Ghrelin通过JNK信号通路抑制LPS诱导的肺泡巨噬细胞凋亡

Ghrelin Affects LPS-induced NR8383 Cell Apoptosis through c-Jun N-terminal Kinase Signaling Pathway

【目的】观察体外条件下加入外源性ghrelin对内毒素(LPS)诱导大鼠肺泡巨噬细胞NR8383凋亡以及吞噬能力的影响,并探讨JNK(c-Jun N-terminal kinase)信号通路在其中所起的作用。【方法】用CCK-8(Cell Counting Kit-8)法检测加入LPS或者ghrelin与LPS共孵育后NR8383的细胞毒性;流式细胞技术、原位末端标记法(TUNEL)检测细胞凋亡率;Western blot检测JNK、phospho-JNK信号通路蛋白以及cleaved caspase-3、Bax、Bcl-2凋亡相关分子蛋白的表达;激光共聚焦技术检测巨噬细胞的吞噬能力;使用ghrelin受体拮抗剂[D-Lys-3]-GHRP-6、JNK特异性抑制剂SP600125分别抑制ghrelin受体及JNK激酶的激活。【结果】CCK-8检测结果显示LPS可显著抑制NR8383增殖,而ghrelin可呈浓度依赖性地阻断这种抑制作用;流式和TUNEL检测进一步证实ghrelin可显著降低LPS所致NR8383凋亡作用(P<0.05),而应用ghrelin受体拮抗剂[D-Lys-3]-GHRP-6可以减弱ghrelin的抗凋亡效果(P<0.05);LPS可以激活JNK激酶活性,并改变其下游凋亡相关蛋白的表达,其中促凋亡蛋白Bax以及cleaved caspase-3表达上调,抗凋亡蛋白Bcl-2表达下调,应用ghrelin可以逆转LPS对JNK激酶的激活,继而下调Bax以及cleaved caspase-3的表达,上调Bcl-2的表达,差异均具有统计学意义(P<0.05);ghrelin还可以维持LPS持续刺激下NR8383的吞噬能力(P<0.05)。【结论】ghrelin通过下调JNK信号通路的激活抑制LPS诱导的NR8383的凋亡,并维持其吞噬能力。

【Objective】 To observe the effects of in vitro exogenous administration of ghrelin on LPS induced apoptosis in rat alveolar macrophage NR8383, and to investigate the potential role of JNK signaling pathway in this impact. 【Methods】 CCK-8 assay was used to examine the cell viability of NR8383 treated by LPS alone or co-incubated by ghrelin and LPS; flow cytometry and TUNEL were used to assessed the apoptosis rate; expression of JNK, phospho-JNK, Bax, Bcl-2, cleaved caspase-3 were detected using Western blot analysis. Laser scanning confocal microscopy was used to observe the phagocytosis of NR8383. Ghrelin receptor antagonist [D-Lys-3]-GHRP-6 and JNK specific inhibitor SP600125 were used to inhibit ghrelin receptor and JNK activation respectively. 【Results】 LPS significantly inhibited the proliferation of NR8383 in a dose-dependent manner, with IC50 value of 250μg / m L. However, ghrelin can block this inhibition casused by LPS. Flow cytometry and TUNEL analysis showed that ghrelin could decrease the apoptosis induced by LPS in NR8383（P〈0.05）, while application of ghrelin receptor antagonist [D-Lys-3]-GHRP-6weakened the antiapoptosis effect of ghrelin（P〈0.05）. LPS activated JNK kinase, increased the expression of Bax and cleaved caspase-3, but decreased Bcl-2 level, which was significantly reversed by pretreatment with ghrelin（P〈0.05）. Ghrelin also apparently maintained the phagocytosis of NR8383 post LPS treatment（P〈0.05）. 【Conclusion】 Ghrelin inhibited LPS inducedNR8383 apoptosis by down-regulating JNK signal pathway, and maintained the phagocytosis of NR8383.