3.8 Animal survival

3.2.3- Lipopolysaccharide (LPS) of E. coli: obtained from Sigma-Aldrich (St Louis, MO, USA).

Death in each group was monitored and recorded during the course of the experiment. Mortality was calculated using Kaplan-Meier test of survival using Graph Pad Prism software (La Jolla, CA).

3.3 Experimental design (Table.1)

3.9 Liver histopathological examination

3.5 Relative reduction of body weight of mice Mice were weighed before the start of the experiment (initial body weight), during the course of the study and after the end of experiment just before euthanasia (final body weight).

3.6 Relative weight of liver After euthanization of mice, livers were dissected out and placed in phosphate buffered saline (pH 7.4) and washed out, then blotted dry on filter paper. Each liver was weighed.

20

a 10

a

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a a

*

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-10 -20 -30

3.7 Clinical chemistry The levels of alanine amino transferase (ALT), alkaline phospatase (ALP), albumin to globulin ratio (A/G ratio), amylase, blood urea nitrogen (BUN) and total bilirubin were measured immediately after blood sampling using an automated VetScan dry chemistry analyzer with Comprehensive Diagnostic Profiles (Abaxis, Union City, CA, USA).

a

*

T9 TQ 10+ (EGCG/LPS)

Blood samples were collected in heparinized micro tubes from the mice by venipuncture in the mandibular region. After euthanization by CO2 asphyxiation, tissue samples of liver (left median lobe) were taken and processed by standard histological techniques.

Treatment of mice with EGCG/LPS caused a significant reduction in body weight compared to vehicle (T1). Animals administered TQ 5 and 10 mg/kg without EGCG/LPS showed no significant change in body weight compared to vehicle (T1). Protection with TQ 5 and 10 mg/kg showed significant restoration of body weight compared to EGCG/LPS group (T3).

T8 TQ 5+ (EGCG/LPS)

3.4 Sample collection

4.1 Change in body weight (Fig.1)

T7 TQ 10/LPS

Thymoquinone + LPS + EGCG

4. RESULTS

T6 TQ 10

T9

Numerical data were analyzed by one way ANOVA test followed by Tukey Cramer multiple comparisons using Graph Pad Prism software (La Jolla, CA). A P-value of less than 0.05 was considered to show a significant difference between vehicle and other groups.

T5 TQ 5/LPS

T8

3.10 Statistical analysis

T4 TQ 5

T6 T7

6 mg/kg single dose IP once daily (8 days) IG 6 mg/kg single dose IP 1500 mg/kg (5 days) IG 5 mg/kg (8 days) IG 5 mg/kg (8 days) IG 6 mg/kg single dose IP 10 mg/kg (8 days) IG 10 mg/kg (8 days) IG 6 mg/kg single dose IP 5 mg/kg (8 days) IG 6 mg/kg single dose IP 1500 mg/kg once daily (5 days) IG 10 mg/kg (8 days) IG 6 mg/kg single dose IP 1500 mg/kg once daily (5 days) IG

T3 EGCG/LPS

T4 T5

once daily (8 days) IG

Liver samples were kept in formalin solution (10%) for 24 hours then washed in tap water for 12 hours followed by absolute ethyl alcohol for dehydration of tissues. Tissues were cleared in xylene and embedded in paraffin blocks. Three micron-thick sections were stained by hematoxylin (RICCA chemical Co., Arlington, TX) and eosin (EMD Chemicals, Gibbstown, NJ). The slides were observed for lesions and analyzed using routine light microscopy.

T2 Veh/LPS

T3

10% DMSO (Vehicle) LPS + 10% DMSO LPS + EGCG Thymoquinone Thymoquinone + LPS Thymoquinone Thymoquinone + LPS Thymoquinone + LPS + EGCG

Dose

T1 Veh

T2

Treatment

Relative change in body weight (%)

Group (n=10) T1

Fig.1: Percent change of the body weight after treatment of mice with EGCG/LPS with or without TQ protection. * Significantly different from vehicle control (T1), a

significantly different from EGCG/LPS group (T3).

P < 0.0001



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4.2 Effect on liver weight (Fig.2)

4.4 Effect on plasma ALP (Fig.4)

No significant change in relative weight of liver was observed in EGCG/LPS treated mice either with or without TQ protection compared to vehicle (T1).

Treatment of mice with EGCG/LPS caused a significant increase in plasma ALP level (2.5 fold) compared to vehicle (T1). Administration of TQ 5 and 10 mg/kg without EGCG/LPS did not cause any significant change in plasma ALP level compared to vehicle (T1). Protection with TQ 5 and 10 mg/kg caused a significant decrease of plasma ALP level compared to EGCG/LPS group (T3).

