Nutr Res Pract.  2023 Jun;17(3):421-437. 10.4162/nrp.2023.17.3.421.

Highly water-soluble diacetyl chrysin ameliorates diabetes-associated renal fibrosis and retinal microvascular abnormality in db/db mice

• Kang YH
• Park SH
• Sim YE
• Oh MS
• Suh HW
• Lee JY
• Lim SS

• KMID: 2542840
• DOI: http://doi.org/10.4162/nrp.2023.17.3.421

Abstract

BACKGROUND/OBJECTIVES
Chronic or intermittent hyperglycemia is associated with the development of diabetic complications. Oxidative stress and inflammation can be altered by hyperglycemia in diverse tissues, including kidneys and eyes, and play a pivotal role in diabetic complications. Our previous studies showed that the water-insoluble 5,7-dihydroxyflvone chrysin effectively combats diabetic damages incurred in diabetic kidneys and retinas. The current study employed the newly-synthesized 5.7-di-O-acetylchrysin, having higher solubility than chrysin, to compare the effects on diabetes-associated renal fibrosis and abnormal retinal neovascularization.
MATERIALS/METHODS
In the in vivo study, db/db mice as animal models of type 2 diabetes were orally administrated 10 mg/kg BW diacetylchrysin, daily for 10 weeks.
RESULTS
Unlike chrysin, oral administration of 10 mg/kg diacetylchrysin did not lower the blood glucose level and 24 h urine volume in db/db mice. Nevertheless, the urinary albumin excretion was markedly reduced. The administration of diacetylchrysin also diminished the deposition of collagen fibers in diabetic glomeruli and tubules by suppressing the induction of connective tissue growth factor and collagen IV in diabetic kidneys. Supplying diacetylchrysin enhanced the membrane type-1 matrix metalloproteinase (MMP) expression reduced in diabetic kidneys, while the tissue inhibitor of MMP-2 induction was attenuated in diacetylchrysin-challenged diabetic kidneys. In addition, supplementing diacetylchrysin to diabetic mice ameliorated renal injury due to glomerulosclerosis and tubular interstitial fibrosis. Furthermore, the reduced retinal inductions of Zonula occludens-1 and vascular endothelial cadherin in db/db mice were elevated in the retinal tissues of diacetylchrysintreated animals. Oral administration of diacetylchrysin curtailed the induction of vascular endothelial growth factor (VEGF) and VEGF receptor 2 in db/db mice, ultimately retarding diabetes-associated retinal neovascularization. Additionally, the retinal formation of acellular capillaries with leaky vessels was reduced in diacetylchrysin-treated db/db mice.
CONCLUSION
Diacetylchrysin may act as a potent pro-health agent for treating renal fibrosisassociated diabetic nephropathy and retinal neovascularization-associated diabetic retinopathy.

Keyword

5,7-diacetylchrysin; renal fibrosis; retinal neovascularization; diabetic nephropathy; diabetic retinopathy

Figure

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  Fig. 1 Chemical structures of chrysin and diacetylchrysin (A), and changes in food intake (B), drinking water intake (C), and body weight (D) of db/m controls and db/db mice after the 10 week-supplementing challenge with diacetylchrysin. The db/db mice were supplemented with 10 mg/kg diacetylchrysin for 10 weeks. The db/m mice were introduced as control animals. The data are presented as mean ± SEM for each treatment group (n = 9).SEM, standard error of mean.*P < 0.05 relative to 10th week or db/m control mice.

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  Fig. 2 Blood levels of fasting glucose (A) and glycated hemoglobin level (HbA1c, B), oral glucose tolerance curve (C), 24 h urine volume (D), and urinary albumin excretion (E) of db/db mice treated with diacetylchrysin. The db/db mice were supplemented with 10 mg/kg diacetylchrysin for 10 weeks. The db/m mice were introduced as control animals. The blood glucose levels and HbA1c, 24 h urine volume, and urinary albumin excretion were measured after the 10 weeks treatment of diacetylchrysin. The values (mean ± SEM, n = 9) not sharing a common small letter are significantly different at P < 0.05.HbA1C, hemoglobin A1c; SEM, standard error of mean.*P < 0.05 relative to db/m control mice.

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  Fig. 3 Effects of diacetylchrysin on formation of collagen fibers in glomeruli (A) and renal tubules (B). The db/db mice were supplemented with 10 mg/kg diacetylchrysin for 10 weeks. The db/m mice were employed as control animals. Glomerular and tubular fibrosis in db/db mouse kidneys was observed by Masson’s trichrome staining (A and B). The collagen fibers were stained in blue. Each photograph is representative of 4 mice. Magnification: 200-fold.

