Sage Journals: Discover world-class research

Abstract

Stem cells are identified as a novel cell therapy for regenerative medicine because of their ability to differentiate into many functional cell types. We have shown earlier a new model of hepatotoxicity in mice by administering (1500 mg/kg) epigallocatechin-3-gallate (EGCG) intragastric (IG) for 5 days after a single intraperitoneal dose (6 mg/kg) of lipopolysaccharide (LPS). In this study, we aimed to study the effect of intrahepatic (IH) injection of mouse embryonic stem cells (MESCs) on the hepatotoxicity induced by EGCG/LPS in mice. Mice were administered EGCG/LPS and rested for 3 days. MESCs were obtained from American Type Culture Collection and cultured in vitro for 4 days. Stem cells were injected IH. Seven days later, a single dose of LPS (6 mg/kg) followed by daily doses of IG administration of EGCG were re-administered for 5 days. At the end of the experiment, blood samples were collected for analysis of biochemical parameters associated with liver. Results showed that the group of mice that were administered MESCs prior to EGCG/LPS showed lower levels of alanine amino transferase, alkaline phosphatase, and bilirubin, higher albumin/globulin ratio, and less remarkable histopathological lesions. Also, that group of mice showed less expression of oxidative stress biomarkers (oxidized low-density lipoprotein Ox.LDL and chemokine CXCL16), less expression of nuclear protein receptors (retinoic acid receptor and retinoid X receptor), and less expression of inflammatory biomarkers (tumor necrosis factor α and transforming growth factor β1) compared with other groups of mice that were not given MESCs. In conclusion, MESCs can ameliorate EGCG/LPS-induced hepatotoxicity in mice.

Keywords

Green tea epigallocatechin-3-gallate lipopolysaccharide stem cells mice hepatotoxicity

Introduction

Stem cells are master cells that can differentiate into any other type of cells. The early studies performed on stem cells utilized pluripotent embryonic stem cells.¹ In almost all tissues, mesenchymal stem cells (MSCs) are present as spindle-shaped, fibroblast-like cells and are responsible for regeneration and cellular homeostasis.² In the last decade, MSCs were successfully isolated from various tissues. The most intensely studied MSCs are those derived from bone marrow.³ Stem cells hold great potential for the treatment of various degenerative diseases and immune disorders, largely because of their differentiation potential and immunoregulatory capacity. In vitro expanded stem cells have already been administered in vivo to both animals in preclinical models and to patients in clinical settings, demonstrating promising clinical utilities.⁴ A remarkable property of stem cells is their powerful capacity for regulating immune responses. As a result, the current stem cell-based therapy has mainly been applied to alleviating immune disorders. The success of stem cells in modulating immune responses and promoting tissue repair in preclinical studies has prompted exploration of these cells in the clinical settings^5

–8 and has led to registration of numerous clinical trials to evaluate the potential of stem cell-based therapy worldwide.⁹ It remains to be seen if these trials will improve clinical applications of stem cells to treat various devastating diseases that affect human health.

Although epigallocatechin-3-gallate (EGCG) is known to be a potent antioxidant that has been used against a variety of ailments such as cancer, cardiovascular disease, nephrotoxicity, and metabolic disorders such as diabetes.¹⁰ Several preclinical studies and clinical case reports have implicated EGCG to be hepatotoxic.^11,12 In our previous studies, we have shown that administration of EGCG at daily doses of 1500 mg/kg intragastric (IG) for 5 days, after a single 6 mg/kg intraperitoneal (IP) dose of lipopolysaccharide (LPS), caused hepatotoxicity in mice.^13,14 We have also shown that green tea and EGCG, under the influence of the inflammatory conditions induced by LPS, cause hepatocellular toxicity, as concluded from overexpression of the oxidative stress biomarkers, namely, oxidized low-density lipoprotein (Ox.LDL) and CXCL16, overexpression of nuclear protein receptors (retinoic acid receptor (RAR) and retinoid X receptor (RXR)), and overexpression of inflammatory biomarkers, namely, tumor necrosis factor (TNFα) and transforming growth factor (TGFβ).^15,16 Mouse embryonic stem cells (MESCs) have been shown to differentiate into hepatocytes when introduced in vivo and hence participate in tissue repair after liver injury.^17,18 In the current study, we aimed to evaluate the effect of intrahepatic (IH) injection of MESCs on the hepatotoxicity induced by EGCG/LPS in mice.

Materials and methods

Animals

Male ND-4 mice were obtained from Harlan Lab (Indianapolis, Indiana, USA) at 5 weeks of age and 23–28 g body weight, housed in micro isolator cages with corn cob bedding with 12 h light/12 h dark cycle at 72°F (22°C) with 35–50% relative humidity. Mice were fed on laboratory chow (TekLad 57001, Madison, Wisconsin, USA) and water ad libitum. All animals were fasted for at least 8 h before any treatment. All animal study protocols were approved by the Institutional Animal Care and Use Committee (IACUC), University of Mississippi, USA.

Chemicals

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. The purity of EGCG was tested using high-performance liquid chromatography.

LPS of Escherichia coli was obtained from Sigma-Aldrich (St Louis, Missouri, USA).

Antibodies and cell lines

Primary antibodies, anti-rabbit (Ox.LDL, CXCL16, RAR, RXR, TNFα, and TGFβ) and anti-rat (CD45, CD90, Sca-1, and rabbit anti-integrin β1), were purchased from (Abcam, Cambridge, Massachusetts, USA).

