The Mechanism Of Notch-1 Signaling On Proliferation And Activation Of Astrocyte After Hypoxia Exposure

Objective:Activation of astrocyte has been implicated in the neonatal hypoxic brain injury. However, the mechanisms that control astrocytic activation and astrogliosis formation remain unknown. Here, we found that Notch-1 is upregulated and activated by hypoxia in astrocytes as confirmed by an increase in NICD and Hes-1 expression. Remarkably, blockade of Notch-1 signaling with the specific inhibitor DAPT suppressed astrocytic proliferation. In addition, both expression and secretion of inflammatory factor IL-1β was inhibited in DAPT-pretreated astrocytes, while no significant change in TNF-α expression was detected. Most interestingly, GFAP and VEGF expression was suppressed by DAPT pretreatment in hypoxic astrocytes and further confirmed in neonatal rats following hypoxic brain injury. Furthermore, inhibition of Notch caused a remarkable decrease in NF-KB/p65 expression and translocation. Moreover, downregulation of either VEGF or NF-κB/p65 reduced astrocytic proliferation. Taken together, these results demonstrated that Notch-1 signaling was activated in hypoxic astrocytes and regulated astrocytic proliferation and activation by suppressing VEGF or NF-KB/p65 signaling pathway. Therefore, Notch signaling is a potential therapeutic strategy in hypoxia brain damage.Methods:We use postnatal Wistar rats as the model organism to explore the mechanism of Notch-1 signaling on proliferation and activation of astrocyte after hypoxia exposure via cell cultures, postnatal rats’ treatment, proliferation index by bromodeoxyuridine (BrdU) labeling and immunostaining, double Immunofluorescence, RT-PCR, western blotting analysis, analysis of IL-1βand VEGF by ELISA, transfection with siRNA,and analysis NF-κB/p65 protein level by using NF-κB/p65 ELISA kit.Results:1.Notch signaling was activated in primary astrocytes after hypoxia exposureTo determine whether Notch-1 is activated in primary astrocytes under hypoxic conditions, we first examined both mRNA and protein for Notch-1 expression using RT-PCR, immunofluorescence staining and Western blot analysis, respectively. Densitometric evaluation of the relative expression showed that Notch-1 mRNA expression was significantly increased after hypoxia. Immunofluorescence staining indicated notable enhancement of Notch-1 in astrocytes under hypoxia as compared with the normoxia control. Consistently, Western blot analysis showed the increase in Notch-1 protein expression. Arising from the increase in Notch-1, we next investigated whether Notch signaling was activated in hypoxic astrocytes. Indeed, we found a significant increase in NICD protein expression in hypoxic astrocytes at 12 h as compared to control. This was further confirmed by the detection of enhanced immunofluorescence intensity of NICD both in the cytoplasm and nucleus, especially in the nucleus.As the main target gene of Notch signaling, Hes-1 mRNA expression was concurrently increased at 6 and 12 h after hypoxia in primary astrocytes and significantly inhibited by DAPT, a potent Notch inhibitor. Similar tendency in changes of Hes-1 protein expression was also observed in cells treated with DAPT under hypoxia as compared to untreatment control.2.Notch signaling blockage with DAPT prevented astrocytic proliferationTo investigate the role of Notch signaling in astrocytic proliferation, we performed BrdU incorporation assay followed by immnumofluorensence staining. The ratio of BrdU positive cells against total cells was increased from 31.7±3.8%to 48.4±4.5% (p<0.01) after 12 h hypoxia stimulation. However, in DAPT-treated cells, the percentage of BrdU positive cells was decreased to 31±5% (p<0.05). Additionally, we performed cell death assay by Annexin V/PI staining. There was no significant difference of the percentage of apoptotic astrocytes between control and hypoxia or between hypoxia and hypoxia+DAPT. The above results ruled out the possibility that the cell death led to the reduction of astrocytic proliferation after DAPT treatment.3.Notch signaling blockage in astrocytes affected the production of inflammatory mediators TNF-α and IL-1βhypoxia resulted in a significant increase in mRNA expression of TNF-α and IL-1β in primary astrocytes. In DAPT-treated astrocytes, mRNA expression of TNF-α had no significant changes while IL-1β mRNA expression was significantly inhibited. The results showed a significant decrease in IL-1β protein expression level detected by ELISA while no obvious changes of TNF-α expression could be found in astrocytes pretreated with DAPT (Data not shown).4.DAPT treatment in hypoxic astrocytes inhibited GFAP and VEGF expressionNotch signaling inhibition in astrocytes led to a significant decrease in mRNA expression of GFAP. Next, we investigate whether Notch signaling inhibition could suppress VEGF expression, both of which play important roles in reactive astrocytes after hypoxic exposure. RT-PCR analysis showed significant inhibition in VEGF mRNA expression in DAPT-treated astrocytes. ELISA confirmed the decrease of VEGF secretion in DAPT-treated astrocytes. The intensity of VEGF in hypoxic astrocytes was strongly enhanced as compared to control cells, but this effect was abrogated by treatment with DAPT in astrocytes. Consistently, brain tissue under hypoxia, but not control brain tissue, displayed an increase in VEGF expression as confirmed by Western blot analysis. However, DAPT treatment evidently abrogated the VEGF protein expression induced by hypoxia.5.The involvement of NF-κB and VEGF pathway in activation and proliferation of hypoxic astrocytesBoth Western blot analysis and ELISA analysis showed a significant increase in NF-KB/p65 in astrocytes exposed to hypoxia, but these effects were significantly blocked by DAPT treatment. To determine whether VEGF and NF-κB are involved in astrocytic proliferation under hypoxia, we separately introduced a VEGF siRNA vector, a p65 siRNA vector and their control siRNA vectors into astrocytes. Western blot analysis indicated notable depletion of VEGF and p65 in the VEGF siRNA and a p65 siRNA transfected cells compared with their controls, respectively. Knockdown of VEGF and NF-κB in astrocytes significantly reduced BrdU incorporation and therefore proliferation of astrocytes by nearly up to 50% and 40% compared with their controls.Conclusions:1. Notch signaling was activated in primary astrocytes after hypoxia exposure2. Notch signaling blockage with DAPT prevented astrocytic proliferation3. Notch signaling blockage in astrocytes affected the production of inflammatory mediators TNF-α and IL-1β4.. DAPT treatment in hypoxic astrocytes inhibited GFAP and VEGF expression5. The involvement of NF-κB and VEGF pathway in activation and proliferation of hypoxic astrocytes...