| Part I: Time course analysis of the protein level and secretion of ANXA1 after ICH.Objective: To investigate the different time points after ANXA1 changes in the cerebrovascular endothelium and serum after ICH.Methods: In vivo experiment, 42 rats(44 rats were used, 42 rats were survived after the surgery) were randomly assigned to 7 groups of 6 rats each, a sham group and 6 experimental groups arranged by time: 6 h, 12 h, 24 h, 48 h, 72 h and 1w after ICH. All the rats in the experiment were killed at the indicated time point after ICH. The brain cortex of 6 rats in each group was extracted and used for double immunofluorescence analysis and western blot analysis, and blood was collected for serum ANXA1 concentration analysis. In vitro experiment was also performed to evaluate the effect of oxy Hb treatment on the protein level of ANXA1 in h CMEC/D3 cells.Results: 1,In order to detect the expression of ANXA1 in BMVECs during SBI after ICH, through immunofluorescent colocalization with the endothelial marker protein v WF, we first tested the protein level of ANXA1 in the cerebral microvasculature. The results demonstrated that, compared with the sham group, the protein level of ANXA1 in the microvascular endothelium were reduced significantly from 6 h after ICH, reached the lowest point at 24 h, and then rebounded gradually, and were similar to that in the sham group at 1 w,Western blot analysis of protein sample from capillaries further verified the ICH-induced decrease in the protein level of ANXA1(P<0.01); 2, Consistent with the in vivo data, western blot assay showed that the protein level of ANXA1 in cultured h CMEC/D3 cells was significantly decreased by Oxy Hb treatment at 6-18 h(P<0.01). 3,In addition, ELISA analysis of the serum of rats were performed and found that the serum content of ANXA1 significantly decreased at 24 h after ICH, and then rebounded to the corresponding level of sham group at 48 h(P<0.05). 4,In vitro,detection of Western blot to examine vascular endothelial cell AXNA1 expression: 6h, 12 h, 18 h experimental group compared with the control group ANXA1 expression was significantly reduced(P<0.01).Conclusion: 1,The expression of ANXA1 in SD Rats’ brain tissue vascular endothelial cells and serum reached the lowest point at 24 h, and the tendency of ANXA1 expression is first decreased and then increased; 2, The expression of ANXA1 in vitro human brain microvascular endothelial cells(h CMEC/D3) was also first decreased and then increased, when it was stimulated by Oxygenated hemoglobin.Part II: Effects of ANXA1 on ICH-induced SBI and the underlying mechanisms.Objective: To establish ICH model in rats and BMVECs(brain microvascular endothelial cells) culture in vitro ICH model observe by improving the ANXA1 dose whether ANXA1 can effect on the prognosis of intracerebral hemorrhage and the protection of blood brain barrier.Methods:In in vivo experiment, 120 rats(124 rats were used, 120 rats were survived) were randomly divided into 5 groups(n = 24 per group): sham group, ICH + vehicle group, ICH + human recombinant ANXA1(rh ANXA1)-Low dosage group(ANXA-L, 0.34 μg/kg body weight, i.v.), ICH + rh ANXA1-Middle dosage group(ANXA-M, 0.67 μg/kg body weight, i.v.), and ICH + rh ANXA1-High dosage group(ANXA-H, 1.34 μg/kg body weight, i.v.). The dosage of rh ANXA1 was determined based on a previous study(Cristante et al., 2013). rh ANXA1 was given at 3 h after collagenase injection, which was chosen based on the time needed for collagenase inducing hemorrhage in this ICH model we established. At 24 h after ICH, all the rats were exsanguinated. The brain cortex of 6 rats per group were extracted for terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL) staining, fluoro-jade B(FJB) staining, and 6 rats per group were used to isolate brain capillaries for western blot analysis and immunoprecipitation analysis. Another 6 rats were used for evens blue staining to evaluate BBB disruption, while the remaining 6 rats per group were subjected to brain edema evaluation.Results: 1, TUNEL staining showed that only a few TUNEL-positive apoptotic cells were observed in the brain microvascular endothelium in sham group, while the apoptotic index was found to be significantly higher in ICH group. As compared with ICH group, rh ANXA1 at 0.67μg /kg body and 1.34μg /kg body exerted significant rescue effects on VEC apoptosis(P<0.01); 2, FJB-positive cells were obviously increased both in cortex and perihematoma brain in ICH group, which was significantly attenuated by 0.67/kg body and 1.34/kg body rh ANXA1 treatment(P<0.01); 3, Furthermore, treatment with 1.34g/kg body weight rh ANXA1 induced a significant up-regulation in the protein level of occluding and zona occludens 1(ZO-1), supporting a previous report that ANXA1 affects tight junction expression(P<0.01); 