Secondary antibodies, goat anti-rabbit secondary antibody (Texas Red, Alexa Fluor 488) and rabbit anti-rat (Texas Red), were purchased from Abcam.

Nuclear stain (4′,6-diamidino-2-phenylindole (DAPI)) was purchased from Abcam.

MESCs (ES-D3) and mouse embryonic fibroblasts (C57BL/6) were purchased from (American Type Culture Collection, Manassas, Virginia, USA).

Experimental design

Mice were administered EGCG/LPS and let to rest for 3 days. Then, MESCs adjusted to 1 × 10⁶ stem cells in a volume of 50 µL of saline were injected IH to each mouse. Stem cells were allowed to settle in mice livers for 7 days, prior to re-administration of a single IP dose of LPS (6 mg/kg) and IG doses of EGCG (1500 mg/kg) in a volume of 100 uL for 5 days. Table 1 shows the different treatments and doses that were administered to each group of experimental animals.

Table 1.

Treatments and doses administered to mice.

Group (n = 10)	Treatment	Dose
S1	10% DMSO (vehicle) +LPS +Saline	Once daily (5 days) IG 6 mg/kg single dose IP (50 µL/animal) IH
S2	EGCG +LPS +Saline	1500 mg/kg daily dose IG (5 days) 6 mg/kg single dose IP (50 µL/animal) IH
S3	EGCG +LPS +MESCs	1500 mg/kg daily dose IG (5 days) 6 mg/kg single dose IP Single dose IH (1 × 10⁶ cells in 50 µL saline)
S4	LPS +MESCs +10% DMSO (vehicle)	6 mg/kg single dose IP Single dose IH (1 × 10⁶ cells in 50 µL saline) Once daily (5 days) IG

DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; IG: intragastric; IP: intraperitoneal; IH: intrahepatic.

Sample collection

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

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).

Relative weight of liver

After euthanization of mice, livers were dissected out and dipped in phosphate-buffered saline (PBS; pH 7.4) and blotted dry on filter paper to remove excessive saline. Each liver was weighed.

Clinical chemistry

The levels of alanine amino transferase (ALT), alkaline phosphatase (ALP), albumin to globulin ratio (A/G ratio), amylase, and total bilirubin (TB) were measured immediately after blood sampling using an automated VetScan dry chemistry analyzer with comprehensive diagnostic profiles (Abaxis, Union City, California, USA).

Animal survival

Death in each group was monitored and recorded during the course of the experiment. Mortality was calculated using Kaplan–Meier test of survival using GraphPad Prism software (La Jolla, California, USA).

Liver histopathological examination

Liver samples were kept in formalin solution (10%) for 24 h then washed in tap water for 12 h 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, Texas, USA) and eosin (EMD Chemicals, Gibbstown, New Jersey, USA). The slides were observed for lesions and analyzed using routine light microscopy.

Statistical analysis

Numerical data were analyzed by one-way analysis of variance test followed by Tukey Cramer multiple comparisons using GraphPad Prism software. A p value of less than 0.05 was considered to show a significant difference between vehicle and other groups.

MESC culture

MESCs were cultured in an undifferentiated condition in 0.1% gelatin-coated plastic dishes in Dulbecco’s Modified Eagle’s medium (Life Technologies, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies), leukemia inhibitory factor (10 ng/mL) (Millipore, Temecula, California, USA), 1% nonessential amino acids (Life Technologies), 1% penicillin–streptomycin (10,000 U/mL; Life Technologies), 0.1 mM/2β-mercaptoethanol (Life Technologies), 1% sodium pyruvate (Life Technologies), and 1% Glutamax (Life Technologies), as described previously.¹⁹ Cells maintained in 60-mm² cell culture dishes (BD Falcon, Franklin Lakes, New Jersey, USA) under 5% CO₂ and 95% humidity at 37°C were passaged every 2–3 days. ESCs at low passage numbers were frozen and stored in liquid nitrogen to replenish the cultures periodically. ESCs were frozen at 5 × 10⁶ cells per mL in FBS containing 10% dimethyl sulfoxide (DMSO).

Immunofluorescence staining

Paraffin tissue sections were heated to 60°C in an oven for 20 min, immediately deparaffinized in 100% xylene, and rehydrated through a graded ethanol series. Antigen retrieval was performed by incubating the tissue sections for 20 min in 0.01 M sodium citrate buffer, pH 6.0, in a microwave oven (500 W). After incubation with blocking buffer (0.1% Triton X-100/PBS containing 1% bis(trimethylsilyl)acetamide (BSA) and 10% horse serum) for 1 h, tissue sections were incubated with the primary antibodies (diluted in 1% BSA/10% horse serum/PBS). Following washing, bound antibodies were detected by goat anti-rabbit TexRed, Alexa Fluor and goat anti-rat TexRed, and Alexa Fluor secondary antibodies. Nuclei were stained with DAPI and slides were mounted in Fluoromount G. Evaluation was performed by Nikon fluorescence microscope (model: Nikon eclipse 90i with a DS-U3 imaging system, Nikon Metrology, Inc., Brighton, Missouri, USA) under blue, green, and red channels. Fluorometric analysis: fluorometric intensity of at least nine microscopic fields was measured for each tissue section or cultured cells using ImageJ/NIH software (National Institute of Mental Health, Bethesda, Maryland, USA).

