Icariin improves angiogenesis in the brain hypoperfusion rats through up-regulating VEGF signaling pathway

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(Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education,Zunyi Medical University,Zunyi Guizhou 563099,China)

[Abstract] Objective To investigate the effects of icariin on angiogenesis after rat chronic cerebral hypoperfusion.Methods Rats received the surgery of bilateral common carotid arteries occlusion (BCCAO).After 9 days of the procedure,rats were divided into six groups:Sham,Icariin (60 mg/kg),BCCAO,BCCAO+icariin (15 mg/kg),BCCAO+icariin (30 mg/kg) and BCCAO+icariin (60 mg/kg) groups,respectively.Rats were treated with difference doses of icariin or distilled water for 28 days.Capillary density was labeled by CD31 antibody through immunofluorescence.Nitric oxide (NO) content in the cerebral cortex was measured by Griess test.The protein expression and the phosphorylation level of main mediators in vascular endothelial growth factor (VEGF) pathway were tested by Western blot assay.Results This study showed that CD31+ capillary density was reduced,NO content was decreased,VEGF signaling pathway was down-regulated in BCCAO rats.However,post-hypoperfusion treatment with icariin for 28 days significantly reversed the abovementioned parameters,including the increased capillary density,augmented NO content and up-regulated VEGF signaling pathway in the cerebral cortex of BCCAO rats.Conclusion Icariin has a positive effect on angiogenesis during chronic cerebral hypoperfusion,and this beneficial effect is perhaps relative to increasing NO content and up-regulating VEGF signaling pathway in cerebral cortex.

[Key words] icariin; angiogenesis; nitric oxide; vascular endothelial growth factor; brain ischemia; rat

Cerebral hypoperfusion acutely could elicit stroke,and chronically leads to cognitive impairment,suggesting as a risk factor for neurodegenerative diseases[1-2].However,therapeutic strategies are limited and one of the most common revascularization procedures in the central nervous system is direct extracranial-intracranial vessel bypass surgery,which is technically challenging and often clinically inefficient[3].The development of a safe therapeutic angiogenesis through that blood vessel formation is induced could benefit patients suffering from common neurovascular diseases,such as vascular dementia and cerebral ischemia.

Icariin,a flavonoid extracted from Chinese herbEpimediumbrevicornum,has a variety of effects on the neurodegenerative diseases including Alzheimer’s disease and ageing,as well as vascular diseases[4-14].However,the effect of icariin on the angiogenesis after chronic cerebral hypoperfusion is still unknown.The purpose of this study was,therefore,to observe the angiogenesis after chronic cerebral hypoperfusion under icariin condition by measuring the capillary density,nitric oxide (NO) content and the expression of main mediators in vascular endothelial growth factor (VEGF) signaling pathway in the cerebral cortex.

1 Material and methods

1.1 Materials Icariin (purity≥8% by HPLC) was purchased from Nanjing Zelang Medical Technology Co.,Ltd (Nanjing,China),and it was dissolved in double-distilled water and mixed by ultrasonic for 8 min.All reagents were reagent-graded and commercially available.

1.2 Animals Adult male Sprague-Dawley rats(weight 280 to 300 g at the beginning of this study) were purchased from the Third Military Medical University (Chongqing,China).All rats were housed in cages under a specific pathogen-free environment with (23±1) ℃,55% relative humidity,12 h light-dark cycle,and allowed free access to water and food.

1.3 Ethic statement The experimental protocols were in accordance with the Guidelines for the Care and Use of Laboratory Animals of China and approved by the Animal Ethic Committee in Zunyi Medical University.

1.4 Experimental procedures

1.4.1 Animal modeling and grouping A neck ventral midline incision in rats was made after anesthetizing with 7% choral hydrate (0.5 ml/kg),following the bilateral common carotid arteries were exposed and carefully separated from vagal nerve.The bilateral common carotid arteries were double occluded with 5-0 silk suture in BCCAO rats.Sham rats were only undergoing the same surgical procedure without ligation.Animals were kept on a heating pad until recovery from anesthesia,and then randomly assigned to the following six groups:Sham,Icariin 60 mg/kg,BCCAO,BCCAO+icariin 15,30 and 60 mg/kg groups,respectively.The model was shaped in 9 days after operation[15].Treated rats were administered with various doses of icariin by gavage.Sham and BCCAO control rats were administered with volume-matched distilled water from the 9thday after surgery for 28 successive days.

1.4.2 Immunohistochemistry staining Three rats per group were anesthetized with 7% choral hydrate (0.5 ml/kg),and were transcardially perfused with phosphate-buffered saline (PBS,0.1 M) and then 4% paraformaldehyde until the tail and limbs were rigid.Brains were removed and post-fixed in the same fixative with 20% sucrose for 2 days at 4 ℃.The frozen brain tissues were cut into 30 μm thick sections in the coronal plane.The following steps were that:(1) washed with PBS and incubated in PBS with 0.3% Triton for 30 minutes,(2) blocked in 20% goat serum (GIBCO Invitrogen) for 1 hour,(3) incubated with primary antibody CD31 (1∶200,Abcam) at 4 ℃ over night,(4) washed and incubated for 1 h at room temperature with fluorescent Alexa-488 secondary antibody (anti-mouse for CD31,1∶500,Abcam),(5) photographed under fluorescence microscope (Olympus BX53,Olympus,Japan).

