探討間葉幹細胞在以N-nitrosodimethylamine誘發成肝炎、肝纖維化及肝癌之大鼠體內的生理、病理角色

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(1)國立臺灣師範大學生命科學系碩士論文. 探討間葉幹細胞在以 N-nitrosodimethylamine誘發成肝炎、肝纖維化及肝癌之大鼠體內的生理、病理角色 Pathophysologic Roles of Mesenchymal Stem Cell Therapy in N-nitrosodimethylamine-Induced Hepatic Inflammation, Fibrosis and Carcinoma in the Rat. 研究生：賴怡安 Yi-An Lai 指導教授：鄭劍廷博士 Chiang-Ting Chien, Ph.D. 賴韻如博士 Yun-Ju Lai, Ph.D.. 中華民國 106 年 8 月.

(2) 目錄 I.中文摘要 ......................................................................................... 4 II. ABSTRACT ................................................................................. 6 III. INTRODUCTION .................................................................... 10 IV. METHODS AND MATERIALS ............................................... 14 1. Animal Model ...............................................................................14 1.1 N-nitrosodiethylamine-induced Rats ..................................14 1.2 Grouping .............................................................................14 1.3 Surgical Preparation ............................................................15 2. Mesenchymal Stem Cells .............................................................15 2.1 MSC isolation .....................................................................15 2.2 Identification .......................................................................15 2.3 MSC culture ........................................................................16 2.4 L-theanine Preconditioning ................................................17 2.5 MTT Assay .........................................................................17 2.6 Wound Healing Assay .........................................................18 2.7 Medium Reactive Oxygen Species Detection ....................18 2.8 Cytokine Array....................................................................18 1.

(3) 3. Blood Biochemical Estimation .....................................................19 4. Blood Reactive Oxygen Species Detection ..................................19 5. Hematoxylin and Eosin Staining ..................................................19 6. Masson Staining ............................................................................20 7. Immunohistochemistry .................................................................20 8. Western Blot ..................................................................................21 9. In Vivo Imaging System (IVIS) ....................................................22 9.1 MSC Labeling .....................................................................23 9.2 Surgical Preparation............................................................23 9.3 Tumor Labeling ..................................................................23 10. Statistical analysis .......................................................................24 V. RESULTS................................................................................... 25 1. Purification and culture of P1 MSC..............................................25 2. L-theanine reduces free radical & promote viability ....................25 3. L-theanine preconditioning promotes MSC migration.................26 4. L-theanine promotes liver regeneration and angiogenesis ...........26 5. Liver function is significantly recovered after MSC treatment ....27 6. MSC treatment reduces ROS amount ...........................................27 2.

(4) 7. H-E staining shows the liver injury status of each group .............27 8. MSC treatment reduces fibrosis, apoptosis, but not pyroptosis and autophagy in DEN-treated livers ......................................................28 9. Western blot analysis conforms that MSC treatment inhibits apoptosis, but not pyroptosis and autophagy in DEN-treated livers 28 10. MSCs and tumor in vivo were traced..........................................29 VI. DISCUSSION ........................................................................... 30 VII. CONCLUSION ....................................................................... 33 VIII. REFERENCES ...................................................................... 34 IX. FIGURES & TABLES .............................................................. 40. 3.

(5) I.中文摘要在台灣、亞洲及南非地區肝細胞癌是最嚴重的癌症。由於肝癌無論是手術治療或非手術治療，其治療效果均與腫瘤期別有密切關係，因此早期診斷、早期治療是非常重要的。然而高達 80％的原發性肝細胞癌病人於五年內會再併發復發性肝細胞癌，其再復發的機制至今仍不十分清楚。因此對於肝癌之形成機轉、復發原因與預防或治療方式之探究是至為重要的。隨著不同時期和程度的肝損傷，過度的活性氧(reactive oxygen species, ROS) 如 O2˙- 、 H2O2 以及 NO 會造成脂質過氧化 (lipid peroxidation)、蛋白質氧化(protein oxidation)、DNA 損傷(DNA damage) 與突變產生(mutagenesis)。這些活性氧(reactive oxygen species, ROS)d 可能主要來自肝細胞之粒線體、活化的巨噬細胞 (activated macrophages,. 或稱 Kupffer cells) 與入侵之中性球 (infiltrating. neutrophils)。活性氧(reactive oxygen species, ROS)會啟動細胞核因子 nuclear factor-kappa B (NF-kB) 與 activator protein-1 (AP-1) 移位至細胞核而造成許多發炎細胞激素的釋放引起發炎。過量活性氧(reactive oxygen species, ROS)透過 Kupffer cell 內的 NADPH oxidase 或是肝細胞之 cytochrome P4502E1 (CYP2E1) 會促進肝臟內之 stellate cells 4.

(6) 活化而造成肝纖維化與肝癌。肝損傷時肝臟內許多細胞受原位細胞或發炎細胞氧化壓力增加引起 Bax/Bcl-2 family 啟動細胞凋亡 (apoptosis)、caspase 1/IL-1調節之發炎性細胞死亡(pyroptosis)或 Beclin-1/LC-3β 調節之細胞自噬 (autophagy)造成肝功能損傷進而形成肝腫瘤。間葉幹細胞(MSC)具有極佳自我更新（self-renewal）及增生（proliferation）能力，MSC 同時也具有旁分泌（paracrine）的功能，其培養基(CM)中可能含有高量的 growth factors、cytokines 及 prostaglandins。這些物質會增加細胞增生、存活及血管生成而提升組織修復。為探究 MSC 之治療效益，我們利用以 N-nitrosodiethylamine (DEN) 刺激肝發炎、硬化與腫瘤之大鼠動物模式，評估 MSC 給予對 DEN 誘發肝發炎、硬化與腫瘤之病理生理效應。研究發現靜脈給予 MSC 後，其主要匯集於受傷的肝臟組織。另外，MSC 的給予改善 DEN 引起之肝發炎、硬化與腫瘤等病理特徵，包括降低 Masson 染色之損傷指標、細胞凋亡、細胞自噬，發炎性細胞死亡表現，並降低肝功能 AST 與 ALT 之數值。綜合研究結果，MSC 有相當之潛力發展成為對肝損傷患者之細胞治療製劑。關鍵字：活性氧、肝損傷、間葉幹細胞. 5.