Results

Identification of MESCs in mouse liver sections

At termination of the experiment, tissue samples were dissected out of mice, after euthanasia, and prepared for immunofluorescence staining and imaging. Four different markers of MESCs were used to identify the presence of stem cells in the livers of the treated mice. Mice in group S1 that were administered DMSO (10%, IG) and LPS (6 mg/kg IP) showed no reaction with the specific antibodies to CD 45, CD 90, Sca-1, or integrin β1. Similarly, mice in group S2 that were administered EGCG (1500 mg/kg IG) and LPS (6 mg/kg IP) showed no evidence of stem cell markers. Both groups S3 and S4 that were administered MESCs IH with and without (EGCG/LPS), respectively, showed reactions with the specific antibodies for CD45, CD 90, Sca-1, and integrin β1 (Figure 1).

Figure 1.

Immunofluorescence staining for identification of stem cells in mouse liver sections using different MESCs markers. Green (Alexa Fluor 488) = CD45, CD90, Sca-1; red (Texas Red) = integrin ß1; and blue (DAPI) = nucleus. Magnification power: 200×. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; DAPI: 4′,6-diamidino-2-phenylindole.

Histopathology of mouse livers

S1 (DMSO/LPS): Liver samples showed normal structure of liver with mild infiltration of inflammatory cells (thick blue arrows).

S2 (EGCG/LPS): Liver samples showed severe congestion in sinusoids and around the central vein (yellow arrows), with multiple areas of vacuolation that may refer to steatosis (yellow circles). Areas of multifocal necrosis, indicated by loss of cellular structure (yellow rectangles), were evident. There was a huge infiltration of inflammatory cells in livers of this group (thick blue arrows).

S3 (EGCG/LPS + MESCs): Liver samples showed some areas of vacuolation that may refer to steatosis (yellow circles), with an infiltration of inflammatory cells (thick blue arrows).

S4 (MESCs/LPS): Liver samples showed normal structure of liver with some infiltration of inflammatory cells (thick blue arrows; Figure 2, Table 2).

Table 2.

Histopathological scores for hepatic lesions.^a

	S1 (DMSO/LPS)	S2 (EGCG/LPS)	S3 (EGCG/LPS + MESCs)	S4 (MESCs/LPS)
Sinusoidal congestion	1	2.5	1	1
Hemorrhage	0	2.5	0	0
Vacuolar change	0	3	2	0
Lipidosis	0	2	1	0
Hepatic necrosis	0	4.5	0	0
Cumulative pathology	1	14.5	4	1

DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells.

^aSeverity scores: 0 = absent; 1 = minimal; 2 = mild; 3 = moderate; 4 = marked; and 5 = severe.

Figure 2.

Representative photomicrographs showing the histopathology of mouse liver samples. CV: central vein; yellow arrows: congestion; blue thick arrows: infiltration of inflammatory cells; yellow circles: vacuolation and steatosis; yellow rectangles: necrosis and loss of nuclei. Magnification power: 200×, H and E stain. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; H and E: hematoxylin and eosin.

Effect of stem cells on biochemical parameters of liver

Effect of stem cells on plasma level of ALT

Treatment of mice with EGCG 1500 mg/kg IG for 5 days after a single dose of 6 mg/kg LPS IP (positive control group; S2) caused a 42-fold increase of the plasma level of ALT compared with the control group S1. IH injection of MESCs (1 × 10⁶) to the mice before treatment with EGCG/LPS (S3) caused plasma ALT to increase to about fourfold compared with the control (S1). Treatment of mice with MESCs/LPS (S4) showed no significant elevation of plasma level of ALT compared with the control (S1). Mice that were treated with MESCs/LPS with and without EGCG (S3 and S4) showed a significant decrease in plasma level of ALT (88% and 98%, respectively) compared with EGCG/LPS group S2 (Figure 3).

Figure 3.

Plasma level of ALT of mice after injection of MESCs and LPS with or without EGCG. *p < 0.01: significantly different from the control (S1); ^ap < 0.01: significantly different from EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide, MESCs: mouse embryonic stem cells; ALT: alanine transaminase.

Effect of stem cells on plasma level of ALP

Treatment of mice with EGCG 1500 mg/kg IG for 5 days after a single dose of 6 mg/kg LPS IP (positive control group; S2) caused a threefold increase of the plasma level of ALP compared with the control group S1. IH injection of MESCs (1 × 10⁶) to the mice before treatment with EGCG/LPS (S3) showed no significant elevation in the plasma level of ALP compared with the control (S1). Treatment of mice with MESCs/LPS (S4) also showed no significant elevation of plasma level of ALP compared with the control (S1). Mice that were treated with MESCs/LPS with and without EGCG (S3, S4) showed a significant decrease in plasma level of ALP (51% and 67%, respectively) compared with EGCG/LPS group S2 (Figure 4).

Figure 4.

Plasma level of ALP of mice after injection of MESCs and LPS with or without EGCG. ^ap < 0.01: significantly different from EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; ALP: alkaline phosphatase.

Effect of stem cells on plasma level of amylase

Treatment of mice with EGCG 1500 mg/kg IG for 5 days after a single dose of 6 mg/kg LPS IP (positive control group; S2) caused a 2.75-fold increase of the plasma level of amylase compared with the control group S1. IH injection of MESCs (1 × 10⁶) in mice subjected to EGCG/LPS (S3) treatment showed twofold increase in plasma amylase compared with the control (S1). Treatment of mice with MESCs/LPS (S4) showed no significant elevation of plasma level of amylase compared with the control (S1). Mice that were treated with MESCs/LPS with and without EGCG (S3 and S4) showed a significant decrease in plasma level of amylase (22% and 70%, respectively) compared with EGCG/LPS group S2 (Figure 5).

Figure 5.

