Dissertations / Theses on the topic 'Advanced lipoxidation end product'

INTRODUCTION AGEs and ALEs (Advanced Glycoxidation/Lipoxidation End products) are covalently modified proteins that can act as pathogenic factors in several chronic diseases, like diabetes and cardiovascular diseases. These covalent adducts are formed by different mechanisms. AGEs are proteins covalently modified by reducing sugars or their oxidative degradation products, involving the Maillard reaction. ALEs are proteins modified by reactive carbonyl species (RCS) generated by lipid peroxidation. AGEs/ALEs can be the basis of many different pathologies, underlining the importance for good analytical methods for identification and characterization for the use of biomarkers, but also as a drug target. However, the identification, characterization and quantification of AGEs/ALEs remains to be very challenging due to heterogeneous precursors (sugars, lipids) leading to heterogeneous AGEs/ALEs, present in low concentrations and being very complex analytes. Various techniques to identify and characterize AGEs/ALEs have been described, making use of an isolation/enrichment step based on reactive groups, like carbonyls. However, not all AGEs/ALEs retain reactive groups and therefore can not be isolated and identified using these techniques, indicating the need for a new strategy. The strategy that has been employed in our laboratory is to use the soluble domain of the RAGE receptor, VC1, to affinity enrich AGEs. Using this approach, AGEs/ALEs will be enriched independently of the protein and type of modification. Moreover, a ligand of RAGE can be identified, which could be a potential biomarker of a disease caused by oxidative stress. RAGE is a type I cell surface receptor that is expressed in several cells, such as endothelial cells, smooth muscle cells, but also dendritic cells and T-lymphocytes and is predominantly located in the lungs. The receptor has been implicated in many different pathologies with a marked oxidative base, such as diabetes, atherosclerosis and neurodegenerative diseases. One of the pathways that can be activated is the Nf-κB pathway. The Nf-κB pathway is the ideal signaling pathway to investigate the binding and activation of RAGE by AGEs or ALEs. For this purpose, a cell line was obtained with and without overexpression of RAGE. Furthermore, the cell lines were transfected with a Nf-κB reporter gene, providing us with a fast and high-throughput assay for the evaluation of a pro-inflammatory response upon stimulation with AGEs/ALEs. AIM OF THE PROJECT The identification and characterization of AGEs/ALEs has proven to be crucial in the onset and development of many pathologies. Therefore, good analytical strategies need to be developed/optimized for better understanding of the exact nature of modification, to understand the role they play in disease progression. Identified AGEs/ALEs can serve as biomarker, as well as drug targets. The VC1 technique was proven to be a promising technique to accommodate the need for enrichment of AGEs for better characterization. The first aim of the project was therefore to investigate whether also ALEs are binder of RAGE, since they share the same structural properties than AGEs, and also have been shown to activate the Nf-κB pathway, implicating a role for receptors, like RAGE. Furthermore, to gain a deeper insight into the molecular mechanisms involved in the protein-protein engagement. Since a successful enrichment strategy was developed, the second aim of this project was focused on identifying AGEs/ALEs in biological samples. The first part was focused on oxidizing healthy human plasma in-vitro using AAPH as a radical initiator, and the incubation of plasma directly with RCS, anticipating the production of AGEs/ALEs. The VC1 technique was then used to identify which AGEs/ALEs are produced. Simultaneously, other variables during the sample preparation and analysis were optimized. As explained before, AGEs/ALEs are present in very low concentrations in biological samples, hence the need for very sensitive methods and instrumentation allowing identification. Since human serum albumin (HSA) is the main protein present in plasma, around 50-60%, and has multiple nucleophilic targets, it represents the best model for characterizing AGEs/ALEs. For this reason, the focus was on extracting HSA from plasma, using the newest generation of tribrid MS for the analysis of AGEs/ALEs in plasma samples. AGEs are ligands for RAGE, meaning, they can bind and activate the receptor, inducing a signaling pathway and pro-inflammatory response. ALEs have also been shown to induce a pro-inflammatory response; however, no specific receptor has been linked to this cellular event. Using a cell line with and without RAGE, we aimed to determine whether ALEs can bind and activate the Nf-κB pathway through RAGE. RESULTS AND DISCUSSION ALEs as binder of RAGE In order to investigate the interaction between RAGE and ALEs, different ALEs were produced in-vitro by incubating HSA with different concentrations of well-known lipid derived RCS and in particular: ACR, MDA and HNE. After 24, 48 and 72 h, aliquots of the incubation mixtures were withdrawn, and the reaction was stopped by removing the excess of RCS by ultrafiltration. Intact protein analysis by direct infusion MS was used to evaluate the extent of HSA modifications and demonstrated that by using a wide range of molar ratios and different time-points a quite wide array of ALEs for each tested RCS was generated. In order to characterize ALEs selectively enriched by RAGE, a VC1 pull-down assay was performed as previously described. HSA and HSA treated with MDA, ACR or HNE were assayed for binding to VC1-resins and to control resin. As expected, unmodified HSA was not retained by the VC1-resin. At increasing molar ratios and incubation time, higher amounts of albumin modified with MDA or ACR were eluted from the VC1 resin, with a predominance of the high molecular weight (HMW) species. The modified albumins were retained by the VC1-resin, but not by the control resin. ALEs in the reaction mixtures and those enriched by VC1 were analyzed by bottom-up MS in order to identify the PTMs and to localize the amino acid residues involved in the protein adduct formation. With regard to MDA, only di-hydropyridine adducts on lysines (DHPK), and N-2-pyrimidyl-ornithine adducts on arginines (NPO) were retained by VC1-domain. The n-propenal modifications of lysine (NPK), largely identified before enrichment, were not identified after the enrichment. ACR induced a set of modifications which were