The Expression And Regulation Of Two-pore Domain Potassium Channel Trek-1and It's Involvement In Astrocyte Function And Neuroprotection Under Simulated Ischemia Condition

Objective:To investigate the expression of two pore domain potassium channel TREK-1on primary cultured neurons and astrocytes under normal and hypoxia conditions and the effects of TREK-1activity on astrocytic functions including glutamate clearance, release of s100β and proliferation of astrocytes and neuronal apoptosis under hypoxia condition. The roles of TREK-1on astocyte function under normal and pathological conditions remain unclear largely due to lack of specific pharmacological agents. In order to specifically inhibit the TREK-1channel activity and provide a helpful technique basis to further investigate the function of TREK-1channel on astrocytes, small interfering RNA targeted for TREK-1were designed, synthesized and transfected to cultured astrocyte.Methods:The expression of TREK-1in cultured neurons and astrocytes were investigated by double immunofluorescence staining respectively. The temporal changes of TREK-1expression at both RNA and protein levels in cultured astrocytes under hypoxia insults were measured with Real-time PCR, immunofluorescence staining and Western Blot; In purified cultured astrocytes, when treated with the TREK-1channel blockers, the changes of Glu concentration in the cultured media were analyzed by the colorimetric method; The S100β levels in the supernatant fluid of cultured medium were measured with the Elisa and the proliferation of astrocytes were measured by FACS and BrdU staining under hypoxia condition. Astrocyte and neuron were co-cultured in vitro according to the reference description. After the co-cultures were treated with the TREK-1channel blockers under hypoxia condition for2h, the apoptosis of neurons were detected with TUNEL staining. TREK-1specific siRNA duplexes (siT1, siT2, siT3) were transfected to cultured astrocyte at the optimal concentration, and the most efficient siRNA were identified by the method of immunocytochemistry staining and Western blot;Results:The TREK-1immunoreactivity was expressed on both neurons and astrocytes under normal condition. During the early phase of hypoxia insults, at1h and6h after hypoxia, the expression of TREK-1was up-regulated at both RNA and Protein levels. However, at the late phase, it began to decrease and returned to normal level gradually. Quinine (200μM) or Bupivacaine (500μM) were shown to suppresse the astrocytic glutamate uptake capacity under both normal and hypoxia condition. Meanwhile, they significantly enhanced hypoxia induced astocytic S100β production and proliferation. In astrocyte and neuron co-cultures, Quinine (200μM) or Bupivacaine (500μM) increased the rate of neuronal apoptosis under hypoxia condition. Chemical synthesis small interfering RNA targeted for TREK-1siT2showed the most powerful knockdown efficiency in the TREK-1expression;Conclusions:Our results suggest that TREK-1activity is closely related with astrocyte function and neuronal survival. Chemical synthesis small interfering RNA transfection is sufficient in knockdowm the expression of TREK-1.This would provide evidence showing astrocytic TREK-1involvement in ischemia pathology which may serve as a potential new therapeutic target in stroke.