(7) II. Abstract Hepatocellular carcinoma (HCC) is the most severe cancer in Taiwan, Asia, and South Africa. The therapeutic strategy to treat HCC, either operative or non-operative methods, is related to the tumor stages. Therefore, an earlier diagnosis and treatment of HCC is important. However, 80% of the patients with primary HCC will be accompanied by relapsing HCC in five years. The mechanism of HCC recurrence is not clearly understood yet. Based on the information, it is necessary to investigate the pathophysiologic mechanisms of HCC formation and recurrence and to discover novel techniques and methods to prevent and cure HCC. Hepatogenesis begins in the setting of chronic liver inflammation and hepatic fibrosis, which are possibly caused by hepatotoxins. Increased oxidative stress, inflammatory cytokines and growth factors subsequently lead to hepatocyte proliferation. Prolonged cell damage by chronic inflammation is critical in cancer development. Overproduction of reactive oxygen species (ROS) including O2˙-, H2O2 and NO, can cause lipid peroxidation, protein oxidation, DNA damage and mutagenesis associated with various stages of liver injury. ROS can trigger translocation of nuclear factor-kappa B (NF-B) and activator protein-1 (AP-1) to nucleus and activation of several inflammatory cytokines and adhesion molecules that contribute to further production of ROS and consecutive cell death. The increased ROS can enhance Bax/Bcl-2 ratio, caspase 3 (CPP32) expression, and poly-(ADP-ribose)-polymerase (PARP) fragments subsequently 6.

(8) resulting in apoptotic cell death, enhance the expression of the autophagypromoting protein Beclin-1 expression, leading to autophagy and caspase 1/IL-1 mediated pyroptosis. Mesenchymal stem cells (MSC) provide excellent self-renewal and proliferation ability. In addition, MSC may release high levels of growth factors, cytokines and prostaglandins in the microenvironment or condition medium (CM) by the paracrine mechanism to enhance cell proliferation, survival and angiogenesis to repair injured tissue. To investigate the therapeutic efficiency of MSC treatment, we used N-nitrosodiethylamine (DEN) as an inducer to develop hepatocellular inflammation, fibrosis and carcinoma on rats, and evaluated the pathophysiological effects of MSC treatment on these rat models. MSC application improves DEN-induced hepatocellular inflammation, fibrosis and carcinoma. The degree of fibrosis by Masson staining was also decreased. Furthermore, the marker of apoptosis, pyroptosis, and autophagy was enhanced by DEN injury and was decreased by MSC treatment. The increased level of AST and ALT activity by DEN injury was also reduced by MSC treatment. In conclusion, MSC has a considerable potential to be a cell therapy for patients with liver injury and HCC.. Key Words：reactive oxygen species, liver injury, mesenchymal stem cells. 7.

(9) Abbrevations. reactive oxygen species. ROS. mesenchymal stem cells. MSC. MSC in hypoxic culture. HMSC. N-nitrosodiethylamine. DEN. Hepatocellular carcinoma. HCC. condition medium. CM. complete condition medium. CCM. interferon gamma. IFNg. vascular endothelial growth factor. VEGF. stromal cell-derived factor 1. SDF-1. hepatocyte growth factor. HGF. aspartate aminotransferase. AST. alanine aminotransferase. ALT. γ-glutamyl transpeptidase. γ-GT. hematoxylin-and-eosin stain. H-E stain. nuclear factor-kappa B. NF-B. activator protein-1. AP-1. poly-(ADP-ribose)-polymerase. PARP. acute kidney injury. AKI. Interleukin-1 beta. IL-1β. phosphate-buffered saline. PBS 8.

(10) fetal bovine serum. FBS. dimethyl sulfoxide. DMSO. alpha-tocopherol. vitamin E. epigallocatechin gallate. EGCg. 3-(4,5-Dimethylthiazol-2-yl)-2,5-. MTT. diphenyltetrazolium bromide Luminol-enhanced. CL. chemiluminescence Fluorescein Isothiocyanate. FITC. in vivo imaging system. IVIS. 2-deoxyglucose. 2-DG. 1,1'-Dioctadecyl-3,3,3',3'-. DiR. tetramethylindotricarbocyanine iodide. 9.

(11) III. Introduction Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the most common type of liver cancer, representing 83% of all cases. The 5-year relative survival rate is approximately 7% and become a leading cause of cancer-related mortality worldwide. It represents the third cause of cancer-related deaths and the first cause of death amongst cirrhotic patients. Hepatitis viral infection, food additives, alcohol, fungal toxins (aflatoxins), toxic industrial chemicals, air and water pollutants are the major risk factors of HCC. 1, 2. . N-nitrosodimethylamine (DEN) is a. potent hepatocarcinogenic nitrosamine presents in tobacco smoke, water, cheddar cheese, cured and fried meals, occupational settings, cosmetics, agricultural chemicals and pharmaceutical agents. 3-5. . DEN produces the. pro-mutagenic products, O6-ethyldeoxyguanosine and O4 and O6ethyldeoxythymidine in livers leading to HCC. 6-8. . These toxic products. produced by DEN may evoke oxidative stress and inflammation in livers and consequently lead to HCC. However, the detailed progress and mechanism for DEN-induced a simulated HCC in rats is not clearly defined. Overproduction of reactive oxygen species (ROS) including O2˙-, H2O2 and NO, can cause lipid peroxidation, protein oxidation, DNA damage and mutagenesis associated with various stages of liver injury 9-12. The main sources of ROS may derive from the mitochondria of hepatocytes, the activated macrophages (Kupffer cells), and the infiltrating neutrophils. 9, 11. . ROS can trigger translocation of nuclear factor-kappa B. (NF-B) and activator protein-1 (AP-1) to nucleus. 9. and activation of 10.

(12) several inflammatory cytokines and adhesion molecules that contribute to further production of ROS and consecutive cell death 9. The increased ROS can enhance Bax/Bcl-2 ratio, caspase 3 (CPP32) expression, and poly(ADP-ribose)-polymerase (PARP) fragments subsequently resulting in apoptotic cell death. 13, 14. and enhance the expression of the autophagy-. promoting protein Beclin-1 expression leading to autophagy. 15-17. . ROS. accumulation leading to subsequent autophagy and apoptosis has been reported in the HCC. 10. . However, it is uncertain that DEN -induced. oxidative stress contributing to apoptosis and autophagy formation in the livers. Furthermore, a new type of programmed cell death, pyroptosis, that is recognized with the caspase 1 activation and IL-1 secretion 18, 19 may be involved in the DEN-induced liver injury. Stem cells have the potential of self-renewal and the ability to differentiate into many types of cells 1-3. Bone marrow-derived stem cells can mobilize into the ischemic injured kidneys and contribute to renal parenchymal turnover and regeneration marrow-derived. mesenchymal. stem. 4-6. . Administration of bone. cells. (BMMSC). has. been. demonstrated to markedly accelerate tubular proliferation, repair the kidney and improve renal function 7-11. Treatment with MSC seems to favor functional and morphological recovery after AKI. Nevertheless, these bone-marrow-derived cells only account for a small percentage of cells of regenerative capacity in the injured liver. Surviving tubular epithelial cells are the predominant cellular components involved in repopulation after ischemic injury. 12. . Previous studies pointed that the condition medium. derived from the MSC culture (CM-MSC) has been reported to serve 11.