Plasma level of amylase in mice after injection of MESCs and LPS with or without EGCG. *p < 0.01: significantly different from the control (S1); ^ap < 0.01: significantly different from EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells.

Effect of stem cells on plasma A/G ratio

Treatment of mice with EGCG 1500 mg/kg IG for 5 days after a single dose of 6 mg/kg LPS IP (positive control group; S2) caused a significant reduction of the plasma A/G ratio (one-half) compared with the control group S1. All other treatments showed no significant change in plasma A/G ratio compared with the control (S1). Mice that were treated with MESCs/LPS with and without EGCG (S3 and S4) showed no significant difference in plasma A/G ratio compared with EGCG/LPS group S2 (Figure 6).

Figure 6.

Plasma A/G ratio of mice after injection of MESCs and LPS with or without EGCG. *p < 0.01: significantly different from the control (S1); ^ap < 0.01: significantly different from EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; A/G: albumin to globulin ratio.

Effect of stem cells on TB

Treatment of mice with EGCG 1500 mg/kg IG for 5 days after a single dose of 6 mg/kg LPS IP (positive control group; S2) caused a threefold increase of the level of TB compared with the control group S1. IH injection of MESCs (1 × 10⁶) to the mice before treatment with EGCG/LPS (S3) caused TB to increase more than twofold compared with the control (S1). Treatment of mice with MESCs/LPS (S4) showed no significant elevation of TB compared with the control (S1) while showed a significant reduction of TB (50%) compared with EGCG/LPS group S2 (Figure 7).

Figure 7.

Level of TB of mice after injection of MESCs and LPS with or without EGCG. *p < 0.01: significantly different from the control (S1); ^ap < 0.01: significantly different from the EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; TB: total bilirubin.

Effect of stem cells on survival of mice

All the mice in the group that was treated with EGCG 1500 mg/kg IG for 5 days after a single dose of 6 mg/kg LPS IP (positive control group; S2) died on day 4 of the treatment (100% mortality). None of the mice in the other groups showed any mortality during the period of the experiment. Animals that showed signs of severe illness (moribund condition) were considered as dead and euthanized according to IACUC guidelines (Figure 8).

Figure 8.

Percent survival of mice after injection of MESCs and LPS with or without EGCG. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells. Horizontal dots/lines indicate survival of mice. Vertical lines indicate mortality of mice.

Effect of stem cells on oxidative and inflammatory markers of liver

Effect of stem cells on Ox.LDL

Treatment of mice with EGCG 1500 mg/kg IG and LPS 6 mg/kg IP (S2) significantly overexpressed Ox.LDL in the liver of mice (about sixfold) compared with the control group S1. Pretreatment of mice with MESCs before (EGCG/LPS) (S3) also significantly overexpressed Ox.LDL in the liver of mice, but to lesser extent (about threefold) compared with the control group S1. Mice in the later group S3 showed a significant reduction of Ox.LDL expression (about 50% reduction) compared with (EGCG/LPS) group S2. Mice in the LPS/MESC-treated group S4 showed no significant change in Ox.LDL expression compared with the control group S1 and showed a significant reduction of Ox.LDL expression compared with (EGCG/LPS) group S2 (Figure 9).

Figure 9.

Expression of Ox.LDL in mouse liver after treatment with EGCG/LPS with or without pretreatment with MESCs. CV: central vein; PV: portal vein; blue (DAPI): nucleus; red (Texas Red): Ox.LDL. *p < 0.01: significantly different from the control (S1); ^ap < 0.01: significantly different from EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; DAPI: 4′,6-diamidino-2-phenylindole; Ox.LDL: oxidized low-density lipoprotein.

Effect of stem cells on CXCL16

Treatment of mice with EGCG 1500 mg/kg IG and LPS 6 mg/kg IP (S2) significantly overexpressed CXCL16 in the liver of mice (about 18-fold) compared with the control group S1. Pretreatment of mice with MESCs before (EGCG/LPS) (S3) also significantly overexpressed CXCL16 in the liver of mice, but to lesser extent (about ninefold) compared with the control group S1. Mice in the later group (S3) showed a significant reduction of CXCL16 expression (about 50% reduction) compared with (EGCG/LPS) group S2. Mice in the LPS/MESC-treated group S4 showed no significant change in CXCL16 expression compared with the control group S1 and showed a significant reduction of CXCL16 expression compared with (EGCG/LPS) group S2 (Figure 10).

Figure 10.

Expression of CXCL16 in mouse liver after treatment with EGCG/LPS with or without pretreatment with MESCs. CV: central vein; PV: portal vein; blue (DAPI): nucleus; red (Texas Red): CXCL16. *p < 0.01: significantly different from the control (S1); ^ap < 0.01: significantly different from EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; DAPI: 4′,6-diamidino-2-phenylindole.

Effect of stem cells on RAR

Treatment of mice with EGCG 1500 mg/kg IG and LPS 6 mg/kg IP (S2) significantly overexpressed RAR in the liver of mice (about 24-fold) compared with the control group S1. Pretreatment of mice with MESCs before (EGCG/LPS) (S3) showed no significant change in the expression of RAR in the liver of mice compared with the control group S1. Mice in the latter group S3 showed a significant reduction of RAR expression (about 80% reduction) compared with (EGCG/LPS) group S2. Mice in the LPS/MESC-treated group S4 showed no significant change in RAR expression compared with the control group S1 and showed a significant reduction of RAR expression compared with (EGCG/LPS) group S2 (Figure 11).

Figure 11.

