IPI549调控小鼠肝脏缺血再灌注损伤的作用研究

Effect of IPI549 on hepatic ischemia-reperfusion injury in mice

摘要:

目的：研究IPI549特异性抑制PI3Kγ对小鼠肝脏缺血再灌注损伤的作用。方法：构建C57BL/6J小鼠肝脏缺血再灌注损伤模型和RAW264.7巨噬细胞缺氧复氧模型，给予IPI549（PI3Kγ抑制剂）干预，以ALT、HE染色、TUNEL判断肝脏损伤情况，Western blot检测组织和原代细胞p110γ（PI3Kγ）蛋白表达变化和RAW264.7细胞缺氧复氧过程TNF-α、JNK、p-JNK蛋白表达情况，ELISA检测细胞上清TNF-α水平。结果：相比于原代肝细胞，枯否细胞明显高表达p110γ。与Sham组比较，IR组肝组织中p110γ的蛋白水平降低；使用PI3Kγ特异性抑制剂（IPI549）干预小鼠，可加重小鼠肝脏缺血再灌注后肝组织损伤和肝脏组织细胞凋亡，而利用GdCl3预处理封闭枯否细胞，可使得IPI549引起的肝组织细胞损伤和凋亡加重得到缓解。在体外实验中，IPI549干预使RAW264.7细胞缺氧复氧过程JNK磷酸化水平升高、TNF-α蛋白表达增加，细胞上清TNF-α分泌增多。结论：PI3Kγ抑制剂IPI549干预使小鼠肝脏缺血再灌注损伤加重，其机制与IPI549抑制枯否细胞中的PI3Kγ影响JNK信号通路的活化，发挥促炎作用有关。

Abstract:

Objective：To investigate the effect of specific inhibition of PI3Kγ by IPI549 on hepatic ischemia-reperfusion injury in mice. Methods：C57BL/6J mice were used to establish a model of hepatic ischemia-reperfusion injury，and RAW264.7 macrophages were used to establish a model of hypoxia and reoxygenation. The mice and RAW264.7 macrophages were treated with the PI3Kγ in－hibitor IPI549，and alanine aminotransferase，HE staining，and terminal deoxynucleotidyl transferase-mediated dUTP nick-end label－ing were used to evaluate hepatic injury. Western blot was used to measure the changes in the protein expression of p110γ （PI3Kγ） in tissue and primary cells and the protein expression of tumor necrosis factor-α（TNF-α），c-Jun N-terminal kinase（JNK），and phos－phorylated JNK （p-JNK） in RAW264.7 cells during hypoxia and reoxygenation. ELISA was used to measure the level of TNF-α in cell supernatant. Results：Kupffer cells had significantly higher expression of p110γ than primary hepatocytes. Compared with the sham group，the ischemia-reperfusion injury group had a significant reduction in the protein expression of p110γ in liver tissue. The mice treated with the PI3Kγ inhibitor IPI549 showed aggravation of liver injury and cell apoptosis after hepatic ischemia-reperfusion，and GdCl3 pretreatment to block Kupffer cells alleviated the aggravation of cell injury and apoptosis in liver tissue caused by IPI549. In vitro experiments showed that IPI549 intervention of RAW264.7 cells increased the phosphorylation level of JNK，the protein expres－sion of TNF-α，and the secretion of TNF-α in cell supernatant. Conclusion：Intervention with the PI3Kγ inhibitor IPI549 can aggra－vate hepatic ischemia-reperfusion injury in mice，possibly by inhibiting PI3Kγ in Kupffer cells to affect activation of the JNK signaling pathway and exerting a pro-inflammatory effect