(13) multiple positive functions in tissue regeneration. MSC actively secrete several essential growth factors including SDF-1, VEGF-A, and HGF 13-16. Such secretion of these growth factors implies that MSC or CM-MSC can aid in regeneration and repair of the injured tissue by promoting angiogenesis,. preventing. apoptosis,. and. stimulating. native. cell. proliferation. Evidence showed MSC with pre-treatment such as lipopolysaccharide (LPS) treatment or hypoxia conditioning environment can cause significant changes in cytokine production but not in migratory capacity. 20. . Some. studies had pre-incubated human mesenchymal stem cells with EGCg (epigallocatechin gallate, extract from green tea) for preventing oxidative stress-induced cellular senescence and the EGCg pre-treatment did reduce cellular senescence in H2O2 treated hMSCs. 21. . Another study incubated. different concentration of EGCg with MSC for 2 hours and found out EGCg-preconditioning inhibited MSC differentiation to adipogenic lineage. 22. . 10 μM of L-theanine was reported as a supplementation of. culture media increased the expression of self-renewal regulatory gene Oct4. 23. . These evidences bring forward a novel cell preconditioning. method, not merely supplied treatments with oral administration which can possibly avoid interaction between different treatments. Previous study showed EGCg had immunomodulatory effects via T regulatory cells in murine model 24. Other studies indicated tea extract can be used in diabetic-uremia treatment. 25. . L-theanine is a component. discovered in tea leaves. It is an amino acid which is structural similar to one of the neurotransmitter, glutamate. 26. . Its oxidative ability and 12.

(14) preventive effects on cognitive dysfunction are well recognized. Several studies showed that L-theanine had protective effects on carbon tetrachloride-induced acute liver injury in mice. 27. and also prevented. alcoholic liver injury through enhancing the antioxidant capability of hepatocytes 28. In present studies, we examined whether administration of MSC, or green tea extract-preconditioned MSC could protect liver from DEN induced liver injury in rats. We also investigated the possible therapeutic effects of MSCs on oxidative stress, infiltrated leukocytes, liver function, status of apoptosis, pyroptosis, and autophagy associated with DENinduced hepatic injury.. 13.

(15) IV. Methods and Materials 1. Animal Model 1.1 N-nitrosodiethylamine-induced Rats Male Wistar rats (200 to 250g) at the age of 7 weeks were provided by BioLASCO Taiwan Co., Ltd. (Ilan, Taiwan) and housed at the Experimental Animal Center, National Taiwan Normal University, at a constant temperature with a consistent light cycle (light from 07:00 to 18:00). Food and water were provided ad libitum. All surgical and experimental procedures were approved by National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee and were in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997). Different liver dysfunctions are induced by a toxic reagent, N-nitrosodiethylamine, DEN (Sigma Aldrich, St. Louis, MO), in 2, 4, and 8 weeks. 1.2 Grouping Sixty animals were randomly divided into ten groups, group 1 : control group (n=6); group 2 : 2 weeks of DEN treatment (n=6); group 3 : 4 weeks of DEN treatment (n=6); group 4 : 8 weeks of DEN treatment (n=6); group 5 : 2 weeks of DEN treatment and MSC (n=6); group 6 : 4 weeks of DEN treatment and MSC (n=6); group 7 : 8 weeks of DEN treatment and MSC (n=6) ; group 8 : 2 weeks of DEN treatment and Ltheanine preconditioned MSC (n=6); group 9 : 4 weeks of DEN treatment and L-theanine preconditioned MSC (n=6); group 10 : 8 weeks of DEN treatment and L-theanine preconditioned MSC (n=6). 14.

(16) 1.3 Surgical Preparation DEN (Sigma Aldrich, St. Louis, MO) were given as an inducer of liver injury at 500 ppm in drinking water throughout the entire experiment (2, 4, 8 weeks). MSC were isolated to the first passage, P1, and administered intravenously from 1×106 to 5×106. The rats were sacrificed 4 weeks after MSC treatment. 2. Mesenchymal Stem Cells 2.1 MSC isolation Male Wistar rats (200 to 250g) at the age of 7 weeks were provided by BioLASCO Taiwan Co., Ltd. (Ilan, Taiwan). Rat MSCs were isolated from adipocytes and all procedures were approved by National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee. Adipocytes obtained from rats were washed with phosphate-buffered saline (PBS) with 1% penicillin/ streptomycin and minced into small pieces around 1x1 cm2. Microvascular tissue which is adjacent on adipocytes were carefully removed and the adipose tissue were enzymatically dissociated by adding isometric 0.15% collagenase type I (Gibco, now part of Thermo Fisher Scientific, Waltham, MA, USA) for 15 minutes at incubator supplemented with 5% CO2. Adipose tissue dissolved in the collagenase type I enzyme solution was centrifuged at 15000 rpm for 5 minutes, the cell pellet was resuspended and transfer to a T75 culture flask. Cells attached at the bottom of the flask were observed after 24 hours of culturing. 2.2 Identification To. detect. the. surface. markers. of. MSC,. we. performed 15.

(17) immunocytochemistry staining followed flow cytometry analysis. MSCs (from passage 1 to passage 4) were seeded into a T75 culture flask for 24 hours and then were resuspended to single-cell suspension followed by fixation with 4% paraformaldehyde for 30 minutes. For flow cytometry detection, the MSCs were incubated with 2 μL of FITC Mouse Anti-Rat CD90 (positive marker, BD Pharmingen, La Jolla, CA) or FITC Mouse Anti-Rat CD45 (negative marker, BD Pharmingen, La Jolla, CA) antibodies. After incubating for 30 minutes, MSCs were washed with PBS three times and analyzed by flow cytometry (BD FACSAria III; BD Biosciences, San Jose, CA, USA). 2.3 MSC culture Primary MSCs were obtained from Wistar rats, seeded into a T75 culture flask, and grown in complete culture medium (CCM: α-MEM (αminimal essential medium; Gibco-BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine). Media were changed every 2 days. The incubator environment is a gas mixture composed of 94% N2, 5% CO2, and 1% O2, combining with two air sensors, one for CO2 and the other for O2. The O2 concentration is achieved and maintained utilizing delivery of nitrogen gas (N2) generated from a liquid nitrogen tank or a tank containing pure N2. If O2 percentage rises above the desired level, N2 gas is automatically injected into the system to displace the excess O 2. MSCs were cultured in condition medium for 48 hours, and the condition medium was filtered by a 0.22 μm filter (Millipore, Billerica, MA, USA) before culturing. The condition medium is composed of α-MEM (α-minimal 16.

