| Chapter One Proteomic Analysis of Protective Effects of GSPB2on Aorta in db/db MiceBackgroundDiabetes mellitus (DM) is a disease of metabolic disorder characterized by hyperglycemia and insulin resistance accompany with insufficiency of secretion or action of endogenous insulin. With the development of economy and the improvement of living standard, and the aging population increased, the prevalence of DM has become a global public health problem, resulting in morbidity and mortality and increased 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 lesions of heart, blood vessels, kidneys, nerves, retina, peripheral nerve, brain and other tissues.Aorta complications are the major cause of mortality in patients with DM. The risk of diabetic patients to atherosclerosis and its complications is2to4times than that of nondiabetic patients. DM has been regarded as an equivalent to coronary disease. The hallmarks of macrovascular damage are accelerated atherosclerosis and remodeling of large arteries characterized by thickening and stiffening of the arterial wall. Thus far, the underlying mechanism remains poorly understood, and there is no cure available for DM and its cardiovascular complications. Therefore, elucidation of the molecular mechanism of aorta damage associated with DM and identification of the target protein critically involved in this process could lead to a more specific strategy in fighting against the vascular complications of DM.Procyanidins are a complex family of polyphenol polymers widely existing in natural products. Grape seed proanthocyanidin extracts (GSPE) derived from grape seeds. Dimeric procyanidin B2is one of the main components of GSPE, composed of two molecules of the flavan-3-ol (-)-epicatechin linked by a4bâ†'8bonds. Studies including ours have shown that procyanidin B2has properties including anti-inflammation, anti-oxidant, anti-AGEs-induced proliferation and migration of aortic smooth muscle cells. Previous data showed that GSPE has protective effect on aorta of STZ induced diabetic rats. However, the molecular mechanism remains unrevealed. 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. 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. In an effort to understand the protective effect and the potential target protein of GSPB2on aorta in type2diabetes (T2DM), we resorted to iTRAQ technique of the aorta using db/db diabetic mice. db/db mice are well-established animal model to study T2DM complications. The purpose of this experiment was to explore the protective mechanisms of GSPB2on aorta in db/db mice by searching the differential protein among CC group, DM group and DMT group, thus provide candidate targets for clinical treatment of diabetic vascular complications.Objective1. To study the characteristic of aorta in db/db mice, observe the morphology changes by light and electron microscope.2. To explore the differentially expressed proteins of aorta in db/db mice by iTRAQ, further defined the physiopathologic mechanisms of diabetic macrovascular.3. To establish the aortic differentially expressed proteins of db/db mice after treatment of GSPB2, evaluate the protective effects of GSPB2on diabetic aortic damage.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 GSPB2-treated group with a dosage of30mg/kg/d (DMT, n=8) for10weeks.1. The measurement of body weight, FBG, TC, TG and AGEs:All mice were weighed every week during the experiment. At the end of the intervention, all mice were fasted overnight and then sacrificed. Fasting blood was collected, fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), and serum advanced glycation end products (AGEs) specific fluorescence determinations were measured.2. Aortic morphology and ultratructure:The aortas were dissected from the controls, db/db mice and db/db mice treated with GSPB2, then fixed in4%paraformaldehyde and embedded in paraffin for HE staining and intima-media thickness measurement, or fixed in glutaraldehyde for ultrastructure observation using electron microscope.3. Proteomic analysis (iTRAQ):About50mg Aortic tissue from each of four mice per group was pooled and homogenized in the presence of liquid nitrogen. In filter-aided sample preparation (FASP) detergents are removed by ultrafiltration, and after protein digestion, peptides are separated from undigested material. About60μg peptides of each