Effects Of IL-1β On The Proliferation And Differentiation Of Hippocampal Neural Stem Cells In Vitro

Background and objective:The pathogenesis of depression is not clearly revealed. There are many theoretical hypotheses on the term of etiology of depression. Damage to hippocampal neurons and aregeneratory as an important etiology contributes to depression is drawing more and more attention nowdays. Hippocampus is one of the important zones where neurons can renew themselves. Hippocampal neural stem cells are the precursor cells of neurons, which have an important impact on the sum total of neurons'regeneration and maintenance. In vitro experiments showed that IL-1βcan inhibit the proliferation of hippocampal neural progenitor cells.Therefore, study the role and mechanism of IL-1βin the process of depression, will not only reveal the pathogenesis of depression, but also provide new targets for the development of new antidepressants.Our preliminary work has confirmed that,intraperitoneal injection of LPS, intraperitoneal injection of reserpine can both cause behavioral depression in rats and cytokine IL-1βelevation in various brain regions. Intraventricular injection of IL-1βcan also cause behavioral depression in rats, Intraventricular injection antagonist of IL-1βmaybe partially improve the behavioral depression. Non-selective adenosine receptor antagonist caffeine and A_2A receptor antagonist can both partially reverse this behavioral depression. In vitro IL-1βcan dose dependently restrain hippocampal neurons viability, reducing the expression of BDNF,so CREB-BDNF pathway may play an important role in this process, NMDA receptor may be involved,too. The study showed that IL-1βis closely related to behavioral depression, on its molecular mechanism may be related to the decreased expression of brain derived neurotrophic factor (BDNF).This experiment aims to further explore the effect and mechanism of IL-1βon the activity of hippocampal neural stem cells including the influence of A_2A receptors and role of the NMDA receptor in this process. Research the effect of IL-1βon the percentage of neural stem cells differentiate into neurons and astrocytes. Materials and methods:1. The isolation,Culture and identification of Hippocampal NSCs3-4 neonatal SD rats (<24h), decapitate the brain in biological safety cabinet, separate two hippocampus on the both sides,remove residual vascellum, cut up hippocampus tissues. Mechanically triturate them into single-cell suspension, add with 10% FBS in DMEM/F12 medium,then place cell suspension in 37℃,5%CO₂ incubator for 30min, removal of fibroblast cells, 24h after the supernatant was discarded and re-adding fresh culture medium containing 10% FBS in DMEM/F12, 72h after,centrifuge and discard the supernatant, chang into neural stem cell culture medium (containing 2% B27, 20ng/ml bFGF and 20ng/ml EGF in DMEM/F12 medium), the medium was renewed on day 3-4 and 7-8 days passage one time. Continuously pure culture for about 21 days, the cells can be used as experiments materials. A highly selective monoclonal antibody Nestin was used to identify Neural stem cells by immunocytochemistry (ICC).2. The effect and mechanisms of IL-1βon NSCs proliferation2.1,IL-1β,incubates neural stem cells for 3 days, remove half of the cells when change medium, measuring cell activity by methyl thiazol tetrazolium assay(MTT assay). Then refill the remaining cells with fresh medium and drugs as before, continuously culture for another 3 days, take out all of the residual cells, measuring cell activity.2.2,10ng/ml IL-1βadding NMDA receptor antagonist MK-801 or A_2A receptor antagonist MSX-3 incubate neural stem cells for 3 days, take out all of the cells,measuring cell activity.2.3,IL-1βwith different experimental concentrations (0,5,10,20)ng/ml incubate neural stem cells for 3 days, collect the supernatant,measure the BDNF concentration in the supernatant by ELISA kits.3. The effect of Caffeine and NGF on NSCs proliferationthe Caffeine or NGF incubate neural stem cells for 3 days, remove half of the cells when change medium, measuring cell activity by methyl thiazol tetrazolium assay(MTT assay). Then refill the remaining cells with fresh medium and drugs as before, continuously culture for another 3 days, then remove all of the cells, measuring cell activity. 