Protection And Mechanism Of Phlorizin On Retinopathy In Db/db Mice

Part OneEffects of Phlorizin on Retinopathy in db/db MiceBackgroundDiabetes is a metabolic disorder characterized by hyperglycemia and insufficiency of secretion or action of endogenous insulin as well as insulin resistance. Nowdays the incidence of diabetes mellitus (DM) has increased worldwild due to various reasons, including the elevation of living standards, the modem life styles and aging. DM has presented a severe social problem, affecting both the health and quality of life among publie.DM not only develops impaired glucose metabolism, but attacks both macrovascular and microvascular damages throughout the body. Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes and the worst ocular complications of diabetes. Nearly all people with type1and more than half with type2diabetes develop retinopathy. DR remains the leading cause of visual loss and acquired blindness among working-age adults in the industrialized world. Person with DR are25times more likely to become blind than individuals without diabetes. With an ongoing pandemic of DM. more concerns have been focused on the DR expressing an consistanl increase of incidence. The present measures to treat DR are systemic therapy (tight control of glucose and blood pressure) in conjuction with ocular approaches (laser photocoagulation, surgical intervention) as needed. However, current therapeutic options for treating DR, as described above, are limited by considerable side effects and are far from satisfactory. In spite of present managements, DR has remained difficult to prevent or arrest. Thus, there clearly is a strong incentive to search for potential candidates combating DR.Phlorizin, a phloretin glucoside, is a dihydrochalcones and is maily distributed in the root bark, stem, leaves and fruit of apple trees. Phlorizin makes the major sources of flavonoid. Phlorizin has been reported to possess various properties, including being anti-inflammatory, anti-tumorigenic, antioxidative, and having the ability to lower glucose concentration and improve memory. Recently a series of studies were conducted using phlorizin to curb diabetic complications. In Ins2Akita mice, chronic phlorizin treatment further normalized cardie dysfunction and alleviated ventricular hypertrophy. Also, phlorizin has been reported to inhibit aorta lesion and hepatic damage in db/db mice. In streptozotocin-induced diabetic rats, phlorizin prevented proteinuria, hyperfiltration and kidney hypertrophy, alleviating the early renal functional and preventing some structural changes in diabetes. T-1095, a derivative of phlorizin, suppressed the development of albuminuria and the expansion of the glomerular mesangial area in db/db mice, indicating that the progression of diabetic nephropathy was prevented. Though having evidence that phlorizin treated vascular comlications of DM, no reports have shown the effect of phlorizin on retinopathy in db/db mice. Therefore, We took db/db mice, generally popular applied, as an animal model for type2DM to observe the retina cells apoptosis, detected by Terminal transferase dUTP nick end labeling (TUNEL) and to measure glial fibrillary acidic protein (GFAP) expression and in retinas using western blotting analysis among the control, db/db mice, and phlorizin treated db/db mice. Meanwhile, the expression of glycogen synthase kinase-3beta (GSK-3β) and phosphorylation GSK-3β were also monitored in retinas. The purpose of this study was to examine the effects of phlorizin on DR and to explore the mechanisms underlying the phlorizin therapy and the onset and progression of DR, thus providing new approaches for the management of DR.Objectives1. The aim of the study was to characterize the early pathological changes of retina in db/db mice, including retinal cells apoptosis and the GFAP expression in db/db mice. 2. The aim of the study was to examine the effects of phlorizin on the retinal cells apoptosis and the GFAP expression in db/db mice.3. The aim of the study was to examine the effects of phlorizin on the expression of GSK-3β and phosphor-GSK-3β in db/db mice, and to explore the mechanism by which phlorizin treated DR, further to provide new approaches for the management of DR.MethodsMale C57BLKS/J db/db(n=16, seven weeks old) and db/m mice (n=8, seven weeks old) were purchased from the Model Animal Research Center of Nanjing University (Jiangsu, China). The mice were kept under observation for one week before the experiments started. C57BLKS/J db/m mice were selected as the control group (CC, n=8). The db/db mice were divided into2groups:an untreated diabetic group (DM, n=8) administered normal saline solution by intragastric gavage and another diabetic group treated with a dosage of20mg/kg of phlorizin (DMT, n=8) for ten weeks. All mice were weighed regularly during the experiment. At the end of the intervention, all mice were fasted overnight and then sacrificed. Fasting blood was collected, and serum advanced glycation end products (AGEs) specific fluorescence determinations were measured. Afterwards, the whole eyes from the controls, db/db mice and db/db mice treated with phlorizin were immediately enucleated and fixed in4%paraformaldehyde and embedded in paraffin. Then the retinas were dissected. The retina sections were then isolated and cut using standard histological procedures. To determine whether retinal cells apoptosis was influenced by diabetic state and treatment of phlorizin. the TUNKL method for detecting DNA breaks in situ was applied