4,We examined the potential role of ANXA1 in these conditions. i.v. administration of rh ANXA1 at 0.375-1.34μg g/kg body weight significantly reduced the degree of evans blue dye extravasation into the brain, effectively rescuing the ICH-enhanced BBB leakage(P<0.01). These findings were further confirmed by western blot assay of brain content of albumin. Notably, as compared with low-dosage rh ANXA1(0.375μg /kg body), high-dosage rh ANXA1(1.34μg /kg body) had a greater effect on improvement of BBB integrity of rats undergoing ICH.Finally, brain water content was found to be significantly higher in brain samples of ICH group than that in rats subjected to the sham group(P<0.05). The mean brain water content was lower in rats with high-dosage rh ANXA1(1.34/kg body) treatment than in the ICH control group(P<0.01).Conclusion: By increasing with the ANXA1 dose which can improve the prognosis of intracerebral hemorrhage and the blood brain barrier integrity.Part Ⅲ: Mechanisms of ANXA1 actions.Objective: By small RNA interference and site-directed mutagenesis studies have been performed in vitro to examine the mechanisms underly the actions of ANXA1.Methods: 1,To further investigate the mechanism whereby recombinant ANXA1 could rescue the permeability deficit seen in ICH rats, we analyzed the interaction between ANXA1 and β-actin in whole cell lysis and the interaction between ANXA1 and FRP2 in plasma membrane protein extraction, which have been previously shown to mediate tight junction organization. By Immunoprecipitation experiments trying to analyse the interaction between ANXA1 and β-actin/FRP2 which is the different between in sham group and treatment group; 2, Immunoprecipitated ANXA1 was subjected to western blot using antibodies against different phosphorylation amino acid residues. Firstly, both wild type EGFP-ANXA1 and the mutant proteins were strongly expressed in transfected h CMEC/D3 cells. Then, we examined the phosphorylation status of the wild type and mutant proteins.; 3,Consequently, we investigated whether a similar action of ANXA1 could be identified in the restoration of BBB function in vitro. The interference efficiency of 3 different si RNAs specific for ANXA1 was test, and the most efficient one was used in the following study. Compared with the control group, whether in oxy Hb group can decrease in BMVEC viability was observed, whether the viability was exacerbated by ANXA1 knockdown and attenuated by wild type EGFP-ANXA1 overexpression. In addition, by using a well-established method to examine the transendothelial permeability of a monolayer of BMVCEs, we check that whether oxy Hb-treated cells more significantly greater FITC-dextran permeability than control h CMEC/D3 cells, which was intensified by ANXA1 knockdown and suppressed by wild type EGFP-ANXA1 overexpression. 4, In control group, immunofluorescence assay revealed clear distribution of ANXA1 apparent alongside F-actin fibrils, as stained by rhodamine-phalloidin, further indicating a previous reported role for ANXA1 in the formation / stabilization of the actin cytoskeleton. Compared with control group, fluorescence intensity of ANXA1 and F-actin fibrils in oxy Hb group was significantly decrease, especially the distribution of F-actin fibrils alongside the cell edge, which is critical for tight junctions formation. Compared with the EGFP group, wild type EGFP-ANXA1 overexpression significantly rescued the oxy Hb-induced disruption of F-actin fibrils, while threonine 24 to alanine mutation significantly abolished the rescue effects of wild type ANXA1. 5, To investigate further the potential roles of serine27 and threonine 24 in the secretion of ANXA1, we examined the impact of mutating these sites on the cultured supernatant content of ANXA1 using GFP-tagged constructs. In addition, we examine the trend of the cultured supernatant content of ANXA1, by the western blot find it whether which are the interaction between wild type EGFP-ANXA1 and FPR2 was enhanced by oxy Hb treatment and almost completely abolished by S27 A mutation.Results: 1, Recombinant ANXA1 Rescued ICH-induced Disruption in the interaction between ANXA1 and β-actin/FRP2; 2, ICH induced phosphorylation of ANXA1, especially at ser27 and Thr24; 3, Rescue Effects of ANXA1 on BMVEC Permeability in a phosphorylation-dependent manner; 4, Mutation of threonine 24 impairs the interaction of ANXA1 to β-actin; 5, Mutation of serine27 impairs the secretion of ANXA1, which is critical for its interaction with FPR2Conclusion: ANXA1 prevents ICH-induced BBB dysfunction in threonine 24 and serine 27 phosphorylation-dependent manners. ANXA1 phosphorylation maybe a self-help strategy in BMVECs after ICH, however, that was almost completely abolished by the ICH-induced loss of ANXA1. |
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