group were labeled with iTRAQ reagents (114for the peptides of CC group,115for the peptides of DMT group, and117for the peptides of DM group respectively) following the manufacturer’s instructions (Applied Biosystems). Strong Cation Exchange (SCX) chromatography was performed to separate the labeled samples into10fractions. Eluted peptides were collected and desalted by an offline fraction collector and C18cartridges. The precursor ion was through collision-induced dissociation by tandem mass spectrometry, product ion was analyzed by secondary MS. The different ionic strength of reporter group represents the difference in the relative abundance of its labeled polypeptides. 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. Use114as the reference (CC group).4. Subcellular localization analysis, functional analysis and ingenuity pathway analysis:The localization analysis of the identified proteins in aortas was performed by using AmiGO (Version1.8). The functional analysis was performed by KOGnitor (http://www.ncbi.nlm.nih.gov/COG/grace/kognitor.html). The global protein changes data in the aorta of db/db mice treatment with GSPB2were analyzed through the use of Ingenuity Pathways Analysis (Ingenuity Systems, CA). Ingenuity software uses the data to navigate the Ingenuity pathways database for interactions between these focus proteins and all the other protein stored in the database to generate biological networks.5. Western blot:Some differentially expressed proteins were validated using western blot analysis.Results1. General DataIn 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 and obesity. GSPB2treated db/db mice also displayed abnormal, but better than the db/db mice.2. Effects of GSPB2on Body Weight, FBG, TC, TG and AGEsThere was no statistical significance of body weight between DM group and DMT group prior to the experiment (p>0.05), but were significantly higher than CC group (p<0.01). At week2of the study, the body weight of DM group was significantly increased. This trend did not change until the end of the experiment (18weeks old). However, the increase of body weight was significantly inhibited by GSPB2administration in the DMT group compared to the DM group (p<0.01).There was no statistical significance of FBG between DM group and DMT group at the beginning of experiment (p>0.05), but were significantly higher than CC group (p<0.01). GSPB2decreased the level of FBG in DMT group, but there was no significant difference (p>0.05).The serum TC, TG and AGEs were measured at the end of experiment. The TC. TG and AGEs of DM group were significantly higher than CC group (p<0.01). GSPB2significantly reduced the serum TC, TG and AGEs of db/db mice (p<0.01).3. Effects of GSPB2on Aortic MorphologyHE staining:Under light microscopy, aortic remodeling and proliferation of vascular smooth cells (VSMC) and endothelial injury were observed in the aorta of db/db mice. Moreover, GSPB2suppressed the endothelial injury, aortic remodeling and proliferation of VSMC and led to light microscopic findings similar to those of the control mice.4. Effects of GSPB2on Aortic Ultrastructure Under electron microscopy, the impaired endothelial cells and the rod-like mitochondria in endothelial cells were seen in aortic tissue in the diabetic mice. Moreover, the rupture of vascular elastic membrane and the increased collagen fibers were observed in the diabetic aorta. Many smooth muscle cells showed pyknotic nucleus, increased heterochromatin, mitochondrial condensation, swelling of the endoplasmic reticulum, and many autophagosome appeared, whereas normal ultrastructure was observed in the aortic tissue of the control db/m mice. GSPB2tended to improve the preservation of the fine structure of aortic tissue.5. Mass Spectrometry Identify the Differentially Expressed ProteinsWe identified1530proteins in this study.557proteins were shown to have significantly different abundance between control group and DM group (±1.5-fold). Of these557proteins, the levels of139proteins were normalized by GSPB2treatment.6. Subcellular Localization Analysis of Differentially Abundant GSPB2Associated Arterial 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.40%were in cytoplasm,18.8%in nucleus,12.1%in plasma membrane,7.9%in endoplasmic reticulum,7.3%in mitochondrion,6.7%in extracellular,6%in centrosome,3%in ribosome,2.4%in Golgi,1.2%in