Stroke was one of the leading causes of disability and the second highest cause of death globally, with certain obviously increasing prevalence in some areas [
Inflammation was a crucial factor associated with the progression and prognosis after cerebral ischemia-reperfusion injury. Previous studies had shown that the abnormal activation of residential microglia which were derived from glial cells and macrophages which were derived from circulating monocytes contributed to the acute cerebral ischemic injury [
Dectin-1 is one of the members of the C-type lectin receptor (CLR) family, which involved in numerous pathophysiological processes including neuroinflammation [
Male C57BL/6 mice (6–8 weeks old, 18–22 g) were directly purchased from the Animal Experimental Center of Three Gorges University. All mice were maintained in a specific-pathogen-free (SPF) environment in 3 cages (8 rats/cage in total) with 4 groups divided individually (2 rats/group), and the environment was 22–25°C with adequate food and water. All animal-related studies complied with the National Institutes of Health guidelines for the care and use of laboratory animals. Animal-related studies were carried out by Ding Fadian in the animal laboratory center of Wuhan Barfil Biotechnology Co., Ltd. Animal-related studies were approved by the Ethics Committee of Mindong Hospital affiliated to Fujian Medical University.
The rats were of the same range of age at the start of the experiment. C57BL/6 were anesthetized with 5% isoflurane, and the anesthetization condition was maintained with 1.5% isoflurane. The mice were fixed on the operating plate in supine positions, and the skin on the neck was disinfected. The submandibular gland could be observed by removing the skin along the middle of the neck in the process of blunt separation. The gland was gently pushed to one side to separate to the tracheal anterior muscle and then separated down along the right sternocleidomastoid tendon. After seeing the carotid sheath, the skin muscle was fixed with a retractor. At this point, the common carotid artery was seen. The common carotid artery away from the bifurcation was ligated, and a slip knot was left at the common carotid artery near the bifurcation, and the external carotid artery near the bifurcation was ligated. A small incision in the common carotid artery was made by microscopic shear. We picked up the MCAO line tied through the blood vessels and gently lifted the silk thread of the common carotid arteries' ligation. Then, we loosened the vascular clamp when the line tied enters the internal carotid artery, and we continued to push the line tied until the marker reaches the location of the vessel bifurcation. Tighten the slipknot. After 60 minutes of ischemia, the plug was removed, and the live mice were ligated to death. The muscle and skin were sutured. Samples were taken 6, 12, and 24 hours later. The success of the model was determined by Zea-Longa’s method [
The sections of the brain of the mice were put in 2% triphenyl-2,3,5-tetrazoliumchloride (TTC) at a 37°C temperature box for 30 minutes. Each slice was fully soaked in TTC and washed with PBS. The result of TTC was photographed with a digital camera blindly. The white areas of sections (infarction area) and red sections (noninfarcted area) were measured using ImageJ, and the total infarct area is the sum of the infarcts (infarction area/(noninfarcted area + infarction area)).
The brain tissues were cut, grinded, and lysed with RIPA lysis and extraction buffer (Cat #P0013B, Beyotime), and protease and phosphatase inhibitor were added (Cat #P105539, Aladdin). Proteins (30
The brain tissue was cut into pieces with scissors and digested with 0.25% trypsin-EDTA complex digestive solution at 37°C for 30 min. Serum terminated the digestion, and the falcon filtered the cells. After centrifugation, the liquid was discarded. PBS-resuspended cells with 1 mL 0.5% bovine serum albumin (BSA) per flow tube were centrifuged at 1000 rpm for 3 min. The supernatant was discarded, and PBS was added to resuspended cells to be measured. Antibodies CD11b (eBioscience™) and F4/80 (eBioscience™) were added and incubated at 4°C in dark for 30 min, after which the cells were measured using the flow cytometer (Beckman Coulter). FSC and SSC were set in the first experiment based on the cell size of macrophages. In the following experiments, these were not changed.
Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Prism Software Inc., San Diego, CA, USA) and SPSS 15.0 for Windows (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as median (interquartile range, IQR) and calculated by Mann–Whitney test. Categorical variables were expressed as percentage and compared by test between groups.