(18) essential medium; Gibco-BRL, Gaithersburg, MD), supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. 2.4 L-theanine Preconditioning L-theanine is a component discovered in tea leaves. It is an amino acid structurally similar to one of the neurotransmitter, glutamate. 26. . Its. oxidative ability and preventive effects on cognitive dysfunction are well recognized 29. To investigate whether L-theanine could enforce the homing effects of MSCs, we preconditioned MSCs with L-theanine (Taiyo Kagaki CO LTD, Japan) for 2 hours and then changed back to the regular medium for the following 24 hours. The L-theanine solution was filtered by a 0.22 μm filter (Millipore, Billerica, MA, USA) before precondition. Free radical counts of L-theanine preconditioned medium was investigated compared with normal condition medium. 2.5 MTT Assay The. MTT. (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium. bromide) (Sigma, St. Louis, MO, USA) assay was utilized as a method to determine the cell vitality. It is hydrophilic and can be transformed to purple hydrophobic formazan salt through reacting with metabolically viable cells. MSCs were incubated in different concentration of L-theanine for 2 hours in the incubator with MTT reagent, and the reagent was removed and replaced with dimethyl sulfoxide, DMSO (Sigma, St. Louis, MO, USA). The optical density value was determined and read by an ELISA reader (MRX Microplate Reader, Dynex Technologies, Inc., Chantilly, VA, USA) at 570 nm 30. 17.

(19) 2.6 Wound Healing Assay MSCs were seeded in a 6-well plate and cultured overnight. An artificial wound was created by disposable 10 μL pipette tips. Detaching MSCs were washed out by serum free condition medium three times after scratched and the attached cells were cultured with serum free condition medium. The MSC migration were imaged under an inverted microscope and photographed at the time 0 and 24 hours of culturing. 2.7 Medium Reactive Oxygen Species Detection To investigate the ROS counts in fresh medium, condition medium, and L-theanine preconditioned medium, we collected these media after 48 hours incubation at the 37C and 5% CO2, and then measured the ROS levels by the Chemiluminescence Analyzing System (Model CLA-ID3; Tohoku Electronic Inn Co., Sendai, Japan) detecting Luminol-enhanced chemiluminescence (CL) signal emitted from the sample surface continuously.To investigate the ROS account in fresh medium, condition medium, and L-theanine preconditioned medium, we collected media which were incubated for 48 hours and measured them by the Chemiluminescence Analyzing System (Model CLA-ID3; Tohoku Electronic Inn Co., Sendai, Japan) detecting Luminol-enhanced chemiluminescence (CL) signal emitted from the sample surface continuously. 2.8 Cytokine Array To analyze the difference between MSCs and L-theanine preconditioned MSCs, two cytokines, interferon gamma (IFNg) and vascular endothelial growth factor (VEGF), were detected by cytokine 18.

(20) array (Glass Slide-based Antibody Arrays L-Series, RayBio, GA, USA) 3. Blood Biochemical Estimation Whole blood (2 ml) were collected after sacrificed, transferred to an EDTA-administered tube, and then spun in a centrifuge at 12,000 rpm for 15 min. The serum were stored at -80°C until analysis. The serum activity of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and γ-glutamyl transpeptidase (γ-GT) were determined by chemistry analyzer (TOSHIBA TBA-40FR, Diamond Diagnostics, MA, USA). 4. Blood Reactive Oxygen Species Detection We presumed liver dysfunction can cause overproduction of reactive oxygen species (ROS) including O2˙-, H2O2 and NO, leading to lipid peroxidation, protein oxidation, DNA damage and mutagenesis. Thus, the ROS generation in response to DEN injury were measured from whole blood. Whole blood were collected from Wistar rats in each group and were measured by the Chemiluminescence Analyzing System as previous mentioned in method 2.8. 5. Hematoxylin and Eosin Staining The histologic features were assessed and scored for inflammation (portal and lobular) by hematoxylin and eosin staining as described below. We de-waxed and hydrated livers in paraffin sections. After staining with hematoxylin for 5 minutes, the slides were washed in tap water for another 5 minutes, and immersed in eosin for 30 seconds. After washing in tap water for 1 minutes, they were dehydrated by xylene. The histological changes were observed after hematoxylin and eosin (H-E) staining. 19.

(21) 6. Masson Staining The histologic features were assessed and scored for fibrosis by Masson trichrome staining. The grade of lobular inflammation was scored as 0 = no foci, 1 = <2 foci/200x field, 2 = 2-4 foci/200x field, and 3 = >4 foci/200x field. Fibrosis was scored as 0 = none, 1 = zone 3 perisinusoidal fibrosis or portal/periportal, 2 = perisinusoidal and portal/periportal fibrosis, 3 = bridging fibrosis, and 4 = cirrhosis 31. 7. Immunohistochemistry We suggest that the high levels of ROS might promote hepatic accumulation of leukocytes, nitrated protein and lipid peroxides. Accordingly, we immunostained apoptosis 9, autophagy 17 and pyroptosis markers in the paraffin-embedded sections of liver tissues. For these oxidative injury measurement, the rats (n=6 in each group) were sacrificed at the end of experiment. A 18-gauge needle connected to an infusion pump (Infors AG, CH-4103, Bottmingen, Switzerland) was inserted into the portal vein. The livers were perfused with 37C Hanks' balanced salt solution (flow rate, 10 ml/min; pH 7.4), and the perfusate was allowed to drain from the inferior vena cava. The livers were removed and fixed in 10% formalin for immunostaining. Hepatic sections were deparaffinized in xylene for 10 minutes and drenched in ethanol for rehydrated at room temperature, followed by immunohistochemically staining to present in vivo markers of apoptosis, pyroptosis, and autophagy. For apoptosis marker staining, primary antibodies included rabbit anti-Bax (1:200, Cell Signaling Technology, Denver, MA, USA), rabbit 20.

