探討天然物對視網膜退化性疾病及葡萄膜炎之治療機轉

The Studies of Therapeutic Mechanisms of Natural Compounds on Retinal Degenerative Disease and Uveitis

林凡立(Fan-Li Lin)

指導教授 ： 蕭哲志

共同指導教授 ： 張宏名(Hung-Ming Chang)

臺北醫學大學/醫學院/醫學科學研究所/博士(2017年)

摘要

眼部疾病常威脅著人們的視力，其中漸進的視網膜細胞死亡以及失調的發炎反應常為主要的病理原因。許多文獻指出天然物具抗凋亡、抗發炎、以及抗氧化的效果進而支持天然物於眼部疾病的運用。本研究中，我們探討兩種天然物成份chrysophanol (Cassia toro)以及theissenolactone C (LC53; Theissenia cinerea)分別應用於視網膜退化以及葡萄膜炎等動物眼部疾病模式之治療效果與相關機制。
視網膜色素變性 (Retinitis pigmentosa, RP) 是一最盛行且持續惡化之遺傳性視網膜退化疾病，其最終可導致失明。化合物N-methyl-N-nitrosourea (MNU)誘發視網膜底層退化之動物模式因可模擬病理上大量photoreceptor凋亡之相關視網膜疾病，常被應用於探討RP或老年黃斑部疾病(age-related macular degeneration, AMD)等疾病機轉或藥物開發。據此，我們探討決明子 (Cassia toro) 中主成分chrysophanol於MNU 誘發之小鼠RP疾病模式之視網膜保護效果。結果指出chrysophanol能保護MNU所誘發之視網膜功能性及結構性的損傷。此外，TUNEL染色結果顯示chrysophanol抑制了MNU所誘發之photoreceptor細胞凋亡，而此抗凋亡的能力可能是透過降低PARP、caspase-3的活化及抑制Bax的合成而來。另一方面，降低的glial fibrillary acidic protein (GFAP)表現指出chrysophanol具有抑制過度gliosis的能力，此舉可能與減少的matrix metalloproteinase-9 (MMP-9)表現及活化相關。細胞實驗結果指出chrysophanol可抑制經toll-like receptor (TLR) 配體lipopolysaccharide (LPS)所刺激初級小膠質細胞之MMP-9活化以及抑制LPS所刺激小膠質細胞株BV2之iNOS及COX-2之生合成。上述實驗結果說明chrysophanol具有抗凋亡及緩解gliosis等視網膜保護效果，其可能對於緩解臨床上RP及相關視網膜退化之疾病具有治療的價值。
除了退化性的視網膜疾病外，發炎介質與發炎反應的異常也被指出參與在各種眼部疾病。葡萄膜炎(Uveitis)即是一種典型眼部發炎性疾病，其為全球所有眼疾中導致失明的主因之一。內毒素誘發葡萄膜炎 (Endotoxin-induced uveitis, EIU) 之動物模式模擬了人類急性葡萄膜炎，其可誘發眼部大量發炎反應。於此，本文探討真菌萃取物Theissenolactone C (LC53)在大鼠EIU疾病模式中的眼保護效果。結果顯示，LC53有效的減緩了EIU所引發的虹膜充血及視功能損傷。此外在EIU後，LC53降低了前房液中蛋白質滲漏、tumor necrosis factor-α (TNF-α)及monocyte chemoattractant protein-1 (MCP-1)的釋放、MMP-2及MMP-9的活化，並減緩了眼組織中氧化性壓力(oxidative stress)的損害。LC53另一方面抑制了EIU後Müller膠細胞的過度活化及monocyte/macrophage的浸潤。分子實驗結果顯示在EIU中，LC53降低了發炎相關蛋白的表現包括HSP90、TLR4的生合成及p65的磷酸化。同時LC53透過抑制凋亡相關蛋白如Bax表現及caspase-3的活化，並加強抗凋亡蛋白Bcl2合成而達到抗凋亡的效果。根據細胞實驗結果，LC53有效的抑制LPS所誘發BV2小膠質細胞株iNOS及COX2的合成表現，另外亦抑制了活性氧壓力(ROS)的大量生成。這些效果可能是透過抑制TLR4/IRAK1/TAK1 訊息路徑進而減少IKKβ、p65、及MAPKs JNK的磷酸化所致。綜合實驗結果，EIU過程中LC53透過其抗發炎、抗凋亡及抗氧化的作用提供眼部保護效果，更顯示LC53運用於臨床acute anterior uveitis或相關眼部發炎疾病的治療價值。
本研究成果指出chrysophanol和theissenolactone C在退化性視網膜疾病模式及發炎性葡萄膜炎中皆提供抗發炎、抗凋亡及降低gliosis之活性，諸多證據支持這些天然物應用在臨床眼科疾病治療的潛力。