Expression of RAR in mouse liver after treatment with EGCG/LPS with or without pretreatment with MESCs. CV: central vein; PV: portal vein; blue (DAPI): nucleus; red (Texas Red): RAR. *p < 0.01: significantly different from the control (S1); ^ap < 0.01: significantly different from EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; DAPI: 4′,6-diamidino-2-phenylindole; RAR: retinoic acid receptor.

Effect of stem cells on RXR

Treatment of mice with EGCG 1500 mg/kg IG and LPS 6 mg/kg IP (S2) significantly overexpressed RXR in the liver of mice (about 11-fold) compared with the control group S1. Pretreatment of mice with MESCs before (EGCG/LPS) (S3) showed no significant change in the expression of RXR in the liver of mice compared with the control group S1. Mice in the latter group S3 showed a significant reduction of RXR expression (about 90% reduction) compared with (EGCG/LPS) group S2. Mice in the LPS/MESC-treated group S4 showed no significant change in RXR expression compared with the control group S1 and showed a significant reduction of RXR expression compared with (EGCG/LPS) group S2 (Figure 12).

Figure 12.

Expression of RXR in mouse liver after treatment with EGCG/LPS with or without pretreatment with MESCs. CV: central vein; PV: portal vein; blue (DAPI): nucleus, red (Texas Red): RXR. *p < 0.01: significantly different from the control (S1); ^ap < 0.01: significantly different from EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; RXR: retinoid X receptor; DAPI: 4′,6-diamidino-2-phenylindole.

Effect of stem cells on TNFα

Treatment of mice with EGCG 1500 mg/kg IG and LPS 6 mg/kg IP (S2) significantly overexpressed TNFα in the liver of mice (about 11-fold) compared with the control group S1. Pretreatment of mice with MESCs before (EGCG/LPS) (S3) showed no significant change in the expression of TNFα in the liver of mice compared with the control group S1. Mice in the latter group S3 showed a significant reduction of TNFα expression (about 80% reduction) compared with (EGCG/LPS) group S2. Mice in the LPS/MESC-treated group S4 showed no significant change in TNFα expression compared with the control group S1 and showed a significant reduction of TNFα expression compared with (EGCG/LPS) group S2 (Figure 13).

Figure 13.

Expression of TNFα in mouse liver after treatment with EGCG/LPS with or without pretreatment with MESCs. CV: central vein; PV: portal vein; blue (DAPI): nucleus; red (Texas Red): TNFα. *p < 0.01: significantly different from the control (S1); ^ap < 0.01: significantly different from EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; TNFα:, tumor necrosis factor α; DAPI: 4′,6-diamidino-2-phenylindole.

Effect of stem cells on TGFβ1

Treatment of mice with EGCG 1500 mg/kg IG and LPS 6 mg/kg IP (S2) significantly overexpressed TGFβ1 in the liver of mice (about 11-fold) compared with the control group S1. Pretreatment of mice with MESCs before (EGCG/LPS) (S3) showed no significant change in the expression of TGFβ1 in the liver of mice compared with the control group S1. Mice in the latter group S3 showed a significant reduction of TGFβ1 expression (about 88% reduction) compared with (EGCG/LPS) group S2. Mice in the LPS/MESC-treated group S4 showed no significant change in TGFβ1 expression compared with the control group S1 and showed a significant reduction of TGFβ1 expression compared with (EGCG/LPS) group S2 (Figure 14).

Figure 14.

Expression of TGFβ1 in mouse liver after treatment with EGCG/LPS with or without pretreatment with MESCs. CV: central vein; PV: portal vein, blue (DAPI): nucleus, red (Texas Red): TGFB1. *p < 0.01: significantly different from the control (S1); ^ap < 0.01: significantly different from EGCG/LPS group S2. DMSO: dimethyl sulfoxide; EGCG: epigallocatechin-3-gallate; LPS: lipopolysaccharide; MESCs: mouse embryonic stem cells; TGFβ1: transforming growth factor β1.

Discussion

Stem cell-based therapy has become a new hope for many patients worldwide. In the current study, we tried to evaluate the efficacy of MESCs in protection against EGCG/LPS-induced hepatotoxicity in mice. For stem cells to show a beneficial effect, two elements are critical, namely, an environment of injury²⁰ and a time period to become functional.^21,22 In the current study, we initiated liver injury by administering a single dose of EGCG/LPS. After implantation of MESCs, we gave a time period of 7 days for the cells to become functional. Stem cells have been shown to get trapped in the lung when administered via the intravenous route,^23,24 therefore, we implanted MESCs directly in the liver. Delivery and presence of MESCs in the liver were further confirmed by specific immunofluorescence of stem cell markers (CD45, CD90, Sca-1, and intergrin ß1). These markers have been reported earlier.^25
–27

In the current study, the positive control group S2 showed a 100% mortality after only 4 days, while mortality was curtailed upon treatment with MESCs. This was a clear indication that stem cells may have reduced the toxicity of EGCG/LPS. The mortality data are similar to an earlier study that showed the effect of treatment of mice with high doses of alcoholic solutions of EGCG,²⁸ the study in which mortality of mice was 85%. Upon histopathology, control mice (S1) that were administered LPS (6 mg/kg, IP) and DMSO (10%, IG) and mice that were treated with MESCs (1 × 10⁶, IH) group S4 showed normal structure of hepatocytes with minimal sinusoidal congestion and minimal infiltration of inflammatory cells. Group S2 that was administered EGCG (1500 mg/kg, IG) for 5 days after a single dose of LPS (6 mg/kg, IP) showed marked sinusoidal congestion, multiple vacuolation, multifocal necrosis, and infiltration of inflammatory cells. Group S3 that was administered 1 × 10⁶ MESCs, IH prior to EGCG/LPS showed remarkably less vacuolation, congestion, and infiltration of inflammatory cells with no evidence of necrosis. It was evident from the histopathology findings that MESCs protected hepatocytes from the necrosis that occurred upon treatment with EGCG/LPS without MESCs. It was shown earlier that treatment with stem cells prevented carbon tetrachloride (CCl₄)-induced hepatic injury in rats and decreased mortality by 50%.²⁹