1.4.3 Nitric oxide concentration test Four rats’ cortexes per group were collected for NO Colorimetric Test.NO production (determined as nitrite and nitrate accumulation) was tested by the Griess reaction using a NO Colorimetric Assay (Beyotime,USA) following the description of the kit.

1.4.4 Western blot assay Cerebral cortexes were isolated and homogenized in RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors (100 μmol/l phenylmethylsulfonyl fluoride) using a Homogenate rod on ice.Supernatant was prepared by centrifugation at 14 000 g for 15 min at 4 ℃.Protein concentration was gauged using the bicinchoninic acid protein assay (Beyotime).Total protein was subjected to electrophoresis on 4-10% gradient sodium dodecyl sulfate-polyacrylamide gels,followed by electrophoretic transfer to PVDF membranes.Membranes were blocked in 5% defatted milk in 15 mM Tris-HCl,pH 7.4,150 mmol/l NaCl,and 0.1% Tris-buffered saline and Tween 20 (TBST) for 2 h at room temperature.The following primary antibodies were used in this study:VEGFA,VEGFR2 and MMP9 (Sangon Biotech),eNOS and p-eNOS at Ser1177 site (Abcam),ERK1/2and p-ERK1/2at Thr202/Tyr204 site (Cell Signaling Technology).Then the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Beyotime).Immunoblots were visualized using chemiluminescence reagent BeyoECL Plus (Beyotime).The image was scanned and band intensity was quantified using Quantity One 1-D analysis software v4.52 (BioRad).

1.5 Statistical analysis Statistical analysis was performed using SPSS.All data values were expressed as mean±SD and were analyzed for statistical significance using a one-way ANOVA,followed by Bonferroni multiple comparisons test.SignificantP-value was considered less than 0.05.

2 Results

2.1 Icariin increased the capillary density in the cerebral cortex of BCCAO rats To investigate the effect of icariin on angiogenesis in brain,we prepared the chronic cerebral hypoperfusion rat model,and using CD31 antibody,a specific marker of endothelial cells,to label the positive cells of the cortex vessels.The results showed that the vessels density in the cortex of BCCAO rat was similar to that in sham rat,however,icariin treatment could increase the vessels density compare with BCCAO control rat,particularly in high dose of icariin [F(5,12)=4.925,P<0.05] (Fig 1).This evidence suggested that icariin had positive effect on the cerebral cortex angiogenesis after hypoperfusion.

A:BCCAO rats were treated with various doses of icariin from the 9thday of surgery lasting for 28 days.The representative coronal sections of parietal cortex were shown.The brain tissues were labeled with CD31 antibody to observe parietal cortex under fluorescence microscope (scale bar=50 μm).B:Quantitative analysis of micro-vessels density in the parietal cortex region.Data were expressed as mean±SD (n=3).Fig 1 Icariin increased the CD31+ capillary density in the cerebral cortex of BCCAO rats

2.2 Icariin increased the NO level in the cerebral cortex of BCCAO rats Because NO has been recognized as a crucial mediator in the angiogenesis,whether the angiogenesis of icariin was relative to NO content was investigated in this study.Upon the principle that nitrate is reduced to nitrite by nitrate reductase,Griess reagent Assay can measure NO production (ascertained as nitrite accumulation).We used Griess kit to measure the NO content in the cortex.The results were shown in Fig 2 that compared with sham rats’ brain,the NO content reduced in BCCAO rats,which was augmented by icariin treatment [F(5,18)=9.603,P<0.01],suggesting that icariin increasing capillary density of BCCAO rats may be relative to increasing NO level.

Treated with icariin for 28 days from the 9thday of surgery,NO content in the cerebral cortex supernatant was measured by Griess Reagent Assay.Data were expressed as mean±SD (n=4). Fig 2 Icariin increased the NO content in the cortex of BCCAO rats

2.3 Icariin activated the VEGF signaling pathway in the cerebral cortex of BCCAO rats Considering the important role of VEGF signaling pathway in angiogenesis, we studied the effect of icariin on the main mediators in VEGF signaling pathway.As Fig 3 showed that the protein expression of VEGFA and VEGFR2 was decreased in BCCAO rats compare with Sham rats.After orally treatment with icariin significantly increased their protein expression compared to BCCAO control rats [VEGFA:F(5,12)=34.834,P<0.01; VEGFR2:F(5,12)=3.328,P<0.05,respectively].

