Department of Vascular Biology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
Department of Pediatrics, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
Gerodontology. 2022 Jun;39(2):139-147. doi: 10.1111/ger.12545. Epub 2021 Feb 18.
This paper describes the effect of Porphyromonas gingivalis (P gingivalis) lipopolysaccharide (LPS) on the expression of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2) in cultured hCMEC/D3 human brain microvascular endothelial cells.
P gingivalis is one of the important pathogens in periodontitis, and periodontitis is a risk factor for brain disorders including cerebrovascular diseases and Alzheimer's disease. However, the mechanisms underlying the pathogenesis of P gingivalis-mediated brain diseases are incompletely understood. Effects of P gingivalis LPS on brain endothelial cells are not known well.
The hCMEC/D3 human brain microvascular endothelial cells were cultured and treated with P gingivalis LPS. The expression of IL-6 and CCL2 mRNA and protein was examined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Effect of inhibitors of Toll-like receptor (TLR) 2, TLR4, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) was also investigated. Phosphorylation of NF-κB p65, p38 MAPK and JNK was examined using Western blotting.
P gingivalis LPS-induced mRNA and protein expression of IL-6 and CCL2 in hCMEC/D3 cells in a concentration-dependent manner at the concentration of 0.5-50 µg/mL. Maximal mRNA expression of IL-6 and CCL2 was found 2 and 4 hours after stimulation, respectively. Induction of IL-6 and CCL2 by P gingivalis LPS was almost completely inhibited by pretreatment of cells with TLR4 inhibitor but not by TLR2 inhibitor. Treatment of cells with P gingivalis LPS for up to 2 hours induced phosphorylation of NF-κB p65, p38 MAPK and JNK. IL-6 induction was decreased by pretreatment of cells with NF-κB inhibitor SN50 or p38 MAPK inhibitor SB203580, while CCL2 induction was reduced by SN50 or JNK inhibitor SP600125.
IL-6 and CCL2 produced upon P gingivalis LPS stimulation may contribute to the inflammatory reactions in brain endothelial cells and subsequent neurological disorders such as cerebrovascular and Alzheimer's diseases.
本文描述牙龈卟啉单胞菌脂多糖（P.gingivalis LPS）对培养的人脑微血管内皮细胞 hCMEC/D3 中人白细胞介素 6（IL-6）和 C-C 基序趋化因子配体 2（CCL2）表达的影响。
牙龈卟啉单胞菌是牙周炎的重要病原体之一，牙周炎是包括脑血管病和阿尔茨海默病在内的脑部疾病的危险因素。然而，牙龈卟啉单胞菌介导的脑部疾病发病机制尚不完全清楚，牙龈卟啉单胞菌 LPS 对脑内皮细胞的影响也知之甚少。
培养 hCMEC/D3 人脑微血管内皮细胞，并以牙龈卟啉单胞菌 LPS 处理。采用定量逆转录聚合酶链反应和酶联免疫吸附试验分别检测 IL-6 和 CCL2 mRNA 和蛋白的表达。还研究了 Toll 样受体（TLR）2、TLR4、核因子-κB（NF-κB）、p38 丝裂原活化蛋白激酶（p38 MAPK）和 c-Jun N 末端激酶（JNK）抑制剂的作用。采用 Western blot 检测 NF-κB p65、p38 MAPK 和 JNK 的磷酸化。
牙龈卟啉单胞菌 LPS 以浓度依赖性方式诱导 hCMEC/D3 细胞中 IL-6 和 CCL2 的 mRNA 和蛋白表达，浓度为 0.5-50μg/ml。刺激后 2 小时和 4 小时发现 IL-6 和 CCL2 的最大 mRNA 表达。牙龈卟啉单胞菌 LPS 诱导的 IL-6 和 CCL2 诱导几乎完全被 TLR4 抑制剂预处理的细胞抑制，但 TLR2 抑制剂不能抑制。用牙龈卟啉单胞菌 LPS 处理细胞长达 2 小时诱导 NF-κB p65、p38 MAPK 和 JNK 的磷酸化。用 NF-κB 抑制剂 SN50 或 p38 MAPK 抑制剂 SB203580 预处理细胞可降低 IL-6 诱导，而用 SN50 或 JNK 抑制剂 SP600125 可降低 CCL2 诱导。
牙龈卟啉单胞菌 LPS 刺激后产生的 IL-6 和 CCL2 可能导致脑内皮细胞炎症反应和随后的脑血管和阿尔茨海默病等神经障碍。
