Dietary Compound Chrysin Inhibits Retinal Neovascularization with Abnormal Capillaries in db/db Mice
<p>Chemical structure of chrysin (<b>A</b>); inhibitory effects of chrysin on viability (<b>B</b>); nuclear condensation (<b>C</b>); and DNA fragmentation (<b>D</b>) in and glucose toxicity of retinal endothelial cells. human retinal microvascular endothelial cells (HRMVEC) were incubated with 33 mM glucose in the absence and presence of 1–20 μM chrysin up to five days. Cells were also incubated with 5.5 mM glucose and 27.5 mM mannitol as osmotic controls. After HRMVEC were cultured in media for two days, cell viability was measured by using MTT assay (<span class="html-italic">n</span> = 3, 100% viability with 5.5 mM glucose). Means not sharing a common letter differ, <span class="html-italic">p</span> < 0.05. Nuclear condensation was examined with Hoechst 33258 in HRMVEC (<b>C</b>) and the DNA fragmentation measured with TUNEL assay and nuclear staining was done with 4′,6-diamidino-2-phenylindole (DAPI) (<b>D</b>). Representative microphotographs were obtained by fluorescent microscopy with fluorescein green filter. Magnification: 200×. Fluorescence intensity was quantified by using an Axiomager (Zeiss, Oberkochen, Germany). microscope system. Respective values not sharing a letter are different at <span class="html-italic">p</span> < 0.05.</p> "> Figure 2
<p>Time course responses of induction of HIF-1α and VEGF by high glucose (<b>A</b>) and inhibition of HIF-1α, VEGF, and VEGFR2 by chrysin (<b>B</b>). HRMVEC were incubated with 33 mM glucose in the absence and presence of 1–20 μM chrysin up to five days. Cells were also incubated with 5.5 mM glucose and 27.5 mM mannitol as osmotic controls. Cell lysates were subject to Western blot analysis using a primary antibody against HIF-1α, VEGF, or VEGFR2. β-Actin protein was used as an internal control. Bar graphs (mean ± SEM, <span class="html-italic">n</span> = 3) in the bottom panels represent densitometric results of upper blot bands. Means not sharing a common letter differ, <span class="html-italic">p</span> < 0.05.</p> "> Figure 3
<p>Inhibition of retinal tissue induction of HIF-1α, VEGF and VEGFR2 by chrysin. The db/db mice were orally supplemented with 10 mg/kg chrysin daily for 10 weeks. The db/m mice were introduced as control animals. Mouse retinal tissue extracts were subject to Western blot analysis with a primary antibody against HIF-1α and VEGF (<b>A</b>). β-Actin protein was used as an internal control. Bar graphs (mean ± SEM, <span class="html-italic">n</span> = 3) in the right panel represent densitometric results of left blot bands. Values not sharing a common letter differ, <span class="html-italic">p</span> < 0.05. Histological sections of mouse retina were immunohistochemically stained using a primary antibody of VEGFR2 (<b>B</b>). The VEGFR2 was identified as FITC-green staining and the sections were counter-stained with 4′,6-diamidino-2-phenylindole (blue) for the nuclear staining. Magnification: 200×. Retinal layers are labeled as follows: neurofiber layer/ganglion cell layer (NFL/GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), and photoreceptor inner segment/outer segment (IS/OS).</p> "> Figure 4
<p>Induction of endothelial junction markers of VE-cadherin, PECAM-1, and ZO-1 by chrysin. The db/db mice were orally supplemented with 10 mg/kg chrysin daily for 10 weeks. The db/m mice were introduced as control animals. Mouse retinal tissue extracts were subject to Western blot analysis with a primary antibody against VE-cadherin and PECAM-1 (<b>A</b>). β-Actin protein was used as an internal control. Bar graphs (mean ± SEM, <span class="html-italic">n</span> = 3) in the right panel represent densitometric results of left blot bands. Values not sharing a common letter differ, <span class="html-italic">p</span> < 0.05. Histological sections of mouse retina were immunohistochemically stained using a primary antibody of ZO-1 (<b>B</b>). The ZO-1 expression was identified as 3,3′-diaminobenzidine staining (brown) and the sections were counter-stained with hematoxyline. Magnification: 200×. Retinal layers are labeled as follows: neurofiber layer/ganglion cell layer (NFL/GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), and photoreceptor inner segment/outer segment (IS/OS).</p> "> Figure 5