lysosome.7. Bioinformatic Functional Analysis of Differentially Abundant GSPB2Associated Arterial ProteinsAmong the functional assignment of the proteins,17%were involved in signal transduction mechanisms,12%in cytoskeleton,9%in posttranslational modification, protein turnover, chaperones,7%in translation, ribosomal structure and biogenesis,6%in intracellular trafficking, secretion, and vesicular transport,4%in cell cycle control, cell division, chromosome partitioning,4%in nucleotide transport and metabolism,4%in lipid transport and metabolism, and the remaining proteins were identified as dispersed across13remaining categories.8. Ingenuity Pathway Analysis of Differentially Abundant GSPB2Associated Arterial ProteinsIngenuity Pathway Analysis (IPA) showed that the primary pathway involved was cell death, lipid metabolism and oxidative stress. The pathways would facilitate the understanding of diabetes biomarkers for further study. 9. Validation of iTRAQ Data on Other Selected ProteinsTo validate the proteomic analysis of aorta using iTRAQ we performed, milk fat globule epidermal growth factor-8(MFG-E8), cysteine and glycine-rich protein1(CSRP1) and gultathione S transferases theta-1(GSTT1) were validated using western blotting analysis. MFG-E8and CSRP1were found to be inhibited whereas GSTT1was enhanced in the DMT group compared to the DM group (p<0.01). The significance of those proteins would be interesting topic for our future studies.Conclusion1. GSPB2intervention could significantly improve the obesity trend of db/db diabetic mice.2. After treated with GSPB2, the TC, TG and AGEs decreased in DMT group than those of DM group.3. GSPB2suppressed the aortic remodeling and endothelial injury, and led to light and electron microscopic findings similar to those of the control mice.4. As a new technique of quantitative proteomics, iTRAQ enables comparative analysis for many groups. We obtained the reliable differentially expressed proteins from CC group, DM group and DMT group.5.557proteins were shown to have significantly different abundance between control group and DM group. Of these557proteins, the levels of139proteins were normalized by GSPB2treatment. Most of the proteins were in cytoplasm, the pathway involved were cell death, lipid metabolism and oxidative stress. The pathways would facilitate the understanding of aortic damage in diabetes.6. Western blotting showed MFG-E8and CSRP1were found to be inhibited whereas GSTT1was enhanced in the DMT group compared to the DM group. The significance of those proteins would be critical candidate targets for future studies of GSPB2on diabetic aorta. Chapter Two Protective Mechanism of GSPB2on Aorta and the Intervention Study of MFG-E8in db/db MiceBackgroundWith the completion of human genome project, life science research has entered the post genomic era. Protein is the executor of physiological functions. Research on the structure and function of proteins will directly clarify the physiological or pathological mechanism. Traditional method on individual protein has been unable to meet the requirement of post genomic era. The proteins are multiple and dynamic at different conditions. Therefore we must explore changes of global and dynamic proteins for comprehensive understanding of complicated conditions. Proteomics is one of the critical researches in post genomic era.Grape seed procyanidin B2is one of the most powerful drugs of free radical scavenger. The ability related to its phenolic hydroxyl of molecular structure. Our previous studies found that GSPB2could inhibit the generation of AGEs-induced reactive oxygen species (ROS) and proliferation of vascular smooth muscle cell, GSPE possess definite protective effect on aorta of type1diabetic rats. However, the protective mechanism of GSPB2and its molecular target on type2diabetes remain poorly understood. The traditional drug research pays more attention on drug phenotypic observation or the activity of single molecular or enzyme, unable to realize the comprehensive analysis of drug targets. But the combination of quantitative proteomics and pharmaceutical research provide a means for potential development of new pharmacological molecular. In part1of this study,557proteins were shown to have significantly different abundance between control group and DM group. Of these557proteins, the levels of139proteins