Glucagon Like Peptide-1 Analogue Promotes Primary Cortical Neuron And Related Mechanism

Part one Primary Culture of Cortical Neurons and DefinitionObjective: Cell culture technique is an important method in basic medical research. Primary cell culture refers to the culturing of cells directly derived from tissue or organs. At present, the morphological characteristics,material metabolism, physiological and pharmacological processes of neurons are mainly focused on the cellular level and molecular level. Primary cell culture can study the structure and function of individual nerve cells, and provides the opportunity to reproduce the growth process in vivo. It is an effective model to reflect the activity of neural cells. This method enables researchers to observe the morphological and functional changes of cells in a long term and easily impose experimental conditions, such as physical,chemical or biological stimulation. With a more direct way, it facilitates the research of various neurological diseases at the cellular and molecular levels.Because the neuron is a highly differentiated cell, they seldom devide after birth, so it is necessary to establish a reliable, stable, simple and easy method for primary neuron culture. In order to solve this problem, our study,based on previous methods, explore a simple and feasible way for primary cortical neurons culture, and thus lay a fundamental for further work in central nervous system disorders within the cellular and molecular level.Methods: Cortical neurons were obtained from the embryonic brains at embryonic day 16(E16) of C57BL/6 mice. Briefly, the cerebral cortex of the embryonic brains were dissected under an optical microscope and incubated in papain(2mg/ml) in D-Hank’s buffered salt solution for 15 min at 37℃. The dispersed cerebral cortical tissues were then neutralized with Dulbecco’s modified Eagle medium(DMEM) containing 10% fetal bovine serum(FBS)and dissociated into single cells. Cells were seeded onto culture dishes coatedwith poly-L-lysine and grown in Neurobasal Medium containing 2% B27 supplement and 0.5 m M glutamine and incubated at 37℃ in a humidified 5%CO2 incubator. Change half of the medium to Neurobasal/B27 serum-free after 2h incubation. Cells would be ready after 24 hours for follow-up tests.NSE and GFAP are specific staining to identify neurons and astrocytes,respectively. GLP-1R was identified by immunofluorescence assay.Results:1 Optical microscope showed neuronal cells were round or oval in shape,cell body was small and bright with obvious surrounding halo. Cells have long neurites with multiple intercellular connections.2 Fluorescence microscope showed green fluorescence with NSE antibody existed in most of the cytoplasm and neurites while green fluorescence with GFAP antibody were rare in field with only in few cell cytoplasm and neurite.3 Anti-β-tubuli Ⅲ staining pictures showed neurons were clear with green cytoplasm and polygonal cell body. Immunofluorescence staining with GLP-1R antibody showed red fluorescence in the field. After image fusion,GLP-1R was widely expressed inside the cells which illustrate that primary cultured neuron cells did express GLP-1R.Part two Effect of liraglutide on neurite lengthObjective: Glucagon-like peptide-1(GLP-1), an incretin hormone, is secreted from the ileal L cells lining the gut in the presence of nutrients in the lumen and potentiate glucose-stimulated insulin secretion after a meal. It could be transcripted and translated according to proglucagon gene, degraded by dipeptidyl peptidase-IV(DPP-IV) and excreted by renal pathway. GLP-1processes glucose-dependent insulinotropic properties to regulate glucose homeostasis by promoting insulin secretion from b-cell, inhibiting inappropriate glucagon secretion from a-cell, delaying gastric empty and suppressing appetite and so on. Meanwhile, GLP-1 could promote b-cell proliferation, inhibit b-cell apoptosis, increase synthesis of insulin and improve b-cell function, thus delayed and even reverse the development oftype 2 diabetes. Glucagon-like peptide based therapy was widely accepted in clinic.GLP-1 exerts its multiple functions by active glucagon-likepeptide-1receptor(GLP-1R) and widely distributed in the whole body, including heart,brain, lung, kidney as well as pancreas. GLP-1 may play an important role in cardiovascular protection through the combination of GLP-1R and the expression of cardiovascular tissue in rodents and humans. Spanish scientists