Investigation Of The Protective Effects Of Phlorizin On Cardiac Damage In DB/DB Mice By Quantitative Proteomics

Investigation of the Protective Effects of Phlorizin on Cardiac Damage in db/db Mice by Quantitative ProteomicsBackgroundDiabetes mellitus (diabetes mellitus, DM) is a common endocrine and metabolic disease caused by absolute or relative insulin secretion defect and characterized by chronic increased blood glucose levels. In recent years, with the improvement of the living standard, modern life styles and aging, the prevalence of diabetes mellitus is rapidly increasing worldwide. It has become a worldwide public health problem and a serious threat to human health and quality of life. DM not only develops sustained elevated circulating glucose, but can be accompanied by a variety of complications attacking both macrovascular and microvascular lesion leading to a variety of complications. Among these, cardiovascular complication has become one of the most harmful complications with the highest morbidity and mortality.Diabetic heart disease is the main cause for death of diabetic patients, especially in type2diabetes. Broadly-defined concept of diabetic heart disease includes coronary heart disease, diabetic cardiomyopathy and diabetic cardiovascular autonomic neuopathy. Patients with diabetes often develop hypertension and atherosclerosis leading to cardiovascular complications. However, some diabetic patients develop heart failure without hypertension and coronary artery disease. This phenomenon was first described by Rubler et al. and was termed "diabetic cardiomyopathy"(DCM). Diabetic cardiomyopathy is characterized by structural and functional changes in the heart, such as elevated left ventricular (LV) mass, myocardial fibrosis, and abnormal diastolic function, eventually progress to arrhythmia, heart failure and cardiogenic shock. However, the mechanistic details of diabetic cardiac damage remain unclear, and this disease has not yet been sufficiently studied. The clinical treatment of diabetic cardiac damage focuses on controlling DM, preventing myocardial damage, arrhythmia, heart failure and antithrombotic therapy. So far, current therapeutic options for the treatment of diabetic heart disease, as described above, have certain limitations and are far from satisfactory. Therefore, there is an urgent need to find the more effective approaches for the intervention of diabetic heart disease.Phlorizin (phloretin-2’-O-glucoside, PHL), a dihydrochalcone derived from apple peels, bark and leaves, is a known antioxidant. Previews studies have shown that PHL has many biological activities and pharmacological actions, such as blood glucose regulation, anti-inflammation, anti-tumor, cognition improvement, antioxidative action and so on. It has been used in medicine, cosmetics, food and other fields. The main pharmacological property of phlorizin is to produce renal glycosuria and block intestinal glucose absorption through inhibition of sodium/glucose cotransporters in the kidney and intestine. In addition, animal experiments showed that long-term oral PHL administration has protective effect on DM complications related to targeting organs such as the kidney, heart, aorta and retina. Although cardioprotective benefits of phlorizin have been reported, little is known about the effect of phlorizin on cardiac damage in type2diabetes mellitus (T2DM).Isotope labeling relative and absolute quantitative (isobaric tags for relative and absolute quantitation, iTRAQ) technology is a new proteomics quantitative research technique in recent years, which has good quantitative effect, high repeatability, and can be used for separate up to four different samples at the same time. This technology can help researchers find differentially expressed proteins, analyze the protein function, and conduct accurate identification and quantitation of all the proteins present in a genome or a complicated hybrid system.In this study, we used phlorizin to treat T2DM in db/db mice, a common used diabetic animal model, for10weeks to observe the effects of PHL on their body weight, blood glucose, blood triglycerides, blood cholesterol and blood AGE levels.We also observed the morphological changes in histopathology and ultrastructure of db/db mouse heart. Additionally, we applied proteomics relative quantitative techniques iTRAQ, bioinformatics methods like Turbo SEQUEST software and international protein index (international protein index, IPI) database retrieval to separate and identify differentially expressed proteins in myocardial tissue of mice in normal control group (CC group), untreated db/db diabetic mice group (DM group) and PHL treatment db/db mice group (DMT group), which aims at revealing mechanism of protective effect of PHL for diabetic cardiac damage in db/db mice. It provides a novel way to find new drug targets for drug research and development and helps more rational and efficient treatment for patients with diabetic heart disease.Objectives1. The aim of the study was to examine the effects of phlorizin on body weight, metabolic parameters and serum AGEs levels, and the pathological changes of myocardium via histology and ultrastructure in diabetic db/db mice.2. The purpose of the study was to identify and quantify the differently expressed proteins in normal control group (CC group), untreated db/db diabetic mice group (DM group) and PHL treatment db/db mice group (DMT group), hoping to clarify the mechaniam underlining the protection of phlorizin against diabetic cardiac damage..MethodsMale C57BLKS/J db/db (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 before the experiments started. C57BLKS/J db/m mice were selected as control group (CC, n=8). The db/db mice were divided into2groups:untreated diabetic group (DM, n=8) administrated