苦参碱对特应性皮炎炎症、氧化应激和伤口愈合的影响

doi:10.11958/20231689

摘要/Abstract

摘要：

目的评估苦参碱对体外特应性皮炎（AD）模型中HaCaT角质形成细胞的炎症、氧化和迁移反应及伤口愈合的影响。方法采用MTT法评估苦参碱的细胞毒性，依据实验结果，将HaCaT细胞分为对照组，模型组，苦参碱0.05、0.1和0.2 mmol/L组，模型组采用10 μg/L肿瘤坏死因子-α（TNF-α）/干扰素-γ（IFN-γ）刺激HaCaT细胞24 h，苦参碱0.05、0.1和0.2 mmol/L组分别用相应浓度的苦参碱预处理细胞1 h，然后用10 μg/L TNF-α/IFN-γ刺激24 h。酶联免疫吸附试验（ELISA）法测定白细胞介素（IL）-6、IL-8和IL-1β和胸腺和活化调节趋化因子（TARC）、巨噬细胞衍生趋化因子（MDC）、调节活化正常T细胞表达和分泌的因子（RANTES）的水平；实时荧光定量PCR检测抗氧化基因表达水平；划痕愈合实验分析细胞迁移；免疫组织化学分析核因子（NF）-κB p65的核易位；Western blot检测p38、细胞外调节蛋白激酶（ERK）和p65蛋白的磷酸化水平。结果与模型组比较，苦参碱0.1和0.2 mmol/L组降低IL-6、IL-1β、IL-8和趋化因子的表达，增强超氧化物歧化酶（SOD）1、SOD2、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GPx）的表达，并促进划痕愈合率（P<0.05）。此外，苦参碱0.1和0.2 mmol/L可抑制p38、ERK和p65蛋白的磷酸化及p65的核易位。结论苦参碱具有抗炎和抗氧化特性，并刺激角质形成细胞AD模型的伤口闭合。

关键词: 苦参碱, 皮炎，特应性, 炎症, NF-κB, 氧化性应激

Abstract:

Objective To evaluate the effects of matrine on the inflammatory, oxidative and migratory responses of HaCaT keratinocytes in an in vitro atopic dermatitis (AD) model. Methods HaCaT cells were divided into the control group (no treatment) and the model group (0.05, 0.1 and 0.2 mmol/L). HaCaT cells in the model group were stimulated with 10 μg/L tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ) for 24 h. Cells in the model group were pretreated with 0.05, 0.1 and 0.2 mmol/L matrine for 1 h and then stimulated with 10 μg/L TNF-α/IFN-γ for 24 h. MTT assay was used to assess the cytotoxicity of matrine. Enzyme-linked immunosorbent (ELISA) assay was performed to determine levels of cytokines (IL-6, IL-8 and IL-1β) and chemokines (TARC, MDC and RANTES). mRNA expression levels of antioxidant genes were detected by real-time quantitative PCR (RT-qPCR). Cell migration was analyzed by scratch closure assay. Immunohistochemistry was commended to analyze the nuclear translocation of NF-κB p65. Western blot assay was used to detect the phosphorylation levels of p38, ERK and p65 proteins. Results Compared with the model group, matrine 0.1 and 0.2 mmol/L significantly decreased the expression levels of IL-6, IL-1β, IL-8 and chemokines, and matrine significantly enhanced the expression levels of superoxide dismutase-1 (SOD1), SOD2, catalase (CAT) and glutathione peroxidase (GPx), and promoted scratch wound healing (P<0.05). In addition, matrine 0.1 and 0.2 mmol/L inhibited the phosphorylation levels of p38, ERK and p65 proteins and the nuclear translocation of p65. Conclusion Matrine possesses anti-inflammatory and antioxidant properties, and stimulates wound closure in eratinocyte AD model, which may be related to the inhibition of the activation of MAPK and NF-κB pathways.