identified only after VC1 enrichment and in particular the N-(3-formyl-3,4-dehydro-piperidinyl) lysine (FDPK) modifications, the Michael adduct on cysteines, the double Michael adduct of lysines, the Michael adduct of histidine, the N-2-(4 hydroxy-tetrahydro-pyrimidyl) ornitine (propane-arginine, HTPO) and the Nε-(3-methylpyridinium)-lysine (MP-lysine). Most of the ALEs generated by HNE were found both before or after enrichment, with the exception of a few Michael adducts which were selectively retained by VC1 (not detected before enrichment). With a view to rationalizing the key factors influencing the RAGE binding of the monitored adducts, in silico studies were performed. They were focused on the adducts on arginine and lysine residues as formed by ACR and MDA since they are numerous, with a very broad range of affinity, thus allowing the development of clear structure-affinity relationships. RAGE-ligand interacting regions are characterized by a rich set of positively charged residues which can bind acidic regions of a protein. The mechanism identified using in silico studies, involves a basic amino acid at the center of carboxylic acids like glutamate and aspartate, which forms a set of ionic bridges. Once the basic amino acid is modified by ACR or MDA to an adduct with a neutral charge, the carboxylic acids become available to freely contact the RAGE positive residues. Identification of AGEs/ALEs in biological samples The VC1 technique has proven to be successful in enriching AGEs and ALEs, so the next step was to exploit this technique in biological samples. In order to identify proteins prone to be modified due to oxidative pathways, and possibly serve as biomarker, healthy human plasma was oxidized using the radical initiator AAPH. Different concentrations of AAPH and different timepoints were tested for the presence of protein carbonyl groups, an indicator for protein oxidation and possibly the formation of AGEs/ALEs. A time and concentration dependent formation of carbonyl groups is observed in plasma. Next, samples were analyzed using a bottom-up approach. Results obtained were showing many oxidation products, such as amino side chain oxidation, however no AGEs/ALEs were identified. Thus, a new approach was adopted, including the incubation of plasma directly with RCS, such as HNE, MDA and ACR. This resulted in the formation of AGEs/ALEs in plasma samples, however, they could not be retained by the VC1 domain. Instead of using the VC1 technique to enrich AGEs/ALEs from biological samples, other variables throughout the experimental set-up were optimized. Previously, peptides were analyzed using the Orbitrap LTQ XL, a very powerful instrument. Nonetheless, the newest generation of tribrid MS offers even higher resolution, and it increases protein coverage due to parallel isolation and detection, and faster analyzers. Moreover, we focused on AGEs/ALEs from HSA and using NaBH4 to reduce and stabilize adducts throughout the analysis. This new approach permitted us to identify many AGEs/ALEs in both healthy human plasma samples, but also AGEs/ALEs only present in heart failure samples. Glycation on lysine residues was the main modification identified, present in both healthy and heart failure plasma samples. Important is the HNE Michael adduct, specifically identified in only heart failure samples. Moreover, the importance of stabilizing adducts is underlined by the fact that the acrolein Michael adduct could only be identified after reduction with NaBH4. Development of a cellular assay to determine pro-inflammatory activity of RAGE binders Another part of this project was focused on elucidating whether AGEs/ALEs induce an inflammatory response in cells. For this purpose, a collaboration was started with the Laboratory of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino. Using a rat epithelial lung cell line overexpressing RAGE, and a control cell line not expressing RAGE, it could be detected if AGEs/ALEs exhibit an effect by binding to RAGE. Moreover, both cell lines were transfected with a Nf-κB reporter gene allowing us a fast and sensitive method for determining if binding of RAGE induces a down-stream signaling pathway. This system implies a firefly luciferase gene downstream from the Nf-κB gene. When the Nf-κB pathway is activated, independently from RAGE, it produces the firefly luciferase enzyme. After adding a luciferin substrate, firefly luciferase is able to convert this substrate into another substrate with light as by-product, which can be measured by a luminometer. IL-1α was used as a positive control, since it induces a strong inflammatory response through Nf-κB. Moreover, known ligands of RAGE able to activate the Nf-κB pathway, were used to validate the cellular experiment, including HSA modified with fructose (AGE), and HMGB1. Results show that Nf-κB is already increased in untreated cells with RAGE and that AGEs induce the Nf-κB pathway independently from RAGE. Moreover, the difference between control and RAGE cells is not significantly increased in the presence of HMGB1 compared to untreated. However, the positive control seemed to induce a much stronger activity in cells with RAGE. Overall, this cellular assay is good for assessing pro-inflammatory activity, however, it is not optimized yet for distinguishing a RAGE-dependent mechanism. CONCLUSION In summary, by using an integrated MS (intact protein and bottom-up approach) and computational approach we have found that some ALEs generated from lipid peroxidation RCS are RAGE binders. We have also found the basic features that ALEs from HNE, MDA and ACR must have to be a RAGE binder: 1) the covalent adducts should greatly reduce or abolish the basicity of the target amino acid, 2) the basic amino acid should be at the center of a set of carboxylic acids which, once the residue is modified, become available to freely contact the RAGE positive residues. Next step was to use the VC1 technique to enrich AGEs/ALEs in biological samples. First, oxidized human plasma was used, however, using the Orbitrap LTQ XL, it was not sufficient to identify AGEs/ALEs. Therefore, analysis was moved to a higher resolution mass spectrometer, which allowed us to identify AGEs/ALEs in plasma samples of heart failure patients, showing the powerfulness of this new generation MS. Important was to understand whether ALEs could induce pro-inflammatory activity through RAGE, since we showed that ALEs are RAGE binders. Unfortunately, the cellular assay that was set up is efficiently in determining Nf-κB dependent pro-inflammatory activity, but not if it is RAGE dependent.