(22) anti Bcl-2 (1:200, Proteintech, Chicago, IL, USA) and rabbit anti-caspase3 (1:200, Chemicon, CA, USA). For pyroptosis marker staining, primary antibodies included rabbit anti-caspase 1 (1:200, Promega, Madison, WI, USA), and mouse anti Interleukin-1 beta (IL-1β, 1:1000; Cell Signaling Technology, Denver, MA, USA). For autophagy marker staining, primary antibodies included rabbit anti Beclin-1 (1:200; Proteintech, Chicago, IL, USA), rabbit anti-LC-3β (1:200, Chemicon, CA, USA). The percentage of protein expression were calculated as a stained area/total area × 100% and analyzed by Image J software. Primary antibody was applied overnight at 4°C, and the tissue sections were washed with phosphate buffer saline tween-20 (PBST) three times before incubating with secondary antibodies for 1 hour at room temperature. The slides were applied by Dako Liquid diaminobenzene, DAB (ImmPACT DAB Peroxidase Substrate; Vector, Burlingame, California, USA) for 1-5 minutes, and washed with double-distilled water (ddH2O), then immersed with hematoxylene for 1-3 minutes. The slides were dehydrated in ethanol series and were mounted in mounting medium (Leica, Wetzlar, Germany). 8. Western Blot The liver protein concentration were determined by a BioRad Protein Assay (BioRad Laboratories, Hercules, CA, USA). 20 g of protein were electrophoresed as described below. The expression of Bax, Bcl-2, caspase 3, caspase 1, IL-1β, Beclin-1 and LC-3β in liver tissue were evaluated by 21.

(23) Western immunoblotting and densitometry as described previously. 19. .. Briefly, the total proteins were homogenized with a prechilled mortar and pestle in extraction buffer, which consists of 10 mM Tris-HCl (pH 7.6), 140 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, 2% -mercaptoethanol, 10 g/ml pepstatin A, and 10 g/ml aprotinin. The mixtures were homogenized completely by vortexing and kept at 4°C for 30 min. The homogenate were then centrifuged at 12,000 rpm for 12 min at 4°C, the supernatant were collected, and the protein concentrations were determined by BioRad Protein Assay (BioRad Laboratories). Antibodies raised against the polyclonal rabbit anti-rat Bax (Chemicon, CA, USA), polyclonal rabbit anti-rat Bcl-2 (Biovision), caspase 3 (Chemicon, CA, USA), rabbit anti-LC-3β (Chemicon, CA, USA), rabbit anti-caspase 1 Promega, Madison, WI, USA), mouse antiInterleukin-1 beta (IL-1β, Cell Signaling Technology, Denver, MA, USA), rabbit anti-Beclin-1 (Proteintech, Chicago, IL, USA) and monoclonal mouse anti-mouse -actin (Sigma, Saint Louis, MI, USA) were used at 1:400. All of these antibodies cross-react with the respective rat antigens. Proteins on SDS-PAGE gels were transferred to nitrocellulose filters and stained as described. The density of the band with the appropriate molecular mass were determined semi-quantitatively by densitometry using Image J software (Research Services Branch of the National Institute of Mental Health, MD, USA). 9. In Vivo Imaging System (IVIS) The existence of DEN-induced tumor and the distribution of the 22.

(24) injected MSCs in vivo is essential to be traced. To support our hypothesis that MSCs play a significant role to ameliorate DEN-induced liver dysfunction, we utilized the Caliper IVIS system (Xenogen IVIS Imaging System 200 Series) to trace MSCs and tumor in vivo. 9.1 MSC Labeling 1×106 MSCs at passage 1 were labeled with DiR reagent (D12731, Invitrogen, Simon Fisher Scientific Inc., USA) for 30 minutes before intravenous transplantation. The distribution of MSCs in rats were traced in vivo by IVIS 2 hours after intravenous transplantation. 9.2 Surgical Preparation For preparation of MSCs, 1×106 MSCs were seeded into a T75 culture flask with condition medium for 24 hours, and the MSCs were collected in a centrifugal tube by 0.5% trypsin (Lonza Walkersville, Inc., USA). After 5 minutes centrifuged at 15,000 rpm, MSCs were labeled with DiR and incubated for 30 minutes then intravenously transplant immediately. Each rat was injected 1mL of condition medium containing 1×106 MSCs. The rats were anaesthetized and settled into the chamber of IVIS system. The control group was injected with 1mL of condition medium. 9.3 Tumor Labeling To identify the existence and distribution of tumor in vivo, the control rats and the DEN treatment rats were labeled with tumor marker, 2deoxyglucose (2-DG). Most of the cancer cells are well-established by the characteristic of elevating rate of metabolism; while 2-DG is a glucose analog which utilizes the GLUT transporters to enter cells, it is a proper reagent to trace tumor in vivo. In this experiment, 2-DG was intravenously 23.

(25) transplanted into rats 5 minutes before being settled into the IVIS chamber. 10. Statistical analysis All values were expressed as mean ± standard error mean (SEM). Differences between groups were evaluated by paired t-test. One-wayANOVA analysis of variance were used for establishing differences between groups with same treatment but different timepoints. Intergroup comparisons were made by Duncan's multiple-range test. Differences were regarded as significant if p < 0.05 is obtained.. 24.

(26) V. Results 1. Purification and culture of P1 MSC The MSCs were isolated from adipocytes as described in materials and methods. The morphology of MSCs are shuttle-shaped as shown in the Figure 1. To confirm and identify the MSCs, cells isolated from adipocytes were stained with anti-CD90 and CD45 antibodies after 48 hours culturing, and detected by flow cytometry. CD90 was used as positive surface marker for MSC and CD45 was used as a negative marker. CD90 was highly expressed in our isolated cells, while CD45 was merely presented. We further investigated the expression levels of CD90 marker from passage 1 to passage 4, and found that passage 1 cells expressed the highest level of CD90 than other generations (94.9% positive in P1, and was followed by P2-93%, P3-79.4%, P4-69.9 %). Therefore, we used the cells from passage 1 for the rest of experiments. 2. L-theanine reduces free radical & promote viability There were various reports indicated that tea extracts have biological and pharmacological activities such as antioxidation 32, anti-carcinogenesis 33, 34. , anti-mutagenesis. 35. , and anti-inflammation. 36. . L-theanine is a free. amino acid in tea extracts. It was reported to act as a neurotransmitter in the brain and has a relaxation-inducing effect in humans 37-40. According to previous evidences, we presumed that the ingredients in tea extracts such as EGCg and L-theanine may strengthen the therapeutic effects of MSCs on damaged tissue. In order to improve the therapeutic effects of MSCs, we first pretreated the MSCs with EGCg and L-theanine. The level of free 25.