<p>Effect of chrysin on expression of VE-cadherin and <span class="html-italic">N</span>-cadherin in high glucose-exposed HRMVEC (<b>A</b>) and histopathological changes in trypsin-digested retinal vessels (<b>B</b>). HRMVEC were incubated with 33 mM glucose in the absence and presence of 1–20 μM chrysin up to five days. Cells were also incubated with 5.5 mM glucose and 27.5 mM mannitol as osmotic controls. Cell lysates were subject to Western blot analysis using a primary antibody against VE-cadherin or <span class="html-italic">N</span>-cadherin (<b>A</b>). β-actin protein was used as an internal control. Bar graphs (mean ± SEM, <span class="html-italic">n</span> = 3) in the right panels represent densitometric results of left blot bands. The db/db mice were orally treated with 10 mg/kg chrysin for 10 weeks, and db/m mice were introduced as control animals. Retinal vessels were stained with H&E stain (<b>B</b>). Acellular capillaries (black arrow) were observed in db/db mice. Magnification: 200×. The number of acellular capillaries was measured to assess the extent of retinopathy. Values in bar graphs (mean ± SEM, <span class="html-italic">n</span> = 3) not sharing a common letter differ, <span class="html-italic">p</span> < 0.05.</p> "> Figure 6
<p>Inhibition of retinal induction of Ang-1, Ang-2, and Tie-2 by chrysin. HRMVEC were cultured in media of 33 mM glucose in the absence and presence of 1–20 μM chrysin for two days (<b>A</b>). Cells were also incubated with 5.5 mM glucose and 27.5 mM mannitol as osmotic controls. The db/db mice were orally supplemented with 10 mg/kg chrysin daily for 10 weeks. The db/m mice were introduced as control animals (<b>B</b>). HRMVEC lysates and mouse retinal tissue extracts were subject Western blot analysis was conducted for the induction of Ang-1, Ang-2, and Tie-2 using a primary antibody against Ang-1, Ang-2, or Tie-2. β-Actin protein was used as an internal control. Bar graphs in the bottom panel represent densitometric results of upper blot bands. Values (means ± SEM, <span class="html-italic">n</span> = 3) not sharing a common letter differ, <span class="html-italic">p</span> < 0.05.</p> "> Figure 7
<p>Blockade of new capillary bed formation (<b>A</b>) and vascular leakage (<b>B</b>) by chrysin. The db/db mice were orally treated with 10 mg/kg chrysin for 10 weeks and db/m mice were introduced as control animals. Typical appearance of ischemic retinopathy was observed by fluorescein-dextran perfused retinal flat-mounts. Retinas were dissected, flat mounted and observed by confocal microscopy. Magnification: 400×.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Retinal Endothelial Cell Culture
2.3. Assays for Nuclear Condensation and DNA Fragmentation
2.4. Western Blot Analysis
2.5. In Vivo Animal Experiments
2.6. Immunohistochemical Staining
2.7. Retinal Trypsin Digestion Assay
2.8. FITC–Dextran Perfused Retinal Flat Mounts
2.9. Data Analysis
3. Results
3.1. Inhibition of Apoptosis of High Glucose-Induced Retinal Epithelial Cells by Chrysin
3.2. Inhibitory Effects of Chrysin on Induction of Pro-Angiogenic Proteins
3.3. Elevation of Induction of Endothelial Proteins by Chrysin
3.4. Blockade of Retinal Acellular Capillary Formation by Chrysin
3.5. Inhibition of Ang/Tie-2 Receptor Induction by Chrysin
3.6. Suppressive Effect of Chrysin on New Vessel Formation and Retinal Vascular Leakage
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
AGE | advanced glycation end products |
Ang | angiopoietin |
BRB | blood retinal barrier |
DR | diabetic retinopathy |
HIF-1 | hypoxia inducible factor-1 |
N-cadherin | neural cadherin |
PECAM-1 | platelet endothelial cell adhesion molecule-1 |
ROS | reactive oxygen species |
VE-cadherin | vascular endothelial cadherin |
VEGF | vascular endothelial growth factor |
VEGFR2 | VEGF receptor 2 |
ZO-1 | zona occulden-1 |
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Kang, M.-K.; Park, S.-H.; Kim, Y.-H.; Lee, E.-J.; Antika, L.D.; Kim, D.Y.; Choi, Y.-J.; Kang, Y.-H. Dietary Compound Chrysin Inhibits Retinal Neovascularization with Abnormal Capillaries in db/db Mice. Nutrients 2016, 8, 782. https://doi.org/10.3390/nu8120782
Kang M-K, Park S-H, Kim Y-H, Lee E-J, Antika LD, Kim DY, Choi Y-J, Kang Y-H. Dietary Compound Chrysin Inhibits Retinal Neovascularization with Abnormal Capillaries in db/db Mice. Nutrients. 2016; 8(12):782. https://doi.org/10.3390/nu8120782
Chicago/Turabian StyleKang, Min-Kyung, Sin-Hye Park, Yun-Ho Kim, Eun-Jung Lee, Lucia Dwi Antika, Dong Yeon Kim, Yean-Jung Choi, and Young-Hee Kang. 2016. "Dietary Compound Chrysin Inhibits Retinal Neovascularization with Abnormal Capillaries in db/db Mice" Nutrients 8, no. 12: 782. https://doi.org/10.3390/nu8120782