Non enzymatic protein covalent modifications are involved in the toxic effects induced by electrophilic xenobiotics as well as by endogenous cytotoxic oxidation by-products. Aim of my Ph.D work was to set-up MS methods for the identification, characterization and quantification of non-enzymatic covalently modified proteins and peptides in biological matrices. To reach this goal both tandem MS and high resolution approaches were employed due to the wealth of structural and molecular information that these techniques can provide. As a first step the MS methods were applied for understanding in both in vitro and ex vivo conditions the mechanism of protein haptenation induced by amoxicillin (AX). The MS approach was then focused to study in ex vivo condition the covalent reaction between histidine dipeptides, such as carnosine, and toxic endogenous intermediates like reactive carbonyl species (RCS). .

Advanced glycated end-products (AGEs) are a heterogenous group of compounds formed through the Maillard reaction. During AGE formation, reactive α-dicarbonyls are formed, such as glyoxal (GO) and methylglyoxal (MG). These α-dicarbonyls are present at elevated concentrations in diabetes, and are frequently implicated in the initiation and progression of diabetic complications. Previous research has linked α-dicarbonyls with formation of reactive oxygen species (ROS) and inflammation. However, much of the prior work has been conducted using concentrations of α-dicarbonyls that are substantially higher than can be generated in vivo, and the biochemistry has been investigated under conditions (e.g. pH) outside the physiological range. The aim of the work presented in this thesis was to test the hypothesis that GO and MG are pro-oxidant and pro-inflammatory at (patho)physiological concentrations in both monocytes and pancreatic β-cells. In this work several new and important observations have been made regarding the action of α-dicarbonyls on oxidative stress and inflammation. 1) The amount of oxidative species production by α-dicarbonyls in glycation reactions with amino acids and proteins may be so low as to be negligible in vivo, despite previous evidence to the contrary. 2) α-dicarbonyls did not appear to generate oxidative stress within inflammatory cells nor pancreatic β-cells by depleting the levels of GSH. 3) At least in the β-cell model, the mechanism of action of the α-dicarbonyls did not involve dysregulation of the antioxidant SOD enzymes. 4) Neither α-dicarbonyl significantly affected insulin production by β-cells, except at cytotoxic concentrations. 5) Treatment of inflammatory cells with α-dicarbonyls induced release of the proinflammatory cytokine IL-8. 6) In both immune cells and pancreatic β-cells, α-dicarbonyls were involved in O2.- generation by activation and/or upregulation of NADPH oxidase. 7) Despite the structural similarities of α-dicarbonyls, they have distinct mechanisms of action with respect to oxidative stress.

The Receptor for Advanced Glycation End products (RAGE) interacts with several classes of structurally unrelated ligands. The activation of RAGE by its ligands results in the cellular activation of several kinases and transcription factors including mitogen activated protein kinases (MAPKs) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) resulting in sustained inflammation, which is involved in pathologies such as diabetes, cancer, Alzheimer's disease, multiple sclerosis and other diseases associated with chronic inflammation. Current mouse models of human disease have shown that RAGE activity can be efficiently suppressed using either soluble RAGE (sRAGE) or anti-RAGE antibodies as inhibitors. Our goal was to generate new monoclonal antibodies against RAGE that can serve as diagnostic as wells as therapeutic tools in RAGE related pathologies. The chapters in this dissertation are a complete documentation of the development of these anti-RAGE antibodies. Additionally, an introductory review of antibodies, which includes structure and function, types of antibodies and production and basic understanding of RAGE and its ligands, has been provided to facilitate the understanding of the chapters. The first chapter details the development and characterization of anti-RAGE antibodies produced from hybridoma. The next chapter explores the effects of the generated antibodies to mammalian cells in in vitro settings and the final chapter applies the generated antibodies in vivo. During the course of this work, the antibodies developed showed binding to RAGE at nano-molar affinities which are comparable to the affinities of current antibodies used for therapeutic purposes, diagnostic and research purposes. We were also able to delineate that the possible mechanism of action of the antibodies is by preventing binding to RAGE. Lastly, we observed that one of the generated antibodies was able to reduce tumor growth in vivo in a melanoma xenograft mouse model.

Cyclooxygenase (COX) is the rate limiting enzyme that catalyses the production of prostanoids, which are crucial to vascular homeostasis. Evidence suggests that endothelial dysfunction and inflammation play a role in vascular complications in aging and diabetes. Previous animal studies by our laboratory at RMIT University reported enhanced COX expression with aging in rat aortas, platelets and monocytes. Potentially, alteration in COX expression may result in an imbalanced prostanoid production favoring the synthesis of vasoconstrictors and hence increase the risk of cardiovascular events in the aging population. The regulation of altered COX expression in aging, however, is not clear. It has been suggested that histone hyperacetylation may be an important mechanism that regulates COX levels during the aging process as increased histone acetylation has been shown to occur with aging. Thus, we hypothesized that COX expression is modulated by histone hyperacetylati on. This was investigated by measuring COX expression in histone hyperacetylated cultured endothelial cells. In the case of diabetes, studies have reported that the development of diabetes and its complications is associated with persistent inflammatory activity, evident with increased inflammatory markers in the circulation. COX-mediated pathways may be involved in this inflammatory process in diabetes. Furthermore, the formation of advanced glycation end products (AGEs) is accelerated in diabetes. AGEs can bind to receptors for AGEs (RAGE), which has also been suggested to play a role in inflammation in diabetes. We hypothesized that COX- and RAGE-mediated pathways contribute to increased inflammation in diabetes and potentiate the development of diabetic vascular complications. This was investigated by measuring changes in COX-mediated pathways in both rat and human diabetic models. The current thesis reports: 1) in cultured endothelial cells, histone hyperacetylation was associated with increased COX expression; 2) an overall increase in inflammation was observed in diabetes involving COX- and RAGE-mediated pathways. This was supported by increased platelet COX-1 and monocyte COX-2 levels in Zucker rats, increased monocyte COX-2 in human Type 1 diabetes and elevated plasma TXB2 and PGE2 levels in both human Type 1 and Type 2 diabetic subjects. Up-regulation of RAGE expression was further found in platelets and monocytes in both human diabetes types. When treated with NSAIDs, plasma prostanoid levels, COX and RAGE expression were reduced significantly in both platelets and monocytes in human diabetic subjects. 3) It is unclear how COX and RAGE expression was regulated, but histone modifications may be one of the mechanisms. Data from cultured cells indicated that increased COX expression was associated with increased histone acetylation levels induced by TSA. Concurrent increases in histone acetylation and COX-2 levels were also observed in human Type 1 diabetes, but similar findings were not observed in human Type 2 diabetes. In addition, we failed to find an age-dependent increase in monocyte histone H4 acetylation in human Type 2 diabetes despite an age-dependent increase in monocyte COX-2 expression. Thus, whether histone hyperacetylation modulates COX expression and in what conditions require further investigation.