(27) radicals of fresh medium, MSC-conditioned medium followed by measuring the free radical levels of EGCg-pretreated MSC-conditioned and. L-theanine-pretreated. MSC-. conditioned. medium. by. Chemiluminescence Analyzing System. We observed the significant reduction of free radical levels in all of the MSC-conditioned medium (Figure 2A). Moreover, the viability of MSC cells was improved by EGCg or L-theanine treatments regardless the treatment concentrations (Figure 2B). We used 10 µM as a moderate concentration of L-theanine pretreatment in the following experiments. 3. L-theanine preconditioning promotes MSC migration To evaluate the effects of L-theanine on the migration of MSCs, the wound healing assay were performed and the migration situation of MSCs in different concentration of L-theanine precondition were analyzed. It is obviously that L-theanine preconditioned MSCs migrated faster than the control group (Figure 3). 4. L-theanine promotes liver regeneration and angiogenesis The MSC-conditioned medium and L-theanine preconditioned medium were collected and analyzed for the secretion of cytokines. The expression of IFNg and VEGF were shown in Figure 4. IFNg, angiotensinogen, and caspase 12 are markers which highly compatible to liver regeneration 41, 42. In previous study of suppression on hepatic stellate cell activity, antioxidant such as alpha-tocopherol (vitamin E), γ-interferon, and hepatocyte growth factor (HGF) all had the inhibit ability. 43. .We. observed that 10T group (10 μM L-theanine preconditioned MSCs) showed a higher expression of IFNg then the MSCs only control group 26.

(28) (MSC-conditioned medium) and medium only control. VEGF plays a role on angiogenesis, and its expression revealed about 20 times higher in 10T group than the control group (Figure 4). 5. Liver function is significantly recovered after MSC treatment To investigate the therapeutic effects of MSC in vivo, we generated a DEN-induced liver-injury rat model. The experimental flow chart was presented in Figure 5. The liver tissues were collected and the appearances were observed (Figure 6). The serum activity of liver function indicators (AST, ALT, and γ-GT) were determined by a chemistry analyzer. Liver markers were significantly increased in DEN-groups as the time prolonged after DEN treatment, and MSC treatments significantly decreased these markers, except for γ-GT (Figure 7). 6. MSC treatment reduces ROS amount To further evaluate the MSC effects on ROS production, we measured the ROS levels in the rat whole blood. Results indicated that the levels of ROS augmented as the time progression after DEN treatment. After MSC treatments, the levels of ROS were decreased compared to the DEN only, not in MSC treatment groups (Figure 7D). 7. H-E staining shows the liver injury status of each group To reveal the pathologic evidences of MSC therapeutic effects, all liver tissues were collected and subjected to hematoxylin-and-eosin (HE) staining (Figure 8A). The increased shattered tissue was noted in the DENinduced groups but not in sham control groups. Oppositely, the shattered situation decreased in MSC treatment groups. H-E staining of portal triad was also displayed in Figure 8B. A portal triad, as known as portal field, 27.

(29) portal area, or portal tract, is a distinctive arrangement in liver, consisting proper hepatic artery, hepatic portal vein, and small bile ductules of cuboidal epithelium. 8. MSC treatment reduces fibrosis, apoptosis, but not pyroptosis and autophagy in DEN-treated livers To explore whether DEN affected hepatic fibrosis, we investigated the fibrosis status using Masson staining for connective tissues (Figure 8C). To confirm the DEN-induced injury stages, we further stained the tumor marker, alpha-fetoprotein (AFP) (Figure 8D). To investigate how did DEN influence liver injury, the apoptosis, autophagy, and pyroptosis markers were also detected, including the following biomarkers, Bax, Bcl-2, caspase 3, Beclin-1, LC-3β, caspase 1, and IL-1β. The results showed a significant inhibition of apoptosis in MSC treatment groups and L-theanine preconditioned groups after DEN-induced liver injury. However, in autophagy and pyroptosis, there were no significant changes observed in both MSC treatment groups and L-theanine preconditioned groups. 9. Western blot analysis conforms that MSC treatment inhibits apoptosis, but not pyroptosis and autophagy in DEN-treated livers To confirm the pathology of IHC (immunohistochemistry) results, hepatic levels of apoptosis-related proteins (Figures 9, 10 and 11), pyroptosis-related proteins (Figures 12 and 13), and autophagy-related proteins (Figures 14 and 15) were determined by Western blot analysis. The results showed marked increases in the expression of Bax and caspase 3 in the DEN-induced groups, while the expression levels of Bcl-2 were decreased. These observations were absent in the MSC treatment groups, 28.

(30) which indicate that MSCs have the function to inhibit apoptotic formation. There were no significant differences for caspase-1 and IL-1β expressions in each group. The expression of autophagy-related proteins Beclin-1 and LC-3β was significantly elevated in DEN-induced groups. However, there were no significant effects on Beclin-1 and LC-3β expression in MSC treatment groups and L-theanine preconditioned MSC groups. 10. MSCs and tumor in vivo were traced Caliper IVIS system is a proper method to trace the MSCs delivery and their interactions with tumors in vivo. We used 2-DG as a tumor marker to detect the existing of tumors in DEN-induced rats (Figure 16A). Indicated in Figure 16B, control rats and DEN-induced rats were treated with DiR fluorescence-labeled MSCs 2 hours before photographed by IVIS. DiR is a lipophilic, near-infrared fluorescent cyanine dye. Results showed that the majority distribution of 2-DG in 8-week of DEN-treated rats were in liver under IVIS detection (Figure 16A). The distribution of MSCs 2 hours after intravenous transplantation showed majority in the bladder but some focused in liver compared to no DEN-damaged group (Figure 16B). These results indicate that DEN-induced liver dysfunction triggers liver to release messages and elevates MSCs homing effects to impaired tissues.. 29.

(31) VI. Discussion Stem cell therapy has become a popularly proposed new strategy nowadays since these cells provide highly potential for all different diseases due to their excellent self-renewal and differentiation ability. Being able to be isolated and purified from bone marrow, placenta, adipose tissue and so on, MSCs are easier to acquire and less ethical concerns compared to other types of stem cells. MSCs also have abilities of antiapoptosis, angiogenesis, tissue repair promotion, anti-inflammation, growth factor production, immunosuppression, and nerve protection. 44, 45. .. Furthermore, previous evidence has regarded that stem cell therapy is a potential therapeutic approach for liver failure 46, 47 . Green tea is a well-known antioxidant ingredient existing the ability to prevent oxidant-induced cell damage 48. Lots of studies also demonstrated that the extracts of green tea inhibit tumor formation and growth in lung, liver, pancreas, esophagus, duodenum and intestine. 4-6, 45, 48. . We. investigated if there is a better therapeutic effect on liver injury by combining mesenchymal stem cells and the green tea ingredients. Instead of oral administration of green tea, we pretreated MSCs with L-theanine, one of the major active principles in green tea extracts, for 2 hours. As the results of morphology and H-E staining showed, livers in DEN-induced liver injury groups developed worse condition than the one in the control group, and those in MSC-treatment groups slightly reversed liver damage. However, the reversal situation of the livers in the L-theanine preconditioned-MSC-treatment groups, were not significantly better than 30.