6

500

4

*

400

Plasma ALP U/L

2


200

a

a

a

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100

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a a

* Significantly different from vehicle control (T1), a significantly different from EGCG/LPS group (T3). P < 0.0001

4.5 Effect on BUN concentration (Fig.5)

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T8 TQ 5+ (EGCG/LPS)

BUN level mg/dL


T8 TQ 5+ (EGCG/LPS)

T7 TQ 10/LPS

T6 TQ 10

T5 TQ 5/LPS

T4 TQ 5

T3 EGCG/LPS

T2 Veh/LPS

T1 Veh

0

T7 TQ 10/LPS

a

T6 TQ 10

a

T5 TQ 5/LPS

a

T4 TQ 5

a

T2 Veh/LPS

a

T1 Veh

Plasma ALT U/L

*

a

Fig.4: Plasma ALP level after treatment of mice with EGCG/LPS with or without TQ protection.

Treatment of mice with EGCG/LPS caused a significant increase in BUN concentration (about 4 fold) compared to vehicle (T1). Administration of TQ 5 and 10 mg/kg without EGCG/LPS did not cause any significant change in BUN concentration compared to vehicle (T1). Protection with TQ 5 and 10 mg/kg caused a significant decrease of BUN concentration compared to EGCG/LPS group (T3).

300

a


4.3 Effect on plasma ALT (Fig.3)

T8 TQ 5+ (EGCG/LPS)

T7 TQ 10/LPS

T6 TQ 10

T5 TQ 5/LPS

T4 TQ 5

T3 EGCG/LPS

Fig.2: Relative weight of livers after treatment of mice with EGCG/LPS with or without TQ protection.

Treatment of mice with EGCG/LPS caused a significant increase in plasma ALT level (about 6 fold) compared to vehicle (T1). Administration of TQ 5 and 10 mg/kg without EGCG/LPS did not cause any significant change in plasma ALT level compared to vehicle (T1). Protection with TQ 5 and 10 mg/kg caused a significant decrease of plasma ALT level compared to EGCG/LPS group (T3).

a

0 T1 Veh

T8 TQ 5+ (EGCG/LPS)

T7 TQ 10/LPS

T6 TQ 10

T5 TQ 5/LPS

T4 TQ 5

T3 EGCG/LPS

T2 Veh/LPS

T1 Veh

0

300

T2 Veh/LPS

Relative weight of liver

8

* Significantly different from vehicle control (T1), a

Fig.5: BUN concentration after treatment of mice with EGCG/LPS with or without TQ protection.


P < 0.0001

T3 EGCG/LPS

0

Fig.3: Plasma ALT level after treatment of mice with EGCG/LPS with or without TQ protection.

* Significantly different from vehicle control (T1), a


P < 0.0001



4.6 Effect on plasma albumin to globulin (A/G) ratio (Fig.6) Treatment of mice with EGCG/LPS caused a significant increase in A/G ratio (cut to one third) compared to vehicle (T1). Administration of TQ 5 and 10 mg/kg without EGCG/LPS did not cause any significant change in A/G ratio compared to vehicle (T1). Protection with TQ 5 and 10 mg/kg caused a significant decrease of A/G ratio compared to EGCG/LPS group (T3).

4.8 Effect on plasma amylase (Fig.8) Treatment of mice with EGCG/LPS caused a significant increase in plasma level of amylase (about 3 fold) compared to vehicle (T1). Administration of TQ 5 and 10 mg/kg without EGCG/LPS did not cause any significant change in plasma level of amylase compared to vehicle (T1). Protection with TQ 5 and 10 mg/kg caused a significant decrease of plasma level of amylase compared to EGCG/LPS group (T3). 2000

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T8 TQ 5+ (EGCG/LPS)

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T2 Veh/LPS

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T1 Veh

Plasma amylase level U/L

2.5

500

0.5

T7 TQ 10/LPS

T6 TQ 10

T5 TQ 5/LPS


T8 TQ 5+ (EGCG/LPS)

T7 TQ 10/LPS

T6 TQ 10

T5 TQ 5/LPS

T4 TQ 5

T3 EGCG/LPS

T2 Veh/LPS

T1 Veh

Fig.6: Plasma albumin to globulin (A/G) ratio after treatment of mice with EGCG/LPS with or without TQ protection.