Acute liver injury is identified with a multifold increase of plasma ALT.³⁰ The current study showed that treatment of mice with EGCG/LPS, without pretreatment with MESCs, significantly elevated ALT (42-fold) compared with the control. Additionally, in obstructive liver injury, elevation of ALP accompanied with an elevation of both TB and amylase has been documented.³¹ In the current study, ALP, amylase, and TB showed significant elevation of about threefold compared with the control along with a significant reduction of A/G ratio (50%). It has long been known that the abnormalities of A/G ratio are seen in various hepatic and renal injuries.³² On the other hand, mice that were pretreated with MESCs prior to EGCG/LPS showed a better liver profile compared with EGCG/LPS-treated mice. The MESC-pretreated mice showed a significant reduction of ALT, ALP, and amylase (88%, 51%, and 22%, respectively) compared with the control, and an improvement in the A/G ratio and TB compared with EGCG/LPS-treated mice; however, these latter were not statistically significant. These findings may indicate that the dose of MESCs used in this study (1 × 10⁶ cells) was not sufficient to bring complete amelioration of liver injury, although some progress was observed. Higher doses of MESCs may be tried in future for better amelioration of this hepatic injury, although the side effects of MESCs could then become another concern. The pattern of liver injury in the current study was similar to what we had shown earlier.^13,14 The ameliorative effects of MESCs in the current study are similar to the those documented earlier where stem cells reversed hepatic fibrosis and modulated the biochemical parameters of liver injury induced by CCL₄ in normal and nude mice.^33
–35 Similar results were also observed in our previous study, where thymoquinone (TQ) modulated the response to EGCG/LPS-induced liver injury.³⁶ TQ has anti-inflammatory and antioxidant properties, thus it was expected that it would have curbed the pro-inflammatory effect of LPS and also have prevented oxidative stress induced by EGCG/LPS. It is difficult to comment on the mechanism by which MESCs caused modulation of hepatic injury in the current study; however, one can speculate that the presence of MESCs may have inhibited the pro-inflammatory cytokines TNFα and/or the pro-oxidant effect of EGCG, thus prevented liver damage. Due to the similarity of results obtained from the current study and those observed with TQ,³⁶ it is tempting to hypothesize that TQ may also stimulate proliferation of MSCs in the liver and hematopoietic stem cells in the bone marrow which in turn may have provided protection against EGCG/LPS-induced liver injury.

Although EGCG is known to be antioxidant, we have shown earlier that at higher concentrations, it behaves as a pro-oxidant.¹⁶ In the current study, administration of MESCs prior to EGCG/LSP significantly reduced the expression of Ox.LDL and CXCL16 (50%) compared with EGCG/LPS-administered group. Ox.LDL is well known to be a product of lipid peroxidation and considered an indirect marker of oxidative stress.³⁷ CXCL16 was shown earlier as a scavenging receptor to Ox.LDL.^38,39 CXCL16 was observed to be overly expressed in some liver diseases.⁴⁰ It is important here to point toward the role of Ox.LDL and CXCL16 in regulating migration and differentiation of stem cells. Reports have shown that expression of Ox.LDL and chemokines such as CXCL16 promotes differentiation of stem cells to repair tissue damage.^41,42 Whether expression of Ox.LDL and CXCL16 played a role in the effect of MESCs on hepatocytes in the current study is not clear.

The nuclear receptors RAR and RXR are important regulators of cell proliferation and differentiation. These receptors were reported to mediate signaling pathway of retinoids in vivo.⁴³ They play a major role in regulating bile acid, cholesterol, fatty acid, steroid, and xenobiotic metabolism and homeostasis. Hepatic stellate cells normally store retinoids, but upon liver injury, release lipid droplets of retinoic acid, thereby upregulate retinoid receptors (RAR and RXR).⁴⁴ Also, retinoic acid signaling was diminished in a case of cholestatic liver fibrosis, concomitantly with overexpression of TGFβ.⁴⁵ Our current study shows that IH inoculation of MESCs prevented expression of RAR and RXR in response to EGCG/LPS-induced injury. These findings are similar to our previous study where RAR and RXR were overly expressed in HepG2 cells upon treatment with GT/LPS in vitro.⁴⁶ Treatment with MESCs prior to EGCG/LSP significantly reduced the expression of TNFα and TGFβ, 88% and 90%, respectively, compared with EGCG/LPS-administered group. TNFα and TGFβ are known as biomarkers for liver disease complications (fibrosis and cirrhosis)^47,48 and as mediators of apoptosis.⁴⁹ The overexpression of TNFα and TGFβ in the current study may be due to the start of healing process that follows hepatocellular necrosis or apoptosis. We had earlier shown similar results with HepG2 cells upon treatment with EGCG/LPS in vitro.¹⁶

The mechanism by which MESCs ameliorated EGCG/LPS-induced hepatotoxicity in mice is not established. Earlier, human umbilical cord blood-derived cells were shown to differentiate into hepatocytes, as indicated by their secretion of albumin, and ameliorated liver injury.⁵⁰ In our study, stem cells, rather than differentiating into hepatocytes, might have elaborated certain substances that contributed to reducing the hepatic injury induced by EGCG/LPS in mice. It was reported earlier that MESCs secreted a wide array of arteriogenic cytokines and contributed to reducing liver fibrosis through paracrine mechanisms.^51,52 It is hard to imagine that a small number of stem cells (1 × 10⁶) would differentiate into hepatocytes and compensate for the severe hepatocellular damage resulting from EGCG/LPS treatment; it seems more likely that secretion of cytokine-like substances may have initiated signals to inhibit such liver damage. Further studies are needed to unveil the mechanism by which liver injury is prevented by stem cells in this model.