(32) those in the MSC-only treatment groups. Moreover, both the levels of ROS and liver function markers indicate that 1. DEN-induced liver injury in rat is an appropriate animal model to study liver malignant diseases, and 2. the MSC-or L-theanine preconditioned-MSC-treatment do help to control and even further to reverse the hepatic dysfunction status. For the active principles of green tea extracts, we not only investigated the effects of 10 µM of L-theanine but also 10 µM of EGCg on the levels of ROS and MSC migration ability. EGCg is one of the active ingredients in green tea. It is recognized to exert protective effects against bladder hyperactivity. 11,. 49. , cancer. 50. , lipogenesis. atherosclerosis 41, and acute liver injury. 49. 51. , inflammation. 41, 50. ,. . Although both EGCg and L-. theanine had good effects on reducing the ROS level (Figure 2A), only Ltheanine induced better migration ability of MSCs in wound healing assay (Figure 3). Therefore, we used 10 µM of L-theanine as the preconditioning ingredient for all other experiments. In addition, the morphology of MSC-treatment groups had better liver appearance compared to DEN-treated group. However, in 8 weeks of DEN treatment with L-theanine preconditioned-MSC group, the recover status did not show as good as in the 8 weeks of DEN treatment with MSC only. This result may be explained by the results of cytokine assay in which we observed the higher IFNg and VEGF expression (Figure 4). Comparing the outcome of IHC staining and Western blot analysis, it is obviously to know DEN and MSC treatment matter to hepatic apoptosis and pyroptosis progress in this experiment, both MSC treatment and Ltheanine preconditioned MSC have significantly reduce the expression of 31.

(33) apoptosis-related proteins. A long-term tracking of MSC in vivo was used by a commercial lipophilic membrane dye, DiR. DiR can be used to label cells without any effect on their homing or proliferation 42, 52.. 32.

(34) VII. Conclusion We investigated the therapeutic effects of MSCs and L-theanine preconditioned-MSCs on the DEN-induced liver injury in a rat model. MSC therapy attenuated DEN-induced hepatic oxidative stress, inflammation, and fibrosis. Both MSC treatment groups and L-theanine preconditioned groups showed a protection effects on hepatic injury and the liver dysfunction via inhibiting fibrosis and apoptosis, but not affecting autophagy and pyroptosis pathways. However, L-theanine preconditioned MSCs do not reveal a better treatment effects than the MSCs.. 33.

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(41) IX. Figures & Tables. Figure 1. Characterization of adipose tissue-isolated MSCs. Primary rat MSCs isolated from adipose tissue were stained with (a) a FITC-conjugated anti-rat CD90 monoclonal antibody or (b) a FITCconjugated anti-rat CD45 monoclonal antibody. The percentage of CD90 positive cells in different passages of (c) primary MSC cultures. (d) In passage 1 of rat MSCs, to the percentage of CD90 positive cells were the highest one among the first four passages of primary cultured MSCs.. 40.

(42) (a). (b). Figure 2. ROS counts decrease in MSC conditioned medium pretreated with EGCg or L-theanine. (a) ROS counts in MSC-conditioned medium, EGCg-preconditioned medium and L-theanine-preconditioned medium were all significantly decreased compared to medium only. (b) Cell viability in different concentrations of L-theanine of MSC-conditioned medium was represented as the mean ± SEM. *p<0.05 versus control group. One-wayANOVA followed by student’s t-test. ROS counts were significantly decreased in MSC-conditioned medium. 41.

(43) Figure 3. L-theanine promotes MSC migration. (a) MSCs were pretreated with EGCg or L-theanine and the wounds were created with a 10 μL tip. The migration situations of MSCs were evaluated after 24 hours of culturing. L-theanine preconditioned-MSCs migrated faster than other two groups. (b) Quantitative analysis of the level of migrated MSCs. Values are presented as mean ± SD. *p<0.05 compared with control group.. 42.

(44) (a). (b). Figure 4. L-theanine increases immunomodulatory function and angiogenesis. (a) Secreted levels of IFNg or (b) VEGF in the medium only or MSCconditioned medium with or without EGCg or L-theanine were detected by ELISA. L-theanine pretreatment increased the secretion of IFN- and VEGF.. 43.

(45) Figure 5. DEN-induced liver injury in the rat model. The experimental flow chart of the rat model establishment. Liver injury was induced by DEN in the 7-week-old rats. 1×106 cells/rat MSCs or L-theanine preconditioned MSCs were given to rat by intravenous injection after 2, 4 or 8 weeks of DEN injury. Liver tissue and blood were collected at the indicated time points for further evaluation.. 44.

(46) Figure 6. Morphology of liver tissues from 10 groups. Appearance of liver tissues from Wistar rats with DEN and MSC treatments. The rats were sacrificed and the liver were collected for photographing. Festered tissues were seen on 8 weeks of DEN-induced liver.. 45.

(47) (a). (b). (c). Figure 7(a)(b)(c) Biochemical markers of liver and bile ductules function. (a)(b) DEN treatment increased the AST, ALT activity in serum (*p<0.05), while MSC or L-theanine preconditioned-MSC treatment decreased AST and ALT activity. (c) γ-GT value was significant enhanced in DEN-induced groups, there was a decreasing trend in MSC treatment groups but no significant differences were shown.. 46.

(48) (d). Figure 7(d). MSCs decrease levels of ROS on DEN-induced rats. (d) Whole blood was collected after sacrificed and then the level of ROS was detected by Chemiluminescence Analyzing System. It was apparently notified that MSC treatment groups and L-theanine preconditioned-MSC groups reduced the levels of free radical induced by DEN treatment. (Mean ± SEM; n=6/group). In conclusion, we observed severe liver injury after DEN treatment but relieved situation after MSC application.. 47.

(49) (a). Figure 8(a). Histological evaluation by H-E staining (400x) All livers were collected after sacrificed under anesthesia, fixed with formalin and waxed. We de-waxed and hydrated livers in paraffin sections, and stained the slides in hematoxylin for 5 minutes and immersed in eosin for 30 seconds, then dehydrated by xylene. (a) Livers of the rats administered only DEN showed shattered images, 48.

(50) while livers in MSC treatment groups showed more normal appearance of liver parenchyma.. 49.

(51) (b). Figure 8(b). H-E staining of liver portal triad (400x) (b) Image shows occlusion of bile ductule in DEN-induced groups. (Red arrow: hepatic artery, Blue arrow: hepatic portal vein, Black arrowhead: small bile ductule) Hepatic sections were re-fix in Bouin's solution, and stain in Weigert's iron hematoxylin working solution, then stain in Biebrich scarlet-acid fuchsin solution. Phosphomolybdic-phosphotungstic acid 50.