were normalized by GSPB2treatment. According to our prior knowledge, database and literature material, we found that MFG-E8was increased by2.4-fold in abundance in db/db mice, which was normalized by GSPB2treatment, protein expression ratio:DMT/DM0.59.Justified to its name, milk fat globule-epidermal growth factor-8(MFG-E8) was initially identified as an indispensable component of the milk fat globule. It is expressed in many histocyte. Studies have shown that MFG-E8was expressed in inflammation and artery plaque, the level of MFG-E8was significantly increased with ages, and elevated in breast cancer and bladder cancer. Its effect on diabetes still remains unknown. Therefore, this part of our study will further explore the effect of MFG-E8on diabetic aortic damage based on the identification of iTRAQ. We evaluated the function of MFG-E8on aorta of T2DM by inhibiting of MFG-E8by RNA interference and exogenous recombinant MFG-E8administration in db/db mice. Approaches targeting MFG-E8could potentially lead to new modality in the prevention and treatment of vascular complications in DM patients.Objective1. Further establish the protective mechanism of GSPB2on aorta of db/db mice, define the pathophysiological mechanism of macro vascular disease in T2DM.2. Construction of lentiviral vector with short hairpin RNA for MFG-E8, and overexpression MFG-E8with recombinant, observe the aortic morphologic changes and further explore the mechanism for MFG-E8involved in the diabetic aortic damage.Methods1. Primary culture of aortic endothelial cell:The aorta of male C57BLKS/J db/m mice (8weeks old) was isolated. Longitudinal dividing the aortic wall and cut into small pieces, stick the lining surface in the cell culture flasks. Then put it in the incubator with endothelial cell medium (ECM, ScienCell) with an atmosphere of5%CO2/95%air. After7-9days, cell fused into a single layer, dealt with0.25%trypsin digestion and passage. Cells from passages3to6were used in this study.2. Construction of MFG-E8RNAi and endothelial cell transfection:Lentiviral vectors with short hairpin RNAs (shRNAs) for MFG-E8were designed and chemically synthesized. The lentiviral vector with green fluorescence protein (GFP) was used as the RNAi control. Aortic endothelial cell were transfected with lentiviral vectors at a multiplicity of infection (MOI=50,75,100) according to the manufacturer’s instructions. Fluorescence microscope was used to measure transfection efficiency at time points of12,24,48and72hours after transfection. The optimal MOI was determined for100. The most effective target site of RNA interference was confirmed by using western blot for the expression levels of MFG-E8after3days.3. Treatment of MFG-E8RNAi and recombinant MFG-E8in db/db mice:Male C57BLKS/J db/db and db/m mice (n=40,7weeks old) were used in this study. C57BLKS/J db/m mice were selected as control group (CC, n=8). The db/db mice were divided into4groups:an untreated diabetic group (DM, n=8), LV-GFP treated db/db group (GFP, n=8), MFG-E8RNAi treated db/db mice group (M-RNAi, n=8) and recombinant MFG-E8treated db/db mice group (rmMFG-E8, n=8). MFG-E8RNAi (M-RNAi group) or LV-GFP (GFP group) was diluted to a total volume of300μl containing4×107TU was injected into the tail vein of fourteen-week-old male db/db mice. Recombinant mouse MFG-E8was diluted in PBS and300ul of the solution (20μg/kg) was injected through the tail vein of fourteen-week-old male db/db mice twice a week for4weeks (rmMFG-E8group). Each group of mice was observed from week13to week18without any administration of hypoglycemic therapy.4. Aortic morphology and ultrastructure:At the end of the intervention, all mice were fasted overnight and then sacrificed. the aortas were dissected, then fixed in4%paraformaldehyde and embedded in paraffin for HE staining and intima-media thickness measurement, or fixed in glutaraldehyde for ultrastructure observation using electron microscope.5. MFG-E8and p-ERK expression in aorta using western blot:Some aortic tissues were kept at-80℃for MFG-E8and p-ERK expression analysis.6. ELISA of MCP-1in db/db mice serum:Sera were separated for ELISA of MCP-1.Results1. Effects of MFG-E8RNAi and recombinant MFG-E8on Aortic MorphologyUnder light microscopy, aortic remodeling and proliferation of vascular smooth cells (VSMC) and endothelial injury were observed in the aorta of DM