科学研究費助成事業　　研究成果報告書

様　式　Ｃ－１９、Ｆ－１９、Ｚ－１９ （共通）

機関番号：

研究種目：

課題番号：

研究課題名（和文）

研究代表者

研究課題名（英文）

交付決定額（研究期間全体）：（直接経費）

１０１０７

若手研究(B)

2015～2013

糖転移ヘスペリジンによる糖尿病網膜症新規治療薬の開発

The development of a new therapy in diabetic retinopathy by application of alpha-glucosylhesperidin

６０４３１４１７研究者番号：

横田　陽匡（Yokota, Harumasa）

旭川医科大学・医学部・助教

研究期間：

２５８６１６０９

平成 年 月 日現在２８ ６ ２３

円 3,000,000

研究成果の概要（和文）：本研究によって分散ヘスペレチンが網膜における虚血再還流によって惹起される神経節細胞死を抑制することを明らかになった。分散ヘスペレチンは虚血再灌流によって発生する酸化ストレスや活性化したミクログリアによる慢性炎症を抑制し、結果として神経網膜におけるアポトーシスを減少させる。過剰な酸化ストレスや慢性炎症を発症基盤とする糖尿病網膜症に対して、分散ヘスペレチンが神経網膜を保護する可能性があると考えられた。またミューラー細胞におけるグリオーシスも抑制することから糖尿病黄斑浮腫に対しても有効である可能性が示唆された。

研究成果の概要（英文）：The present study for the first time demonstrated that water-dispersible hesperetin (WD-Hpt) decreased the number of cell death in the ganglion cell layer after ischemia reperfusion (I/R) injury in the retina. This beneficial protective effect of WD-Hpt on the retina derived from reduction of oxidative stress. Consequently, this led to alleviation of activated microglia and reduction of chronic inflammation. Therefore, WD-Hpt is likely to protect neural cells in the retina from diabetes by reducing oxidative stress and chronic inflammation. Moreover, WD-Hpt was shown to reduce I/R-induced gliosis in Muller cells. This also indicates that WD-Hpt can be used for the treatment in diabetic macular edema.

研究分野： 眼科学

キーワード： 糖尿病網膜症　虚血性網膜症　神経保護　血管保護　分散ヘスペレチン　糖転移ヘスペリジン

２版

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Fig. 1 Reduction of ischemiareperfusion (I/R)-inducedreactive oxygen species (ROS)formation by water-dispersiblehesperetin (WD-Hpt).a Western blot analysis ofnitrotyrosine formation in theI/R retinas treated with normalsaline (NS) or WD-Hpt. I/Rincreased the nitrotyrosineformation in mice treated withNS. This effect was reduced byWD-Hpt treatment (n = 3;**P\ 0.01 vs NS control (con);!P\ 0.05 vs NS I/R). b Thedihydroethidium (DHE)imaging of superoxideformation at 6 h after I/R. WD-Hpt reduced I/R-induced DHEreaction (n = 6; **P\ 0.01 vsNS con; !!P\ 0.01 vs NS I/R).GCL ganglion cell layer, IPLinner plexiform layer, INL innernuclear layer, OPL outerplexiform layer, ONL outernuclear layer. Scale bar 50 lm

Fig. 2 The inhibitory effect of WD-Hpt on activated microglia.a Fluorescent microscopic imaging of retinal sections labeled withIba1, microglial marker, at 24 h after I/R. I/R resulted in microgliawith a large cell body and shorter processes (NS I/R) compared tomicroglia with a small cell body and longer process in control retina(NS con). WD-Hpt mitigated the alteration of the morphology ofmicroglia in I/R retina (WD-Hpt I/R). Scale bar 200 lm (left line)

and 50 lm (middle line). b Expression of IL-1b in BV-2 cellsstimulated by lipopolysaccharide (LPS). Hpt reduced increasedexpression of IL-1b at a concentration of 100 lM. Total RNA wasextracted and IL-1b mRNA levels were assayed by real-timequantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 3; *P\ 0.05 vs con; !P\ 0.05 vs LPS)

Neuroprotective effect of water-dispersible hesperetin in retinal ischemia reperfusion injury 55

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Glial activation is another prominent feature of retinal

I/R injury, diabetic retinopathy and other forms of ischemicretinopathy [4–6]. To see whether WD-Hpt can alleviate

this aspect of retinal injury, we examined the expression of

GFAP, known to increase during glial activation. As shownin Fig. 4a, the immunoblotting demonstrated that, com-

pared with the contralateral control eyes, GFAPs were

markedly increased in the NS I/R retina. By contrast,GFAP levels in the WD-Hpt I/R retina were almost com-

parable to those in the control retina. In addition, GFAP

immunoreactivity in the NS I/R retina was localized tofilamentous processes in the nerve fiber layer to the outer

limiting membrane, corresponding to the distribution ofastrocytes and Muller cells (Fig. 4b). GFAP immunola-

beling in the radial Muller cell processes in the WD-Hpt

I/R retina was much weaker than in the NS I/R retina,

suggesting that WD-Hpt reduced glial injury in the I/Rretina.

