| BackgroundDiabetes mellitus(DM) is a disease of metabolic disorder characterized by hyperglycemia and insulin resistence accompany with insufficiency of secretion or action of endogenous insulin. It occurs under abnormal genetic and environmental factors, with the development of society and economy and improvement of living standard. The prevalence of DM in the world is rising. Lifestyle changes and social ageing population have accelerated this trend. DM has become a public health problem after following the cardiovascular and cerebrovascular disease and tumor. DM is alsothe main cause of death, disability and medical expenses. The damage of DM is not only for its persistent hyperglycemia, but also the multi-system damage caused by hyperglycemia and metabolic disorders such as chronic heart, blood vessels, kidneys, nerves, retina, peripheral nerve, brain and other tissues.Diabetic nephropathy(DN) is a serious and progressive complication of diabetes and occurs in30-40%of diabetic patients. DN is emerging as the leading cause of end-stage renal failure in developed country and the second cause in our country. The pathophysiologic changes of DN include hemogynamic alterations such as hyperfiltration and hyperperfusion. The structural changes of kidney in diabetes consist of glomerular and tubular hypertrophy, followed by thickening of glomerular and tubular basement membranes and progressive accumulation of extracellular matrix in the mesangium and the interstitium, progressive glomerulosclerosis and tubulointerstitial fibrosis, and worsening of renal function. The pathogenesis of DN remains uncertain, and the overall mechanisum are complex and multifactorial, including oxidative stress, accumulation of advanced glycated end product(AGEs), genetic susceptibility. However, although positive effects on the development and progression of DN through strict control of blood glucose, blood pressure, and blood lipids, smoking cessation, in particular, blockade of the renin-angiotensin system have been reported, these treatments have not been enough to prevent the high incidence of end-stage kidney damage caused by diabetes. Therefore, it is important to develop novel therapeutics that allow for the prevention and retardation of DN.Phlorizin is a natural phenolic compounds. It can be obtained from the apple roots, barks, stems, leaves and fruits. Molecular structure of phlorizin is C21H24O10. Phlorizin first isolated from the bark of the apple tree by French chemists in1835, is a member of the chalcone class of organic compounds. Rearch data show that95%polyphenols in apple trees is phlorizin. It has been reported that phlorizin have possessed a variety of potent proterties, including lowering blood glucose, anti-oxidation, anti-aging, anti-tumor. Phlorizin is also endothelial protective by suppressing endothelial adhesion molecule expression and platelets activation. In addition, phlorizin also has a variety of biological activity, such as the regulation of blood pressure, protection of heart and removing free radicals. Although phlorizin has been reported to be effective in reducing the blood glucose level in DM, little is know about its effects on renal complication. In this study, we used phlorizin to treat diabetic (db/db) mice to explore the effects of phlorizin on DN and to determine the underlying mechanism, db/db mice are well-established animal model to study T2DM complications.Through this experiment, possible protective mechanisms of phlorizin on DN can be found. This study provide new ideas and approaches for the clinical treatmet of DN.Objectives1. To observe the effects of phlorizin on FBG, TC, TG, AGEs, BUN, Cr, UA and24h urinary protein in db/db diabetic mice to explore the mechanism. And to explore the mechanisms of phlorizin’s kidney preventing effects. 2. To study the characteristic of kidney in db/db mice, observe the renal pathological changes by light and electron microscope and the effects of phlorizin on the kidney of db/db mice.MethodsMale C57BLKS/J db/db mice (n=16,7weeks old) and db/m mice (n=8,7weeks old) were purchased from Model Animal Research Center of Nanjing University (Jiangsu, China). The mice were kept under observation for one week prior to the start of the experiments. C57BLKS/J db/m mice were selected as control group (CC, n=8). The db/db mice were divided into2groups:an untreated diabetic group (DM, n=8) administrated with normal saline solution by intragastric administration and phlorizin-treated group with a dosage of20mg/kg/d (DMT, n=8) for10weeks. All mice were weighed every week during the experiment. At the end of the intervention,24h urine specimins of all mice were collected in metabolic cages. All mice were fasted overnight and then sacrificed. Fasting blood was collected, and the kidneys were dissected. Renal tissues and sera were kept at-80℃for further analysis. Fasting blood glucose, TC, TG, AGEs,24h urinary protein, BUN, Cr and UA were measured. Morphological changes of the kidney were examined by light microscopy and H-800electron microscope.Results1. General characteristicsIn the course of experiment, the mice of CC group showed good condition with smooth furs. No diabetic symptoms were shown. The mice of DM group showed polydipsia, polyphagia and hyperdiuresis with filthy furs, rapid growth and obesity. Phlorizin treated db/db mice also displayed abnormal, but better than the db/db mice.2. Effects of phlorizin on body weightsBaseline body weights were similar in mice