Dans un contexte de vieillissement de la population et d’accroissement des maladies chroniques liées à l’âge comme le diabète, de nouveaux biomarqueurs de l’état de santé à long terme doivent être étudiés. Les produits de glycation avancée (AGE) sont des molécules témoins de la charge métabolique accumulée au cours du temps, dénommée "mémoire métabolique". Les AGE jouent un rôle important dans les lésions à long terme dans le diabète et dans le déclin du métabolisme global lié au vieillissement. L’accumulation cutanée des AGE peut être mesurée par autofluorescence (AF) de manière instantanée et non invasive grâce à l’AGE-READER. Les objectifs de cette thèse étaient d’évaluer la valeur de l’AF cutanée des AGE en tant que marqueur de mémoire métabolique chez des personnes âgées de la cohorte des 3-Cités et parallèlement d’évaluer la valeur pronostique de l’AF pour les complications du diabète chez des patients porteurs de diabète de type 1. Chez les personnes âgées, nous avons montré que l’AF reflétait les statuts glycémique et rénal 10 ans avant la mesure. Chez les patients atteints de diabète de type 1, l’AF était associée à la présence d’une neuropathie 4 ans plus tard. De plus, dans cette même population, nous avons décrit l’évolution de l’AF sur 4 ans de suivi. Nous avons montré que les principaux déterminants de son évolution étaient la fonction rénale et le traitement par pompe à insuline. Enfin nous avons trouvé que l’augmentation de l’AF sur 4 ans de suivi était associée à la survenue de la maladie rénale. Ces travaux soulèvent de nouvelles perspectives de recherche quant à l’intérêt de l’AF à différents âges clés de la vie en tant que biomarqueur de pathologies qui évoluent sur des dizaines d’années
In the context of the ageing of the population and the increase of age related diseases such as diabetes, new biomarquers of the long-term health status should be considered. Advanced glycation end products (AGE) are molecules indicators of the metabolic burden over time, called “metabolic memory”. AGE play an important role in long term diabetes injuries and in the global decline of the metabolism related to ageing. Skin accumulation of AGE can be measured by autofluorescence instantly and non-invasivly with a tool called AGE-READER. The objectives of my dissertation were to evaluate the value of the skin autofluorescence (sAF) of AGE as marker of metabolic memory in elderly people from the 3-City cohort and in parallel, in patients with type 1 diabetes, evaluate the prognostic value of sAF for diabetes complications. In the elderly population, we showed that sAF reflected glycemic and renal status of 10 years before. In patients with type 1 diabetes, sAF was associated to the presence of neuropathy 4 years later. Moreover, in this same population, we described the evolution of sAF in 4 years of follow-up and we showed that the principal determinants of the evolution of sAF were kidney function and insulin pump therapy. Finally, we also found that increase of sAF in 4 years was associated with the occurrence of kidney disease. This work rises new research opportunities about the interest of sAF at differents key ages as biomarker of pathologies which evolve in several decades

博士
高雄醫學大學
醫學研究所博士班
93
Diabetic nephropathy (DN) is characterized by glomerulopathy and tubulointerstitial expansion followed by renal fibrosis, including tubulointerstitial fibrosis. For example, we have shown that advanced glycation end-product (AGE)-induced mitogenesis and collagen production are dependent on the JAK2 pathway in normal rat kidney interstitial fibroblasts (NRK-49F cells). Leptin is a Janus kinase 2 (JAK2)-activating cytokine via the long form leptin receptor (Ob-Rb). Leptin and connective tissue growth factor (CTGF) are involved in renal fibrosis. However, the relationship between leptin and CTGF in terms of AGE-induced effects remains unknown. Moreover, angiotensin II (Ang II) and CTGF are important in the pathogenesis of DN. Thus, we studied the role of leptin, Ang II, JAK2 and CTGF in AGE-induced effects in NRK-49F cells. We found that leptin and AGE increased mitogenesis and type I collagen protein expression at 3 d and 7 d, respectively. AGE also increased leptin mRNA and protein expression at 2 d and 3 d. AGE increased CTGF mRNA and protein expression at 3 d and 5 d. AG-490 (JAK2 inhibitor) abrogated AGE-induced leptin mRNA and protein expression. AG-490 and Ob-Rb antisense oligodeoxynucleotides (ODN) abrogated AGE-induced CTGF mRNA and protein expression. AG-490 and CTGF antisense ODN abrogated AGE-induced mitogenesis and collagen protein expression. Additionally, leptin dose (0.2-1 �慊/ml) and time (1-2 d)-dependently increased CTGF protein expression. AG-490 abrogated leptin (1 �慊/ml)-induced CTGF protein expression at 2 d. AG-490 and CTGF antisense ODN abrogated leptin-induced mitogenesis and collagen protein expression at 3 d. Additionally, we found that Ang II (10-7 M) increased mitogenesis and type I collagen production at 3 d. We also found that AGE increased angiotensinogen protein at 2 d, which was attenuated by AG-490 (a JAK2 inhibitor). Ang II (10-7 M) increased CTGF mRNA and protein expression at 1 d and 2 d, which were attenuated by AG-490. Moreover, losartan (a type I angiotensin receptor blocker) and captopril (an angiotensin converting enzyme inhibitor) attenuated AGE-induced CTGF mRNA/protein expression while attenuating AGE-induced mitogenesis and type I collagen production. Moreover, AG-490 and CTGF antisense (but not sense) oligodeoxynucleotide attenuated Ang II (10-7 M)-induced mitogenesis and type I collagen production at 3 d. We conclude that AGE-induced mitogenesis and type I collagen production are dependent on the Ang II-JAK2-CTGF and leptin-JAK2-CTGF pathways while Ang II and leptin-induced mitogenesis and type I collagen production are dependent on the JAK2-CTGF pathway. Moreover, because previous studies by others showed that Ang II can induce leptin, we speculate that AGE-induced mitogenesis and type I collagen production may be dependent on the AGE-Ang II-leptin-JAK2-CTGF pathway in these cells.

碩士
中華醫事學院
生物科技研究所
94
In Taiwan, diabetes mellitus and end-stage renal disease is the 4th and 8th leading cause of death, respectively. Moreover, diabetic nephropathy (DN) is the chief cause of new dialysis patients in our country. Based on others and our previous studies, we suggested that nitric oxide (NO), hyperglycemia and advanced glycation end product (AGE) are three of the most significant factors in the pathogenesis of DN. However, the interactions between the signal transduction pathway of NO/cGMP-dependent protein kinase (PKG) and AGE-mediated renal tubulointerstitial fibrosis remain poorly understood. In the present study, the mechanisms by which tetrahydrobiopterin (BH4) modulates the AGE-induced hypertrophic growth in renal tubular epithelial (LLC-PK1) cells were examined. We found that AGE time- and dose-dependently decreased nitric oxide (NO) production and GTP cyclohydrolase I (GTPCH I)/inducible NO synthase (iNOS) activation. These effects were not observed when cells were treated with non-glycated BSA. NO and iNOS stimulated by BH4 and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prevented AGE-induced JAK2/STAT1/STAT5, Raf-1/ERK, and JNK/p38 MAPK activation. Nevertheless, addition of 2,4-diamino-6-hydroxypyramidine (DAHP) that inhibits GTPCH I activity may enhance AGE-induced these effects. The ability of iNOS/NO to inhibit AGE-induced hypertrophic growth was verified by the observation that BH4 and SNAP inhibited both cyclin-dependent kinase inhibitors (CDKI) p21Waf1/Cip1 and p27Kip1 expression. Furthermore, BH4 significantly decreased extracellular matrix (ECM) proteins fibronectin and collagen IV synthesis in AGE-treated cells. These findings suggest that BH4 supplementation is renoprotective partly by attenuating AGE-induced renal tubular hypertrophy by increasing GTPCH I/iNOS activation and reducing CDKI/ECM expression.

