過氧化氫對大鼠星狀神經膠細胞間隙接合之影響

Abstract

中樞神經系統受傷或一些引起神經退化的疾病，都會造成中樞神經系統內局部的氧化壓力上升，而首當其衝受影響的就是中樞神經系統內含量最多的星狀神經膠細胞。星狀神經膠細胞間有發達的間隙接合構造，可以快速且同步地調節中樞神經系統內的微環境，以減緩氧化壓力的影響。我們利用H2O2來模擬氧化壓力的傷害，探討初級培養的星狀神經膠細胞間間隙接合功能上及分布上的變化。我們先以GFAP的免疫螢光染色確認細胞的純度，再以螢光染劑DCF-AM確定H2O2會進入細胞及造成細胞內氧化壓力的上升。我們發現，H2O2在3~20分鐘的短時間處理下，就能造成螢光追蹤劑藉由間隙接合在細胞間傳遞的面積增加16%~21%。在間隙接合的組成蛋白Cx43的磷酸化程度上，H2O2的處理會造成整體Cx43蛋白質的去磷酸化。另外，我們利用蔗糖梯密度離心的方法，將脂筏蛋白質和其他胞內的蛋白質分離，觀察到Cx43和caveolin-3會共同分布在脂筏會分布的位置，且此分布情形並不受H2O2的影響。在免疫螢光染色，我們發現脂筏凹陷蛋白Caveolin-3和間隙接合蛋白Cx43有同位染色現象，而且H2O2處理之後亦不影響此同位染色的情形。為了進一步確認磷酸激酶的作用對Cx43磷酸化狀態的影響，我們先利用PKC的抑制劑chelerythrine處理細胞，結果抑制PKC會造成間隙接合傳遞螢光追蹤劑的功能下降。但以p368-Cx43的抗體去檢測PKC在Cx43上的作用點，發現H2O2使p368-Cx43的染色有下降趨勢。而H2O2的處理也會使活化態的Src磷酸激酶減少，而以Src的抑制劑PP2處理也可以促使間隙接合的功能上升，這結果暗示H2O2的處理可能是透過抑制Src的活性來增進間隙接合的功能。綜合以上結果，我們在初級培養的星狀神經膠細胞內，觀察到H2O2的處理會造成間隙接合的傳遞功能上升，並使間隙接合組成蛋白Cx43的磷酸化程度下降。H2O2的處理並不影響Cx43在Caveolin-3組成脂筏內之分布，但卻可使活化態的Src減少，暗示H2O2並不是透過改變Cx43的分布來增進間隙接合的功能，而是藉由抑制Src磷酸激酶的活性來促進間隙接合通道的開啟。

In central nervous system, both injury and neurodegenerative disease could locally cause oxidative stress. Astrocytes are the most populated cells in central nervous system and are affected first by oxidative stress. Gap junctions in astrocytes could modulate the microenvironment in central nervous system and reduce harmful effect by oxidative stress. We used H2O2 to simulate oxidative stress of central nervous system injury and investigate function and distribution changes of gap junctions in primary astrocyte cultures. We used the fluorescent dye, DCF-DA, to confirm the permeability of H2O2 in astrocytes. The area of fluorescent dye permeation through gap junction increased 16%~21% 3~20 minutes after treatment of H2O2 concomitant with Cx43 dephosphorylation. Co-fractionation of Cx43 and caveolin-3 was determined by a discontinuous sucrose gradient centrifugation, and this co-fractionation was not disrupted by H2O2 treatment. By immunocytochemistry, Cx43 and caveolin-3 were colocalized at cell-cell contacts, and this colocalization was not changed by H2O2 treatment, either. Treatment of PKC inhibitor, chelerythrine, prevented H2O2-induced gap junctional intercellular communication as assessed by fluorescent dye coupling. Furthermore H2O2 also decreased immunoreactivity of p368-Cx43 in astrocytes. Besides, H2O2 caused a decrease in the levels of p-Src, the active Src kinase, and pretreatment of Src kinase inhibitor, PP2, alone, could enhance gap junctional intercellular communication. These results suggested that H2O2 may inhibit Src kinase activity to promote gap junctional intercellular communication. In conclusion, we have demonstrated that H2O2 could increase gap junctional intercellular communication and decrease the phosphorylation state of Cx43 in astrocytes and this effect may be mediated through a pathway related to Src kinase inhibition. Although Cx43 and caveolin-3 were codistributed at lipid raft, this codistribution was not affected by H2O2.