and GFP group. Moreover, MFG-E8RNAi suppressed the endothelial injury, aortic remodeling and proliferation of VSMC and led to light microscopic findings similar to those of the control mice. In aorta of recombinant MFG-E8protein-treated group, the deteriorative changes were observed. The blood vessel was thicker, characterized by more inflammatory cells infiltrated such as neutrophil and lymphocyte and focal necrosis. Endothelial cell swelling and perivascular inflammation were also observed.2. Effects of MFG-E8RNAi and recombinant MFG-E8on Aortic UltrastructureUnder electron microscopy, the impaired endothelial cells and the rod-like mitochondria in endothelial cells were seen in aortic tissue in the diabetic or GFP treated mice. Moreover, the rupture of vascular elastic membrane and the increased collagen fibers were observed in the diabetic aorta. Many smooth muscle cells showed pyknotic nucleus, increased heterochromatin, mitochondrial condensation, swelling of the endoplasmic reticulum, and many autophagosome appeared, whereas normal ultrastructure was observed in the aortic tissue of the control db/m mice. MFG-E8RNAi tended to improve the preservation of the fine structure of aortic tissue, but in the rmMFG-E8group, the deteriorative changes and the migration of smooth muscle cells in the vascular elastic membrane were observed.3. Effects of MFG-E8RNAi and Recombinant MFG-E8on the Expression of MFG-E8Western blotting analysis showed protein expression of MFG-E8in DM or GFP group was significantly higher than that of control db/m group (p<0.01); after treatment with MFG-E8RNAi, the expression of MFG-E8decreased compared to GFP treated db/db mice (p<0.01), which confirmed the transfection efficiency in db/db mice in vivo. However, the rmMFG-E8group displayed increased expression of MFG-E8compared to that of both control db/m group and DM group (p<0.01).4. Effects of GSPB2, MFG-E8RNAi and Recombinant MFG-E8on ERK1/2phosphorylation levelsTo understand the mechanism of atherosclerosis in T2DM, we further examined the ERK1/2phosphorylation (p-ERK1/2) in aorta of db/db mice by Western blotting. The expression of p-ERK1/2in db/db mice were higher than that of control db/m mice (p <0.05); after treatment with GSPB2, the expression of p-ERK1/2was decreased compared to that of untreated db/db mice. MFG-E8RNAi decreased in the phosphorylated ERK1/2(p<0.01). Treatment with recombinant MFG-E8increased ERK1/2phosphorylation (p<0.01).5. Effects of GSPB2. MFG-E8RNAi and Recombinant MFG-E8on the Levels of MCP-1The expression of MCP-1in serum was measured by ELISA. The expression of MCP-1in serum was significantly increased in DM group compared with that of control group (p<0.01). GSPB2and MFG-E8RNAi decreased the level of MCP-1(p <0.01). Recombinant MFG-E8significantly increased the expression of MCP-1(p<0.01).Conclusion1. Construct a reliable lentiviral vectors for MFG-E8interference, and verify the transfection efficiency in cell and animal. 2. GSPB2and MFG-E8RNAi significantly inhibit the protein expression of MFG-E8, thus reduce aortic injury of T2DM, and recombinant MFG-E8aggravate the aortic damage.3. GSPB2and MFG-E8RNAi significantly decrease the protein expression of p-ERK, and recombinant MFG-E8increase the p-ERK.4. GSPB2and MFG-E8RNAi significantly decrease the expression of MCP-1in serum, and recombinant MFG-E8increase the MCP-1. BackgroundDrug addiction is a kind of stubborn and easily recurrence of mental illness which has the characteristics of long-term memory. With the development of the biological mechanism of addictive behaviors research, drug addiction has been got more and more attention. Under the effects of addictive drugs, the long-range adaptive changes in nervous system, and behavioral, cell and molecular and gene expression changes. Studies have shown that addictive drugs inducing the formation of behavioral sensitization involving new gene transcription, protein synthesis and modification of histone acetylation.Metabotropic glutamate receptors (mGluRs) are coupled to G-protein, regulate intracellular second messenger and lead to metabolic changes. According to the amino acid sequence homology, its pharmacological characteristics and the signal transduction mechanism, mGluRs can be divided into three groups:mGluRs â… group, mGluRs â…¡ group and mGluRs â…¢ group. mGluRs â…¡ group could prevent