4. The effect of IL-1βon NSCs differentiate into astrocytes and neuronspolylysine coats plate, adjust the density of neural stem cells, different experimental concentrations of IL-1β(0,5,10,20)ng/ml with 10% FBS were added to neural stem cell culture medium. 3 days later, renew the supernatant, then continuously culture for another 3 days,remove all the supernatant, By fluorescence immunocytochemical staining and flow cytometry detect the ratio of astrocytes and neurons stained by GFAP orβ-Tubulin-Ⅲantibody.5. Data analysisExcel 2003 and SPSS 17.0 statistical software were applicated for statistical analysis. Datas were presented as mean±standard deviation((x|-)±S), compared with analysis of variance. A level of P<=0.05 was accepted as statistically significant, P<=0.01 was accepted as statistically highly significant.Results1. The effect and mechanisms of IL-1βon NSCs proliferation1.1. Culture,Morphology observatifition and identification of Neural stem cellsNeural stem cells are observed under inverted phase-contrast microscope. On the primary cells are single-cell suspension, bright and round, and aggregate to form spheroid-like bodies, which called neurosphere. 24h after in the supernatant seeing a large number of organizations debris,so it is difficult to observe neurospheres, discard most of the supernatant and add fresh DMEM/F12 medium containing 10% FBS. 24h after neurospheres consisted of 4-8 cells appear in the vision field, cells proliferation continue, on the 4th day change the culture medium into the neural stem cell culture medium, the diameter of the neurospheres is about 50um-100um. On the 7th day, the diameter of the neurospheres aggregates to about 100-200um. Mechanically triturate and trypsin-dispersed cell culture , the neural stem cells were passaged and diepersed into smaller groups. Then cells proliferate in the same way to form neurospheres. About 21 days after the third generation, neural stem cells can be used as experimental materials.The neurospheres of NSCs are plated on Polylysine coated 96 cells cultural plates for 24 hours. Nestin antibody was applied to identify NSCs by the method of immunocytochemistry. In the microscope field , all the neurospheres are wholly stained positively.1.2. IL-1βdecreases the proliferation of NSCsIL-1β(0,5,10,20) ng/ml incubate hippocampal neural stem cells for 3 days. Take out half of cells to analyse cells viability and proliferation rate by using MTT assay, then supplement fresh nutrient medium continue to cultivate for 3 days. Remove surplus all cells, using the same method analyses cells viability.Cells viability after IL-1βtreatment are significantly decreased when compared with the control group by MTT on 3rd day and 6th day(P<0.05).The results showed that different experimente concentration of IL-1βincubate neural stem cells for 3 or 6 days, can dose-dependently reduce the activity of hippocampal NSCs. 1.3. NMDA receptor antagonist reverse the effect of IL-1βon NSCs proliferationIL-1β10ng/ml and NMDA receptor antagonist MK-801(0,2.5,5,10,20,40)um/ml incubate neural stem cells for 3 days, the control group is normally cultured neural stem cells without IL-1βand MK-801.The results showed that On the 3rd day cells viability of IL-1β10ng/ml,MK-801 (5,10,20, 40)um/ml groups were significantly increased when compared with the IL-1β10ng/ml, MK-801(0)um/ml group by MTT(P<0.05). Cells viability of IL-1β10ng/ml,MK-801(0)um/ml group was significantly decreased when compared with the control group(P<0.01). The results reveal that MK-801 can reverse the effect of IL-1β.1.4. The role of A_2A receptor antagonists in IL-1βinhibitory effect on NSCsIL-1β10ng/ml and A_2A receptor antagonist MSX-3(0,125,250,500,1000,2000)ng/ml incubate neural stem cells for 3 days , the control group is normally cultural neural stem cells without IL-1βand MSX-3.The results showed that on 3rd day cells viability of the IL-1β10ng/ml and MSX-3 (0um/ml) group was significantly decreased compared with the control group(P<0.01) .cells viability of IL-1β10ng/ml, MSX-3 (500ng/ml) was significantly increased when compared with the IL-1β10ng/ml and MSX-3 (0ng/ml) group by MTT(P <0.05).When compared with the control group cells viability of IL-1β10ng/ml,MSX-3(0,125,250,1000,2000)ng/ml was significantly decreased (P <0.05). The results reveal that MSX-3 can partially reverse the effect of IL-1β. 1.5. IL-1βinhibit the release of BDNF of hippocampal NSCsIL-1βat the dose of (0,5,10,20)ng/ml was added respectively to the cultured NSCs.The concentration of BDNF in supernatant after IL-1βtreatment 3 days was significantly decreased when compared with the control group by enzyme linked immunosorbent assay (ELISA) (P <0.05).2. The effect of IL-1βon the differentiation of neural stem cells2.1. IL-1βpromote neural stem cells differentiation into astrocytesMechanically triturate and trypsin-dispersed cell culture, the Neural stem cells were diepersed into single cells suspension. 