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  Fig. 4 Effects of diacetylchrysin on the expressions of CTGF (A), collagen IV (B), MT1-MMP (C), and TIMP-2 (D) in db/db mice. The db/db mice were orally treated with 10 mg/kg diacetylchrysin or 10 mg/kg chrysin for 10 weeks. The db/m mice were introduced as control animals. Western blot analysis with tissue extracts was conducted with a primary antibody against CTGF, collagen IV, MT1-MMP, and TIMP-2. β-Actin protein was used as an internal control. The bar graphs (mean ± SEM, n = 3) in the right panel represent quantitative results obtained from a densitometer. Values not sharing a common small letter are significantly different at P < 0.05.CTGF, connective tissue growth factor; MT1-MMP, membrane type 1-matrix metalloproteinase; TIMP-2, tissue inhibitor of metalloproteinase 2; SEM, standard error of mean.

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  Fig. 5 Inhibition of renal interstitial fibrosis (arrows) in glomeruli (A) and renal tubules (B) of db/db mice by diacetylchrysin. The db/db mice were supplemented with 10 mg/kg diacetylchrysin for 10 weeks. The db/m mice were employed as control animals. Glomerular and tubular tissue sections were stained by PAS reagent and counterstained with hematoxylin (A and B). Each photograph is representative of four mice. Magnification: 200-fold.PAS, Periodic acid-Schiff.

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  Fig. 6 Effects of diacetylchrysin on the expression of endothelial junction markers. The db/db mice were orally supplemented with 10 mg/kg diacetylchrysin 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 ZO-1 (A), VE-cadherin (D), and PECAM-1 (E). β-Actin protein was used as an internal control. Bar graphs (mean ± SEM, n = 3) in the right panel represent densitometric results of left blot bands. Values not sharing a common small letter differ, P < 0.05. Histological sections of mouse retina were immunohistochemically stained using a primary antibody of ZO-1 and red Cy3-conjugated secondary antibody for visualizing the ZO-1 induction (B). In another experiment with 10 mg/kg chrysin administration, the ZO-1 expression was identified as 3,3′-diaminobenzidine staining (brown) and the sections were counterstained with hematoxylin (C). Each photograph is representative of four mice. Magnification: 200-fold. Retinal layers are labeled as follows: NFL/GCL, IPL, INL, OPL, ONL, and photoreceptor IS/OS.ZO, Zona occluden; VE, vascular endothelial; PECAM, platelet endothelial adhesion molecule; SEM, standard error of mean; NFL, neurofiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment; OS, outer segment.

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  Fig. 7 Inhibition of retinal tissue induction of VEGF (A) and VEGFR2 (B) by diacetylchrysin. The db/db mice were orally administered 10 mg/kg diacetylchrysin daily for 10 weeks. The db/m mice were employed as control animals. Mouse retinal tissue extracts were subject to Western blot analysis with a primary antibody against VEGF and VEGFR2 (A and B, respectively). β-Actin protein was used as an internal control. Bar graphs (mean ± SEM, n = 3) in the right panel represent densitometric results of left blot bands. Values not sharing a common small letter differ, P < 0.05. Immunocytochemical analysis of VEGFR2 in db/db mice treated with 10 mg/kg chrysin was achieved by histological staining of mouse retina sections using a primary antibody of VEGFR2. FITC-conjugated secondary antibody was used for visualizing the VEGFR2, and the nuclear staining was done with 4′,6-diamidino-2-phenylindole (C). Each photograph is representative of 4 mice. Magnification: 200-fold. Retinal layers are labeled as follows: NFL/GCL, IPL, INL, OPL, ONL, and photoreceptor IS/OS.VEGF, vascular endothelial growth factor; VEGFR2, VEGF receptor 2; SEM, standard error of mean; FITC, fluorescein isothiocyanate; NFL, neurofiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment; OS, outer segment.

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  Fig. 8 Inhibition of abnormal neovascularization by diacetylchrysin. The db/db mice were orally supplemented with 10 mg/kg diacetylchrysin daily for 10 weeks. The db/m mice were employed as control animals. To reveal acellular capillaries (red arrow) in db/db mice treated with 10 mg/kg diacetylchrysin, retinal vessels were subjected to H&E staining (A). Magnification: 200-fold. To detect new capillary bed formation (yellow arrow) and vessel leakage (red arrow) by diacetylchrysin exposure (B), typical appearance of retinal capillaries was observed by FITC-dextran-perfused retinal flat-mounts. Retinas were dissected, flat mounted, and observed by confocal microscopy. Magnification: 400-fold. Each photograph is representative of 4 mice.H&E, hematoxylin and eosin; FITC, fluorescein isothiocyanate.

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  Fig. 9 Schematic diagram showing the effects of diacetylchrysin on nephropathy and retinopathy in the type 2 diabetic mouse model. The arrows indicate activation by hyperglycemia or diacetylchrysin.

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