of the DM group and the DMT group, and higher than the mice of the CC group. At as early as week2of the study, the body weight in db/db mice group was significantly heavier than that of the control mice(P<0.05). This trend did not change until the end of the experiment. However, the body weight was significantly inhibited at2th week,4th week,6th week,8th week, and10th week after phlorizin administration in the DMT group compared to the DM group (P<.05).3. Effects of phlorizin on FBG, TC, TG, and AGEsThere was no statistical significance of FBG and AGEs between DM group and DMT group at the beginning of experiment (p>0.05), but were significantly higher than CC group (p<0.05). The serum FBG, TC, TG and AGEs were measured at the end of experiment. The FBG, TC, TG and AGEs of DM group were significantly higher than CC group (p<0.05). Phlorizin significantly reduced the serum FBG, TC, TG and AGEs of db/db mice (p<0.05).4. Effects of phlorizin on24h urinary protein, BUN, Cr, and UAThe24h urinary protein and serum BUN, Cr and UA were measured at the end of experiment. The24h urinary protein, BUN, Cr and UA of DM group were significantly higher than CC group (p<0.05). Phlorizin significantly reduced the24h urinary protein, BUN and Cr (p<0.05). However, compared DM group, DMTgroup exhibited a slight decrease in serum UA that did not achieve statistical significance (p>0.05).5. Effects of phlorizin on morphology of kidneyThe microscopic images of the sections of the control group appeared normal. The main findings of light microscopy were a variable degree of mesangila expansion and an increase in the total glomerular size in DM group in comparison with CCgroup. Phlorizin treatment significantly reduced the extent of mesangila expansion and total glomerular area in db/db mice.6. Effects of phlorizin on ultrastructure of kidneyUnder electron microscopy, the glomerular base membranes(GBM) were observed to be irregularly thickened and diffuse mesangial matrix deposits were seen in DM group. In addition, increased foot process width, foot process fusion, decreased podocyte number and podocyte hypertrophy were seen. In DMT group, GBM thickness and podocyte number were similar to those in CC group.ConclusionPhlorizin can decrease the body weight of the db/db mice. Phlorizin also decreased the FBG, TC, TG and AGEs of the db/db mice. In addition, phlorizin reduced the24h urinary protein, lowered BUN and Cr, ameliorated the pathological damage of diabetic nephropathy. The protective effects of phlorizin on diabetic nephropathy can be attributed to its antiglycation, decreasing blood lipid and antioxidant effect. Perhaps new medications can be found to effectively treat diabetic nephropathy through penetrating study on phlorizin. Part TwoProteomic Analysis of Protective Effects of Phlorizin on Diabetic Nephropathy in db/db MiceBackgroundWith the completion of the human genome framework, life science has turned to the post-genomic era. Gene is just genetic information carrier, while the protein is the major executor of life activities. So proteomics has become the core content of post-genomic era. Proteomics is the biggest prospects in the field of pharmaceutical research. Researching on proteins of cells, organs and body fluids takes the important role in the drug development. It includs both proteins translated directly from genetic material and the modified proteins arising from extensive post-translational processing. Quantitative proteomics is the major part of functional proteomics, qualitatively and quantitively identifying the whole proteins of the cells. Quantitative proteomics focusing on the dynamic structural and functional alterations of the proteins. Theremore, quantitative proteomics profiling is applied to identify the differentially expressed proteins under disease or after drug intervention. With the development of proteomics, a new quantitative approach based on the mass spectrum using isobaric tag for relative and absolute quantitation (iTRAQ) has become the main method with its unique superiority. In2004, American Applied Biosystem Company developed iTRAQ. It can carry out four samples under the same experimental conditions. The sample can be labeled almost all proteins by iTRAQ, so phosphorylated-proteins and glycosylated-proteins also can be researched. The labeling process can be completed at room temperature for1hour on the simpler procedure. iTRAQ enables the detection and quantitation of differentially expressed proteins, and analyzes the protein function. It has been widely used in finding the pathogenesis, disease markers, the differentially expressed proteins at different state, and so on. iTRAQ labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful quantitative proteomics technique, which has been showen lessen variation, enhance throughput, and enable accurate quantitative analysis.Phlorizin has been proved to possess multiple pharmacological activities, such as anti-oxidative, regulation of blood sugar level, and so on. Phlorizin has demonstrated protective effects on kidney in the db/db mice. But the molecular targets and the underlying mechanism of phlorizin are unclear. In the second part, we identified differentially expressed proteins in kidneys of the control db/m mice, diabetic db/db mice and phlorizin treated db/db mice using the iTRAQ labeling combined with LC-MS/MS, followed by urbo SEQUEST program software and international protein index (IPI) mouse protein database. The purpose of this