碩士
高雄醫學大學
醫學研究所碩士班
106
Diabetic nephropathy is a major cause of end stage renal disease, associated with inflammation, receptor for advanced glycation end product (RAGE) and TGF-receptor (TGF-R). NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3) inflammasome is pro-inflammatory. NLRP3 is activated by deubiquitinases or signal (e.g. ATP)-induced mitochondrial ROS to oligomerize with ASC and pro-caspase 1 to form an inflammasome, which activates IL-1β and IL-18. NLRP3 knockout and IL1R1 inhibitor-treated mice are protected from diabetic nephropathy (DN). However, the roles of NLRP3 inflammasome in DN were not proven because NLRP3 also has inflammasome-independent effects to active of the TGF-β-Smad3 pathway, but the roles of the cross-talk between monocyte and renal cells in terms of NLRP3 are not known. Thus, we studied the roles of NLRP3 in DN. We found that in Mes-13 (mouse mesangial) cells, high glucose (HG, 30 mM) increased NLRP3, ASC, and active caspase-1 and IL-1β levels. Concomitantly, HG increased TGF-β1 and UCHL5 levels. Advanced glycation end-products increased NLRP3, active caspase-1/IL-1β and TGF-β1, but not COX2 levels. In THP1 (human monocyte) cells, HG increased NLRP3, UCHL5 and TGF-β1, but not ASC levels. AGE increased NLRP3, ASC, UCHL5, active caspase 1 and COX2 levels in THP1 cells. Transwell studies showed that AGE-treated Mes-13 cells increased THP1 cell migration. Finally, we found that NLRP3 has a high level of tubular expression in the streptozotocin-diabetic mice at the 8th week. Thus, HG-and AGE-induced NLRP3-related pathways in Mes-13 and THP1 cells and NLRP3 (inflammasome-dependent or inflammasome-independent) may be a novel target for the treatment of DN.

博士
高雄醫學大學
醫學研究所
88
Advanced glycation end-product (AGE) is important in the pathogenesis of diabetic nephropathy, which is characterized by cellular hypertrophy/hyperplasia leading to renal fibrosis. However, the signal transduction pathways of AGE remain poorly understood. JAK (Janus kinase)/STAT (signal transducers and activators of transcription) pathway has been associated with cellular proliferation in some extra-renal cells. Because interstitial fibroblast proliferation may be important in renal fibrosis, we studied the role of the JAK/STAT pathway in NRK-49F (normal rat kidney fibroblast) cells cultured in AGE. We found that AGE dose-dependently increased cellular mitogenesis in NRK-49F cells at 5 and 7 d. AGE (100 g/ml) induced tyrosine phosphorylation of JAK2 (but not JAK1, JAK3, and TYK2) at 15-60 min. In addition, AGE also induced tyrosine phosphorylation of STAT1 and STAT3 at 1-2 h and 0.5-4 h, respectively. Being a transcription factor, AGE also increased the DNA-binding activities of STAT1 and STAT3. AG-490 (a specific JAK2 inhibitor) inhibited tyrosine phosphorylation of JAK2 and the DNA-binding activities of STAT1 and STAT3. The same results were obtained using the specific “decoy” oligodeoxynucleotides (ODNs) that prevented STAT1 and STAT3 from binding to DNA. Meanwhile, the STAT1 or STAT3 decoy ODN and AG-490 were effective in reversing AGE-induced cellular mitogenesis. To reveal the relationship between the JAK/STAT activity and cell cycle progression, we examined the effects of AGE on STAT5 activity and cell cycle progression in NRK-49F cells. We found that AGE rapidly induce tyrosine phosphorylation and protein-DNA binding activity of STAT5 at 5 min and 15 min, respectively. Among several G1 cyclins and cyclin-dependent kinases (cdks), cyclin D1 expression and cdk4 activity were both increased by AGE. Nevertheless, the levels of cdk inhibitors were not affected by AGE. Furthermore, our results showed that cyclin D1 expression and cdk4 activity were all inhibited by AG-490 and STAT5 decoy ODN. Interestingly, STAT5 decoy ODN and AG-490 both significantly blocked AGE-induced cell cycle progression and cellular mitogenesis. In this study, we also examined whether JAK/STAT are involved in AGE-regulated extracellular matrix (ECM) production in NRK-49F cells. We found that AGE time- and dose-dependently increased type I collagen and fibronectin productions in these cells. However, AGE did not increase TGF- bioactivity and TGF-1 mRNA expression. Additionally, AGE increased RAGE (receptor for AGE) protein expression and MAPK (ERK1/ERK2) activity. AGE-induced RAGE expression was dose-dependently inhibited by antisense RAGE ODN and captopril. AGE-induced type I collagen production and JAK2-STAT1/STAT3 activation were decreased by AG-490, antisense RAGE ODN and captopril. Meanwhile, STAT1 and STAT3 decoy ODNs also suppressed the induction of collagen by AGE. In contrast, they did not inhibit AGE-induced fibronectin synthesis. We concluded that the JAK2-STAT1/STAT3 signal transduction pathway is necessary for AGE-induced cellular mitogenesis in NRK-49F cells. In addition, STAT5 appears to modulate cyclin D1 synthesis and cdk4 activity, thereby contributing to AGE-induced cellular mitogenesis. On the other hand, we showed that RAGE-JAK2-STAT1/STAT3 pathway and MAPK kinase were involved in AGE-induced ECM production. Furthermore, renin-angiotensin system (RAS) may play an important role in AGE-induced biological effects.