with normal saline solution by intragastric gavage and diabetic group treated with phlorizin at a dosage of20mg/kg/d (DMT, n=8) for10weeks. All mice were weighed every week. At the end of the experiments, all mice were fasted overnight and sacrificed. Fasting blood was collected. Fasting blood glucose (FBG), blood triglycerides (TC), blood cholesterol and serum advanced glycation end products (AGEs) specific fluorescence determinations were measured. Whole heart from the controls, untreated db/db mice and phlorizin treated db/db mice were immediately enucleated and were fixed in4%paraformaldehyde and then embedded in paraffin. Some parts of hearts were dissected for histological procedures. The rest parts of hearts tissue were kept at-80℃for further proteomic studies.Heart tissue (50mg) from each of four mice per group was prepared and digested with trypsin. A total of60ug of peptides from each group were labeled with iTRAQ reagents following the manufacturer’s instructions. The control group peptides were labeled with Reagent114; the DMT group, Reagent116; and the DM group, Reagent117. The labeled samples were then separated into10fractions using PolySulfoethyl A strong cation-exchange (SCX) columns. Mass spectrometric analysis was performed using a liquid chromatography system and an LTQ-Velos ion trap mass spectrometer. Differentially expressed genes were analyzed using Ingenuity Pathway Analysis.The data packet containing the differentially expressed proteins identified in the iTRAQ experiment was converted by IPA to "fold change" and uploaded into IPA. Each identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base.Results1. General observationMice in CC group grew in good condition with smooth furs and active state duing experiments. The db/db mice exhibited sedentary, polydipsia, polyuris and polyphagia with shaggy furs and a rapidly increased body size. Phlorizin treated db/db mice also exhibited abnormal condition, but much better than untreated db/db mice.2. The effect of PHL diabetes on body weight, FBG, TG, TC and AGES in db/db miceDuring the observation period, the DM and DMT groups gained substantially more weight than the control group (P<0.05). Nevertheless, phlorizin treatment significantly reduced body weight gain in db/db mice at the second week after phlorizin administration (P<0.05). After10weeks, serum FBG, TG, and TC levels in the DM group were significantly higher than those in the control group (P<0.05). However, phlorizin treatment dramatically reduced these values in the DMT group compared with the DM group (P<0.05). In addition, db/db mice had significantly elevated serum AGE levels. After phlorizin treatment, AGE levels in db/db mice were reduced.(P<0.05)3. The effect of PHL on histological observation of in db/db miceOn H&E-stained sections, the myocardium in DM group exhibited significant myocardial hypertrophy and myofiber disarray accompanied by damaged nuclei and increased degeneration. However, phlorizin treatment attenuated this cardiomyocyte hypertrophy to a similar level to the control group.4. The effect of PHL on ultrastructural observation of myocardium in db/db miceMyocardial ultrastructure could be visualized by electron microscopy. In the control group, the myofibrils were arranged in a striated pattern, and the mitochondria were positioned in rows along the myofibrils. Although the sarcomere was of the same length, some cardiomyocyte mitochondria in the DM group showed cristae loss. Large areas of the myocardium exhibited a complete disruption of myofibril and mitochondrial arrangements. The shape of the nuclei was altered, and the nuclear membrane was disrupted. However, due to the protective effect of phlorizin in the DMT group, the number of degenerated mitochondria was significantly decreased, and the myofibril disorder was markedly attenuated.5. Differentialy expressed proteins identified by Mass spectrometryUsing the iTRAQ approach followed by LC-MS/MS identification and IPI database searching, we analyzed the effect of phlorizin on the myocardial protein profile of db/db mice. Of the113differentially expressed proteins,29were elevated in the DM group compared with the control group but were still decreased by phlorizin treatment. An additional84proteins were decreased in the DM group compared with the control group, but these were restored by the phlorizin treatment. 6. Features of differentialy expressed proteins after PHL interventionAccording to the features of differentialy expressed proteins, we made functional classification of proteins involved in metabolic disorders in db/db mice into altered proteins in cardiac lipid metabolism, altered proteins related to myocardial mitochondria and altered proteins involved in cardiomyopathy.7. Bioinformatics analysis of differentially expressed proteinsThe top-ranked biological process and involved disease include cardiovascular disease,lipid metabolism, cardiovascular system development and function, endocrine system disorder, free radical scavenging and energy production, which may play important role during diabetic cardiac damage.The top protein network was generated by pathway analysis of differentially expressed proteins. There was a cluster of35proteins in the network, of which24are included on our list such as Dapk3, Titin, Prkaa, Des, ILK, Nampt and so on. This network may provide important information of interaction between proteins involved in lipid metabolism, mitochondrial function, and cardiomyopathy.8. Validation of iTRAQ data for selected candidate proteinsWe selected two proteins for Western blot analysis to validate the iTRAQ data. Calnexin was found to be decreased, whereas integrin-linked protein kinase (ILK) was increased in the DMT group compared with the DM group. Quantification of band intensity showed