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. 2022 Dec;36(12):e23213.
doi: 10.1002/jbt.23213. Epub 2022 Sep 16.

Bradykinin/bradykinin 1 receptor promotes brain microvascular endothelial cell permeability and proinflammatory cytokine release by downregulating Wnt3a

Linqiang Huang  1 , Mengting Liu  1   2 , Wenqiang Jiang  1 , Hongguang Ding  1 , Yongli Han  1 , Miaoyun Wen  1 , Ya Li  1   3 , Xiaoyu Liu  1   2 , Hongke Zeng  1
Affiliations

Bradykinin/bradykinin 1 receptor promotes brain microvascular endothelial cell permeability and proinflammatory cytokine release by downregulating Wnt3a

Linqiang Huang et al. J Biochem Mol Toxicol. 2022 Dec.

Abstract

Stroke is a life-threatening disease with limited therapeutic options. Damage to the blood-brain barrier (BBB) is the key pathological feature of ischemic stroke. This study explored the role of the bradykinin (BK)/bradykinin 1 receptor (B1R) and its mechanism of action in the BBB. Human brain microvascular endothelial cells (BMECs) were used to test for cellular responses to BK by using the Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine staining, enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence, cellular permeability assays, and western blotting to evaluate cell viability, cytokine production, and reactive oxygen species (ROS) levels in vitro. A BBB induced by middle cerebral artery occlusion was used to evaluate BBB injuries, and the role played by BK/B1R in ischemic/reperfusion (I/R) was explored in a rat model. Results showed that BK reduced the viability of BMECs and increased the levels of proinflammatory cytokines (interleukin 6 [IL-6], IL-18, and monocyte chemoattractant protein-1) and ROS. Additionally, cellular permeability was increased by BK treatment, and the expression of tight junction proteins (claudin-5 and occludin) was decreased. Interestingly, Wnt3a expression was inhibited by BK and exogenous Wnt3a restored the effects of BK on BMECs. In an in vivo I/R rat model, knockdown of B1R significantly decreased infarct volume and inflammation in I/R rats. Our results suggest that BK might be a key inducer of BBB injury and B1R knockdown might provide a beneficial effect by upregulating Wnt3a.

Keywords: B1R; blood-brain barrier; bradykinin; inflammation; stroke.

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Conflict of interest statement

All authors declare no conflict of interest.