碩士
慈濟大學
藥理暨毒理學碩士班/博士班
102
Aging, long-term hyperglycemia or advanced glycation end product (AGE)-enrich diet will cause AGEs accumulation in the body. The accumulation of AGEs induces diabetic complication, renal impairment or atherosclerosis. Therefore, level of AGEs is considered a biomarker for assessing the occurrence of diabetic complications or cardiovascular disease. Previous studies indicated that the concentration range of free AGEs was less than 1000 nM in human plasma. Developing a general and sensitive assay for quantification of free AGEs is necessary. In this study, we developed a highly-sensitive assay using benzoyl chloride derivatization and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for Nε-carboxymethyl-lysine (CML). For free CML analysis, plasma was filtrated by Amicon Ultra-0.5 centrifugal filter and the filtrate was derivatized with benzoyl chloride. The derivatized sample with internal standard analyzed by HPLC-MS/MS. The method was validated for precision, accuracy, recovery and linearity. The chromatographic time is 6.5 min and the retention time of di-benzoyl-CML is 2.8 min. Limit of detection (LOD) and limit of quantification (LOQ) was 14.2 nM and 25 nM, respectively. The linear correlation was greater than 0.99 in the range from 0 nM to 5 μM. Intra- and inter-day accuracy were -7.0 %–2.6 %, and intra- and inter-day precision were less than 9.0%. Carryover was not detected and matrix effect was compensated by internal standard. Every parameter was validated according to the FDA Guidance for Industry and ICH Q2(R1) guideline. Addition of D5-benzoyl-derivatized standard showed better results than external standard calibration on quantitation. The benzoyl-derivatization elevates sensitivity and shortens analysis time. Adding D5-benzoyl-derivatized standard provides the same results as using isotope-labeled AGE and reduce the analysis cost. To our knowledge, this is the first report of free AGEs determination in human plasma using benzoyl chloride derivatization and HPLC-MS/MS.

碩士
高雄醫學大學
生物化學研究所
91
In Taiwan and many other countries, diabetes mellitus is the major factor of end-stage renal disease (ESRD). The pathology of diabetic nephropathy is characterized by cellular hyperplasia, hypertrophy and expansion of extracellular matrix which result in renal fibrosis and ESRD. Although the pathogenesis of diabetic nephropathy is not clear yet. But in the previous studies, high glucose and advanced glycation end-product (AGE) have been shown to play the important roles of diabetic nephropathy. Rosiglitazone is a new drug for diabetes. It is the agonist of peroxisome proliferator activated receptor gamma (PPAR-gamma). PPAR-gamma heterodimerizes with 9-cis-retinoic acid receptor (RXRα) and, after ligand binding, functions as transcription factors in the regulation of glucose metabolism. However, we want to know if Rosiglitazone would get the effects through the other signal transduction pathways and its effect on diabetes nephropathy. We use two cell lines : kidney fibroblast of normal rat (NRK-49F) and the mesangial cell of normal mouse (MES-13). We found that Rosiglitazone could effectively reverse the increase of extracellular matrix induced by high glucose and AGE in NRK-49F and MES-13 cells. It also can reverse the increase of DNA synthesis by high glucose and AGE in NRK-49F cells. In NRK-49F and MES-13 cells, we also found that Rosiglitazone could reverse the increased expression of collagen type IV. We also observed that Rosiglitazone could reverse the hypertrophy of MES-13 cells induced by high glucose and AGE. In the molecular level, we found that in NRK-49F cells, Rosiglitazone could reverse the increase of cyclin D1, JAK2 and SOCS-3 induced by AGE in 2 hours. Then we used immunoprecipitations for the JAK2 protein and get the same result. Thus, we have demonstrated the effects of Rosiglitazone on cell cycle and the JAK signaling pathway in NRK-49F cells. In MES-13 cells, we found that rosiglitazone could inhibit the increased expression of TGF-β receptor type II and cyclin D1 induced by high glucose and AGE. Regard the effects of p21Waf1 and p27Kip1, our experiments showed that high glucose increased the expression of p21Waf1 and p27Kip1 while Rosiglitazone could reverse p27Kip1 expression. We also found that AGE could decrease the expression of p27Kip1 while Rosiglitazone could reverse this effect. Thus, rosiglitazone can improve the increase of ECM and cellular hypertrophy by decreasing cyclin D1, TGF-β signaling and p27Kip1. We concluded that Rosiglitazone can improve the effects induced by high glucose and AGE in NRK-49F and MES-13 by modulating cyclin D1, JAK2, TGF-β receptor type II and p27Kip1.

博士
高雄醫學大學
醫學研究所
96
Diabetic nephropathy is the complication of diabetes mellitus. In Taiwan, diabetes mellitus and end-stage renal disease is the 5th and 8th leading cause of death, respectively. Diabetic nephropathy is a major cause of morbidity and mortality, which comprise of 20-40% of the diabetic patients. The pathogenesis of diabetic nephropathy is characterized by the proliferation of mesangial cells in the early stage but hypertrophy of renal cell at the late stage of diabetes mellitus. Moreover, the thickening of glomerular basement membrane and the accumulation of extracellular matrix leading to renal fibrosis is related of high glucose and the advanced glycation end-products (AGE) . However, the signal pathway associated with AGE in diabetic nephropathy is poorly understood. Recently, the evidence shows that Wnt pathway is associated with the formation of renal fibrosis. According to the previous studies, the glycogen synthase kinase-3β (GSK-3β) , one of the key mediator of Wnt pathway, plays a role in the regulation of glucose metabolism. Thus, we studied the role of β-catenin, and GSK-3β in AGE-induced effects in the proximal tubule-like LLC-Pk1 cells, and their relationship to the diabetic nephropathy. We found that AGE (50, 75, 100 μg/ml) dose-dependently decreased β-catenin protein expression at 48 h incubation, but AGE did not decrease β-catenin protein expression until 48 h. Furthermore, we found that AGE (100 μg/ml) time-dependently (8-48 h) increased GSK-3β-Tyr216 (active GSK-3β) and time-dependently (4-24 h) decreased GSK-3β-Ser9 (inactive GSK-3β) protein expression. Meanwhile, AGE (100 μg/ml) activated GSK-3β kinase activity at 8-48 h. SB216763 (a GSK-3β inhibitor) attenuated AGE (100 μg/ml)-inhibited β-catenin protein expression at 48 h. SB216763 also attenuated AGE (100 μg/ml)-inhibited cell proliferation and attenuated AGE (100 μg/ml)-induced collagen production and type IV collagen protein expression at 48 h. We also found that JAk2, PI3K, MAPK, and PKC pathway inhibitor attenuated the AGE-inhibited β-catenin protein expression, but only JAk/STAT and MAPK pathway attenuated the AGE-inhibited cyclin-D1 protein expression. In immunohistochemistry experiments, we found that b-catenin level decreased in proximal tubules in 3-month STZ-induced diabetic rats and in patient with diabetic nephropathy. We concluded that AGE-inhibited cell proliferation and collagen production are dependent on GSK-3β in LLC-PK1 cells. Moreover, AGE-inhibted β-catenin and cyclin D1 protein expression are also dependent on GSK-3β. It was also the first report that b-catenin levels decreased in renal tissue of patients with diabetic nephropathy.

