Bidirectional Regulation of Keratinocyte Proliferation and Apoptosis by Metformin via NLRP3 Inflammasome Pathway

doi:10.12114/j.issn.1007-9572.2024.0042

Abstract

Abstract:

Background

Lichen planus is a chronic inflammatory disease of the skin and mucosa. Due to its unknown etiology, many patients have poor treatment effect, which seriously affects the quality of life. It is necessary to further study the pathogenesis of lichen planus to provide a new target for drug screening.

Objective

To investigate the effects of metformin on keratinocyte proliferation and apoptosis through NLRP3 inflammasome pathway.

Methods

Experiment period: 2020 to 2023. In vitro experiment, human immortalized keratinocytes (HaCaT) were divided into 4 groups: control group, lipopolysaccharide group (LPS group: 5 μg/mL), metformin group (Met group: 10 mmol/L), LPS combined with metformin group (LPS+Met group: metformin was treated with 10 mmol/L after LPS 5 μg/mL stimulation for 2 hours). In vivo experiments, BALB/c mice were used as research objects to induce psoriatic dermatitis model by applying imiquimod on the back skin, and metformin cream was prepared for treatment. The mice were randomly divided into 3 groups: control group, imiquimod group (IMQ group), and imiquimod plus metformin group (IMQ+Met group). Each group has 10 mice. Mice in the control group were smeared with petroleum jelly on the back, mice in the IMQ group were smeared with imiquimod ointment on the back, mice in the IMQ+Met group were smeared with metformin cream after 12 h of IMQ ointment. Once a day for seven consecutive days. Cell counting kit-8 (CCK-8) and flow cytometry were used to detect the effects of metformin on the proliferation and apoptosis of HaCaT cells. Western Blotting, enzyme-linked immunosorbent assay (ELISA) and Caspase-1 activity assay were used to detect the expression and activity of NOD-like receptor protein 3 (NLRP3) inflammasome pathway protein in HaCaT cells treated with metformin. Finally, hematoxylin-eosin (HE) staining and immunohistochemistry were used to detect the anti-inflammatory effect of metformin on imiquimod-induced psoriatic dermatitis in mice.

Results

The results of CCK-8 experiment showed that the 48 h survival rate of HaCaT cells in LPS, Met and LPS+Met groups were lower than that in control group, while the 48 h survival rate of HaCaT cells in LPS+Met group was higher than that in LPS group (P<0.05). Flow cytometry results indicated that the proportions of G2/M phase and apoptosis of HaCaT cells at 48 h in LPS and Met groups were higher than those in control group, while the proportion of G2/M phase and apoptosis of HaCaT cells at 48 h in LPS+Met group were lower than those in LPS group (P<0.05). Western Blotting results demonstrated that the expression of Caspase-1 p40, Caspase-1 p20, interleukin (IL) -1β and IL-18 proteins in NLRP3 inflammasome pathway of HaCaT cells in LPS and Met groups were higher than those in control group. The expression of Caspase-1 p40, Caspase-1 p20, IL-1β and IL-18 of HaCaT cells in LPS+Met group were lower than those in LPS group (P<0.05). ELISA results showed that the levels of IL-1β, IL-18 and relative activity of Caspase-1 in NLRP3 inflammasome pathway of HaCaT cells in LPS and Met groups were higher than those in control group. The levels of IL-1β, IL-18 and relative activity of Caspase-1 of HaCaT cells in LPS+Met group were lower than those in LPS group (P<0.05). Metformin application in IMQ+Met group significantly improved the imiquimod skin damage observed by HE staining. Immunohistochemical results reported that the expressions of NLRP3, Caspase-1, IL-1β and IL-18 were significantly decreased in IMQ+Met group.

Conclusion

Metformin bidirectional regulates the proliferation and apoptosis of skin keratinocytes through the NLRP3 inflammasome pathway, and can improve the skin damage induced by imiquimod in psoriasis mice, which is expected to provide a theoretical basis for the clinical use of metformin in the treatment of lichen planus.

Key words: Metformin, NLRP3 inflammasome, HaCaT cells, Cell proliferation, Cell apoptosis, Lichen planus, Psoriasis, Mice

摘要：

背景

扁平苔藓是一种皮肤-黏膜慢性炎症性疾病。因其病因不明，许多患者治疗效果欠佳，严重影响生活质量，有必要深入研究扁平苔藓的发病机制，为其药物筛选提供新靶点。

目的

探讨二甲双胍通过NLRP3炎症小体通路对皮肤角质形成细胞增殖和凋亡的调控作用。

方法

实验时间为2020—2023年。体外实验以人永生化角质形成细胞株HaCaT为研究对象，分为4组：对照组、脂多糖（LPS）组（5 μg/mL）、二甲双胍组（Met组：10 mmol/L）、LPS与二甲双胍联合组（LPS+Met组：LPS 5 μg/mL刺激2 h后，再给予二甲双胍10 mmol/L处理）。体内实验以BALB/c小鼠为研究对象，利用咪喹莫特涂抹小鼠背部皮肤诱导了银屑病样皮炎模型，并制备了二甲双胍乳膏进行治疗，将小鼠随机分为3组：对照组、咪喹莫特组（IMQ组）、咪喹莫特与二甲双胍联合组（IMQ+Met组），每组10只；对照组小鼠于背部涂抹凡士林，IMQ组小鼠于背部涂抹咪喹莫特软膏，IMQ+Met组小鼠于背部涂抹IMQ软膏12 h后再涂抹二甲双胍乳膏；1次/d，连续7 d。采用细胞增殖试剂盒（CCK-8）和流式细胞术检测二甲双胍对HaCaT细胞增殖和凋亡的影响；采用Western Blotting、酶联免疫吸附实验（ELISA）和半胱氨酸天冬氨酸蛋白酶1（Caspase-1）活性实验检测二甲双胍处理HaCaT细胞后NOD样受体蛋白3（NLRP3）炎症小体通路的蛋白表达情况及活性水平；最后采用皮肤组织切片苏木素-伊红（HE）染色和免疫组化检测二甲双胍对咪喹莫特诱导银屑病小鼠皮炎的抗炎效果。

