黄芩苷对脂多糖诱导人牙龈成纤维细胞表达炎症因子的影响

高娟; 伍燕; 郑之峻; 王青云; 刘曙; 党妮

doi:10.16766/j.cnki.issn.1674-4152.002755

黄芩苷对脂多糖诱导人牙龈成纤维细胞表达炎症因子的影响

doi: 10.16766/j.cnki.issn.1674-4152.002755

高娟¹,
伍燕¹,
郑之峻¹,
王青云¹,
刘曙¹,
党妮^2, ,

1.
贵阳市口腔医院正畸科, 贵州贵阳 550002
2.
西安市中医医院口腔科, 陕西西安 710000

基金项目:

贵州省卫生健康委科学技术基金项目 gzwjkj2020-1-162

详细信息

通讯作者:
党妮，E-mail：dangni871229@163.com

中图分类号: R781.4 R329.2
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- 被引次数: 0
出版历程
- 收稿日期: 2021-10-15
- 网络出版日期: 2023-02-07

Effect of baicalin on expression of inflammatory factors in human gingival fibroblasts induced by lipopolysaccharide

1.
Department of Orthodontics, Guiyang Stomatological Hospital, Guiyang, Guizhou 550002, China

摘要

摘要: 目的从PI3K/Akt/NF-κB信号通路探讨黄芩苷对脂多糖(LPS)诱导的人牙龈成纤维细胞(HGFs)损伤的影响及机制。方法运用LPS诱导建立人牙龈成纤维细胞损伤模型，设置正常对照组、模型组和模型+黄芩苷(1、10和20μg/mL)组，每组设3个复孔，经不同浓度的黄芩苷干预后，Western blotting检测p-Akt、Akt、NF-κB p65等蛋白的表达，采用qPCR法检测炎性因子TNF-α、IL-1β、IL-6等基因的表达。用PI3K抑制剂LY294002作用半小时后，再用黄芩苷干预，qPCR和Western blotting法检测Akt、p-Akt、NF-κB p65、TNF-α、IL-1β、IL-6等基因和蛋白的表达。结果与模型组比较，黄芩苷低、中、高剂量能有效降低TNF-α、IL-6、IL-1β mRNA的表达水平，促进p-Akt蛋白表达，抑制NF-κB p65核蛋白表达，并呈剂量依赖性(均P < 0.05)。与模型组比较，模型+黄芩苷组p-Akt表达升高(P＜0.05)，NF-κB p65表达降低(P＜0.01)。PI3K抑制剂LY294002作用后，模型+黄芩苷组p-Akt/Akt表达量(0.63±0.18)较模型组(0.56±0.14)升高(P＜0.05)，模型+黄芩苷组NF-κB p65、IL-6、IL-1β、TNF-α表达量较模型组降低(均P＜0.01)。黄芩苷对LY294002作用后，p-Akt/Akt、NF-κB p65、IL-6、IL-1β、TNF-α表达量与模型组比较，差异无统计学意义。结论黄芩苷可抑制LPS诱导的人牙龈成纤维细胞损伤引起的炎症反应，其作用机制可能与促进Akt的磷酸化，抑制NF-κB p65核转录和炎性因子的释放有关。
- 黄芩苷 /
- 脂多糖 /
- 人牙龈成纤维细胞 /
- 炎症反应
Abstract: Objective To investigate the effects of baicalin on the injury of human gingival fibroblasts (HGFs) induced by Lipopolysaccharide (LPS) through PI3K/Akt/NF-κB signaling pathway and its mechanism. Methods LPS induced HGFs injury models were established and divided into normal control group, model group and model + baicalin (1, 10 and 20 μg/mL) groups. Each group was set with 3 repetitions. After treatment with different concentrations of baicalin, Western blotting was used to detect the expression of p-Akt, Akt and NF-κB p65, and the expressions of inflammatory factors TNF-α, IL-1β and IL-6 were detected by qPCR. After treated with PI3K inhibitor LY294002 for half an hour, baicalin was used to intervene, and the genes and proteins expressions of Akt, p-Akt, NF-κB p65, TNF-α, IL-1β and IL-6 were detected by qPCR and Western blotting. Results Compared with the model group, low, medium and high dose baicalin could effectively reduce the mRNA expression levels of TNF-α, IL-6 and IL-1β, promote the protein expression of P-Akt and inhibit the nuclear protein expression of NF-κB p65 in a dose-dependent manner (all P < 0.05). Compared with the model group, the expression of p-Akt in the model + baicalin group was increased (P < 0.05), and the expression of NF-κB p65 was decreased (P < 0.01). After the treatment of PI3K inhibitor LY294002, the expression level of p-Akt /Akt in the model + baicalin group was increased (0.63±0.18) compared with that in the model group (0.56±0.14, P < 0.05), and the expressions of NF-κB p65, IL-6, IL-1β and TNF-α in the model + baicalin group were decreased compared with those in the model group (all P < 0.01). After baicalin treated LY294002, the expression levels of p-Akt/Akt, NF-κB p65, IL-6, IL-1β and TNF-α were not significantly different from those of the model group. Conclusion Baicalin can inhibit the inflammatory response induced by LPS-induced human gingival fibroblast injury, and its mechanism may be related to promote the phosphorylation of Akt, inhibit NF-κB p65 nuclear transcription and the release of inflammatory factors.
- Baicalin /
- Lipopolysaccharide /
- Human gingival fibroblasts /
- Inflammatory response