experiment was to explore the still unknown molecular networks and underlying mechanisms of the protective effects of phlorizin on diabetic nephropathy.Objective1. The purpose of the study was to obtain the differentially expressed proteins of kidney in db/db mice by iTRAQ, further explore the possible underlying mechanisms of diabetic nephropathy.2. To establish the renal differentially expressed proteins of db/db mice after treatment of phlorizin, evaluate the protective effects of phlorizin on diabetic renal damage.MethodsThe renal tissue was dissected among the control, diabetic db/db mice, phlorizin treated db/db mice. The processes of proteomic analysis were described as follows: The separated renal tissue sample were prepared according to the iTRAQ procedure, including tissue disruption, sample clean-up, and pretein solubilization. The protein concentrations were estimated by Bradford arrary. Then the renal tissues were kept at-80℃for further analysis. About20ug peptides of each group were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were digested and iTRAQ labeled, according to ABI manufacturer’s instructions (Applied Biosystems). About60ug peptides of each group were labeled with iTRAQ reagents (114for the peptides of CC group,116for the peptides of DMT group, and115for the peptides of DM group respectively). All the labeled samples were finally mixed together, separated by Strong Cation Exchange (SCX) chromatography into10fractions and finally desalted by an offline fraction collector and C18cartridges (Sigma). After being labeled, 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, which was based on the observation of the peak for reporter ions encoded in the low mass-to-charge ratio portion of the MS/MS spectrum. In the second part, mass spectrometric analysis was performed by 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 by data-dependent acquisition conditions. 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. A threshold was set to1.5with a P-value<0.05yielding at least a50%change in abundance compared to the reference (Use114as the reference). All identified proteins were classified according to the molecular function, biologicai process, and cellular component by AmiGO (http://amigo.geneontology.org/cgi-bin/amigo/go.cgi Version1.8).Results1.SDS-PAGE imageProteins brand loaded on the gels separared by SDS-PAGE were observed clearly, available for the following experiments.2. iTRAQ labeling statistical validationOur results showed that only10percent of the peptides were significantly different between two groups. Protein labeling experiments showed good uniformity.3. Mass spectrometry identify the differently expressed proteinsUsing the iTRAQ labeling compined with LC-MS/MS, we analyzed the effect of phlorizin on the renal protein profile of diabetic db/db mice. The relative quantitation was determined as two pairwise ratios:the DM group against the control group(iTRAQ115/iTRAQ114) and the DMT group against the control group(iTRAQ116/iTRAQ114). Differentially expressed proteins with more than1.5fold change relative to the control were used for the analysis. We identified2842proteins in this study. proteins were shown to have significantly different abundance between control group and DM group (±1.5-fold). A total of121proteins were normalized by phlorizin treatment. Of the121differentially expressed proteins,42were elevated in the DM group compared with the control group. However, the expression levels of these proteins were decreased by phlorizin treatment. An additional79proteins were decreased in the DM group compared with the control group, but these were restored by phlorizin treatment.4. Subcellular localization analysis of differentially abundant phlorizin associated renal proteinsThe localization analysis of the identified proteins was performed using AmiGO (Version1.8). Among these proteins, some are located in one or more subcompartments of the cell, were in cytoplasm, in nucleus, in plasma membrane, in endoplasmic reticulum, in mitochondrion, in extracellular, in centrosome, in ribosome, in Golgi, in lysosome.5. Ingenuity pathway analysis of differentially abundant phlorizin associated renal proteinsIngenuity Pathway Analysis (IPA) showed that the primary pathway involved was lipid metabolism, free radical scavenging and molecular transport, renal and urological disease. The pathways would facilitate the understanding of diabetes biomarkers for further study.Conclusion1. As an advanced technique of quantitative proteomics, iTRAQ enables comparative analysis for many groups. iTRAQ can analyze four samples synchronously with higher accuracy and sensitivity. With the help of iTRAQ, we obtained the reliable differentially expressed proteins from CC group, DM group and DMT group.2.2842proteins were shown to have significantly different abundance between control group and DM group. A total of121differentially expressed proteins were identified in kidney of db/db mice after phlorizin treatment by LC-MS/MS methods. Of these,42proteins were down-regulated while79were up-regulated after phlorizin administration. Some of the proteins were in the pathways involved in lipid metabolism, free radical scavenging, and molecular transport. Our results suggested that these processes may be linked to the pathogenesis of diabetic nephropathy. Phlorizin prevent diabetic nephropathy by regulating the expression of a series of proteins. |
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