结果

CCK-8实验结果显示：LPS组、Met组、LPS+Met组HaCaT细胞48 h存活率均低于对照组，而LPS+Met组HaCaT细胞48 h存活率高于LPS组（P<0.05）。流式细胞术结果显示：LPS组、Met组HaCaT细胞48 h G2/M期细胞、细胞凋亡比例均高于对照组，而LPS+Met组HaCaT细胞48 h G2/M期细胞、细胞凋亡比例低于LPS组（P<0.05）。Western Blotting结果显示：LPS组、Met组HaCaT细胞NLRP3炎症小体通路Caspase-1 p40、Caspase-1 p20、白介素（IL）-1β、IL-18蛋白表达均高于对照组，而LPS+Met组HaCaT细胞NLRP3炎症小体通路Caspase-1 p40、Caspase-1 p20、IL-1β、IL-18蛋白表达低于LPS组（P<0.05）。ELISA实验结果显示：LPS组、Met组HaCaT细胞NLRP3炎症小体通路IL-1β、IL-18水平、Caspase-1相对活性均高于对照组，而LPS+Met组HaCaT细胞NLRP3炎症小体通路IL-1β、IL-18水平、Caspase-1相对活性低于LPS组（P<0.05）。皮肤组织切片HE染色显示：IMQ+Met组小鼠二甲双胍涂抹明显改善了咪喹莫特对皮肤的损害。免疫组化结果显示：IMQ+Met组小鼠则显著降低了NLRP3、Caspase-1、IL-1β和IL-18的表达。

结论

二甲双胍通过NLRP3炎症小体通路双向调节皮肤角质形成细胞的增殖和凋亡，并能够改善咪喹莫特诱导的银屑病样小鼠的皮肤损害，有望为二甲双胍临床上用于治疗扁平苔藓提供理论依据。

关键词: 二甲双胍, NLRP3炎症小体, HaCaT细胞, 细胞增殖, 细胞凋亡, 扁平苔藓, 银屑病, 小鼠

CLC Number:

R 977.15　R 329.25

TIAN Ke,LENG Qiufeng,LYU Jing, et al. Bidirectional Regulation of Keratinocyte Proliferation and Apoptosis by Metformin via NLRP3 Inflammasome Pathway[J]. Chinese General Practice, 2025, 28(06): 742-750. DOI: 10.12114/j.issn.1007-9572.2024.0042.
田珂,冷秋枫,吕晶等. 二甲双胍通过NLRP3炎症小体通路对皮肤角质形成细胞增殖和凋亡的双向调节研究[J]. 中国全科医学, 2025, 28(06): 742-750. DOI: 10.12114/j.issn.1007-9572.2024.0042.

Figures/Tables 10

References 24

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组别	次数	细胞存活率
对照组	3	100.00±0
LPS组	3	62.42±6.21^a
Met组	3	68.66±3.40^a
LPS+Met组	3	75.68±4.35^ab
F值		243.284
P值		<0.001

组别	次数	细胞存活率
对照组	3	100.00±0
LPS组	3	62.42±6.21^a
Met组	3	68.66±3.40^a
LPS+Met组	3	75.68±4.35^ab
F值		243.284
P值		<0.001

组别	次数	G₂/M期细胞比例	细胞凋亡比例
对照组	3	9.50±1.31	1.26±0.15
LPS组	3	28.96±0.94^a	16.91±1.00^a
Met组	3	20.31±1.27^a	12.92±0.98^a
LPS+Met组	3	14.44±0.88^ab	7.47±1.61^ab
F值		338.179	241.729
P值		<0.001	<0.001

组别	次数	G₂/M期细胞比例	细胞凋亡比例
对照组	3	9.50±1.31	1.26±0.15
LPS组	3	28.96±0.94^a	16.91±1.00^a
Met组	3	20.31±1.27^a	12.92±0.98^a
LPS+Met组	3	14.44±0.88^ab	7.47±1.61^ab
F值		338.179	241.729
P值		<0.001	<0.001

组别	次数	NLRP3	Caspase-1 p40	Caspase-1 p20	IL-1β	IL-18
对照组	3	1.00±0	1.00±0	1.00±0	1.00±0	1.00±0
LPS组	3	1.00±0.03	1.73±0.03^a	1.78±0.04^a	1.75±0.05^a	1.50±0.05^a
Met组	3	1.03±0.05	1.44±0.05^a	1.61±0.04^a	1.60±0^a	1.58±0.04^a
LPS+Met组	3	0.98±0.03	0.98±0.03^ab	1.20±0.04^ab	1.19±0.05^ab	1.03±0.04^ab
F值		2.407	615.153	644.506	443.592	319.418
P值		0.265	<0.001	<0.001	<0.001	<0.001