脂多糖通过 Akt 通路下调 ZO-1 蛋白的表达。
Lipopolysaccharide downregulates the expression of ZO-1 protein through the Akt pathway.
机构信息
Capital Institute of Pediatrics, Beijing, China.
Department of Neonatology, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing, China.
出版信息
BMC Infect Dis. 2022 Oct 5;22(1):774. doi: 10.1186/s12879-022-07752-1.
BACKGROUND
Neonatal bacterial meningitis is a common neonatal disease with high morbidity, and can cause serious sequelae when left untreated. Escherichia coli is the common pathogen, and its endotoxin, lipopolysaccharide (LPS) can damage the endothelial cells, increasing the permeability of the blood-brain barrier (BBB), leading to intracranial inflammation. However, the specific mechanism of bacterial meningitis induced by LPS damaging BBB remains unclear. In this study, the mouse brain microvascular endothelial (bEND.3) cells were used as a research object to investigate whether LPS damage BBB through the PI3K/Akt pathway.
METHODS
The bEND.3 cells were stimulated with different concentrations of LPS for 12 h, and the expression of tight junction proteins (ZO-1, claudin-5, occludin) was detected using western blotting. The cells were challenged with the same concentration of LPS (1ug/ml) across different timepoints (0, 2 h, 4 h, 6 h, 12 h, 24 h). Expression of TJ proteins and signal pathway molecules (PI3K, p-PI3K, Akt, p-Akt) were detected. The distribution of ZO-1 in bEND.3 cells were detected by immunofluorescence staining.
RESULTS
A negative correlation is observed between ZO-1 and LPS concentration. Moreover, a reduced expression of ZO-1 was most significant under 1 ug/ml of LPS, and the difference was statistically significant (P < 0.05). Additionally, there is a negative correlation between ZO-1 and LPS stimulation time. Meanwhile, the expression of claudin-5 and occludin did not change significantly with the stimulation of LPS concentration and time. The immunofluorescence assay showed that the amount of ZO-1 on the surface of bEND.3 cells stimulated with LPS was significantly lower than that of the control group. After LPS stimulation, p-Akt protein increased at 2 h and peaked at 4 h. The titer of p-PI3K did not change significantly with time.
CONCLUSION
LPS can downregulate the expression of ZO-1; however, its effect on claudin-5 and occludin is minimal. Akt signal pathway may be involved in the regulation of ZO-1 expression induced by LPS in bEND.3 cells.
背景
新生儿细菌性脑膜炎是一种常见的新生儿疾病，发病率高，未经治疗可导致严重后遗症。大肠杆菌是常见的病原体，其内毒素脂多糖（LPS）可损伤血管内皮细胞，增加血脑屏障（BBB）的通透性，导致颅内炎症。然而，LPS 损伤 BBB 引起细菌性脑膜炎的确切机制尚不清楚。本研究以小鼠脑微血管内皮（bEND.3）细胞为研究对象，探讨 LPS 是否通过 PI3K/Akt 通路损伤 BBB。
方法
用不同浓度 LPS 刺激 bEND.3 细胞 12 h，采用 Western blot 检测紧密连接蛋白（ZO-1、claudin-5、occludin）的表达。用相同浓度 LPS（1μg/ml）刺激细胞不同时间点（0、2 h、4 h、6 h、12 h、24 h），检测 TJ 蛋白和信号通路分子（PI3K、p-PI3K、Akt、p-Akt）的表达。免疫荧光染色检测 bEND.3 细胞中 ZO-1 的分布。
结果
ZO-1 与 LPS 浓度呈负相关。此外，1μg/ml LPS 作用下 ZO-1 表达降低最显著，差异有统计学意义（P<0.05）。同时，ZO-1 与 LPS 刺激时间呈负相关。而 claudin-5 和 occludin 的表达随 LPS 浓度和时间的刺激变化不明显。免疫荧光结果显示，LPS 刺激后 bEND.3 细胞表面 ZO-1 量明显低于对照组。LPS 刺激后 2 h p-Akt 蛋白增加，4 h 达高峰。p-PI3K 滴度随时间变化无明显变化。
结论
LPS 可下调 ZO-1 的表达；然而，对 claudin-5 和 occludin 的作用较小。Akt 信号通路可能参与 LPS 诱导 bEND.3 细胞 ZO-1 表达的调节。
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