T4 TQ 5

0

0.0

T3 EGCG/LPS

Plasma albumin/globulin ratio

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Fig.8: Plasma level of amylase after treatment of mice with EGCG/LPS with or without TQ protection.

* Significantly different from vehicle control (T1),

* Significantly different from vehicle control (T1), a significantly different from EGCG/LPS group (T3). P < 0.0001

a

4.9 Survival of mice (Fig.9)


P < 0.0001

4.7 Effect on total bilirubin (Fig.7) Treatment of mice with EGCG/LPS caused a significant increase in total bilirubin (more than 8 fold) compared to vehicle (T1). Administration of TQ 5 and 10 mg/kg without EGCG/LPS did not cause any significant change in total bilirubin compared to vehicle (T1). Protection with TQ 5 and 10 mg/kg caused a significant decrease in total bilirubin compared to the EGCG/LPS group (T3).

No mortality was observed in the mice treated with the vehicle (T1) or other treatments except in group (T3) that were treated with EGCG/LPS. In this later group mortality started on the day 3 of treatment when 10% of animals died. On the day 4, the mortality was 50% that increased to 60% on day 5, the terminal day of experiment. Animals that showed signs of severe illness (moribund condition) were considered as dead and sacrificed according to IACUC approval. Pretreatment of mice with TQ 5 and 10 mg/kg (T8, T9) prevented the mortality of mice upon treatment with EGCG/LPS.

3

120

Percent survival

Total bilirubin mg/dL

*

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1

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80 60 40 20 0


T8 TQ 5+ (EGCG/LPS)

T7 TQ 10/LPS

T6 TQ 10

T5 TQ 5/LPS

T4 TQ 5

T3 EGCG/LPS

T2 Veh/LPS

T1 Veh

0

Fig.7: Plasma level of total bilirubin after treatment of mice with EGCG/LPS with or without TQ protection. * Significantly different from vehicle control (T1), a

T1 Veh T2 Veh/LPS T3 EGCG/LPS T4 TQ 5 T5 TQ 5/LPS T6 TQ 10 T7 TQ 10/LPS T8 TQ 5+ (EGCG/LPS) T9 TQ 10+ (EGCG/LPS)

100


P < 0.0001

0

2

4

6

Days Exposed

Fig. 9: Percent survival of mice after treatment with EGCG/LPS with or without TQ protection.

4.10 Histopathology of liver (Fig.10) Photomicrographs of liver samples showed: 1- Vehicle (T1): No signs of hepatocellular injury. 2- EGCG / LPS (T3): Hepatocytes exhibited a foamy or finely vacuolated cytoplasm and sometimes swollen. The changes are compatible with microvesicular fatty change,



glycogen accumulation or hydrophic change due to intracellular water accumulating or intracellular edema. A few Hepatocytes demonstrated pyknotic nuclei suggesting early necrosis (circles).

T1

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3- TQ 5 mg/kg + EGCG / LPS: Mild sinusoidal and central vein congestions (arrows) reflecting normal levels of hepatic sinusoidal perfusion. 4- TQ 10 mg/kg + EGCG / LPS: Mild sinusoidal congestion (arrows) reflecting normal levels of hepatic sinusoidal perfusion.

T3

CV

CV

T8

T9

CV

CV

Fig. 10: Photomicrographs of liver samples showing normal and injured livers (H & E stain), magnification power = 100X. CV= Central vein, PV= Portal vein.

5. DISCUSSION In earlier studies we showed hepatotoxicity associated with a combination of high dose of EGCG with a subtoxic dose of lipopolysaccharide (LPS). in vitro [31, 32] and in vivo [33, 34]. In the current study we attempted to explore the effect of sensitization of mice with thymoquinone (TQ) before induction of liver toxicity by EGCG/LPS. Plasma concentrations of some biomarkers for assessment of this protection were determined. Alanine amino transferase (ALT) is one of the early biomarkers of hepatotoxicity [35]. It tends to rise quickly post hepatic injury [36]. The use of EGCG (1500 mg/kg IG for 5 days) / LPS (6 mg/kg IP single dose) without TQ caused ALT to rise to 6 fold compared to control, while pre and concurrent treatment with TQ ( 5 and 10 mg/kg IG for 8 days) with EGCG/LPS caused ALT to remain in the normal range. A similar trend occurred with alkaline phosphatase (ALP). It showed a 6 fold elevation with