that the results from density of bands are almost consistent with the iTRAQ data. This indicates that the iTRAQ data are reliable.Conclusion1. PHL treatment can significantly prevent the obesity trend of db/db mice and reduce the FBG. TG, TC and serum AGEs level of db/db mice.2. PHL treatment can significantly improve the hypertrophy of cardiomyocytes and damage of the nucleus in myocardium of db/db mice. It can also reduce the number of injury mitochondria in cardiomyocyte, and attenuate irregular arrangement of myofibrils.3. As a novel technology of quantitative proteomics, iTRAQ has been widely used to tag peptides for multiplexed protein quantification and provides increased experimental throughput and lower variability. Thus, using iTRAQ is available to study the mechanism of the protection of phlorizin against diabetic cardiac damage in this study.4. A total of113proteins differentially changed with phlorizin treatment in myocardium of db/db mice were identified using LS-ESI-MS/MS methods. Of these,29were elevated in the DM group compared with the control group but were still decreased by phlorizin treatment. An additional84proteins were decreased in the DM group compared with the control group, but these were restored by the phlorizin treatment. The top-ranked biological processes and top protein networkwas generated by pathway analysis suggested that the altered proteins are mainly related to cardiac lipid metabolism, mitochondrial energy production and development of cardiomyopathy. Part TwoCaveolar Fatty Acids and Acylation of Caveolin-1BackgroudCaveolae are cholesterol and sphingomyelin-rich plasma membrane microdomains presented in most types of mammalian cells and tissues. Caveolae were originally identified as50-100nm flask-shaped, non-clathrin-coated invagination of the plasma membrane and found to be involved in endocytosis and potocytosis. However, later studies revealed that these microdomains concentrate a variety of signaling molecules which provide a platform for signal transduction.Thus, changes in the components of caveolae may have a profound effect on cellular functions. Caveolin-1, a22-kDa protein, is the principal structural component of caveolae and a determinant for caveolae formation.A deficiency of caveolin-1in mice eliminated caveolae, which subsequently impaired nitric oxide and calcium signaling in the cardiovascular system, causing aberrations in endothelium-dependent relaxation, contractility, and maintenance of myogenic tone.In addition to cholesterol and sphingomyelin, caveolae also contain a variety of fatty acids. While it is generally believed that covalent attachment of myristic and/or palmitic acid occurs on a wide variety of cellular proteins and the acylation of protein is critical for membrane targeting, the fatty acid compositions of caveolae, the fatty acids bound to caveolin-1, and the effect of acylation of caveolin-1on caveolin-1targeting to caveolae remain poorly understood. Our results revealed that caveolae contain a limited subset of fatty acids, highly enriched with saturated fatty acids, which is quite different from the fatty acid compositions in whole cells. We further demonstrated that the primary fatty acid associated with caveolin-1is stearic acid, not myristic acid as previous speculated.ObjectivesThe aim of the study is to use gas chromatography/mass spectrometry (GC/MS) to identify and quantify the fatty acid compositions of caveolae and fatty acids covalently bound to caveolin-1in Chinese hamster ovary (CHO) cells, a cell system with high caveolin-1expression, and then characterize the fatty acid esterification of caveolin-1with isotope-labeled fatty acids. Another purpose of the study is to observe weather the fatty acid esterification of caveolin-1can affect its targeting to caveolae.MethodsCaveolae were isolated from Chinese hamster ovary (CHO) cells. The caveolar fatty acids were extracted with Folch reagent, methyl esterificated with BF3, and analyzed by gas chromatograph-mass spectrometer (GC/MS). The caveolin-1bound fatty acids were immunoprecipitated by anti-caveolin-1igG and analyzed with GC/MS. For characterization of fatty acid esterification of caveolin-1, CHO cells were cultured in Ham’s F-12medium containing5%FBS,2mmol/L L-glutamine,100U/ml penicillin and100μg/ml streptomycin to80%confluency in10cm culture dish. The cells were starved18h in Ham’s F-12medium containing1%BSA and then labeled with2.5mCi of3H-palmitic acid or25μCi of14C-stearic acid for3h at37o C in the presence/absence of30times of non-labeled palmitic acid (16:0), stearic acid (18:0) or oleic acid (18:1). The cells were dissolved in MBST/OG buffer and immunoprecipitated with anti-caveolin-1/protein A. Non-immune rabbit IgG was used as negative control. The immunoprecipitated caveolin-1was separated by SDS-PAGE and transferred to PVDF membrane. The fatty acid esterification of caveolin-1was detected by autoradiogram at-80°C for6weeks using a Kodak MS film.ResultsIn contrast to the whole CHO cell lysate which contained a variety of fatty acids, caveolae mainly contained three types of fatty acids,0.48μg palmitic acid,0.61μg stearic acid and0.83μ g oleic acid/caveolae preparation/5x107cells. Unexpectedly, GC/MS analysis indicated that caveolin-1was not acylated by myristic acid; instead, it was acylated by stearic acid (0.23μg) and palmitic acid (0.15μg). In addition, the targeting of caveolin-1to caveolae was not affected by the presence of palmitic acid or stearic acid.ConclusionCaveolae contained a special set of fatty acids, highly enriched with saturated fatty acids, and caveolin-1was acylated by palmitic acid and stearic acid. The unique fatty acid compositions of caveolae and acylation of caveolin-1may be important for caveolae formation and for maintaining the function of caveolae.