Key words: matrine, dermatitis, atopic, inflammation, NF-kappa B, oxidative stress

中图分类号:

R758.2

图/表 8

参考文献 19

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基因名称	引物序列（5′→3′）	产物大小/bp
SOD1	上游：ACGGTGGGCCAAAGGATGAA	151
	下游：TCATGGACCACCAGTGTGCG	151
SOD2	上游：AGAAGCACAGCCTCCCCGAC	152
	下游：GGCCAACGCCTCCTGGTACT	152
CAT	上游：CCAACAGCTTTGGTGCTCCG	180
	下游：GGCCGGCAATGTTCTCACAC	180
GPx	上游：TCGGTGTATGCCTTCTCGGC	150
	下游：CCGCTGCAGCTCGTTCATCT	150
GAPDH	上游：TTGGTATCGTGGAAGGACTCA	270
	下游：TGTCATCATATTTGGCAGGTTT	270

基因名称	引物序列（5′→3′）	产物大小/bp
SOD1	上游：ACGGTGGGCCAAAGGATGAA	151
	下游：TCATGGACCACCAGTGTGCG	151
SOD2	上游：AGAAGCACAGCCTCCCCGAC	152
	下游：GGCCAACGCCTCCTGGTACT	152
CAT	上游：CCAACAGCTTTGGTGCTCCG	180
	下游：GGCCGGCAATGTTCTCACAC	180
GPx	上游：TCGGTGTATGCCTTCTCGGC	150
	下游：CCGCTGCAGCTCGTTCATCT	150
GAPDH	上游：TTGGTATCGTGGAAGGACTCA	270
	下游：TGTCATCATATTTGGCAGGTTT	270

组别	细胞因子
组别	IL-6		IL-1β		IL-8
对照组	0.82±0.14		31.5±3.3		9.7±1.8
模型组	6.73±0.57^a		115.2±6.5^a		78.8±6.1^a
0.05 mmol/L组	6.48±0.55		107.8±6.7		76.4±5.7
0.1 mmol/L组	5.51±0.42^b		72.3±4.8^b		49.3±3.8^b
0.2 mmol/L组	4.46±0.35^b		36.6±3.6^b		17.6±2.2^b
F	91.210^＊＊		169.000^＊＊		168.100^＊＊
组别		趋化因子
组别		MDC		TARC		RANTES
对照组		65±7		19±3		11±2
模型组		341±15^a		443±20^a		151±17^a
0.05 mmol/L组		337±18		431±24		147±19
0.1 mmol/L组		208±11^b		346±18^b		79±13^b
0.2 mmol/L组		110±13^b		277±15^b		38±7^b
F		271.500^＊＊		291.300^＊＊		68.470^＊＊

组别	细胞因子
组别	IL-6		IL-1β		IL-8
对照组	0.82±0.14		31.5±3.3		9.7±1.8
模型组	6.73±0.57^a		115.2±6.5^a		78.8±6.1^a
0.05 mmol/L组	6.48±0.55		107.8±6.7		76.4±5.7
0.1 mmol/L组	5.51±0.42^b		72.3±4.8^b		49.3±3.8^b
0.2 mmol/L组	4.46±0.35^b		36.6±3.6^b		17.6±2.2^b
F	91.210^＊＊		169.000^＊＊		168.100^＊＊
组别		趋化因子
组别		MDC		TARC		RANTES
对照组		65±7		19±3		11±2
模型组		341±15^a		443±20^a		151±17^a
0.05 mmol/L组		337±18		431±24		147±19
0.1 mmol/L组		208±11^b		346±18^b		79±13^b
0.2 mmol/L组		110±13^b		277±15^b		38±7^b
F		271.500^＊＊		291.300^＊＊		68.470^＊＊

组别	SOD1	SOD2	CAT	GPx
对照组	1.00±0.04	1.00±0.05	1.00±0.04	1.00±0.03
模型组	0.51±0.02^a	0.42±0.07^a	0.27±0.03^a	0.30±0.04^a
0.05 mmol/L组	0.53±0.03	0.44±0.09	0.30±0.05	0.34±0.02
0.1 mmol/L组	0.88±0.06^b	5.85±0.96^b	0.52±0.06^b	0.56±0.06^b
0.2 mmol/L组	1.25±0.09^b	12.34±1.28^b	0.84±0.08^b	1.07±0.09^b
F	102.800^＊＊	156.500^＊＊	105.500^＊＊	135.000^＊＊