[瘦素通过PI3K/Akt信号通路对气道平滑肌细胞凋亡的影响]

[The effects of leptin on apoptosis of airway smooth muscle cells via the PI3K/Akt signaling pathway].

作者信息

Liu Wen-jing, Zhu Shu-yang, Chen Yu-ling, Wu Xia, Ni Wen-jing, Chen Yun-feng, Zhao Ling

机构信息

Department of Respiratory Medicine, Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.

出版信息

Zhonghua Jie He He Hu Xi Za Zhi. 2012 Dec;35(12):915-8.

PMID:23328183

Abstract

OBJECTIVE

To observe the effects of leptin on the expression of Akt, Pho-Akt, Bcl-2, Bax, caspase-3 and the apoptosis of airway smooth muscle cells (ASMCs), and to explore the possible mechanisms.

METHODS

ASMCs were derived from rat airway tissue and cultured in vitro. The cells were randomly divided into 5 groups including a control group, leptin at concentrations of 50, 100, 200 µg/L groups (group Lep50, Lep100, Lep200), and PI3K specific antagonist with Lep200 group. Then the cells of different groups were incubated for 24 h. An apoptosis detection kit was used for annexin V and PI staining. The expression of Akt, phosphorylation Akt, Bcl-2, Bax, caspase-3 were measured by Western blot.

RESULTS

The apoptosis rates of ASMCs in group Lep50, Lep100 and Lep200 were (3.97 ± 0.39)%, (1.88 ± 0.72)% and (0.77 ± 0.11)%, respectively, all significantly lower than that in the control group (7.38 ± 0.49)% (F = 89.57, P < 0.05). Furthermore, the concentration of leptin was negatively related to the apoptosis rate (r = -0.711, P < 0.05). The apoptosis rates of PI3K specific antagonist with Lep200 group (3.29 ± 0.36)% was higher than that of group Lep200 (0.77 ± 0.11)% (F = 89.57, P < 0.01). After the intervention of leptin, the expression of Bcl-2 was upregulated and positively correlated with leptin concentration (r = 0.939, P < 0.05); Bax was downregulated and negatively related to the leptin concentration (r = -0.908, P < 0.05); while the Bcl-2/Bax ratio was raised after leptin treatment (F = 20.56, P < 0.05). Leptin inhibited the activation of caspase-3 in the negative way. (r = -0.961, P < 0.05). The results also showed that leptin significantly increased phosphorylation of Akt that positively related to leptin concentration (r = 0.958, P < 0.05). Compared with group Lep200, the expression of Pho-Akt and Bcl-2 in PI3K specific antagonist with Lep200 group were downregulated (F = 32.93, 19.48, respectively, P < 0.05), while the expression of Bax and caspase-3 was increased (F = 10.10, 29.86, respectively, P < 0.05); the Bcl-2/Bax ratio was lower in group Lep200 as compared to the PI3K specific antagonist with Lep200 group (F = 20.56, P < 0.05).

CONCLUSION

Leptin can significantly inhibit ASMC apoptosis partially via the PI3K/Akt signaling pathway.

摘要

目的

观察瘦素对气道平滑肌细胞（ASMCs）中Akt、磷酸化Akt（Pho - Akt）、Bcl - 2、Bax、caspase - 3表达及细胞凋亡的影响，并探讨其可能机制。

方法

从大鼠气道组织分离培养ASMCs。将细胞随机分为5组，包括对照组、50、100、200 μg/L瘦素组（Lep50组、Lep100组、Lep200组）以及Lep200与PI3K特异性拮抗剂合用组。然后对不同组细胞进行24小时孵育。采用凋亡检测试剂盒进行膜联蛋白V和碘化丙啶（PI）染色。通过蛋白质免疫印迹法检测Akt、磷酸化Akt、Bcl - 2、Bax、caspase - 3的表达。

结果

Lep50组、Lep100组和Lep200组ASMCs的凋亡率分别为（3.97 ± 0.39）%、（1.88 ± 0.72）%和（0.77 ± 0.11）%，均显著低于对照组（7.38 ± 0.49）%（F = 89.57，P < 0.05）。此外，瘦素浓度与凋亡率呈负相关（r = -0.711，P < 0.05）。Lep200与PI3K特异性拮抗剂合用组的凋亡率（3.29 ± 0.36）%高于Lep200组（0.77 ± 0.11）%（F = 89.57，P < 0.01）。瘦素干预后，Bcl - 2表达上调且与瘦素浓度呈正相关（r = 0.939，P < 0.05）；Bax表达下调且与瘦素浓度呈负相关（r = -0.908，P < 0.05）；而瘦素处理后Bcl - 2/Bax比值升高（F = 20.56，P < 0.05）。瘦素以负向方式抑制caspase - 3的激活（r = -0.961，P < 0.05）。结果还显示，瘦素显著增加Akt的磷酸化，且与瘦素浓度呈正相关（r = 0.958，P < 0.05）。与Lep200组相比，Lep200与PI3K特异性拮抗剂合用组中磷酸化Akt和Bcl - 2的表达下调（F分别为32.93、19.48，P < 0.05），而Bax和caspase - 3的表达增加（F分别为10.10、29.86，P < 0.05）；与Lep200与PI3K特异性拮抗剂合用组相比，Lep200组的Bcl - 2/Bax比值更低（F = 20.56，P < 0.05）。