the cell damage of diabetic peripheral nervous system. mGluRs â…¡ group can induce increased secretion of pancreatic enzymes after glutamate activation, which provides a new thought for the treatment of diabetes. mGluRs â… group includes mGluRl and mGluR5. mGluRs antagonist can alleviate behavioural changes caused by drug addiction, including cocaine addiction. However, its mechanism of treating addiction has not yet been fully understood. The activation of group â… mGluRs. mGluR5in particular, increases protein synthesis at synapses, and mGluR5-induced excessive protein synthesis has been implicated in the pathology of fragile X syndrome. However, fewer studies have investigated the role of mGluR1in cocaine addiction.mGluRs â… group was coupled to extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways to increase translation and protein synthesis. The formation and consolidation of memories require de novo protein synthesis. The development of drug-associated memories and addictive behavior also requires new protein synthesis. However, the upstream signaling mechanisms by which learning and drug exposure lead to protein synthesis remain poorly understood. One possibility is that learning and cue-drug pairing cause the release of glutamate, which activates group â… mGluRs to increase protein synthesis.The ventral tegmental area (VTA) of the midbrain and its dopaminergic projection play a critical role in reward processing and addictive behavior. Both mGluR1and mGluR5are expressed in midbrain dopamine neurons, although mGluR1is expressed at much higher density. We hypothesized that mGluRl-dependent protein synthesis in the ventral tegmental area (VTA) is required for cocaine-induced conditioned place preference (CPP). In the present study, we tested this hypothesis using a combination of electrophysiological, biochemical and behavioral approaches. These results provide evidence that mGluR1-dependent protein synthesis is critically involved in behavioral effects of cocaine.ObjectiveTo explore the underlying mechanism of mGluR1on cocaine addiction and behavioral changes, and provide new avenues for treating drug addiction,Methods1. Male Sprague-Dawley rats were used for slice electrophysiology (P18-30of age,50-1OOg). Whole-cell voltage-clamp recording were made from VTA dopamine neurons in midbrain slices. Inhibitory postsynaptic currents (IPSCs) were evoked by stimulating inhibitory synaptic afferents.2. Male Sprague-Dawley rats (10-11weeks,300-350g) were anesthetized and placed in a stereotaxic device. Guide cannulae were bilaterally implanted2.8mm above the VTA using aseptic techniques. After the surgery, rats were allowed to recover for about1week. CPP experiments were performed using three-chamber CPP apparatus. The CPP protocol consisted of the following sessions:(1) Pre-test (day1):animals were allowed to explore both chambers for20min and time spent in each side was recorded. Rats showing unconditioned side preference (≥180s disparity) were excluded (n=4).(2) Conditioning (day2-9):Rats received bilateral intra-VTA infusions of vehicle, JNJ16259685or cycloheximide via the pre-implanted cannulae. Injector cannulae (33-gauge) were inserted into the guide cannulae. The intra-VTA infusions were made via C313C connectors to2μl-Hamilton micro-syringes. Vehicle (1μl per side), JNJ16259685(0.1ng,1μl per side) or cycloheximide (100ng,1μl per side) was manually injected at a rate of1μl over2min, and the injectors were kept in place for an additional2min to ensure adequate drug diffusion from the injector tip. Thirty minutes after the microinjections, rats received cocaine or saline conditioning.Cocaine conditioning. Rats received saline injection (0.9%NaCl,1ml/kg, i.p.) on days2,4,6,8and were immediately confined to one chamber for20min. On days3,5,7and9, rats received cocaine injection (15mg/kg, i.p.) and were immediately confined to the opposite chamber for20min.Saline conditioning. Rats received daily saline injection and were immediately confined to one chamber for20min on days2,4,6, and8and were confined to the opposite chamber for20min on days3,5,7, and9.