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. 2019 Jun;25(6):704-713.
doi: 10.1111/cns.13101. Epub 2019 Jan 24.

Propofol improved hypoxia-impaired integrity of blood-brain barrier via modulating the expression and phosphorylation of zonula occludens-1

Wei Chen  1   2 , Xing-Zhu Ju  2   3 , Yan Lu  1   2 , Xiao-Wei Ding  1   2 , Chang-Hong Miao  1   2 , Jia-Wei Chen  1   2
Affiliations

Propofol improved hypoxia-impaired integrity of blood-brain barrier via modulating the expression and phosphorylation of zonula occludens-1

Wei Chen et al. CNS Neurosci Ther. 2019 Jun.

Abstract

Aims: Hypoxia may damage blood-brain barrier (BBB). The neuroprotective effect of propofol has been reported. We aimed to identify whether and how propofol improved hypoxia-induced impairment of BBB integrity.

Methods: Mouse brain microvascular endothelial cells (MBMECs) and astrocytes were cocultured to establish in vitro BBB model. The effects of hypoxia and propofol on BBB integrity were examined. Further, zonula occludens-1 (ZO-1) expression and phosphorylation, hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression, intracellular calcium concentration and Ca2+ /calmodulin-dependent protein kinase II (CAMKII) activation were measured.

Results: Hypoxia-impaired BBB integrity, which was protected by propofol. Hypoxia-reduced ZO-1 expression, while induced ZO-1 phosphorylation. These effects were attenuated by propofol. The expression of HIF-1α and VEGF was increased by hypoxia and was alleviated by propofol. The hypoxia-mediated suppression of ZO-1 and impaired BBB integrity was reversed by HIF-α inhibitor and VEGF inhibitor. In addition, hypoxia increased the intracellular calcium concentration and induced the phosphorylation of CAMKII, which were mitigated by propofol. The hypoxia-induced phosphorylation of ZO-1 and impaired BBB integrity was ameliorated by calcium chelator and CAMKII inhibitor.

Conclusion: Propofol could protect against hypoxia-mediated impairment of BBB integrity. The underlying mechanisms may involve the expression and phosphorylation of ZO-1.

Keywords: blood-brain barrier; hypoxia; mouse brain microvascular endothelial cells; propofol; zonula occludens-1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Propofol protected hypoxia‐impaired BBB integrity. A, Evaluation of in vitro BBB integrity by measuring TEER over the course of 7 days coculturing of MBMECs and mouse astrocytes at normoxia condition (95% air, 5% CO2). Data showed TEER peak point at day 6, suggesting the construction of in vitro BBB model. B, Hypoxia (5% O2, 5% CO2, 90% humidity) impaired BBB integrity, which was protected by 100 μmol/L propofol pretreatment. TEER values were expressed as Ω*cm2, presented as mean ±standard deviation, and summarized from five separate experiments. Statistical comparisons were made by paired Student's t test, one‐way ANOVA followed by Tukey's post hoc test (Student's Newman‐Keuls test)
Figure 2
Figure 2
Propofol reversed hypoxia‐mediated ZO‐1 expression and phosphorylation. A, Hypoxia‐reduced protein expression of ZO‐1, which was increased by 100 μmol/L propofol. The upper panel was a representative experiment, and the lower panel was the summary of densitometric data from five separate experiments. GAPDH served as loading control. Data were expressed as normalized ratio of protein band density of ZO‐1 against GAPDH and were presented as mean ±standard deviation. B, Hypoxia‐induced phosphorylation of ZO‐1, which was inhibited by 100 μmol/L propofol. The upper panel was a representative experiment, and the lower panel was the summary of densitometric data from five separate experiments. GAPDH served as loading control. Data were expressed as normalized ratio of protein band density of phosphorylated ZO‐1 against GAPDH and were presented as mean ±standard deviation. Statistical comparisons were made by paired Student's t test, one‐way ANOVA followed by Tukey's post hoc test (Student's Newman‐Keuls test)
Figure 3
Figure 3
Role of HIF1α/VEGF in propofol‐ and hypoxia‐modulated ZO‐1 expression. A, Hypoxia increased HIF1α level, which was attenuated by 100 μmol/L propofol. The upper panel was a representative experiment and the lower panel was the summary of densitometric data from five separate experiments. GAPDH served as loading control. Data were expressed as normalized ratio of protein band density of HIF1α against GAPDH and were presented as mean ±standard deviation. B, Hypoxia‐reduced VEGF level, which was increased by 100 μmol/L propofol. The upper panel was a representative experiment, and the lower panel was the summary of densitometric data from five separate experiments. GAPDH served as loading control. Data were expressed as normalized ratio of protein band density of VEGF against GAPDH and were presented as mean ±standard deviation. C, Hypoxia‐reduced ZO‐1 expression was reversed by propofol, HIF1α inhibitor and VEGF inhibitor. The upper panel was a representative experiment, and the lower panel was the summary of densitometric data from five separate experiments. GAPDH served as loading control. Data were expressed as normalized ratio of protein band density of ZO‐1 against GAPDH and were presented as mean ±standard deviation. D, Hypoxia‐reduced BBB integrity was reversed by propofol, HIF1α inhibitor and VEGF inhibitor. TEER values were expressed as Ω*cm2, presented as mean ±standard deviation, and summarized from five separate experiments. Statistical comparisons were made by paired Student's t test, one‐way ANOVA followed by Tukey's post hoc test (Student's Newman‐Keuls test)
Figure 4
Figure 4
Role of calcium/CAMKII in propofol‐ and hypoxia‐modulated ZO‐1 phosphorylation. A, Hypoxia‐induced intracellular calcium concentration, which was attenuated by propofol and calcium chelator. Data were expressed as fluorescence intensity, presented as mean ±standard deviation, and summarized from five separate experiments. B, Hypoxia‐induced CAMKII phosphorylation, which was attenuated by propofol, calcium chelator and CAMKII inhibitor. The upper panel was a representative experiment, and the lower panel was the summary of densitometric data from five separate experiments. GAPDH served as loading control. Data were expressed as normalized ratio of protein band density of phosphorylated CAMKII against CAMKII, which was normalized with GAPDH, and were presented as mean ±standard deviation. C, Hypoxia‐induced phosphorylation of ZO‐1, which was inhibited by propofol, calcium chelator and CAMKII inhibitor. The upper panel was a representative experiment, and the lower panel was the summary of densitometric data from five separate experiments. GAPDH served as loading control. Data were expressed as normalized ratio of protein band density of phosphorylated ZO‐1 against GAPDH and were presented as mean ±standard deviation. D, Hypoxia‐reduced BBB integrity was reversed by propofol, calcium chelator, and CAMKII inhibitor. TEER values were expressed as Ω*cm2, presented as mean ±standard deviation, and summarized from five separate experiments. Statistical comparisons were made by paired Student's t test, one‐way ANOVA followed by Tukey's post hoc test (Student's Newman‐Keuls test)
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