Reduction of I/R-induced apoptosis by WD-Hpt

Oxidative stress initiates an intrinsic apoptotic pathway

leading to neuronal cell death in the I/R retina [46]. To seewhether WD-Hpt was able to reduce apoptosis in the I/R

retina, we performed immunohistochemistry of tubulin B3

(neuron-specific marker) and cleaved caspase-3 at 24 hafter I/R, and TUNEL staining at 3 days after I/R. As

shown in Fig. 5, the expression of cleaved caspase-3 wasincreased exclusively in the GCL and INL layers of the I/R

retina. Similarly, TUNEL-positive cells were also present

Fig. 3 Protective effect of WD-Hpt on neuronal cell in the GCLduring I/R. Confocal imaging of flat-mounted retina labeled withNeuN antibody at 7 days after I/R shows a significant decrease indensity of NeuN-positive cells in the GCL of the NS I/R retina

compared with the NS con retina. WD-Hpt treatment significantlydecreased the loss of NeuN-positive GCL neurons after I/R (n = 4;**P\ 0.01 vs NS con; !!P\ 0.01 vs NS I/R). Scale bar 100 lm

Fig. 4 The mitigation of glial activation by WD-Hpt in the retinaduring I/R injury. a Western blot analysis shows the increase of glialfibrillary acidic protein (GFAP) at 5 days after I/R, which wasreduced by WD-Hpt treatment (?, I/R treated groups, n = 4 for eachof the treatments; -, control, n = 3 for each of the treatments;

**P\ 0.01 vs NS con; !P\ 0.05 vs NS I/R). b Immunohistochem-istry analysis of retinal sections labeled with GFAP. GCL ganglioncell layer, IPL inner plexiform layer, INL inner nuclear layer, OPLouter plexiform layer, ONL outer nuclear layer. Scale bar 50 lm

56 A. Shimouchi et al.

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Fig. 1 Reduction of ischemiareperfusion (I/R)-inducedreactive oxygen species (ROS)formation by water-dispersiblehesperetin (WD-Hpt).a Western blot analysis ofnitrotyrosine formation in theI/R retinas treated with normalsaline (NS) or WD-Hpt. I/Rincreased the nitrotyrosineformation in mice treated withNS. This effect was reduced byWD-Hpt treatment (n = 3;**P\ 0.01 vs NS control (con);!P\ 0.05 vs NS I/R). b Thedihydroethidium (DHE)imaging of superoxideformation at 6 h after I/R. WD-Hpt reduced I/R-induced DHEreaction (n = 6; **P\ 0.01 vsNS con; !!P\ 0.01 vs NS I/R).GCL ganglion cell layer, IPLinner plexiform layer, INL innernuclear layer, OPL outerplexiform layer, ONL outernuclear layer. Scale bar 50 lm

Fig. 2 The inhibitory effect of WD-Hpt on activated microglia.a Fluorescent microscopic imaging of retinal sections labeled withIba1, microglial marker, at 24 h after I/R. I/R resulted in microgliawith a large cell body and shorter processes (NS I/R) compared tomicroglia with a small cell body and longer process in control retina(NS con). WD-Hpt mitigated the alteration of the morphology ofmicroglia in I/R retina (WD-Hpt I/R). Scale bar 200 lm (left line)

and 50 lm (middle line). b Expression of IL-1b in BV-2 cellsstimulated by lipopolysaccharide (LPS). Hpt reduced increasedexpression of IL-1b at a concentration of 100 lM. Total RNA wasextracted and IL-1b mRNA levels were assayed by real-timequantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 3; *P\ 0.05 vs con; !P\ 0.05 vs LPS)

Neuroprotective effect of water-dispersible hesperetin in retinal ischemia reperfusion injury 55

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T Takumi H, Nakamura H, Shimizu T, Harada R, Kometani T, Nadamoto T, Mukai R, Murota K, Kawai Y, Terao J. Bioavailability of orally administered water-dispersible hesperetin and its effect on peripheral vasodilatation in human subjects: implication of endothelial functions of plasma conjugated metabolites. Food Funct 2012;3(4):389-98

U Nagaoka T, Sato E, Takahashi A, Yokota H, Sogawa K, Yoshida A. Impaired retinal circulation in patients with type 2 diabetes mellitus: retinal laser Doppler velocimetry study. Invest Ophthalmol Vis Sci 2010;51(12):6729-34

U Zheng L, Gong B, Hatala DA, Kern TS. Retinal ischemia and reperfusion causes capillary degeneration: similarities to diabetes. Invest Ophthalmol Vis Sci 2008;48(1):361-7

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1 Neuroprotective effect of water-dispersible hesperetin in retinal ischemia reperfusion injury. Shimouchi A,Yokota H, Ono S, Matsumoto C, Tamai T, Takumi H, Narayanan SP, Kimura S,Kobayashi H, Caldwell RB, Nagaoka T, Yoshida A. Jpn J Ophthalmol 2016;60:51-61

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