Icariin increased the NO level in BCCAO rats’ brain in this study,so we investigated the upstream enzyme,eNOS.And found in Fig 3 that the phosphorylation of eNOS at Ser1177 site decreased in rats’ cortex after BCCAO,icariin treatment could reverse the phosphorylation level of eNOS at Ser1177 site to maintain its activation [F(5,12)=19.14,P<0.01].But no changes of total protein level of eNOS had been found among each group.

Icariin was given to rats from the 9thday after BCCAO for 28 days,the protein level of VEGFA and VEGFR2,as well as MMP9 in the cortex was measured,both the phosphorylation of eNOS at Ser1177 and ERK1/2 at Thr202/Tyr204 and their total protein were also measured by western blot assay.The representative bands were shown in the upper panel.Antibody to β-actin was used to verify equal loading.Statistics of western blot was indicated in the down panel.Data were expressed as mean±SD (n=3).Fig 3 Icariin up-regulated VEGF signaling pathway in the cortex of BCCAO rats

ERK activation is significantly involved in proliferation,morphogenesis,and migration of endothelial cells by VEGF pathway stimulation.Both ERK and NO are major factors in the activation of MMPs.Therefore,we observed ERK1/2protein expression and phosphorylation at Thr202/Tyr204 site,MMP9 protein level in this study.But the results showed that both the phosphorylation of ERK1/2and total protein of MMP9 reduced in rats’ cortex after BCCAO,treatment with icariin significantly suppressed the decrease [p-ERK1/2:F(5,12)=4.45,P<0.05; MMP9:F(5,12)=19.146,P<0.01,respectively],strongly suggesting that icariin improved capillary density relative to activation of VEGF signaling pathway.

3 Discussion

Bilateral common carotid artery occlusion modelis generally used in animal studies examining chronic cerebral hypoperfusion,so we introduced BCCAO rats to investigate the effect of icariin.We found that icariin is adequate to improve angiogenesis after chronic cerebral hypoperfusionin rats by measuring the capillary density using immunofluorescence assay.Simultaneously,we discovered that the mechanism is partly relative to augment NO content and up-regulated VEGF signaling pathway by icariin under chronic cerebral hypoperfusion condition.

Consistent with previous study reported that the number of micro-vessels declined at 2,3,and 4 weeks but increased at 6 weeks after BCCAO[16],in our study BCCAO rats showed the almost similar micro-vessels number assessed with CD31 antibody,a specific marker of endothelial cells,to the sham rats after 5 weeks of cerebral hypoperfusion.BCCAO rats treated with icariin for 28 days from the 9thday of surgery had more CD31+labeled micro-vessels in the cerebral cortex,suggesting that icariin is adequate to induce angiogenesis after hypoperfusion.

Angiogenesis is a complex pathophysiological process that involves a number of angiogenic factors,such as VEGF,transforming growth factor β1,platelet-derived growth factor,and basic fibroblast growth factor.VEGF(also designated as VEGF-A),a specific mitogen for endothelial cell,binding to the receptor tyrosine kinase VEGFR2 triggers multiple signal transduction pathways,which regulate endothelial cell responses that control vascular development[17].BCCAO 5 weeks ago,the protein level VEGFA was significantly decreased and VEGFR2 was prone to decrease not reach significant in this study; icariin treated for 28 days from the 9thday of surgery could improve both the two proteins expression compared to BCCAO rats.

Nitric oxide (NO) could stimulate angiogenesis by up-regulation the expression of VEGF,which in turn stimulates NO release by activating endothelialnitric oxide synthase (eNOS)[18-19].NO has multiple roles in angiogenesis,including inhibiting apoptosis,enhancing endothelial cell proliferation,and enhancing endothelial migration[18].Our results discovered that the NO content reduced in the cortex of BCCAO rat,the activation of eNOS decreased by assessed with the phosphorylation level at Ser1177 site,indicating that angiogenesis is attenuated when NO is reduced.Icariin treated rats showed significantly increased in NO content and enhanced activation eNOS in rat’s cortex after BCCAO,suggesting that the angiogenesis of icariin is in part relative to elevated NO-VEGF level.

It has been reported that VEGFA signaling through activated VEGFR2 involves the MAP-kinase pathway (MEK-ERK) which mediates endothelial cell proliferation and neuroprotection[17],VEGFA aslso recruits a proangiogenic protein matrix metalloproteinase 9 (MMP-9) to induce revascularization[20].MMP family contributes to angiogenesis by helping to detach pericytes from vessels undergoing angiogenesis[21],in which MMP9 is considered important in angiogenesis.In this study,ERK activation which assessed with the phosphorylation at Thr202/Tyr204 site as well as MMP9 protein level were reduced in BCCAO rats,icariin treatment could suppress the decline.The results indicated that icariin possesses the ability to up-regulate VEGF/VEGFR2 pathway in BCCAO rat’s cortex.

We found in this study that icariin improves the angiogenesis in rat’s cortex after chronic cerebral hypoperfusion,and the effect may correlate with enhancing NO content and up-regulating VEGF signaling pathway,indicating that icariin could become a new component for treating chronic cerebral hypoperfusion.