木犀草苷对阿尔茨海默病模型细胞凋亡和炎性因子表达的研究

doi:10.11958/20221922

天津医药 ›› 2023, Vol. 51 ›› Issue (7): 701-706.doi: 10.11958/20221922

木犀草苷对阿尔茨海默病模型细胞凋亡和炎性因子表达的研究

王翠(), 杨畅, 金玉, 高蜜, 张雯, 王琼, 金海涛()

1 武汉市中医医院脑病科（邮编430000）
2 武汉市中医医院内分泌科（邮编430000）

收稿日期:2022-11-21 修回日期:2023-01-15 出版日期:2023-07-15 发布日期:2023-07-18
通讯作者: ^△金海涛 E-mail:hhbdf62@163.com
作者简介:王翠（1985），女，主治医师，主要从事针灸治疗脑血管病方面研究。E-mail：wangcui1985w@163.com
基金资助:
武汉市中医药科研项目(WZ22C65)

Study of cynaroside on apoptosis and expression of inflammatory factor in model cells of Alzheimer’s disease

WANG Cui(), YANG Chang, JIN Yu, GAO Mi, ZHANG Wen, WANG Qiong, JIN Haitao()

1 Department of Encephalopathy, Wuhan Hospital of Traditional Chinese Medicine, Wuhan 430000, China
2 Department of Endocrinology, Wuhan Hospital of Traditional Chinese Medicine, Wuhan 430000, China

Received:2022-11-21 Revised:2023-01-15 Published:2023-07-15 Online:2023-07-18
Contact: ^△JIN Haitao E-mail:hhbdf62@163.com

王翠, 杨畅, 金玉, 高蜜, 张雯, 王琼, 金海涛. 木犀草苷对阿尔茨海默病模型细胞凋亡和炎性因子表达的研究[J]. 天津医药, 2023, 51(7): 701-706.

WANG Cui, YANG Chang, JIN Yu, GAO Mi, ZHANG Wen, WANG Qiong, JIN Haitao. Study of cynaroside on apoptosis and expression of inflammatory factor in model cells of Alzheimer’s disease[J]. Tianjin Medical Journal, 2023, 51(7): 701-706.

摘要/Abstract

摘要：

目的探究木犀草苷通过下调有丝分裂原活化蛋白激酶激酶激酶3（MEKK3）对阿尔茨海默病（AD）模型细胞凋亡和炎性因子表达的影响及可能机制。方法体外培养PC12细胞，经不同剂量（6.25、12.5、25 mg/L）木犀草苷孵育24 h后，建立β淀粉样肽（Aβ）_25-35诱导的AD细胞模型；另转染MEKK3小干扰RNA或过表达载体至PC12细胞，经25 mg/L木犀草苷孵育24 h后，建立Aβ_25-35诱导的AD细胞模型。细胞计数试剂盒-8（CCK-8）检测细胞增殖抑制率，流式细胞术检测细胞凋亡率，酶联免疫吸附试验（ELISA）检测炎性因子[肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6、IL-1β]水平，蛋白免疫印迹法（Western blot）检测MEKK3蛋白表达。结果木犀草苷可降低Aβ_25-35诱导的PC12细胞增殖抑制率、凋亡率、炎性因子（TNF-α、IL-6、IL-1β）水平，且降低细胞中MEKK3蛋白表达（P<0.05）；敲减MEKK3可降低Aβ_25-35诱导的PC12细胞增殖抑制率、凋亡率、炎性因子（TNF-α、IL-6、IL-1β）水平，过表达MEKK3呈相反作用；上调MEKK3表达可减弱木犀草苷对Aβ_25-35诱导的PC12细胞增殖抑制率、凋亡率和炎性因子水平的影响。结论木犀草苷可能通过下调MEKK3抑制AD模型细胞凋亡及炎性因子水平，具有治疗AD的潜在价值。

关键词: 木犀草苷, 阿尔茨海默病, PC12细胞, MAP激酶激酶激酶类, 细胞凋亡, 炎性因子

Abstract:

Objective To explore the effect and possible mechanism of cynaroside on apoptosis and expression of inflammatory factor in model cells of Alzheimer's disease (AD) by down-regulating mitogen-activated protein kinase kinase kinase 3 (MEKK3). Methods PC12 cells were cultured in vitro and incubated with different doses (6.25 mg/L, 12.5 mg/L, 25 mg/L) of cynaroside for 24 h to establish an AD cell model induced by Aβ_25-35. In addition, MEKK3 small interfering RNA or overexpression vector was transfected into PC12 cells, and Aβ_25-35 induced AD cell model was established after incubation with 25 mg/L luteolin for 24 h. CCK-8 was used to detect inhibition rate of cell proliferation, and flow cytometry was used to detect apoptosis rate. ELISA was used to detect inflammatory factors (TNF-α, IL-6, IL-1β). The protein expression of MEKK3 was detected by Western blot assay. Results Cynaroside could decrease the proliferation inhibition rate, apoptosis rate, inflammatory cytokines (TNF-α, IL-6, IL-1β) and MEKK3 protein expression in PC12 cells induced by Aβ_25-35 (P<0.05). Knockdown of MEKK3 could reduce the proliferation inhibition rate, apoptosis rate and inflammatory cytokines (TNF-α, IL-6, IL-1β) of Aβ_25-35-induced PC12 cells, while overexpression of MEKK3 showed the opposite effect. Upregulation of MEKK3 expression attenuated effects of cynaroside on the proliferation inhibition rate, apoptosis rate and inflammatory factors of Aβ_25-35-induced PC12 cells. Conclusion Cynaroside may inhibit apoptosis and expression of inflammatory factors of AD model cells by down-regulating MEKK3, which has potential value in the treatment of AD.

Key words: Galuteolin, Alzheimer disease, PC12 cells, MAP kinase kinase kinases, apoptosis, inflammatory factor

中图分类号:

R742

图/表 11

图1 流式细胞术检测木犀草苷对Aβ25-35诱导的PC12细胞凋亡的影响

Fig.1 Effect of cynaroside on apoptosis of AD model cells detected by flow cytometry

表1 木犀草苷对AD模型细胞增殖抑制率与凋亡率的影响

Tab.1 Effects of cynaroside on proliferation inhibition rate and apoptosis rate of AD model cells

组别	增殖抑制率	凋亡率
对照组	0.02±0.01	6.35±0.16
模型组	44.15±1.84^a	23.83±0.88^a
模型+木犀草苷低剂量组	32.36±1.84^b	20.37±0.89^b
模型+木犀草苷中剂量组	24.06±1.28^bc	16.29±1.12^bc
模型+木犀草苷高剂量组	12.45±0.71^bcd	11.93±0.56^bcd
F	1 482.537^＊＊	675.957^＊＊

表2 木犀草苷对AD模型细胞炎性因子表达和MEKK3蛋白表达水平的影响

Tab.2 Effects of cynaroside on the expression of inflammatory cytokines and MEKK3 protein in AD model cells

组别	TNF-α/（ng/L）		IL-6/（ng/L）
对照组	100.06±5.79		64.78±4.99
模型组	400.08±14.63^a		442.40±32.12^a
模型+木犀草苷低剂量组	332.88±20.38^b		303.79±15.73^b
模型+木犀草苷中剂量组	244.86±12.78^bc		222.45±14.55^bc
模型+木犀草苷高剂量组	148.91±11.88^bcd		126.42±9.40^bcd
F	721.179^＊＊		621.541^＊＊
组别		IL-1β/（ng/L）		MEKK3
对照组		93.60±5.55		0.15±0.02
模型组		275.42±20.46^a		0.67±0.04^a
模型+木犀草苷低剂量组		235.50±13.86^b		0.51±0.03^b
模型+木犀草苷中剂量组		176.98±12.97^bc		0.39±0.04^bc
模型+木犀草苷高剂量组		125.28±8.96^bcd		0.22±0.02^bcd
F		286.230^＊＊		411.612^＊＊