Figures

Figure 1
Figure 1
BK reduced cell viability in a concentration‐ and time‐dependent manner. (A) BMECs were treated with different concentrations of BK for 24 h, and cell viability was detected by the CCK‐8 assay. (B) BMECs were treated with 400 nM BK for the indicated time period, and cell viability was measured by the CCK‐8 assay. BK, bradykinin; BMECs, brain microvascular endothelial cells; CCK‐8, Cell Counting Kit‐8; OD, optical density.
Figure 2
Figure 2
BK enhanced ROS production, cellular permeability, and inflammation, and reduced cell proliferation and tight junction formation by BMECs. BMECs were treated with BK (low concentration, 100 nM or high concentration, 400 nM). ROS production was determined by flow cytometry, and the respective images are shown (A, left panel). Summarized results from three independent experiments are shown (A, right panel). Cell permeability was tested by sodium fluorescein analysis (B, left panel) and the lucifer yellow assay (B, right panel). Cell proliferation was measured by EdU staining. (C, IL‐6, MCP‐1, and IL‐18 expressions were detected by IF (D) and ELISA (E). (F) The cell apoptosis rate was measured using FCM. (G) LDH levels were determined by ELISA. The levels of claudin‐5, occludin, β‐catenin, Wnt3a, and B1R expression were determined by western blotting (H, left panel). The statistical results of western blot studies conducted in three independent experiments (H, right panel). *p < 0.05; **p < 0.01; ***p < 0.001 versus blank. B1R, bradykinin 1 receptor; BK, bradykinin; BMECs, brain microvascular endothelial cells; DCF‐A, 2′,7′‐dichlorodihydrofluorescein diacetate; EdU, 5‐ethynyl‐2′‐deoxyuridine; ELISA, enzyme‐linked Immunosorbent assay; FCM, flow cytometry; GAPDH glyceraldehyde‐3‐phosphate dehydrogenase; IF, immunofluorescence; IL‐6, interleukin 6; LDH, lactate dehydrogenase; MCP‐1, monocyte chemoattractant protein‐1; ROS, reactive oxygen species.
Figure 3
Figure 3
Wnt3a counteracted the effects of BK on BMECs. BMECs were treated with a high concentration (400 nM) of BK or a high concentration of both BK and Wnt3a (100 ng/ml each). ROS production was measured by flow cytometry, and the respective images are shown (A, left panel). The summary results of three independent experiments are shown (A, right panel). Cell permeability was tested by sodium fluorescein analysis (B, left panel) and the lucifer yellow assay (B, right panel). (C) Cell proliferation was measured by EdU staining. IL‐6, MCP‐1, and IL‐18 expressions were detected by (D) IF and (E) ELISA. (F) Cell apoptosis was detected by using the FCM method. (G) LDH levels were determined by ELISA. The levels of claudin‐5, occludin, β‐catenin, Wnt3a, and B1R expression were determined by western blotting (H, left panel). The statistical results of western blot studies conducted in three independent experiments (H, right panel). *p < 0.05; **p < 0.01; ***p < 0.001 versus blank. # p < 0.05; ## p < 0.01 versus BK‐high. B1R, bradykinin 1 receptor; BK, bradykinin; BMECs, brain microvascular endothelial cells; DCF‐A, 2′,7′‐dichlorodihydrofluorescein diacetate; EdU, 5‐ethynyl‐2′‐deoxyuridine; ELISA, enzyme‐linked Immunosorbent assay; FCM, flow cytometry; GAPDH glyceraldehyde‐3‐phosphate dehydrogenase; IF, immunofluorescence; IL‐6, interleukin 6; LDH, lactate dehydrogenase; MCP‐1, monocyte chemoattractant protein‐1; ROS, reactive oxygen species.
Figure 4
Figure 4
Knockdown B1R protected against I/R damage in model rats. A total of 24 rats were assigned to three separate groups: Sham (n = 8), I/R model (n = 8), and adenovirus targeted B1R‐treated I/R model (n = 8). (A) The infarction area was detected by TTC staining. (B) BBB permeability was measured by Evans blue staining. (C) Pathological changes were detected by H&E staining. (D) MCP‐1 expression was determined by IHC. (E) Apoptosis was measured by TUNEL staining. B1R, bradykinin 1 receptor; BBB, blood–brain barrier; H&E, hematoxylin and eosin; IHC, immunohistochemistry; I/R, ischemia/reperfusion; MCP‐1, monocyte chemoattractant protein‐1; TTC, 2,3,5‐triphenyltetrazolium chloride; TUNEL,TdT‐mediated dUTP nick‐end Labeling.
Figure 5
Figure 5
Knockdown of B1R suppressed I/R‐induced inflammation and induced tight junction formation in I/R model rats. A total of 24 rats were assigned to three groups: Sham (n = 8), I/R model (n = 8), and adenovirus targeted B1R treated I/R model (n = 8). (A) IL‐6, IL‐18, and MCP‐1 expression were determined by ELISA. (B) The levels of Wnt3a and BK were determined by ELISA. (C) The levels of claudin‐5, occludin, and B1R expression were determined by western blotting. *p < 0.05; **p < 0.01; ***p < 0.001, versus blank. # p < 0.05; ## p < 0.01, versus BK‐high. B1R, bradykinin 1 receptor; BK, bradykinin; ELISA, enzyme‐linked Immunosorbent assay; GAPDH glyceraldehyde‐3‐phosphate dehydrogenase; IL‐6, interleukin 6; I/R, ischemia/reperfusion; MCP‐1, monocyte chemoattractant protein‐1.
Figure 6
Figure 6
Working model. BK‐induced cell permeability and inflammation and reduced tight junction formation by BMECs by binding to the B1 receptor. Wnt3a expression was suppressed by BK and Wnt3a reduced the deleterious effects of BK. Suppression of the BKB1 receptor significantly decreased the infarct volume and inflammation in I/R rats by regulating the Wnt/β‐catenin pathway. BK, bradykinin; BMECs, brain microvascular endothelial cells; I/R, ischemia/reperfusion.
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