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图 1 黄芩苷对人牙龈成纤维细胞LPS损伤后p-Akt、Akt、NF-κB p65表达的影响

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图 2 黄芩苷对LY294002作用人牙龈成纤维细胞LPS损伤后Akt、p-Akt和NF-κB p65表达的影响

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表 1 PCR引物序列

基因名称		引物序列(5'-3')	长度(bp)
TNF-α	上游引物	GCCACCACGCTCTTC-TGTC	164
	下游引物	GCTACGGGCTTGTCACTCG
IL-6	上游引物	AGACTTCACAGAGGATACCACCCAC	624
	下游引物	CAATCAGAATTGCCATTGCACAA
IL-1β	上游引物	ACAAGGAGAGACAAGCAACGACAA	193
	下游引物	TTTCCATCTTCTTCTTTGGGTATTG
β-actin	上游引物	GATTCCTATGTGGGCGACGA	205
	下游引物	AGGTCTCAAA CATGATCTGGGT

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表 2 不同剂量黄芩苷对人牙龈成纤维细胞LPS损伤后TNF-α、IL-6、IL-1β mRNA表达的影响(x±s)

组别	n	TNF-α/ β-actin	IL-6/ β-actin	IL-1β/ β-actin
正常对照组	6	1.00±0.00	1.00±0.00	1.00±0.00
模型组	6	3.45±0.57^a	3.68±0.35^a	2.59±0.47^a
黄芩苷低剂量组	6	2.20±0.46^b	2.72±0.18^b	2.16±0.46^b
黄芩苷中剂量组	6	1.92±0.32^c	2.15±0.46^c	1.95±0.32^b
黄芩苷高剂量组	6	1.66±0.24^c	1.84±0.13^c	1.62±0.14^c
F值		456.870	473.252	485.043
P值		< 0.001	< 0.001	< 0.001
注：与正常组比较，^aP＜0.01；与模型组比较，^bP＜0.05，^cP＜0.01。

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表 3 黄芩苷对人牙龈成纤维细胞LPS损伤后NF-κB p65、Akt、pAkt表达的影响(x±s)

组别	n	p-Akt/Akt	NF-κB p65
正常对照组	6	1.00±0.00	1.00±0.00
模型组	6	0.38±0.16^a	1.98±0.46^a
黄芩苷低剂量组	6	0.88±0.15^b	1.42±0.18^b
黄芩苷中剂量组	6	1.15±0.23^c	1.25±0.17^c
黄芩苷高剂量组	6	1.56±0.20^c	0.96±0.15^c
F值		503.071	488.025
P值		< 0.001	< 0.001
注：与正常组比较，^aP＜0.01；与模型组比较，^bP＜0.05，^cP＜0.01。

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表 4 黄芩苷对LY294002作用人牙龈成纤维细胞LPS损伤后Akt、p-Akt和核蛋白NF-κB p65表达的影响(x±s)

组别	n	p-Akt/Akt	NF-κB p65
正常对照组	6	1.00±0.00	1.00±0.00
模型组	6	0.56±0.14^a	1.86±0.51^a
模型+黄芩苷组	6	0.95±0.21^b	1.15±0.16^d
模型+LY组	6	0.63±0.18^c	1.69±0.22^c
模型+LY+黄芩苷组	6	0.48±0.15^c	2.05±0.17^c
F值		524.460	578.322
P值		< 0.001	< 0.001
注：与正常组比较, ^aP＜0.01;与模型组比较，^bP＜0.05，^dP＜0.01；与模型+黄芩苷组比较，^cP＜0.01。

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表 5 黄芩苷对LY294002作用人牙龈成纤维细胞LPS损伤后IL-6、IL-1β、TNF-α mRNA表达的影响(x±s)

组别	n	TNF-α/ β-actin	IL-6/ β-actin	IL-1β/ β-actin
正常对照组	6	1.00±0.00	1.00±0.00	1.00±0.00
模型组	6	1.72±0.19^a	1.98±0.14^a	2.00±0.21^a
模型+黄芩苷组	6	1.22±0.15^b	1.35±0.16^b	1.36±0.15^b
模型+LY组	6	1.84±0.37^c	2.03±0.35^d	2.47±0.58^c
模型+LY+黄芩苷组	6	1.68±0.50^c	1.82±0.14^d	2.20±0.27^c
F值		504.611	585.430	576.092
P值		＜0.001	＜0.001	＜0.001
注：与正常组比较, ^aP＜0.01;与模型组比较，^bP＜0.01；与模型+黄芩苷组比较，^cP＜0.01，^dP＜0.05。

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