to retinal tissue. Meanwhie, the expressions of GFAP, GSK-3β and phosphor-GSK-3β in retinas were also monitored. Moreover, some retina tissue were kept at-80℃until further protcomic analysis.Results1. General characteristicsDuring the study, the CC group mice showed good condition and grew up sound with smooth furs. No diabetic related symptoms were shown in this group. The db/db mice featured polydipsia, polyphagia and polyuris with shaggy furs and a rapid weight gain. Phlorizin treated db/db mice also displayed abnormal, much better than the db/db mice.2. Effects of phlorizin on body weight, FBG and AGEsBaseline body weights were similar between DM group and DMT group (P>0.05). The elevated body weights in DM group and DMT group were found comparing with the CC group (P<0.01). At as early as week2of the study, the body weights in DM group and DMT group showed signs of increase. This trend did not change until the end of the experiment. However, the body weight was significantly inhibited at10weeks,12weeks,14weeks,16weeks, and18weeks after phlorizin administration in the DMT group compared to the DM group (P<0.01).Baseline FBG and AGEs were similar between DM group and DMT group (P>0.05). The elevated FBG and AGEs in DM group and DMT group were found comparing with the CC group (P<0.01). Phlorizin administration in the DMT group for week10caused a reduction in FBG and AGEs, as compared wth DM group (P<0.05).3. Effect of phlorizin on diabetes-induced retinal cells apoptosisTUNEL array was performed to detect the retinal cells apoptosis following counterstained with hematoxylin. TUNEL-positive cell was defined as cell with brown-coloured in nucleus after staining. Almost no TUNEL-positive cell staining was detected anywhere in the retina in the CC group. The nucleuses of the cell were in the state of normal structure, showing blue under the hematoxylin staining. The number of TUNEL-positive cells in db/db mice was significantly higher than those of the CC group (P<0.05). The nucleuses of the apoptosis cells were in the state of irregular structure, showing brown staining. TUNEL-positive cells were predominantly located in the ganglion cell layer and in the vascular endothelium. However, Treating db/db mice with phlorizin significantly reduced the number of TUNEL-positive cells(P<0.05).4. Effect of phlorizin on GFAP expression with western blottingGFAP expression, monitored with western blotting analysis, increased in retinas of db/db mice compared with the control group. In contrast, phlorizin treatment downregulated the retinal GFAP expression in db/db mice.5. Effects of phlorizin on the expressions of GSK-3β and phospho-GSK-3βThe expression of phospho-GSK-3β with western blotting decreased in db/db mice when comparing with the control, however, this effect was partially ameliorated with phlorizin treatment.Conclusion1. Phlorizin-treated db/db mice experienced total body weight reduction.2. Phlorizin caused significant decrease both in FBG and in AGEs of db/db mice.3. Phlorizin remarkably inhibited diabetic retina cells apoptosis and downregulated GFAP expression in retinas, suggesting that phlorizin could be of potential benefit in preventing diabetic retinal damage.4. Phlorizin could ameliorate phospho-GSK-3β expression in retinas and suppress the GSK-3β activity, which led to the inbibition of retinal cells apoptosis and the protection of DR. Part TwoiTRAQ-based Proteomic Analysis on the Protective Mechanism of Phlorizin on Retinopathy in db/db MiceBackgroundThe completion of the draft version of human sequence, as an important part of the Human Genome Project, marked the start of a novel era-the postgenone era. It has been recognized that the gene is the only carrier for genetic information and that the proteins facilitate all biological function and carry out the duties specified by the information encoded in genes. Thus, researches concerning the structure and function of proteins help to explore the mechanisms underline the life response to psychology and pathology. However, traditional research methods for one-by-one protein makes it hard to meet the needs of the functional genome, therefore, exploring the nature of life depends on the research of proteins focusing on the global view, dynamic level and network information, hence the advent of proteomics. Proteomics refers to the global analysis of entire protein complement of an organism at a given time. Thus, proteomics remains a milestone of life science in the postgenome era.With the continuing improvement of proteomic research, it is hard for the proteomes of qualitative analysis to meet the needs for the rapidly growth. Thus, more attention has been paid to the proteomes of dynamic quantitative analysis, rather than that of stationary qualitative analysis. Hence quantitative proteomics was approaching. Quantitative proteomics is one of the major components of functional proteomics that identifies systematically the proteins in complex samples and determines their quantity or quantitative change. Quantitative proteomics pays more attention to the dynamic alterations of proteins in cell, suggesting the informations on the structure and function of given cell. Therefore, quantitative proteomics profiling is often applied to study and to identify the differentially protein expression occurring with state of disease as well as drug intervention. Thus, quantitative proteomics is anticipated to provide insights into the function of biological processes, facilitate the identification of diagnostic or prognostic disease markers, and