EGCG/LPS and no elevation when TQ was administered prior and concurrently. On the same track, the results showed that without administration of TQ, EGCG/LPS elevated total bilirubin (TB) more than 8 fold compared to control, while with TQ co-administration this effect diminished. Taking the observations of ALP and TB together, it appears that EGCG/LPS treatment causes obstructive liver disorder. Often, in obstructive liver diseases, the plasma alkaline phosphatase increases. This includes diffuse intrahepatic obstructive disease which may be caused by some drugs or biliary cirrhosis [37]. In addition, liver injury causes elevation of bilirubin indicating lack of capacity of liver to conjugate bilirubin. Our results showed that treatment of mice with EGCG/LPS without TQ caused albumin to globulin (A/G) ratio to fall to one third of the value of control, while it



remained unchanged with the use of TQ with EGCG/LPS. Albumin to globulin (A/G) ratio is an accepted parameter to appraise various liver and kidney diseases [38]. Treatment of mice with EGCG/LPS showed a significant rise in plasma amylase about 3 folds compared to the control group, while it remained normal in the groups treated with TQ. Usually, amylase tends to rise in cases of pancreatitis and pancreatic tumors, but it can also increase in cases of liver cirrhosis [39], cardiomyopathy [40] and also renal disorder leading to impairment of amylase excretion [41]. Taken together, these observations point toward liver injury induced by EGCG/LPS in terms of high ALT, ALP, TB and amylase and low A/G ratio. These biomarkers are known to be indicative of liver injury [42]. Examination of liver tissue samples from mice treated with EGCG/LPS showed marked pallor and fatty changes (Steatosis or liposis) in the centrolobular hepatocytes with the appearance of some pyknotic nuclei reflecting a reversible form of cellular damage that can lead to irreversible hepatocellular necrosis. These changes are compatible with microvesicular fatty change, glycogen accumulation or hydrophic change due to intracellular water accumulating or intracellular edema. Steatosis and oxidative stress are considered to be significant cofactors in liver injury in biliary cirrhosis [43]. On the other hand pre and concomitant treatment with TQ showed no lesions except mild congestion of sinusoids and central vein, which means improvement of the liver condition upon TQ intervention. It is clear from the comparison of the survival data of mice in the group treated with EGCG/LPS and the other groups in which TQ was added, that TQ remarkably improved the survival rates of mice from 40% (in the group of EGCG/LPS) to 100% (in the groups that were protected with TQ). This is also evident from body weight data where the weight loss was much lower in the TQ groups. In addition, treatment of mice with EGCG/LPS caused BUN to rise about 4 fold compared to the control group, while normal values obtained with the pre and concurrent treatment with TQ. Elevated BUN although does not indicate liver toxicity, but may be indicative of possible renal impairment[44] caused by EGCG/LPS. Amelioration with TQ treatment was observed suggesting that TQ has a renal protective effect. It was documented earlier that prophylactic treatment of mice with TQ, before carbon tetrachloride (CCl4) injection, ameliorated hepatotoxicity of CCl4 as evidenced by the significant reduction of the elevated serum liver enzymes, and significant increase of the hepatic GSH content[45, 46]. Also, TQ prevented the ischemia/reperfusion induced alterations in reduced glutathione (GSH) and SOD in gastric mucosa as well as in liver and kidneys in vivo [47, 48]. Inhibition of both cyclo-oxygenase and lipoxygenase pathways is a key factor mediating the anti-inflammatory effects of TQ [49].

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In our study, TQ probably blunted the inflammatory response to LPS by inhibiting both these pathways resulting in inhibition of inflammatory cytokines. It may also have thwarted the pro-oxidant effect of high doses of EGCG by enabling the CAT, GSH-Px and SOD enzyme activities as well as affected CYP3A1 expression and thus reduced the oxidative stress as reported earlier [48]. In the last cited study a dose of 10 mg/kg of TQ showed protective effect; in the current study 5 mg/kg and 10 mg/kg of TQ showed similar hepatoprotective effect. It would be of interest to determine the lowest effective dose of TQ in this model.

6. CONCLUSIONS The results of the current study substantiate our model of EGCG/LPS induced hepatotoxicity in mice. It also showed that TQ has a hepatoprotective effect in this model. We did not determine the oxidative stress parameters and therefore cannot confirm that these protective effects are due to the antioxidant property of TQ; however, it may very well be the case. Further investigations of oxidative stress/antioxidant specific parameters are needed to confirm these primary assumptions.

ACKNOWLEDGMENTS This research is supported by USDA, grant number 586402-1-612. Authors would like to thank Ms. Penny Bolton and her vivarium staff for animal caring during the experiment.

CONFLICT OF INTEREST All authors declare no conflict of interest with the work done in this study.

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