(3) CPP test (day10):all of the animals were allowed to explore freely for20min among the three chambers and time spent on each side is recorded.3. VTA slices from male Sprague-Dawley rats (P18-30of age) were used for western blot. Slices were treated with vehicle or various drugs (see Results section). Western blot were performed using the antibody of phospho-ERK and phospho-mTOR.4. Rats were anesthetized with isoflurane and rapidly decapitated-1hour after the CPP tests. The brains were immediately removed and placed in oxygenated ACSF at4℃. Midbrain slices were prepared, and the VTA was dissected out and homogenized in lysis buffer. Western blot was performed using the antibody of phospho-ERK, phospho-mTOR, phospho-p70S6K, phospho-S6, phospho-eIF4E and eEF1A.Results1. DHPG-induced depression of IPSCs in VTA dopamine neurons is mediated mainly by mGluRl. Bath application of group â… mGluR agonist DHPG (100μM,10min) induced long-term depression (LTD) of evoked IPSCs (n=7, p<0.01vs. baseline). The mGluR1-selective antagonist LY367385(100μM) blocked DHPG-induced depression of IPSCs (n=7, p<0.05vs. control). Another mGluRl-selective antagonist, JNJ16259685(100nM) blocked DHPG-induced depression of IPSCs (n=6, p<0.05vs. control). The mGluR5-selective antagonist MTEP (10μM) did not affect DHPG-induced depression of IPSCs (n=8, p>0.05vs. control).2. Protein synthesis is required for DHPG-induced I-LTD in the VTA.The protein synthesis inhibitor anisomycin (30μM; n=8, p<0.05vs. control) or cycloheximide (80μM; n=6,p<0.05vs. control) blocked DHPG-induced I-LTD but did not significantly affect the early component of DHPG-induced depression of IPSCs(p>0.05vs. control).3. DHPG-induced-I-LTD requires the activation of ERK1/2and mTOR signaling pathways.The MEK inhibitor U0126(20μM) exerted only a modest effect on DHPG-induced I-LTD that did not reach statistical significance (n=8, p>0.05vs. control). U0124(20μM), an inactive analog of U0126, had no effect on DHPG-I-LTD (n=7, p>0.05vs. control). The mTOR inhibitor rapamycin (Rapa,100nM) also had no significant effect on DHPG-induced I-LTD (n=7, p>0.05vs. control), whereas co-application of rapamycin (100nM) and U0126(20μM) blocked DHPG-induced I-LTD (n=8,p <0.05vs. control).4. DHPG induced ERK and mTOR phosphorylation and activation in the VTA via mGluR1. DHPG (100μM,10min) increased p-ERK1/2levels in VTA homogenates compared, this effect was blocked by the JNJ16259685(JNJ,100nM, p<0.001). DHPG also increased p-mTOR levels in VTA homogenates compared with control, this effect was blocked by JNJ16259685(100nM,p<0.001).5. Intra-VTA infusions of the mGluRl antagonist JNJ16259685or protein synthesis inhibitor cycloheximide during the conditioning phase attenuated the acquisition of CPP to cocaine. Intra-VTA infusions of JNJ16259685or cycloheximide significantly attenuated CPP in cocaine-conditioned rats (n=7-10, p<0.001) but did not affect CPP scores in saline-conditioned rats (n=7-10, p>0.05). Intra-VTA infusions of JNJ16259685or cycloheximide did not significantly affect locomotor activity in cocaine-or saline-conditioned rats (p>0.05, n=7-10).6. Western blot was performed on VTA samples collected-1hour after the CPP tests. Cocaine CPP activates ERK and mTOR signaling pathways, p-ERK1/2and p-mTOR levels in the VTA were significantly increased in cocaine-conditioned rats compared with those in saline-conditioned rats, and the increase was blocked by intra-VTA infusions of JNJ16259685(p<0.001). p-p70S6K, p-S6and p-eIF4E levels in the VTA were significantly increased in cocaine-conditioned rats compared with those in saline-conditioned rats, and these increases were blocked by intra-VTA infusions of JNJ16259685during the conditioning phase(p<0.01,p<0.001). eEF1A was significantly increased in cocaine-conditioned rats compared with that in saline-conditioned rats (p<0.001), and this increase was blocked by intra-VTA infusions of JNJ16259685or cycloheximide (Cyc) during the conditioning phase (p<0.001).ConclusionIntra-VTA microinjections of mGluRl antagonist JNJ16259685significantly attenuated or blocked the acquisition of cocaine-induced conditioned place preference (CPP). These results suggest that mGluR1antagonism inhibits de novo protein synthesis; this effect may block the formation of cocaine-cue associations and thus provide a mechanism for the reduction in CPP to cocaine. mGluR1was coupled to extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR) signaling pathways to increase translation. This study might provide new avenues for treating drug addiction. |
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