碩士
高雄醫學大學
生物化學研究所
88
Proximal tubule cells and fibroblasts are important in renal fibrosis in diabetic nephropathy (DN). Transforming growth factor-beta (TGF-beta)、 Insulin-like growth factor-I (IGF-I), high glucose (HG) and advanced glycation end-product (AGE) are also important in DN. Thus, we studied the interactions between LLC-PK1 (proximal tubule) and NRK-49F (fibroblasts) cells in terms of cellular growth (3H-thymidine incorporation, cell numbers and 3H-leucine incorporation, cellular hypertrophy), extracellular matrix (collagen synthesis by 3H-proline incorporation), TGF-beta/IGF-I expressions and the effects of antioxidants on the above events. We found that in NRK-49F cells, AGE-BSA (50、100g/ml)/HG (500mg/dl) increased 1.2 fold while LLC-PK1- conditioned media (CM) decreased cellular mitogenesis 30~40%. AGE/HG had no effects while LLC-PK1-CM increased cellular hypertrophy 1.2 fold. AGE/HG and LLC-PK1-CM also increased collagen synthesis 1.2~1.3 and 1.3~1.4 fold. Neutralizing TGF-beta antibody reversed LLC-PK1-CM-induced (but not AGE/HG-induced) growth inhibition, cellular hypertropy and collagen synthesis in these cells. Captopril and antioxidants (N-acetylcysteine, taurine) were also effective. In LLC-PK1 cells, AGE/HG decreased 30~40% while NRK-49F-conditioned media (CM) increased cellular mitogenesis 1.4~1.5 fold. AGE/HG increased 1.2 fold while NRK-CM had no effects on cellular hypertrophy. Both AGE/HG and NRK-CM increased collagen synthesis 1.2 fold. Additionally, neutralizing IGF-I antibody reversed NRK-CM-induced (but not AGE/HG-induced) proliferation and collagen synthesis. Captopril and antioxidants (N-acetylcysteine, taurine) were also effective. We conclude that AGE/high glucose induces TGF-beta/oxidants in LLC-PK1 cells to affect NRK-49F cells in terms of growth inhibition, cellular hypertrophy and collagen production. Additionally, AGE/high glucose induces IGF-I/oxidants in NRK-49F cells to affect LLC-PK1 cells in terms of mitogenesis and collagen production.

碩士
國立清華大學
化學系
104
The human S100 calcium-binding protein A11 (S100A11) is a member of S100 protein family. Once S100A11 proteins bind to calcium ions at EF-hand motifs, S100A11 will change its conformation promoting interaction with target proteins. The receptor for advanced glycation end products (RAGE) consists of three extracellular domains, including V domain, C1 domain and C2 domain. In this case, V domain is the target for mS100A11 binding. RAGE binds to the ligands result in cell proliferation, cell growth and several signal transduction cascades. We used NMR and fluorescence spectroscopy to demonstrate the interactions between S100A11 and V domain. The Tranilast molecule is a drug used for treating allergic disorders. We found out that V domain and Tranilast would interact with S100A11 by using 1H-15N HSQC NMR titrations. According to the results, we obtained two binary complex models from the HADDOCK program, S100A11-RAGE V domain and S100A11-Tranilast, respectively. We superimposed these two models with the same orientation of S100A11 homodimer and demonstrated that Tranilast molecule would block the binding site between S100A11 and V domain. We further utilized the WST-1 assay to indicate that Tranilast indeed can inhibit the cell proliferation which is induced by the S100A11-V domain interaction. These results will be potentially useful in the development of derivative or new anti-cancer drugs for RAGE-dependent diseases.

碩士
高雄醫學大學
生物化學研究所
88
In Taiwan, diabetes mellitus and end-stage renal disease is the 5th and 7th leading cause of death, respectively. Furthermore, diabetic nephropathy (DN) is a major cause of morbidity and mortality, which occurs in 20-40﹪of the diabetic patients. Therefore, the study of the pathogenesis of DN is an important endeavor in biomedicine. In this regard, high glucose, advanced glycation end-product（AGE）, ketone body transforming growth factor（TGF-β）and angiotensin Π have been proposed to be the essential factors. The hallmark of DN is cellular hyperplasia, hypertrophy and an expansion of extracellular matrix in the glomeruli and renal tubules. It often progresses to renal fibrosis（glomerulosclerosis and tubulointerstitial fibrosis）. Although most studies focused only on glomerular cells, tubulointerstitial change may be more closely correlated with the decline in renal function than glomerulopathy in DN. Thus, we studied the role of TGF-β in the effects of AGE, high glucose and ketone body on cellular growth, cellular hypertrophy and collagen synthesis in human proximal tubular cell line (HK-2). We found that AGE (100 μg/ml, but not BSA), high glucose (500 mg/dl) and ketone body (10 mM) decreased cellular mitogenesis while increasing cellular hypertrophy and collagen synthesis in HK-2 cells at 48hr. Similarly, exogenous TGF-β (1 ng/ml) also decreased cellular mitogenesis while increasing cellular hypertrophy and collagen synthesis at 48hr. AGE, glucose and ketone body also increased TGF-β protein expression (by ELISA) and TGF-β bioactivity (by mink lung epithelial cell inhibition assay). We also found that anti-TGF-β antibody can reverse the effects of AGE, high glucose and ketone body. In addition, AGE/HG and ketone body increased TGF-β responsiveness by increasing type II TGF-β receptor protein expression. We found that AGE, high glucose and ketone body activated Smads-DNA binding activity at 2-4 hr (by electrophoretic mobility shift assay). In contrast, all three treatments did not have effects on receptor-regulated Smads (Smad2 and Smad3) protein expression, but decreased inhibitory Smad (Smad7) protein expression. We also found that Smad2 and 3 antisense oligonucleotides and transcription factor decoy can reverse the effects of AGE, high glucose and ketone body. These results indicate that TGF-β pathway and transcription factor (Smads) play important roles in the pathogenesis of diabetic nephropathy. Angiotensin converting enzyme inhibitors (e.g., captopril) is effective in treating DN because they antagonize the hemodynamic and growth effects of angiotensin II on the kidney. In this regard, we found that captopril reversed AGE/HG and ketone body induced effects in HK-2 cells partly by decreasing endogenous TGF-β protein expression. Thus, angiotensin II is a renal growth factor which can induce cellular hypertrophy and extracellular matrix production in HK-2 cells by upregulating TGF-β. In conclusion, AGE, high glucose and ketone body may affect cellular growth and induce collagen synthesis in HK-2 cells by inducing the expression of TGF-β. The effects of TGF-β are in turn mediated by activation of Smad2 and 3 due to a decrease in Smad7 protein.