Conclusions

It can be concluded from this study that treatment of mice with EGCG/LPS causes hepatotoxicity. It also concluded that MESCs have a role in protection of mouse liver against EGCG/LPS-induced liver injury. Stem cell therapy may provide a future hope for patients in need for transplantation therapy. It provides easier and more economically convenient approach for therapy of those patients. Further studies are needed to clarify the mechanism(s) involved in hepatoprotection by MESCs.

Footnotes

Acknowledgments

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.

Funding

This research is partly supported by United States Department of Agriculture (USDA), grant number (58-6402-1-612).

References

Vodyanik

Smuga-Otto

. Induced pluripotent stem cell lines derived from human somatic cells. Science 2007; 318: 1917–1920.

Friedenstein

Chailakhyan

Latsinik

. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 1974; 17: 331–340.

Barry

Murphy

. Mesenchymal stem cells: clinical applications and biological characterization. Int J Biochem Cell Biol 2004; 36: 568–584.

Prockop

Brenner

Fibbe

. Defining the risks of mesenchymal stromal cell therapy. Cytotherapy 2010; 12: 576–578.

Ren

Zhang

Zhao

. Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2008; 2: 141–150.

Shi

. Mesenchymal stem cells: a new strategy for immunosuppression and tissue repair. Cell Res 2010; 20: 510–518.

Uccelli

Moretta

Pistoia

. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008; 8: 726–736.

Uccelli

Prockop

. Why should mesenchymal stem cells (MSCs) cure autoimmune diseases? Curr Opin Immunol 2010; 22: 768–774.

Ren

Chen

Dong

. Concise review: mesenchymal stem cells and translational medicine: emerging issues. Stem Cells Translpl Med 2012; 1: 51–58.

10.

Kim

Quon

MJ,

Kim

. New insights into the mechanisms of polyphenols beyond antioxidant properties; lessons from the green tea polyphenol, epigallocatechin 3-gallate. Redox Biol 2014; 2: 187–195.

11.

Molinari

Watt

Kruszyna

. Acute liver failure induced by green tea extracts: case report and review of the literature. Liver Transpl 2006; 12: 1892–1895.

12.

Gloro

Hourmand-Ollivier

Mosquet

. Fulminant hepatitis during self-medication with hydroalcoholic extract of green tea. Eur J Gastroenterol Hepatol 2005; 17: 1135–1137.

13.

Saleh

Ali

Abe

. Effect of Green tea and EGCG on Liver: In vivo and In vitro study. Journal of Clinical Toxicology 3: 5.

14.

Saleh

Ali

Abe

. Effect of green tea and its polyphenols on mouse liver (GT and Mouse Liver). Fitoterapia 2013; 90: 151–159.

15.

Saleh

Ali

Hamada

. Effect of green tea on hepatic cells under the influence of inflammatory conditions: in vitro study. Am J Pharmacol Toxicol 2013; 8: 209–217.

16.

Saleh

Ali

Abe

. Possible hepatocellular toxicity of EGCG under the influence of an inflammagen. J Appl Biomed 2014; 12: 291–299.

17.

Yamamoto

Quinn

Asari

. Differentiation of embryonic stem cells into hepatocytes: biological functions and therapeutic application. Hepatology 2003; 37: 983–993.

18.

Heo

Factor

Uren

. Hepatic precursors derived from murine embryonic stem cells contribute to regeneration of injured liver. Hepatology 2006; 44: 1478–1486.

19.

Cao

Lin

Xie

. In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery. Circulation 2006; 113: 1005–1014.

20.

Johansson

Momma

Clarke

. Identification of a neural stem cell in the adult mammalian central nervous system. Cell 1999; 96: 25–34.

21.

Duan

Catana

Meng

. Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo. Stem Cells 2007; 25: 3058–3068.

22.

Chinzei

Tanaka

Shimizu-Saito

. Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes. Hepatology 2002; 36: 22–29.

23.

Barbash

Chouraqui

Baron

. Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation 2003; 108: 863–868.

24.

Lee

Pulin

Seo

. Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell 2009; 5: 54–63.

25.

Shinohara

Avarbock

Brinster

. β1-and α6-integrin are surface markers on mouse spermatogonial stem cells. Proc Nat Acad Sci 1999; 96: 5504–5509.

26.

Asakura

Seale

Girgis-Gabardo

. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002; 159: 123–134.

27.

Herrera

Bruno

Buttiglieri

. Isolation and characterization of a stem cell population from adult human liver. Stem cells 2006; 24: 2840–2850.

28.

Lambert

Kennett

Sang

. Hepatotoxicity of high oral dose (-)-epigallocatechin-3-gallate in mice. Food Chem Toxicol 2010; 48: 409–416.

29.

Zhao

D-C

Lei

J-X

Chen

. Bone marrow-derived mesenchymal stem cells protect against experimental liver fibrosis in rats. World J Gastroenterol 2005; 11: 3431.

30.

