石蒜碱通过TLR4/NF-κB途径抑制LPS诱导的原代小胶质细胞炎症反应

doi:10.11958/20220323

天津医药 ›› 2022, Vol. 50 ›› Issue (9): 897-901.doi: 10.11958/20220323

• 细胞与分子生物学 • 下一篇

石蒜碱通过TLR4/NF-κB途径抑制LPS诱导的原代小胶质细胞炎症反应

抗晶晶¹(), 曹翔¹^,²^,^△()

1 黄河科技学院医学院生化教研室（邮编450063）
2 南京大学医学院附属鼓楼医院神经内科

收稿日期:2022-02-28 修回日期:2022-03-30 出版日期:2022-09-15 发布日期:2022-09-05
通讯作者: 曹翔 E-mail:kangjingjingkuaile@163.com;xiangcao1988@163.com
作者简介:抗晶晶（1987）,女,副教授,主要从事中药抗炎抗肿瘤机制研究。E-mail： kangjingjingkuaile@163.com
基金资助:
国家自然科学基金资助项目(82171310);河南省民办普通高等学校学科专业建设资助项目(医学检验技术)

Lycorine inhibited LPS-induced primary microglial inflammatory response via TLR4/NF-κB signaling pathway

KANG Jingjing¹(), CAO Xiang¹^,²^,^△()

1 Department of Biochemistry, School of Medicine, Huanghe College of Science and Technology, Zhengzhou 450063, China
2 Department of Neurology, Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School

Received:2022-02-28 Revised:2022-03-30 Published:2022-09-15 Online:2022-09-05
Contact: CAO Xiang E-mail:kangjingjingkuaile@163.com;xiangcao1988@163.com

抗晶晶, 曹翔. 石蒜碱通过TLR4/NF-κB途径抑制LPS诱导的原代小胶质细胞炎症反应[J]. 天津医药, 2022, 50(9): 897-901.

KANG Jingjing, CAO Xiang. Lycorine inhibited LPS-induced primary microglial inflammatory response via TLR4/NF-κB signaling pathway[J]. Tianjin Medical Journal, 2022, 50(9): 897-901.

摘要/Abstract

摘要：

目的观察石蒜碱（LYC）对脂多糖（LPS）诱导的原代小胶质细胞炎症反应和表型变化的影响,并探讨其作用机制。方法将培养的原代小胶质细胞分为对照组、LYC组（0.1、0.5、1和5 μmol/L LYC）、LPS组（0.1 mg/L LPS）和LPS（0.1 mg/L LPS）+LYC组（5 μmol/L LYC）。倒置显微镜观察细胞形态变化;流式细胞术检测原代小胶质细胞的纯度及LPS诱导原代小胶质细胞向两种表型转化的情况;CCK-8法检测细胞活性;实时荧光定量PCR（qPCR）检测白细胞介素（IL）-1β、IL-6、肿瘤坏死因子-α（TNF-α）以及不同表型小胶质细胞表面标志物mRNA表达;Griess法检测一氧化氮（NO）含量;免疫印迹法检测细胞TLR4、p-NF-κB p65蛋白表达情况。结果原代小胶质细胞纯度达95%以上。对照组及各浓度LYC组细胞活性差异无统计学意义。后续实验以5 μmol/L的LYC为药物浓度。与LPS组相比,LPS+LYC组的阿米巴样小胶质细胞数量明显减少,IL-1β、IL-6、TNF-α mRNA表达水平以及NO含量降低,CD86 mRNA表达水平和表型比例降低,CD206 mRNA表达水平和表型比例升高,TLR4和p-NF-κB p65蛋白表达水平降低（P<0.01）。结论 LYC通过TLR4/NF-κB信号通路抑制LPS诱导的原代小胶质细胞炎症反应,并促进其向“抑炎型”极化。

关键词: 石蒜碱, 小神经胶质细胞, 脂多糖, Toll样受体4, NF-κB, 炎症反应

Abstract:

Objective To investigate the effect and potential mechanism of lycorine (LYC) on inflammatory response and phenotype of primary microglia induced by lipopolysaccharide (LPS). Methods The cultured primary microglia cells were divided into the control group, the LYC group (0.1, 0.5, 1 and 5 μmol/L LYC), the LPS group (0.1 mg/L LPS) and the LPS+LYC group (0.1 mg/L LPS+5 μmol/L LYC). The morphological changes of cells were observed under inverted microscope. Flow cytometry was used to detect the purity of primary microglia cells and the effect of LPS-induced cell proportion on the two phenotypes of primary microglia cells. Cell activity was detected by CCK-8 method. The mRNA expressions of interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α) and microglia surface markers of different phenotypes were detected by real-time fluorescence quantitative PCR (qPCR). The content of nitric oxide (NO) was determined by Griess method. The expression levels of TLR4 and p-NF-κB p65 protein were detected by Western blot assay. Results The purity of primary microglia was over 95%. There was no significant difference in the cell activity between the control group and the LYC group. The concentration of LYC 5 μmol/L was used in subsequent experiments. Compared with LPS group, the number of amoeba-like microglia was significantly reduced in the LPS+LYC group, expression levels of IL-1β, IL-6, TNF-α mRNA and NO content decreased, the expression level of CD86 mRNA and phenotype ratio decreased, the expression level of CD206 mRNA and phenotype ratio increased, and expression levels of TLR4 and p-NF -κB P65 protein were also decreased (P<0.01). Conclusion Treatment with LYC inhibits LPS-induced primary microglial inflammatory response and promotes the anti-inflammatory polarization through TLR4/NF-κB signaling pathway.