關鍵字

色素性視網膜炎 ； 葡萄膜炎 ； 視網膜發炎 ； 小膠質細胞 ； 天然萃取物 ； 金屬基質蛋白分解酶神經膠ˊ質增生

並列摘要

Ocular diseases are the threat to the eyesight, in which progressive apoptosis of retinal cells and dysregulated inflammatory reaction are the mainly pathogenic factors. Much evidence reveals that natural products protect against ocular diseases through their putative properties including anti-apoptotic, anti-inflammatory, and anti-oxidative effects. In the present study, we investigated the therapeutic mechanisms of two natural compounds, chrysophanol (Cassia toro) and theissenolactone C (LC53) (Theissenia cinerea), in the animal model of retinal degenerative disease and ocular inflammatory uveitis, respectively.
Retinitis pigmentosa (RP) is the most common progressive hereditary retinal degeneration causing blindness. N-methyl-N-nitrosourea (MNU)-induced animal retinal degeneration in the outer retina is a useful animal model for photoreceptor degeneration diseases including RP and age-related macular degeneration (AMD). Accordingly, we evaluated the retinal-protective effects of chrysophanol, an active component of Cassia seed (Cassia toro), in the MNU-induced mouse model of RP. We found that chrysophanol preserved the retina functional and morphological deficiencies in MNU-induced retinal degeneration. Furthermore, TUNEL assays revealed that chrysophanol suppressed MNU-induced photoreceptor cell apoptosis, and this effect may through the inactivation of PARP, Bax, and caspase-3. In addition, decreased glial fibrillary acidic protein (GFAP) immunolabeling demonstrated that chrysophanol reduced the reactive gliosis, which may positively correlated with the attenuation of the matrix metalloproteinase (MMP)-9-mediated gelatinolysis. In vitro studies indicated that chrysophanol ameliorated TLR-activator lipopolysaccharide (LPS)-induced MMP-9 activation in primary microglia and inhibited LPS-induced iNOS and COX-2 expression in the BV2 mouse microglia cell line. The data revealed that chrysophanol provided retina-protective effects through its anti-apoptotic and anti-gliosis activities, suggesting that chrysophanol might have therapeutic value for retarding human retinitis pigmentosa and other degenerative retinopathies.
In addition to the degenerative retinopathies, numerous molecular and physiologic abnormalities associated with inflammation have been found to evolve in ocular diseases. Uveitis, an ocular inflammatory disorder, is one of the leading causes of legal blindness around the world. Endotoxin-induced uveitis (EIU), which largely generates inflammatory responses in the eye, mimics the pathologies in human acute uveitis. Accordingly, we examined the ocular-protective effects of fungal ingredient theissenolactone C (LC53) in the rat model of EIU. The data showed that LC53 significantly suppressed the EIU-induced hyperemia in the iris and restored the retina functional deficiency. Furthermore, LC53 decreased the EIU-induced protein leakage, the secretion of tumor necrosis factor (TNF)-α and monocyte chemoattractant protein-1 (MCP-1), the activation of MMP-2 and MMP-9 in aqueous humor, and oxidative stress burden in the eye tissue. The reactivity of Müller glial cells and the accumulation of monocyte/macrophage were down-regulated by LC53 during EIU. In eye homogenates, LC53 decreased the expression of immune-responded proteins HSP90, TLR4, and phosphorylation of p65. Moreover, LC53 showed the anti-apoptotic effects by suppressing the Bax, cleaved-caspase-3 expression accompanied with the enhanced Bcl-2 expression after EIU. According to the microglia studies, LC53 significantly inhibited the expression of iNOS, COX-2, and ROS production in LPS-stimulated microglial BV2 cells. These effects may through the down-regulation of the TLR4/IRAK1/TAK1 signaling followed by the reduction of IKKβ, p65, and MAPKs JNK phosphorylation. The results revealed that LC53 possessed ocular-protective effects through its anti-inflammatory, anti-apoptosis, and anti-oxidative properties during EIU, suggesting a therapeutic potential for acute anterior uveitis (AAU) and related ocular inflammatory diseases.
In conclusion, we found both chrysophanol and LC53 provided the anti-inflammatory, anti-apoptotic, and anti-gliosis properties in animal model of degenerative retinopathy and inflammatory uveitis suggesting a potential therapeutic value of these natural compounds.

並列關鍵字

retinitis pigmentosa ； N-methyl-N-nitrosourea ； retinal degeneration ； uveitis ； lipopolysaccharide ； ocular inflammation ； TLR4 ； gliosis ； microglia ； matrix metalloproteinase ； chrysophanol ； LC53

參考文獻

83. Sacchetti, M., Mantelli, F., Merlo, D. & Lambiase, A. Systematic Review of Randomized Clinical Trials on Safety and Efficacy of Pharmacological and Nonpharmacological Treatments for Retinitis Pigmentosa. J Ophthalmol 2015, 737053 (2015).

1. Carreras, F.J., Rodriguez-Hurtado, F. & David, H. Ophthalmology in Luanda (Angola): a hospital based report. Br J Ophthalmol 79, 926-933 (1995).

2. Iwase, A. et al. Prevalence and causes of low vision and blindness in a Japanese adult population: the Tajimi Study. Ophthalmology 113, 1354-1362 (2006).

3. Adeoti, C.O. Prevalence and causes of blindness in a tropical African population. West Afr J Med 23, 249-252 (2004).

4. Baumgartner, W.A. & Baumgartner, A.M. Rationale for an experimental treatment of retinitis pigmentosa: 140-month test of hypothesis with one patient. Med Hypotheses 81, 720-728 (2013).