博士
高雄醫學院
醫學研究所
86
Diabetes mellitus and renal disease is the 5th and 7th leading cause of death in Taiwan. Hence, they consume a major portion of medical resources in our country. Furthermore, diabetic nephropathy is a major cause of diabetic morbidity and mortality. Unfortunately, diabetic patients will almost always enter end-stage renal disease (ESRD) once they develop proteinuria. Therefore, the study for the pathogenesis of diabetic nephropathy has become a major topic in biomedical research.Hyperglycemia and advanced glycation end-product (AGE) are two of the essential factors in diabetic nephropathy. The pathology of diabetic nephropathy is characterized by cellular hyperplasia, hypertrophy and the expansion of extracellular matrix which result in renal fibrosis and ESRD. These processes are intimately associated with cytokines/growth factors, esp. transforming growth factor-b (TGF-b). The distal nephron is also important in diabetic nephropathy, although most studies regarding diabetic nephropathy were focused on glomerulopathy and occasionally proximal tubule. Therefore, we performed a series of studies in high glucose and AGE-cultured distal tubule-like MDCK cells. We found that, unlike other renal cells, high glucose did not increase TGF-b production, but it did increase the responsiveness of MDCK cells to TGF-b, which includes: inhibition of cellular mitogenesis, induction of cellular hypertrophy, increase of cell cycle-regulatory retinoblastoma protein (pRb) dephosphorylation and inhibition of cdc2 kinase activity. Affinity-labeling experiments showed that high glucose may increase TGF-b responsiveness by increasing type I and II TGF-b receptor protein expressions. This is the first demonstration that distal tubule is unique in that it responds to high glucose by increasing TGF-b (which may be derived from paracrine sources) responsiveness but not the production of endogenous TGF-b.Regarding the roles of intracellular signal transduction pathways in diabetic nephropathy, protein kinase C (PKC) had been shown to be important. However, the roles of various PKC isoenzymes in diabetic tubulopathy is still not known. Thus, we showed that high glucose induced PKC activation, PKCi and PKCe activation, cytosolic translocation of PKCi(l) and membrane translocation of PKCe. As for the roles of various transcription factors, only AP-1 had been suggested to be involved in diabetic glomerulopathy. Thus, we first showed that high glucose induced activation of transcription factors AP-1 and NF-kB in MDCK cells concomitantly with the induction of type II TGF-b receptor mRNA. We speculate that the above changes in the signal transduction pathways may be involved in the induction of type II TGF-b receptor mRNA, although this speculation awaits further confirmation.Regarding the effects of AGE, we found that, unlike high glucose, AGE inhibited cellular mitogenesis while inducing cellular hypertrophy in the MDCK cells. Moreover, AGE induced the production of bioactive TGF-b in these cells. Importantly, neutralizing anti-TGF-b1 antibody reversed the above AGE-induced effects. Therefore, endogenous TGF-b1 may mediate the above AGE- induced effects in the MDCK cells.Our experiments showed that distal tubular cells behave differently from glomerular and proximal tubular cells in high glucose and AGE cultures. Whereas AGE induced bioactive TGF-b, high glucose only induced the expression of type I and II TGF-b receptors. We conclude that the complex interaction between high glucose, AGE, TGF-b, TGF-b receptors and cell cycle-regulatory proteins (pRb and cdc2) may play important roles in diabetic nephropathy. In addition, PKCi( l), PKCe, AP-1 and NF-kB may mediate some of the above high glucose and AGE-induced effects.

碩士
國立中興大學
食品暨應用生物科技學系所
102
Advanced glycation end products (AGEs) are produced from the Maillard reaction and present in food and biological systems. The present studies show that high levels of circulating AGEs are associated with diabetic complications, such as nephropathy, retinopathy and atherosclerosis.It is worth noting that male diabetic patients may also have reproductive dysfunction, and this dysfunction may result from the oxidative stress induced by AGE-mediated the receptor for AGEs (RAGE) activation. Sustained intake of food rich in AGEs may lead to an increase in endogenous AGEs and chronic oxidative damage. Taken together, it is interesting to clarify the effect of AGE diet on function of testes, kidney and pancreas in normal and diabetic anamals. Silymarin is a flavonoid with hepatoprotective characteristics and powerful antioxidant activity.Therefore, the other section of study was further investigated to clarify the biological actions of silymarin in animals fed an AGE diet. Our study is divided into two sections as follows: In part 1, we assessed the effect of different formula of AGE diet on testes, kidney and pancreas in male BALB/c mice and Sprague-Dawley rats, respectively, and the effect of the biological actions of silymarin. The results showed that AGE diet may lead to accumulation of AGEs in vivo, resulting in oxidative damage of testes and epididymis as well as a decrease in sperm count and motility. To investigate the intervention effect of silymarin, we found that silymarin had potential for prevention of AGE diet-induced oxidative damage. Based on the results of first part, AGE diet was shown to cause accumulation of AGEs. Because the circulating AGEs are closely related to the progression of diabetic complications, it is speculated that diabetic damage of testes and epididymis may be augmented by AGE diet. In part 2, we investigated the effect of AGE diet on STZ-induced type 1 diabetes mellitus (DM) SD rat as well as the intervention effect of silymarin. The results showed that DM-induced oxidative damage led to a decrease of sperm count and motility, and AGE diet may aggravate related lesions, such as an increase of abnormal sperm rate. However, silymarin elevated activity of antioxidant enzymes of testes and ameliorated the lipid peroxidation in pancreas. Silymarin showed the potential for improving DM-induced oxidative damage in vivo; however, it did not significantly improve the STZ and AGE diet-induced decline of sperm count and diffuse spermatic degeneration/necrosis of epididymis. We speculate that the related damage of animals may be difficult to restore; thus, we cannot observe the expected results in related analyses. In conclusion, diversity and preparation method of diet, experimental models, and animal strain might be the reasons why partial results are inconsistent. Based on all results, we summerize that AGE diet-induced oxidative stress are closely related to abnormalities of sperm in rodents. Additionally, administration of silymarin indeed significantly promoted antioxidant actions within testes, kidney and pancreas in rodents, suggesting that silymarin has potential to inhibit the generation of lipid peroxides and improvement of oxidative damage in vivo.