匹诺塞林通过抑制自噬减轻慢性间歇性缺氧诱导的BEAS-2B细胞损伤

doi:10.16138/j.1673-6087.2024.02.05

摘要/Abstract

摘要：

目的：探讨匹诺塞林（pinocembrin, PIN）对慢性间歇性缺氧（chronic intermittent hypoxia, CIH）诱导的BEAS-2B人支气管上皮细胞损伤的影响及可能的调控机制。方法：培养BEAS-2B细胞，通过细胞计数试剂8 (cell counting kit 8, CCK8)确定最适CIH干预时长和最适PIN处理浓度，将BEAS-2B细胞分为对照组（CON组）、CIH组、CIH+PIN组3组。通过CCK8检测不同处理组BEAS-2B细胞增殖活性，蛋白质免疫印迹法（Western blotting, WB）检测缺氧诱导因子-1α（hypoxia-inducible factor 1α, HIF-1α）、凋亡相关基因[裂解的胱天蛋白酶3（cleaved caspase-3）、B细胞淋巴瘤2(B cell lymphoma 2, Bcl-2)、Bcl-2关联X蛋白(Bcl-2 associated X protein, Bax）]及自噬相关基因[轻链3(light chain 3, LC3)-Ⅱ、beclin-1、P62]的蛋白表达水平。加入自噬抑制剂3-甲基腺嘌呤（3-methyladenine, 3-MA）后，将细胞分为CON组、CIH组、CIH+3-MA组、CIH+PIN组、CIH+3-MA+PIN组5组。检测各组HIF-1α、凋亡及自噬相关基因蛋白表达水平。结果：CCK8检测结果显示，随着缺氧时间的延长，BEAS-2B细胞增殖活性在24~48 h显著降低，PIN可以上调CIH下BEAS-2B细胞增殖活性。WB结果显示，与CON组相比，CIH组HIF-1α、Bax、LC3-Ⅱ、beclin-1蛋白表达增加（均P<0.05），P62及抑凋亡基因Bcl-2蛋白表达明显下调（P<0.05）。加入3-MA后，HIF-1α蛋白表达减少，自噬及凋亡被抑制，加入PIN后表现出大致相同的趋势。结论：PIN可以减轻CIH诱导的BEAS-2B细胞损伤，从而发挥保护作用，其机制可能与下调自噬水平，抑制细胞凋亡有关。

关键词: 匹诺塞林, 慢性间歇性缺氧, 自噬, 凋亡

Abstract:

Objective To investigate the effect of pinocembrin (PIN) on BEAS-2B human bronchial epithelial cell injury induced by chronic intermittent hypoxia (CIH) and its possible regulatory mechanism. Methods The optimal CIH intervention duration and PIN treatment concentration were determined by cell counting kit 8 (CCK8) in BEAS-2B cells. BEAS-2B cells were divided into control group (CON group), CIH group and CIH+PIN group. The proliferation activity of BEAS-2B cells in different treatment groups was detected by CCK8. Western blotting (WB) was used to detect the protein expression levels of hypoxia-inducible factor 1α (HIF-1α), apoptosis related gene [cleaved caspase 3, B cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax)] and autophagy related genes [light chain 3(LC3)-Ⅱ, beclin-1, P62]. After adding the autophagy inhibitor 3-methyladenine (3-MA), the cells were divided into five groups: CON group, CIH group, CIH+3-MA group, CIH+PIN group, and CIH+3-MA+PIN group, and the expression levels of HIF-1α, apoptosis and autophagy related genes in each group were detected. Results CCK8 showed that the proliferation activity of BEAS-2B cells significantly decreased between 24-48 h with the prolongation of hypoxia time. PIN could up-regulate the proliferative activity of BEAS-2B cells under CIH. The results of WB showed that compared with CON group, the protein expressions of HIF-1α, Bax, LC3-Ⅱ and beclin-1 were increased in CIH group (P<0.05). The expression of P62 and anti-apoptosis gene Bcl-2 protein was significantly down-regulated (P<0.05). After adding 3-MA, the protein expression level of HIF-1α was reduced, autophagy and apoptosis were inhibited. The same trend was observed after adding PIN. Conclusions PIN can reduce CIH-induced BEAS-2B cell damage and exert a protective effect. The mechanism may be related to down-regulating autophagy level and inhibiting cell apoptosis.

Key words: Pinocembrin, Chronic intermittent hypoxia, Autophagy, Apoptosis

中图分类号:

R563.5

何嫣婕, 何美娟, 王韵, 朱春雪, 黄汉鹏. 匹诺塞林通过抑制自噬减轻慢性间歇性缺氧诱导的BEAS-2B细胞损伤[J]. 内科理论与实践, 2024, 19(02): 115-120.

HE Yanjie, HE Meijuan, WANG Yun, ZHU Chunxue, HUANG Hanpeng. Pinocembrin alleviates BEAS-2B cell damage induced by chronic intermittent hypoxia through inhibiting autophagy[J]. Journal of Internal Medicine Concepts & Practice, 2024, 19(02): 115-120.

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项目	CON组	CIH组	CIH+PIN组	F	P
HIF-1α	1	1.236±0.019¹⁾	0.959±0.022¹⁾²⁾	97.59	<0.000 1
裂解的胱天蛋白酶3	1	1.278±0.025¹⁾	0.906±0.049¹⁾²⁾	31.76	<0.000 6
Bax	1	1.371±0.029¹⁾	1.024±0.031¹⁾²⁾	138.60	<0.000 1
Bcl-2	1	0.875±0.024¹⁾	1.133±0.047¹⁾²⁾	9.20	<0.05
P62	1	0.807±0.016¹⁾	1.163±0.039¹⁾²⁾	20.88	<0.002
beclin-1	1	1.340±0.039¹⁾	0.874±0.062¹⁾²⁾	44.61	<0.000 3
LC3-Ⅱ	1	1.291±0.027¹⁾	0.912±0.086¹⁾²⁾	8.137	<0.05

项目	CON组	CIH组	CIH+3-MA组	CIH+PIN组	CIH+3-MA+PIN组	F	P
HIF-1α	1	1.432±0.039¹⁾	1.221±0.039¹⁾²⁾	1.267±0.040¹⁾²⁾	1.200±0.047¹⁾²⁾	49.96	<0.000 1
裂解的胱天蛋白酶3	1	1.756±0.069¹⁾	1.427±0.087¹⁾²⁾	1.507±0.105¹⁾	1.439±0.093¹⁾²⁾	20.92	<0.000 1
Bcl-2	1	0.582±0.030¹⁾	0.755±0.035¹⁾²⁾	0.890±0.033¹⁾²⁾	0.973±0.054¹⁾²⁾	44.73	<0.000 1
LC3-Ⅱ	1	1.445±0.020¹⁾	1.038±0.126¹⁾²⁾	1.063±0.137¹⁾²⁾	0.775±0.134¹⁾²⁾	13.68	<0.000 5
beclin-1	1	1.419±0.020¹⁾	1.129±0.058¹⁾²⁾	1.158±0.028¹⁾²⁾	1.080±0.040¹⁾²⁾	26.64	<0.000 1
P62	1	0.605±0.015¹⁾	0.879±0.023¹⁾²⁾	0.976±0.019¹⁾²⁾	1.074±0.030¹⁾²⁾	100.40	<0.000 1