contribute to the discovery of proteins as therapeutic targets. This field is growing rapidly and new applications and protocol design are driving technological innovations. Recently a novel, MS-based approach for the relative quantification of proteins, using isobaric tag for relative and absolute quantitation (iTRAQ) is presented, which is regarded as one of the important array for quantitation in proteomics due to its advances. iTRAQ technology, a popular relative quantitative measurement, has provided a means for subtractive analysis where one can target a particular cell mechanism, which in turn provides information about the function of those proteins that are upregulated or downregulated. iTRAQ technology has many roles to play in proteomics research, including exploring the onset of diseases, uncovering the new biomarkers, drug targets validation and new drug development.As a kind of natural flavonoid compound, phlorizin is widely distributed in the root bark, stem, leaves and fruit of apple trees. It has been demonstrated that phlori/.in possesses a variety of potent properties, pharmacological activities, and clinical benefits, including anti-oxidation, free radical scavenging, regularing glucose concentrations and blood pressure, cardioprotection, anti-tumor, and memory improvement. Recently a series of studies were conducted using phlorizin to curb diabetic complications, including diabetes-induced myocardial hypertrophy, aorta lesion, albuminuria and diabetic retinal pericytes lesions. Our previous study addressed in the part one showed that phlorizin had a protective effect on retinopathy in db/db mice. But the mechanism underlying the protection against retinopathy lesions remains unclear, and so does the molecular targets by which phlorizin treated diabetic retinopathy.There is a close relationship between drug treatment, protein expression, and resulting physiological effects. Most of the time, pharmacological intervention results in the regulation or modulation of gene-product expression, in a similar way that complex disease processes, e,g, diabetes induced metabolic dysfunction, alter global protein expression. From this, it could be ascertained that an ideal drug is one that restores global protein expression of a disturbed system to a normal state. Drug researches traditionally focuses on the phenotypic observation only, which make it hard to detect the pharmacological activity and to identify the targets of drug. However, quantitative proteomics, coupling with pharmacy study, provides a means for the potential development of new pharmacological molecules. In practice, it is desirable to analyse proteins expression alterations after drugs administration using quantitative proteomics with high throughout. Thus, quantitative proteomics could help to explore the mechanisms behind the drug, uncover the therapeutic targets, and contribute to the processing of drug actions. From this, quantitative proteomics could provide new approaches for researching and developing drugs.In the part2of this study, We separated and identified differently expressed proteins among the control db/m mice, db/db mice and phlorizin treated db/db mice using iTRAQ technology, followed by urbo SEQUEST program software and international protein index (IPI) mouse protein database. The purpose of this experiment was to explore the mechanisms by which phlorizin curbed diabetic retinopathy, to uncover discovery of proteins as therapeutic targets, and to develop potential for treating diabetes induced retinal lesion.Objective1. The aim of the study was to characterize the differently expressed proteins, further to dermine the mechanism of the onset of diabetic retinopathy.2. The objective of this study was to identify retinal proteomic alterations associated with db/db mice and phlorizin treatment and to uncover discovery of proteins as promising drug target. Also, this study was performed on purpose of determining the mechaniam underlying the protection of phlorizin against diabetic retinopathy and in order to provide novel approaches for the treatment of diabetic retinopathy.MethodsThe retinas tissue was dissected among the control, db/db mice, phlorizin treated db/db mice,4from each group, respectively. The processing of proteomic analysis is as follows. Firstly, the separated retinas tissue sample were prepared for following array using universal procedure, including tissue disruption, sample clean-up, and protein solubilization. After that, the concentrations of the proteins can be estimated using Bradford array. Then the retinas tissue were kept at-80°until further analysis. About20ug of peptides of each group were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to detect the similarities of protein bands loaded on the gels among the three groups for further study. Then, the proteins were digested followed by iTRAQ labeling, according to ABI manufacturer’s instructions (Applied Biosystems). About60ug peptides of each group were labeled with iTRAQ reagents (114for the peptides of C group,116for the peptides of DMT group, and117for the peptides of DM group, respectively). All three labeled samples were finally combined together following by separated using Strong Cation Exchange (SCX) chromatography into10fractions and by desalted by an offline fraction collector and C18cartridges (Sigma). Of note, After labeling, an amount of peptides among each group were performed on mass spectrometric analysis using MALDI-TOF/TOF (ABI4800, USA) to evaluate the effectiveness of reagents labeling