To explore the potential mechanism of dectin-1 in the cerebral ischemia-reperfusion injury, we investigated the expression levels of dectin-1 in the infracted brain tissue at three appropriate time points after the ischemia-reperfusion injury. Protein expression levels of dectin-1 were shown with an increasing trend at 6 hours, 12 hours, and 24 hours after ischemia-reperfusion. Meanwhile, the result of TTC showed that the rate of ischemic area/tissue was dramatically increased (see Figure
The expression level of dectin-1 and infarct area. (a, b) The sham-operated group and the middle cerebral artery occlusion (MCAO) group (after 6 hours, 12 hours, and 24 hours).
Flow cytometry analysis of macrophage in the model of cerebral ischemia-reperfusion injury. (a) It is labeled with CD11b-FITC and F4/80-PE showing uniform and complete labeling of M1-polarized macrophages: (A) sham-operated group; (B) MACO 6 h; (C) MACO 12 h; (D) MACO 24 h (b) Ratio of infarct size. (c) Positive correlation between Dectin-1 and infarct size.
Based on previous studies, the interference of dectin-1 by genetic ablation or antibody blockade led to a considerable improvement in cardiac function, accompanied by an increase of apoptosis-related protein [
Dectin-1 was involved in cerebral infarction after cerebral ischemia-reperfusion injury by mediating apoptosis. (a) The sham-operated group and the middle cerebral artery occlusion (MCAO) group (after 6 hours, 12 hours, and 24 hours). Dectin-1, Bax, and cleaved caspase-3 increased, and antiapoptosis molecule, Bcl-2, decreased at three appropriate time points compared to the sham-operated group. (b) The quantitative analysis of western blot.
The proinflammatory cytokines IL-6 and TNF-
Currently, the study results showed that the dectin-1 expression levels were significantly increased in the brain tissue during the process of ischemia-reperfusion injury. At the same time, the expression of dectin-1 was significantly correlated with apoptosis-related proteins in cerebral ischemia/reperfusion models. Interestingly, the result of flow cytometry indicated that cerebral infarction was associated with the polarization of M1 macrophages and the high expression level of dectin-1. The levels of apoptosis-related proteins and the ratio of M1 macrophages supported the hypothesis that dectin-1 played an important role in cerebral infarction and apoptosis in the process of ischemia-reperfusion injury.
The high expression level of dectin-1 was observed on the bone marrow-derived macrophages and neutrophils during ischemic stroke. The protein deletion and expression tests showed that dectin-1 was significantly related to M1 macrophage polarization and neutrophil infiltration in myocardial infarcted tissue [
During myocardial ischemia-reperfusion injury, macrophages were shown to be the predominant cell type and displayed functional heterogeneity, with proinflammatory macrophages (M1 macrophages) infiltrating at the first time and then a high level of anti-inflammatory macrophages (M2 macrophages) at the second reaction, which was tended to the anti-inflammatory-related immune cells. M1 macrophages expressed TNF-
In the rat model of pneumonia, high expression of dectin-1, which was a key receptor recognized and cleared by macrophages, induced the differentiation of macrophage M1 [
In conclusion, dectin-1 is an important immune-cell-related receptor, of which the expression levels are increased in ischemia-reperfusion injury and correlated with the prognosis of ischemia-reperfusion injury. Dectin-1-mediated M1 macrophage polarization and brain cell apoptosis are involved in reperfusion injury. The research in this paper is still not thorough enough, and a part of the results is just correlation studies. In the future, we may obtain a more scientific research result through the construction of the cell model and the intervention of the protein expression.
The analyzed datasets generated during this study are available from the corresponding author within 12 months of publication.
Animal-related studies were approved by the Ethics Committee of Mindong Hospital affiliated to Fujian Medical University.
Chen Zongyun and Lin Bixia are co-first authors.
The authors declare that there are no conflicts of interest regarding the publication of this article.
ZK and YXY contributed to the project design and experiment design. CZY contributed to the writing and critical revision of the manuscript. LBX contributed to the design and was involved in the critical revision of the manuscript. DFD and HXP contributed to the experimental operation and results’ analysis of the manuscript. CHB contributed to discussion of the experimental result and article translation of the manuscript. DY and LQC contributed to the analysis and interpretation, as well as critical revision of the manuscript. All authors read and approved the final manuscript. Chen Zongyun and Lin Bixia contributed equally to this work.
The authors thank Wuhan Barifil Biotechnology Co., Ltd., for providing them with the site and technical support for animal experiments. This study was supported by the Fujian Medical University Sailing Fund Project (2018QH1221) and Fujian Provincial Department of Finance (BPB-2020ZK).