图2 Western blot检测木犀草苷对AD模型细胞中MEKK3蛋白表达的影响 A：对照组；B：模型组；C—E：模型+木犀草苷低、中、高剂量组。

Fig.2 Western blot detection of the effect of cynaroside on the expression of MEKK3 protein in AD model cells

图3 Western blot检测转染对MEKK3蛋白表达的影响 A：si-NC组；B：si-MEKK3组；C：pcDNA组；D：pcDNA-MEKK3组。

Fig.3 Western blot detection of the effect of transfection on MEKK3 protein expression

图4 流式细胞术检测敲减MEKK3对AD模型细胞凋亡的影响

Fig.4 The effect of knockout MEKK3 on apoptosis of AD model cells detected by flow cytometry

表3 敲减MEKK3对AD模型细胞增殖抑制率、凋亡率及炎性因子表达的影响

Tab.3 Effects of knockdown of MEKK3 on proliferation inhibition rate, apoptosis rate and expression of inflammatory factors in AD model cells

组别	增殖抑制率/%	凋亡率/%	TNF-α/（ng/L）	IL-6/（ng/L）	IL-1β/（ng/L）
模型+si-NC组	44.19±1.96	23.65±1.58	399.83±23.91	448.76±20.57	272.70±35.62
模型+si-MEKK3组	14.56±0.83	13.35±0.97	198.54±19.89	144.50±22.08	133.54±10.81
t	41.762^＊＊	16.667^＊＊	19.416^＊＊	30.248^＊＊	11.215^＊＊

图5 流式细胞术检测过表达MEKK3对AD模型细胞凋亡的影响

Fig.5 Effects of overexpression of MEKK3 on apoptosis of AD model cells detected by flow cytometry

表4 过表达MEKK3对AD模型细胞增殖抑制率、凋亡率及炎性因子表达的影响

Tab.4 Effects of overexpression of MEKK3 on proliferation inhibition rate, apoptosis rate and expression of inflammatory factors in AD model cells

组别	增殖抑制率/%	凋亡率/%	TNF-α/（ng/L）	IL-6/（ng/L）	IL-1β/（ng/L）
模型+pcDNA组	42.35±2.05	21.74±1.50	387.96±25.39	439.55±31.42	280.14±22.90
模型+pcDNA-MEKK3组	60.18±3.72	36.28±2.15	562.30±40.15	641.07±45.19	435.80±30.57
t	12.593^＊＊	16.639^＊＊	11.010^＊＊	10.984^＊＊	12.226^＊＊

图6 流式细胞术检测上调MEKK3对木犀草苷介导的AD模型细胞凋亡的影响

Fig.6 Effects of up-regulation of MEKK3 on cell apoptosis in AD model cells mediated by cynaroside detected by flow cytometry

表5 上调MEKK3恢复木犀草苷对AD模型细胞增殖抑制率、凋亡率及炎性因子表达的影响

Tab.5 Effects of up-regulation of MEKK3 to restore on inhibition rate of proliferation, apoptosis rate and expression of inflammatory factors in AD model cells

组别	增殖抑制率/%	凋亡率/%	TNF-α/（ng/L）	IL-6/（ng/L）	IL-1β/（ng/L）
模型+木犀草苷+pcDNA组	12.48±0.78	11.67±0.62	142.99±17.36	126.56±9.61	133.38±15.76
模型+木犀草苷+pcDNA-MEKK3组	33.27±1.87	21.07±0.83	348.56±19.04	347.98±20.81	238.75±17.50
t	30.782^＊＊	27.220^＊＊	23.935^＊＊	28.979^＊＊	13.423^＊＊

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木犀草苷对阿尔茨海默病模型细胞凋亡和炎性因子表达的研究

Study of cynaroside on apoptosis and expression of inflammatory factor in model cells of Alzheimer’s disease

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