Romaní-Pérez for the first time proposed that GLP-1 plays a key role in the production of surface proteins, establishment and maturity of lung structure.The study also found liraglutide may inhibit the excessive activation of pulmonary tissue RAAS and inhibition of TGF-β1 to improve the effect of pulmonary interstitial fibrosis. Based on renal function, the experimental results confirm that the incretin hormone repairs renal injury in patients with type 2 diabetes mellitus and its mechanism including inhibition of rennin-angiotensin system activation, reduction of urinary albumin, relieving the inflammatory reaction and improving renal circulation and reducing renal fibrosis and etc.With the discovery of all kinds of function of GLP-1 except pancreatic ones, the research of GLP-1 on brain tissue is more of concern. Recent studies in mice showed that among neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, atrophy of amyotrophic lateral sclerosis and apoplexy, GLP-1 protected from memory and synaptic plasticity impair and reduced inflammatory. GLP-1 could effectively protect hippocampus neurons from death induced by glutamate and apoptosis induced by iron in rat. The learning ability of glp-1r knockout mice are lower than wild type mice and knockout mice are more susceptible under the kainate induced seizures. In addition, there are also studies reported that Exendin-4, GLP-1R agonist, has a protective effect in the number and length of neurite, cytoskeletal reorganization and electrophysiological aspects of human neuroblastoma. All kinds of studies have shown that GLP-1 analogues have a positive effect on the nervous system, but most of which are based on animal behaviorassessment. With rare study in vitro, we are not clear what happens in cellular level and molecular level and what mechanism of the changes. A neural circuit is a functional entity of interconnected neurons that is able to regulate its own activity using a feedback loop. It contains a series of branched neurons which interact with their neighbors via synapses to dendrites on other neurons.Patients suffering essential tremor exhibited low dendritic spine density and reduction of dendritic complexity compared with healthy subjects. So neurite growth is a critical step in neuronal development, regeneration,differentiation, and response to injury. Therefore, our study applies the length of neurite as a measure of the neural function to carry on the subsequent experiment.Methods: Cells were cultured by different concentrations of liraglutide(10 n M、100 n M、1μM) after 24 hours inoculation,control group was treated with an equal volume of PBS. Neurite length were measured using immunofluorescence imaging, data were compared by SPSS13.0 statistical software.Result:1 Liraglutide promotes neurites outgrowth in a dose dependent-manner.After treatment with different concentrations of liraglutide(10 n M, 100 n M,and 1 l M) for 24 h, cortical neurons display much longer neurite compared with control group. Using computer image analysis, the length of neurite was30.21 ±6.64μm in 10 n M liraglutide group, 34.90 ± 6.15μm in 100 n M liraglutide group, and 29.37 ± 5.10μm in 1μM liraglutide group. Compared with control group(21.29 ± 4.07μm), it was significantly increased. These results suggested that liraglutide could promote the neurite outgrowth of cortical neurons in a dose-dependent manner, among which 100 n M might be the suitable dosage to carry out the subsequent experiments.2 An MEK–ERK inhibitor U0126 inhibits liraglutide-induced neurite outgrowth of cortical neurons. Neurons were randomly divided into four groups for control group, liraglutide group, MEK-ERK pathway inhibitors-U0126 group and liraglutide+U0126 group(synergy group). Theneurite length was significantly reduced in the synergy group, compared with liraglutide group(liraglutide+U0126, 23.89 ± 7.02μm; liraglutide, 31.95±5.69μm, P<0.05). However, there was no obvious difference of the neurite length between control and U0126 treatment groups(control, 20.43 ± 3.90μm;U0126,21.01 ± 3.20μm, P > 0.05). Moreover, significant difference existed between the synergy group and control group(liraglutide + U0126, 23.89 ±7.02μm; control, 20.43 ±3.90μm, P<0.05).Part three Study of mechanism on liraglutide-promoted neurite growthObjective: Extracellular signal regulated kinases(ERK) as one of the important members in mitogen activated protein kinase(MAPK) family is mainly activated by hypertrophic agonist and