base on the observation that the peak for reporter ions are encoded in the low mass-to-charge ratio portion of the MS/MS spectrum. In part2of present study, Mass spectrometric analysis was performed using a micro liquid chromatography system (MDLC, GE Healthcare) and a LTQ-Velos ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA). The separation column was a0.15mm×150mm capillary packed with Zorbax300SB-C18particles (Agilent Technologies). Mobile phase A (0.1%formic acid in water) and the mobile phase B (0.1%formic acid in ACN) were selected. MS data were acquired using data-dependent acquisition conditions:each MS event was followed by zoom/MS2scans on the five top-most intense peaks. For protein identification and statistical validation, the acquired MS/MS spectra were automatically searched against the non-redundant International Protein Index (IPI) mouse protein database (version3.72) using the Turbo SEQUEST program in the BioWorksTM3.1software suite. Data filtering parameters were chosen to generate false positive protein identification rates of<1%, as calculated by searching the MS2scans against a forward reversed database of proteins. A threshold was set to1.5with a P-value<0.05yielding at least a50%change in abundance compared to the reference (114, control group). All identified proteins were classified by their molecular function, biologicai process, and cellular component using AmiGO (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi Version1.8). Finally, some candidated differentially expressed proteins in the retinal were validated with Western blotting analysis.Results1. Mass spectrometry identify the differently expressed proteinsProtein profiling was analyzed using iTRAQ approach followed by LC-MS/MS identification and IPI database searching. A total of1,651proteins were identified. Of them,1636proteins meet the standard. Also,8972unique peptides were observed by mass analysis. Among them,348proteins were differentially expressed in diabetic retina in comparison to control, comprised of177proteins that were increased and171proteins that were decreased. Moreover, in order to examine the effect of phlorizin on the proteome change, proteome analysis was also conducted on the phlorizin treated diabetic retina. Of the significantly changed proteins between DMT group and DM group,33proteins were down-regulated with the treatment of phlorizin, while27proteins up-regulated.2. Subcellular localization analysis phlorizin associated retina proteins in db/db miceThe localization analysis of the identified proteins in retinas using AmiGO (Version1.8) is shown in our study. The proteins are associated with many subcellular locations, including cytoplasm, nucleus, plasma membrane, mitochondrion and endoplasmic reticulum. The two largest proportion of changed proteins were locates in cytoplasm(33.87%) and nucleus(33.87%). Of note, γ-crystallin was in cytoplasm and in nucleus, while glutaredoxin-3in cytoplasm only.3. Subcellular bioinformatic functional analysis phlorizin associated retina proteins in db/db miceMost of these back-regulated proteins were involved in oxidative stress, apoptosis, energy metabolism and signaling transduction. Among the functional assignment of the proteins,55.00%were in metabolic processes,16.67%in cytoskeleton,6.67%were in stress response,6.67%were in immune response,6.67%were in transport,3.33%in extracellular matrix. Obviously, proteins involved in metabolic processes constituted the largest functional group.4. Validation ofiTRAQ data on selected candidate proteinsTo provide confirmation of differentially expressed protein, two candidate proteins were validated using Western blotting analysis, y-crystallin was found to be inhibited whereas Glrx-3was enhanced in the DMT group compared to the DM group. This result verified the reliability of the iTRAQ results.Conclusion1. As a novel technology of quantitative proteomics, iTRAQ enables an analysis of up to four samples simultaneously in one experiment with more advantages, including high-throughout, accuracy, and high-sensitivity. Thus, in our study with the help ofiTRAQ, it is possible to explore the mechanism underline the protection of phlorizin against diabetic retinopathy and to uncover discovery of proteins as drug tagets.2. A total of60proteins differentially changed occurring with phlorizin treatment in retinas of db/db mice were identified using LC-ESI-MS/MS methods. Of these,33proteins were downregulated while27were upregulated with phlorizin administration. Most of these differentially changed proteins were located in cytoplasm and were related to special functions, including signal transduction, cytoskeleton, metabolic process, apoptosis and oxidative stress. Our finding indicted the above-mentioned mechanisms and processes are linked to the onset and pathogenesis of diabetic retinopathy.3. Phlorizin treatment significantly down-regulated gamma-crystallin in retina of db/db mice while up-regulated glutaredoxin-3. The findings confirmed using Western blotting analysis were consistent with the results from iTRAQ array. Therefore, it could be supposed that gamma-crystallin and glutaredoxin-3might be the critical proteins related to the protection of phlorizin against diabetic retinal lesions.4. Our present study has provided valuable results in highlighting the protection of phlorizin against diabetic retinapathy to better understand the diabetic retinal damage and phlorizin treatment processes and to discover potential therapeutic targets for curbing diabetic-induced retinal lesions.