Niemelä

Alatalo

. Biomarkers of alcohol consumption and related liver disease. Scand J Clin Lab Invest 2010; 70: 305–312.

31.

Bowlus

Gershwin

. The diagnosis of primary biliary cirrhosis. Autoimmun Rev 2014; 11: 00053–00056.

32.

Gray

Barron

. The electrophoretic analyses of the serum proteins in diseases of the liver. J Clin Invest 1943; 22: 191.

33.

Abdel Aziz

Atta

Mahfouz

. Therapeutic potential of bone marrow-derived mesenchymal stem cells on experimental liver fibrosis. Clin Biochem 2007; 40: 893–899.

34.

Banas

Teratani

Yamamoto

. IFATS collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury. Stem Cells 2008; 26: 2705–2712.

35.

Fang

Shi

Liao

. Systemic infusion of FLK1+ mesenchymal stem cells ameliorate carbon tetrachloride-induced liver fibrosis in mice. Transplantation 2004; 78: 83–88.

36.

Saleh

Ali

Wilson

. Thymoquinone protects against EGCG/LPS induced hepatotoxicity in mice. Int J Pharmacol Clin Trials 2014; 26: 1165–1174.

37.

Sakurai

Sawamura

. Stress and vascular responses: endothelial dysfunction via lectin-like oxidized low-density lipoprotein receptor-1: close relationships with oxidative stress. J Pharmacol Sci 2003; 91: 182–186.

38.

Gutwein

Abdel-Bakky

Schramme

. CXCL16 is expressed in podocytes and acts as a scavenger receptor for oxidized low-density lipoprotein. Am J Pathol 2009; 174: 2061–2072.

39.

Minami

Kume

Shimaoka

. Expression of SR-PSOX, a novel cell-surface scavenger receptor for phosphatidylserine and oxidized LDL in human atherosclerotic lesions. Arterioscler, Thromb Vasc Biol 2001; 21: 1796–1800.

40.

H-B

Gong

Y-P

Cheng

. CXCL16 participates in pathogenesis of immunological liver injury by regulating T lymphocyte infiltration in liver tissue. World J Gastroenterol 2005; 11: 4979.

41.

Sordi

Malosio

Marchesi

. Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood 2005; 106: 419–427.

42.

. CXCR4 positive bone mesenchymal stem cells migrate to human endothelial cell stimulated by ox-LDL via SDF-1α/CXCR4 signaling axis. Exp Mol Pathol 2010; 88: 250–255.

43.

Kastner

Grondona

Mark

. Genetic analysis of RXRα developmental function: convergence of RXR and RAR signaling pathways in heart and eye morphogenesis. Cell 1994; 78: 987–1003.

44.

Wang

. Chronic alcohol intake interferes with retinoid metabolism and signaling. Nutr Rev. 1999; 57: 51–59.

45.

Ohata

Lin

Satre

. Diminished retinoic acid signaling in hepatic stellate cells in cholestatic liver fibrosis. Am J Physiol-Gastrointest Liver Physiol 1997; 35: G589.

46.

Saleh

Ali

Hamada

. Effect of green tea on hepatic cells under the influence of inflammatory conditions: in vitro study. Am J Pharmacol Toxicol 2013; 8: 209.

47.

Hammam

Mahmoud

Zahran

. A possible role for TNF-alpha in coordinating inflammation and angiogenesis in chronic liver disease and hepatocellular carcinoma. Gastrointest Cancer Res 2013; 6: 107–114.

48.

. Effects of probiotics on nonalcoholic fatty liver disease: a meta-analysis. World J Gastroenterol 2013; 19: 6911–6918.

49.

Josephs

Bahjat

Fukuzuka

. Lipopolysaccharide and d-galactosamine-induced hepatic injury is mediated by TNF-α and not by Fas ligand. Am J Physiol-Regul, Integr Comp Physiol 2000; 278: R1196–R1201.

50.

Sharma

Cantz

Richter

. Human cord blood stem cells generate human cytokeratin 18-negative hepatocyte-like cells in injured mouse liver. Am J Pathol 2005; 167: 555–564.

51.

Silva

Litovsky

Assad

. Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation 2005; 111: 150–156.

52.

Kinnaird

Stabile

Burnett

. Local delivery of marrow-derived stromal cells augments collateral perfusion through paracrine mechanisms. Circulation 2004; 109: 1543–1549.

Stem cell intervention ameliorates epigallocatechin-3-gallate/lipopolysaccharide-induced hepatotoxicity in mice

Abstract

Keywords

Introduction

Materials and methods

Animals

Chemicals

Antibodies and cell lines

Experimental design

Sample collection

Relative reduction of body weight of mice

Relative weight of liver

Clinical chemistry

Animal survival

Liver histopathological examination

Statistical analysis

MESC culture

Immunofluorescence staining

Results

Identification of MESCs in mouse liver sections

Histopathology of mouse livers

Effect of stem cells on biochemical parameters of liver

Effect of stem cells on plasma level of ALT

Effect of stem cells on plasma level of ALP

Effect of stem cells on plasma level of amylase

Effect of stem cells on plasma A/G ratio

Effect of stem cells on TB

Effect of stem cells on survival of mice

Effect of stem cells on oxidative and inflammatory markers of liver

Effect of stem cells on Ox.LDL

Effect of stem cells on CXCL16

Effect of stem cells on RAR

Effect of stem cells on RXR

Effect of stem cells on TNFα

Effect of stem cells on TGFβ1

Discussion

Conclusions

Footnotes

Acknowledgments

Conflict of interest

Funding

References