(52) solution, aniline blue solution, acetic acid solution were orderly applied then clear in xylene.. 51.

(53) (c). Figure 8(c). Masson staining of liver tissues (400x) (c) In fibrosis-specific Masson staining, the degree of hepatic fibrosis in DEN-induced group was observed.. 52.

(54) (d). Figure 8(d). High expressions of AFP were observed in DEN-induced groups (400x) (d) In general, HCC tissue or serum level frequently expresses high level of α-fetoprotein (AFP). We detected higher expressions of AFP in DEN treatment groups (red arrow), which indicated that we successfully established the DEN-induced carcinoma model. 53.

(55) Figure 9. Relationships between apoptosis-related protein Bax and DEN or MSC treatment. Liver tissue slices obtained as described in figure 9 were subjected to (a) IHC staining for detecting Bax (Red arrow, 400x). The positive staining signals were measured by ImageJ and the quantitative results were showed in (b). (Mean ± SEM; n=6/group; *p<0.05 versus control group; a<0.05 versus 2 week of DEN-induced group; b<0.05 versus 4 week of DEN-induced group; c<0.05 versus 8 week of DEN-induced group) Liver tissue extracts were also analyzed by Western blot for the protein levels of (c) Bax. The expression of Bax were increased significantly after DEN treatment and decreased with the MSC treatment. Data was showed in mean ± standard deviation (SD) of 3-5 separate experiments.. 54.

(56) Figure 10. Relationships between apoptosis-related protein Bcl-2 and DEN or MSC treatment. Liver tissue slices obtained as described in figure 10 were subjected to (a) IHC staining for detecting Bcl-2 (Red arrow, 400x). The positive staining signals were measured by ImageJ and the quantitative results were showed in (b). (Mean ± SEM; n=6/group; *p<0.05 versus control group; a<0.05 versus 2 week of DEN-induced group; b<0.05 versus 4 week of DEN-induced group; c<0.05 versus 8 week of DEN-induced group) Liver tissue extracts were also analyzed by Western blot for the protein levels of (c) Bcl-2. The expression of Bcl-2 were decreased significantly after DEN treatment and increased with the MSC treatment. Expression of Bcl-2 (red arrow) analyzed in IHC or Western blot all showed opposite pattern to Bax. Data was showed in mean ± standard deviation (SD) of 3-5 separate experiments. 55.

(57) Figure 11. Relationships between apoptosis-related protein caspase 3 and DEN or MSC treatment. Liver tissue slices obtained as described in figure 11 were subjected to (a) IHC staining for detecting caspase 3 (Red arrow, 400x). In caspase 3 IHC results, it was similar to the Bax expression, MSC application significantly reduced the expression of caspase 3 (Figure 9c). This indicated that DEN treatment induced apoptosis and MSC treatments inhibited the apoptotic process. The positive staining signals were measured by ImageJ and the quantitative results were showed in (b). (Mean ± SEM; n=6/group; *p<0.05 versus control group; a<0.05 versus 2 week of DEN-induced group; b<0.05 versus 4 week of DEN-induced group; c<0.05 versus 8 week of DEN-induced group) Liver tissue extracts were also analyzed by Western blot for the protein levels of (c) caspase 3. The expression of caspase 3 were increased significantly after DEN 56.

(58) treatment and decreased with the MSC treatment. Data was showed in mean ± standard deviation (SD) of 3-5 separate experiments.. 57.

(59) Figure 12. Relationships between pyroptosis-related protein caspase 1 and DEN or MSC treatment. Liver tissues slices were subjected to IHC staining for pyroptosis markers (a) caspase 1 (Red arrow, 400x). The quantitative results were showed in (b). Western blot analysis of liver tissue extracts was also applied to detect the expression levels of (c) caspase 1. Western blot analysis reveals DEN induced liver injury by pyroptosis, whereas the MSC treatment and L-theanine preconditioned MSC seems to reduce the pyroptosis-related protein expression. (Mean ± SEM; n=6/group; *p<0.05 versus control group; c<0.05 versus 8 week of DEN-induced group). 58.

(60) Figure 13. Relationships between pyroptosis-related protein IL-1β and DEN or MSC treatment. Liver tissues slices were subjected to IHC staining for pyroptosis marker (a) IL-1β (Red arrow, 400x). The quantitative results were showed in (b). Western blot analysis of liver tissue extracts was applied to detect the expression levels of (c) IL-1β. Western blot analysis revealed DEN also induced liver injury by the pyroptosis pathway. MSC treatment seems to reduce the pyroptosis-related proteins expression. (Mean ± SEM; n=6/group; *p<0.05 versus control group; c<0.05 versus 8 week of DEN-induced group). 59.

(61) Figure 14. Relationships between autophagy-related protein Beclin-1 and DEN or MSC treatment. Liver tissues slices were subjected to IHC staining for autophagy marker (a) Beclin-1 (Red arrow, 400x). The quantitative results were showed in (b). In Beclin-1 IHC results, MSC application significantly reduced the expression of Beclin-1. This indicated that DEN treatment induced autophagy and MSC treatments inhibited the autophagy process. Western blot analysis of liver tissue extracts was also applied to detect the expression levels of (c) Beclin-1. In western blot analysis, there was a trend in increasing expression in DEN-induced groups and reducing in 4 weeks of DEN treatment and MSC and in L-theanine preconditioned groups. (Mean ± SEM; n=6/group, *p<0.05 versus control group; c<0.05 versus 8 week of DEN-induced group). 60.

(62) Figure 15. Relationships between autophagy-related protein LC-3β and DEN or MSC treatment. Liver tissues slices were subjected to IHC staining for autophagy marker (a) LC-3β (Red arrow, 400x). The quantitative results were showed in (b). Western blot analysis of liver tissue extracts was also applied to detect the expression levels of (c) LC-3β. The expression of LC-3β was increased significantly after DEN treatment and decreased with the MSC treatment. (Mean ± SEM; n=6/group; *p<0.05 versus control group; a<0.05 versus 2 week of DEN-induced group; b<0.05 versus 4 week of DEN-induced group; c<0.05 versus 8 week of DENinduced group). 61.

(63) (a). Figure 16(a). IVIS system tracks and images tumor in vivo. (a) IVIS imaging of rats in control group and 8 weeks of DEN treatment after tumor marker AFP injection into the tail vein. The liver of 8-week DEN treated rat was directly imaged and was characterized with a high expression of AFP. Tumor site was indicated by the blue fluorescence.. 62.

(64) (b). Figure 16(b). IVIS system tracks and images MSC in vivo. (b) MSCs were labeled with DiR surface marker and were injected into the tail vein. MSC homing ability revealed the damaged liver site indicated by the blue fluorescence.. 63.

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