10% FBS + neural stem cell culture medium re-suspended cells,adjust cells density and culture in Polylysine coated cell culture plate, different experiments concentrations of IL-1β(0,5,10,20)ng/ml was added to supernatant in order to induce NSCs to defferentiate,on the 3rd day the supernatant liquid was half discarded and new supernatant liquid were added as before, on the 6th day, detect the ratio of astrocytes by immunocytochemistry or flow cytometry.Immunocytochemistry and flow cytometry results show that: The percentage of astrocytes in group IL-1β(10,20)ng/ml was significantly inceased when compared with the control group IL-1β0ng/ml, The results reveal that IL-1βcan dose-dependently significantly promote NSCs defferentiate into astrocytes (P<0.05).2.2. IL-1βhas no direct effect on neural stem cells differentiation into neuronsThe methods are same as described above,the only difference is that antibodyβ-TubulinⅢis used to probe the ratio of neurons by immunocytochemistry.Immunocytochemistry results show that the percentage of neurons in groups of IL-1β(5,10,20)ng/ml have no significant difference when compared with the control group IL-1β0ng/ml(P>0.05), IL-1βhas no direct effect on the ratio of neurons among the NSCs differentiated cells(P>0.05). 3. The effect of Caffeine and NGF on NSCs'proliferation viability3.1. The effect of caffeine on NSCs'viability in vitroOur preliminary work has confirmed that: caffeine and A_2A adenosine receptor antagonists can partially reverse IL-1βinduced behavioral depression, indicating that caffeine can partially reverse IL-1βeffect. But further experiments show that caffeine can directly inhibite neuronal activity in vitro rather than reverse neuronal inhibition of IL-1β. To further confirm the effect of caffeine on neural stem cell proliferation and whether caffeine as adenosine receptors antagonist is involved in IL-1β-mediated neuronal injury, do experiments as follows.Caffeine (0,0.1,1,10) mM/ml were added to incubate hippocampal neural stem cells for 3 days, take out half of cells to analyse cell viability and proliferation rate by using MTT assay, then supplement fresh nutrient medium and continue to cultivate for another 3 days, remove surplus all cells, the same methods analyse cells viability.cells viability after caffeine(1,10mM/ml)treatment was also significantly decreased when compared with the control group by MTT on the 3rd day and 6th day (P <0.05).The results suggest that caffeine can significantly decrease hippocampal NSCs'viability in a dose dependent manner in vitro.3.2. The effect of NGF on NSCs'viability in vitroNGF is one kind of neurotrophic factors, widespread in the central nervous system like BDNF. NGF plays important role in central nervous system development and function. In order to obtain as many neurons differentiated from NSCs, there must be sufficient reserves of NSCs. Modulate microenvironment in vivo to see if NGF can promote NSCs to proliferate.NGF(0,20,50ng/ml )were added to incubate hippocampal NSCs for 3 days, take out half of cells to analyse cells viability and proliferation rate by using MTT assay, then supplement fresh nutrient medium continue to cultivate for 3 days, remove surplus all cells, the same method analyses cell viability.On the 3rd and 6th day cells viability after NGF(50ng/ml)treatment was significantly increased when compared with the control group by MTT method(P <0.05), The results suggest that NGF can significantly increase NSCs'viability. Conclusion1. IL-1βdecreased hippocampal neural stem cells proliferation activity in vitro,the mechanism may be correlated with decreased BDNF concentration in the supernatant. NMDA system and the adenosine system, may be also involved in this process.2. IL-1βcan promote neural stem cell to differentiate into astrocytes, but has no significant effect on the differentiation into neurons.3. Caffeine can inhibit neural stem cell proliferation activity in vitro. NGF can promote neural stem cell proliferation in vitro.