closely related with regulation of cell growth, differentiation and proliferation. Phosphorylated extracellular signal regulated kinases(p-ERK) is the active form of ERK, including P-ERKl and P-ERK2, also known as p42/p44 MAPK, mainly composed of nuclear protein kinase during the process of cell growth, differentiation and transcription signal. Related growth factor receptors and tyrosine kinase in the cytoplasm firstly activate Ras which activates c-raf and MEK. Activated p-ERK is transferred into the nucleus, and in the nucleus of a number of nuclear proteins and transcription factors by of these transcription factors phosphorylation and activate the transcription of a series of, which is involved in cell growth, growth, proliferation, differentiation, and apoptosis. AS an important nuclear transcription factor, cyclic adenosine monophosphate(c AMP)response sequence binding protein(CREB) is regulates gene transcription of c AMP response element(CRE) in promoter. It is a key component in intracellular signaling pathways to promote neuronal regeneration, synapse formation and learning and memory ability.Many studies showed that GLP-1 analogues liraglutide protected human neuroblastoma cell line SH-SY5 Y by activation of Akt, MEK1/2 under stimulation of methylacetone aldehyde. GLP-1 protected beta cell of diabetic mouse due to cytokine induced apoptosis and ameliorated memory impairment of Alzheimer mice through the MEK-ERK pathway. Much evidence alsosuggested that the ERK signaling pathway could induce CREB phosphorylation and p38 MAPK pathways involved in this process. In order to figure out whether liraglutide affect neurons function through this signaling pathway, we performed the study and used U0126(MEK-ERK pathway inhibitors) to testified this hypothesis.Methods:In order to check how MEK-ERK signaling pathway related genes in liraglutide promoted neurite outgrowth work, primary neurons were randomly divided into four groups for control group, liraglutide group,MEK-ERK pathway inhibitors-U0126 group and liraglutide+U0126 group,ERK and p-ERK and CREB expression were detected by Western blot and ELISA, receptively.Result:1 MEK-ERK signaling pathway was involved in liraglutide-induced neurite outgrowth. Western Blot result showed that phosphorylation of ERK(p-ERK) level was increased significantly in liraglutide group(P<0.05) while down-regulated after synergizing with U0126(P < 0.05). However, p-ERK level was no significant in U0126 group compared with control(P>0.05).2 Phosphorylated CREB is a key molecule in liraglutide-mediated neurite elongation. The level of phosphorylated CREB(p-CREB) was examined by ELISA assay. Quantification of p-CREB expression showed no significant difference between U0126 and control groups(U0126, 5.58 ± 0.61 ng/ml;control, 5.28 ± 0.82 ng/ml, P>0.05). However, liraglutide increased p-CREB expression and partially inhibited by U0126(liraglutide, 11.31 ± 1.35 ng/ml;U0126+ liraglutide,7.84 ± 2.78 ng/ml, P<0.05).Part four Liraglutide promotes cortical neurons outgrowth under high glucose culture and relevant mechanismObjective: Clinical and basic studies have found that diabetes and neurological diseases are closely related. The risk of dementia in patients with diabetes increased significantly, usually accompanied by cognitive dysfunction, such as learning and memory damage. Epidemic study showed that 30.65% diabetic patients associated with insulin resistance will developAlzheimer’s disease. Increased free radicals as a result of high blood glucose,enhanced oxidative stress, accumulation of advanced glycation end products in the walls of blood vessels, endothelial cell dysfunction, increased aldose reductase activity lead to an increase in intracellular continuously hyperosmolarity, ultimately resulting in cell death. Target of rapamycin mammalian(m TOR) is an important regulatory gene in cell growth,proliferation, differentiation and apoptosis in the physiological and pathological processes. These functions can be realized by regulating the synthesis and degradation of protein, cell energy synthesis and metabolism,cell cycle and so on. Many central nervous system studies have shown that there are abnormal m TOR signal transduction pathways and neuronal cell loss in some neurodegenerative diseases. Cell death includes two forms, namely,cell apoptosis and cell necrosis. The specific regulation mechanism of apoptosis is not very clear at present, but the balance between anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax plays an important role in the process of apoptosis.In order to explore the effect and mechanism of GLP-1 on neuronal cells,our study cultured primary cortical neurons of fetal rat in vitro and observed the effects of different concentrations of GLP-1 analogues(liraglutide) on neurite length and MEK-ERK signaling pathway related in gene expression.With the following study that neurons were exposed to high glucose environment in order to mimic the diseases condition of diabetic neural disorder, we still wanted to figure out the effects of liraglutide on nerve cell activity, and focused on the action of the drug on apoptosis, which provided new ideas for GLP-1 drugs with treatment in diabetes and neural disease.Methods: The primary neurons were treated under high glucose medium,with different concentrations of liraglutide(10-9M、10-8M、10-7M、10-6M,10-5M,10-4M), control group with an equal volume of PBS, cell activity was detect by using CCK-8 kit. Then the cells were randomly divided into control group,high glucose group, high glucose+liraglutide group. Cell apoptosis was observed by Hoechst 33342 staining. Akt, m TOR, HIF-1a, Bcl-2, Bax andclaudin-5 expression were detected by Western blot and RT-PCR.Result:1 Liraglutide protects neurons from high glucose-induced damage. Cell viability was measured by CCK-8 kit. The result showed that cell viability in high glucose group was 31.78±2.81 with base line in control group 100%. Cell viability of different concentration liraglutide group were(10-9M:32.20±4.03;10-8M:43.98±4.06; 10-7M:74.92±5.59; 10-6M:63.29±8.18; 10-5M:52.00±6.01;10-4M:30.89±2.08) lower than the ones in control group. It shows a dose dependent manner with peak effect in 10-7M group, which was used in the following experiment. This suggested that high glucose might impair cell viability and liraglutide could partly alleviate this effect.2 Hoechst 33342 nuclear staining showed that liraglutide reduced apoptosis of neural cells cultured in high glucose.Under high glucose cells showed the typical morphological features of apoptosis, cell body became empty and flat, nuclear shrinkage. Part of cells were broken and hyperchromatic. Phenomenon was mild in liraglutide group.Counting 200 cells, the apoptosis number in control group was 24(apoptosis rate of 12%), the apoptosis number in high glucose group was 62(apoptosis rate of 31%), the apoptosis number in high glucose+liraglutide group was 41(apoptosis rate of 20.5%).3 Liraglutide regulated nerve cell apoptosis factor such as Bcl-2, Bax through Akt/m TOR/HIF-1a pathway. Western blot and RT-PCR result showed that expression of Akt、m TOR、HIF-1a and Bcl-2 in high glucose group were lower than control group, while Akt、m TOR、HIF-1a and Bcl-2 in the high glucose+liraglutide group were higher than high glucose group but still lower than control group. The expression of Bax has the opposite trend to other factors showing that increased in the high glucose group and decreased in the liraglutide+high glucose group. All the changes have statistic difference(P<0.05).4 High glucose and liraglutide had no effect on the expression of claudin-5. Claudin-5 is an important indicator of blood brain barrier function,but the study in vitro showed that the expression of protein and m RNA had no statistic differences among three groups(P > 0.05).Conclusions:1 The primary cultured cells from fetal mice brain are rich in neurons which had good morphologic performance and had little glial cells. The cells could be used as an in vitro model of research and showed the existence of GLP-1 receptor.2 Liraglutide could promote the neurite outgrowth of cortical neurons in a dose-dependent manner and the effect could be partially blocked by U0126,which indicated that indicate that MEK/ERK pathway was involved in liraglutide-mediated neurite outgrowth.3 Liraglutide promoted the neurite outgrowth via ERK phosphorylation and the downstream nuclear factor p-CREB also played an important role.4 Cells viability could be inhibited by high glucose but liraglutide could be partially alleviated it in certain concentrations. Hoechst 33342 nuclear staining showed that liraglutide decreased the apoptosis nuclear staining in high glucose group compared with control. Consistent with apoptosis staining,liraglutide elevated Bcl-2 expression and lowered Bax expression by Akt/m TOR/HIF-1a pathway. In addition, there was no relevance among claudin-5, high glucose and liraglutide.