Key words: Lycorine, microglia, lipopolysaccharides, Toll-like receptor 4, NF-kappa-B, inflammatory response

中图分类号:

R392.12

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）
IL-1β	上游：AAGCCTCGTGCTGTCGGACC	140
IL-1β	下游：TGAGGCCCAAGGCCACAGG	140
IL-6	上游：GCTGGTGACAACCACGGCCT	107
IL-6	下游：AGCCTCCGACTTGTGAAGTGGT	107
TNF-α	上游：CAAGGGACAAGGCTGCCCCG	109
TNF-α	下游：GCAGGGGCTCTTGACGGCAG	109
CD86	上游：GACCGTTGTGTGTGTTCTGG	148
CD86	下游：GATGAGCAGCATCACAAGGA	148
CD206	上游：TTCGGTGGACTGTGGACGAGCA	108
CD206	下游：ATAAGCCACCTGCCACTCCGGT	108
GAPDH	上游：GCCAAGGCTGTGGGCAAGGT	112
GAPDH	下游：TCTCCAGGCGGCACGTCAGA	112

基因名称	引物序列（5′→3′）	产物大小（bp）
IL-1β	上游：AAGCCTCGTGCTGTCGGACC	140
IL-1β	下游：TGAGGCCCAAGGCCACAGG	140
IL-6	上游：GCTGGTGACAACCACGGCCT	107
IL-6	下游：AGCCTCCGACTTGTGAAGTGGT	107
TNF-α	上游：CAAGGGACAAGGCTGCCCCG	109
TNF-α	下游：GCAGGGGCTCTTGACGGCAG	109
CD86	上游：GACCGTTGTGTGTGTTCTGG	148
CD86	下游：GATGAGCAGCATCACAAGGA	148
CD206	上游：TTCGGTGGACTGTGGACGAGCA	108
CD206	下游：ATAAGCCACCTGCCACTCCGGT	108
GAPDH	上游：GCCAAGGCTGTGGGCAAGGT	112
GAPDH	下游：TCTCCAGGCGGCACGTCAGA	112

组别	IL-1β mRNA		IL-6 mRNA
对照组	1.026±0.137		1.047±0.118
LYC组	1.135±0.507		1.032±0.156
LPS组	1 357.000±186.300^a		3 644.000±391.400^a
LPS+LYC组	631.900±83.950^ab		1 549.000±267.200^ab
F	159.500^＊＊		212.000^＊＊
组别		TNF-α mRNA		NO含量
对照组		1.036±0.128		1.185±0.366
LYC组		1.136±0.508		2.238±0.949
LPS组		397.700±62.410^a		28.770±2.472^a
LPS+LYC组		248.800±45.230^ab		18.830±4.847^ab
F		103.100^＊＊		93.640^＊＊

组别	IL-1β mRNA		IL-6 mRNA
对照组	1.026±0.137		1.047±0.118
LYC组	1.135±0.507		1.032±0.156
LPS组	1 357.000±186.300^a		3 644.000±391.400^a
LPS+LYC组	631.900±83.950^ab		1 549.000±267.200^ab
F	159.500^＊＊		212.000^＊＊
组别		TNF-α mRNA		NO含量
对照组		1.036±0.128		1.185±0.366
LYC组		1.136±0.508		2.238±0.949
LPS组		397.700±62.410^a		28.770±2.472^a
LPS+LYC组		248.800±45.230^ab		18.830±4.847^ab
F		103.100^＊＊		93.640^＊＊

组别	CD86 mRNA	CD206 mRNA	CD86（%）	CD206（%）
对照组	0.921±0.062	1.190±0.419	9.150±0.387	8.463±0.697
LYC组	0.741±0.207	0.972±0.278	9.020±0.486	8.850±0.265
LPS组	6.003±1.094^a	3.682±0.584^a	36.150±2.525^a	16.680±1.396^a
LPS+LYC组	3.427±0.542^ab	6.200±0.695^ab	22.400±2.754^ab	25.150±1.234^ab
F	63.910^＊＊	89.730^＊＊	186.600^＊＊	246.500^＊＊

石蒜碱通过TLR4/NF-κB途径抑制LPS诱导的原代小胶质细胞炎症反应

Lycorine inhibited LPS-induced primary microglial inflammatory response via TLR4/NF-κB signaling pathway

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组别	TLR4	p-NF-κB p65
对照组	0.992±0.153	1.000±0.064
LYC组	1.391±0.180	1.139±0.063
LPS组	2.547±0.016^a	2.214±0.242^a
LPS+LYC组	1.665±0.207^ab	1.246±0.384^b
F	52.930^＊＊	17.070^＊＊

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