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为了探讨海马神经元中是否有Toll样受体4(Toll-like receptor 4,TLR4)介导的Akt/FoxO3a/Bim信号通路,及该通路在海马神经元凋亡中的作用和机制,本实验运用脂多糖(lipopolysaccharide,LPS)作用于原代培养的大鼠海马神经元,利用不同的工具药,以减弱或加强TLR4/Akt/FoxO3a/Bim通路的作用。应用CCK-8法检测细胞活力,Western blot法检测神经元p-Akt(Ser473)、Akt、p-FoxO3a(Thr32)、FoxO3a、Bim、活化的Caspase-3蛋白的表达变化,real-time PCR法检测海马神经元Bim的mRNA表达变化,免疫荧光法观察FoxO3a核易位情况,流式细胞术检测细胞凋亡率。结果显示:LPS作用于海马神经元不同时间后,各组细胞活力均下降(P<0.05),且具有一定的时间依赖性;LPS作用后p-Akt(Ser473)、p-FoxO3a(Thr32)表达减少,FoxO3a易位进入胞核,而促凋亡蛋白Bim及活化的Caspase-3表达增加,且海马神经元的凋亡率也增加(P<0.05);PI3K特异性抑制剂LY294002预处理后p-Akt(Ser473)、p-FoxO3a(Thr32)表达较LPS组降低,而促凋亡蛋白Bim及活化的Caspase-3表达升高,细胞凋亡率也明显升高(P<0.05);TLR4抗体预处理后p-Akt(Ser473)、p-FoxO3a(Thr32)较LPS组增多,FoxO3a易位进入胞核的活动减弱,而促凋亡蛋白Bim、活化的Caspase-3及细胞的凋亡率均减少(P<0.05)。以上结果表明,海马神经元中有TLR4介导的Akt/FoxO3a/Bim通路;神经元可通过该通路引起自身的凋亡。
To investigate whether there is Toll-like receptor 4 (TLR4) -mediated Akt / FoxO3a / Bim signaling in hippocampal neurons and the role of this pathway in hippocampal neuronal apoptosis, this experiment The effect of TLR4 / Akt / FoxO3a / Bim pathway on the primary cultured rat hippocampal neurons was investigated by lipopolysaccharide (LPS) using different tools. Cell viability was detected by CCK-8 assay. The expression of p-Akt (Ser473), Akt, p-FoxO3a (Thr32), FoxO3a, Bim and activated Caspase- The mRNA expression of Bim in hippocampal neurons was detected. The FoxO3a nuclear translocation was observed by immunofluorescence and the apoptosis rate was detected by flow cytometry. The results showed that after LPS treatment, the viability of cells in each group decreased (P <0.05) and the time-dependent manner was observed. The expressions of p-Akt (Ser473) and p-FoxO3a (Thr32) (P <0.05). PI3K-specific inhibitor LY294002 pretreatment p The expressions of Akt (Ser473) and p-FoxO3a (Thr32) were significantly lower than those in LPS group, while the expression of Bim and activated Caspase-3 were significantly increased (P <0.05) After pretreatment, the expression of p-Akt (Ser473) and p-FoxO3a (Thr32) increased compared with LPS group, and the activity of FoxO3a translocated into the nucleus decreased. However, the apoptotic rate of Bim, activated Caspase- Decrease (P <0.05). The above results show that TLR4-mediated Akt / FoxO3a / Bim pathway in hippocampal neurons; neurons can cause their own apoptosis through this pathway.