Abstract
To elucidate the protective mechanism of lobetyolin on oxygen–glucose deprivation/reperfusion (OGD/R)-induced damage in BV2 microglial cells. The OGD/R model was established using a chemical modeling method to simulate in vivo brain ischemia in lobetyolin-pretreated BV2 cells. The optimum lobetyolin dosage, chemical concentration, and OGD/R modeling duration were screened. The changes in cell morphology were observed, and the levels of immune response-related factors, including tumor necrosis factor-α (TNF-α), interleukin-6, inducible nitric oxide synthase (iNOS), and cluster of differentiation (CD)206, were detected using the enzyme-linked immunosorbent assay. The expression of chemokine-like-factor-1 (CKLF1), hypoxia-inducible factor (HIF)-1α, TNF-α, and CD206, was detected using western blotting. The gene expression of M1 and M2 BV2 phenotype markers was assessed using quantitative polymerase chain reaction (qPCR). The localization of M1 and M2 BV2 markers was detected using immunofluorescence analysis. The results showed that lobetyolin could protect BV2 cells from OGD/R-induced damage. After OGD/R, CKLF1/C–C chemokine receptor type 4 (CCR4) levels increased in BV2 cells, whereas the CKLF1/CCR4 level was decreased due to lobetyolin pretreatment. Additionally, BV2 cells injured with OGD/R tended to be M1 type, but lobetyolin treatment shifted the phenotype of BV2 cells from M1 type to M2 type. Lobetyolin decreased the expression of TNF-α and HIF-1α but increased the expression of transforming growth factor-β (TGF-β) in BV2 cells, indicating a dose–effect relationship. The qPCR results showed that lobetyolin decreased the expression of CD16, CD32, and iNOS at the gene level and increased the expression of C–C-chemokine ligand-22 and TGF-β. The immunofluorescence analysis showed that lobetyolin decreased CD16/CD32 levels and increased CD206 levels. Lobetyolin can protect BV2 cells from OGD/R-induced damage by regulating the phenotypic polarization of BV2 and decreasing inflammatory responses. Additionally, CKLF1/CCR4 may participate in regulating lobetyolin-induced polarization of BV2 cells via the HIF-1α pathway.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Abbreviations
- OGD/R:
-
Oxygen glucose deprivation/re-oxygenaion
- CKLFI:
-
Chemokine-like-factor 1
- CCR4:
-
C-C chemokine receptor type 4
- HIF-1α:
-
Hypoxiainduciblefactor-1α
- TGF-β:
-
Transforming growth factor beta
- TNF- α:
-
Tumor necrosis factor- α
- CD-16:
-
Cluster of differentiation-16
- CD32:
-
Cluster of differentiation-32
- CD206:
-
Cluster of differentiation-206
- CCL-22:
-
CC-chemokine ligand-22
- IL-1β:
-
Interleukin-1β
- IL-2:
-
Interleukin-2
- IL-4:
-
Interleukin-4
- IL-6:
-
Interleukin-6
- IL-10:
-
Interleukin-10
- IL-12:
-
Interleukin-12
- IL-23:
-
Interleukin-23
- IL-1Ra:
-
Interleukin-1Ra
- iNOS:
-
Inducible nitric oxide synthase
- VEGF-A:
-
Vascular endothelial growth factor-A
- CCK-8:
-
Cell Counting Kit-8
- ELISA:
-
Enzyme-Linked immunosorbent assay
- qPCR:
-
Quantitative PCR Detecting System
- WB:
-
Western blot
- Eda:
-
Edaravonc
- CCI:
-
Chronic Cerebral ischemia
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Acknowledgements
This research was supported by grants from National Natural Science Foundation of China (U21A20410), (81874420), (82274388), (82004236); Key Program of Shanxi Province (International Science and Technology Cooperation) Major regional innovation cooperation project (201803D421006) , (201903D421018) .
Funding
This research was supported by grants from National Natural Science Foundation of China (U21A20410), (81874420), (82274388), (82004236); Key Program of Shanxi Province (International Science and Technology Cooperation) Major regional innovation cooperation project (201803D421006),(201903D421018).
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Junlong Zhang, Wenbin He, and Jie Wang developed the study conception and design. Wenyi Wei, Xin Liu and Jie Wang performed the experiment. Shifeng Chu, Jing Yang and Qinqing Li carried out the data analysis. Junlong Zhang, Wenbin He, Pulin Liu and Jie Wang developed the statistical analysis. The first draft of the manuscript was written by Junlong Zhang, Wenbin He, and Jie Wang. Xin Liu and Shifeng Chu conducted the written review. All authors read and approved the final manuscript.
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Wang, J., Liu, X., Wei, W. et al. Regulation of oxygen–glucose deprivation/reperfusion-induced inflammatory responses and M1-M2 phenotype switch of BV2 microglia by lobetyolin. Metab Brain Dis 38, 2627–2644 (2023). https://doi.org/10.1007/s11011-023-01292-6
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DOI: https://doi.org/10.1007/s11011-023-01292-6