Akt通路及细胞凋亡在瘦素介导的大鼠慢性吗啡镇痛耐受中的作用

摘要

目的：探讨Akt（又称蛋白激酶B）及活化的caspase-3在瘦素介导的大鼠慢性吗啡镇痛耐受中的作用。方法：在SD大鼠建立慢性吗啡镇痛耐受模型；采用Western blotting法测定脊髓Akt和cleaved caspase-3的水平；免疫组织化学染色法检测脊髓磷酸化Akt （ p-Akt ）及cleaved caspase-3阳性细胞；免疫双染检测p-Akt 及cleaved caspase-3阳性细胞的定位。结果：鞘内慢性注射吗啡（15μg）7 d可明显上调大鼠脊髓p-Akt及cleaved caspase-3的蛋白水平；在注射吗啡前30 min，鞘内注射瘦素拮抗剂（3μg）7 d能显著地抑制慢性吗啡处理对p-Akt和cleaved caspase-3的上调作用；p-Akt只定位在脊髓神经元，而cleaved caspase-3仅定位在星形胶质细胞；瘦素拮抗剂、Akt抑制剂及caspase-3抑制剂均能抑制慢性吗啡镇痛耐受。结论：脊髓Akt通路及活化的caspase-3参与瘦素介导的大鼠慢性吗啡镇痛耐受。%AIM:To explore the roles of Akt ( also called protein kinase B ) and active caspase-3 in the leptin-mediated chronic morphine antinociceptive tolerance in rats .METHODS: A model of chronic morphine antinociceptive tolerance was established in the SD rats .The protein levels of spinal Akt and cleaved caspase-3 were tested by Western blotting.The technique of immunohistochemical staining was used to detect the immunoreactivity positive cells of phospho -rylated ( p)-Akt and cleaved caspase-3 in the spinal cord .Double staining of immunohistochemistry was used to examine the cellular location of the p-Akt and cleaved caspase-3 positive cells.RESULTS: The chronic intrathecal injection of morphine (15 μg) for 7 d markedly upregulated the spinal protein levels of p-Akt and cleaved caspase-3 in the rats.Thirty min before injection of morphine , intrathecal injection of leptin antagonist (3μg) for 7 d significantly attenuated the upreg-ulation of the protein levels of p-Akt and cleaved caspase-3 induced by chronic morphine treatment .The p-Akt was exclu-sively observed in the spinal neurons .The cleaved caspase-3 was only localized with the spinal astrocytes .Intrathecal injec-ting the inhibitors of leptin , Akt and caspase-3 ameliorated the chronic antinociceptive tolerance .CONCLUSION: The spinal Akt pathway and active caspase-3 are involved in the leptin-mediated chronic morphine antinociceptive tolerance in rats.