Table 22.1 The one-way ANOVA results of RPE cells viability (A), NO level, IOD value of iNOS expressed in RPE cells of each group (x¯ ± s)

To examine whether the blocking of NO production by hesperidin is mediated by the process of iNOS expression, iNOS protein levels were measured by immunohistochemistry. Hesperidin significantly inhibited high glucose induced iNOS protein in RPE cells (Table 22.1). These results suggest that iNOS is suppressed by hesperidin in high glucose activated RPE cells.

22.4 Discussion

NO is a free radical gas that is synthesized from l-arginine by three different isoforms of NO synthase (NOS). NO plays an important role in homeostatic vasodilatation and regulation of blood flow, but excess release induces tissue disorders because of increased oxidative stress, especially caused by the production of peroxynitrite. More and more physiological studies have indicated the involvement of NO in impulse transduction in the outer retina and in the modulation of visual signal during retinal information processing. The increased plasma NO levels in patients with type 2 diabetes indicate that NO may be associated with the pathogenesis of DR (Izumi et al. 2006). Ocular blood flow is regulated by NO derived from the endothelium and efferent nitrergic neurons. Endothelial dysfunction impairs ocular hemodynamics by reducing the bioavailability of NO and increasing the production of reactive oxygen species (ROS). On the other hand, NO formed by inducible NOS (iNOS) expressed under influences of inflammatory mediators evokes neurodegeneration and cell apoptosis, leading to serious ocular diseases (Toda and Nakanishi-Toda 2007). While not typically expressed in the central nervous system (CNS) under physiological conditions, iNOS is transcriptionally induced by various stimuli associated with pathologic insults including, hypoxia, bacterial components, viral proteins, and cytokines. Once expressed, iNOS catalyzes the nicotinamide adenine dinucleotide phosphate-dependent oxidation of L-arginine to nitric oxide (NO)

and citrulline. Unlike other NOS isoforms, iNOS has continuously high and longlasting activity and, depending on the temporal expression and cellular context, can either be toxic or protective. Continued presence of NOS-immunoreactivity in the photoreceptors from 16–17 weeks of fetal life to adulthood indicates other functions besides their definitive involvement in the photoreceptor function of transduction and information processing (Shashi and Tapas 1999).

Retinopathy is one of the most severe ocular complications of diabetes and is a leading cause of acquired blindness in young adults. Sustained hyperglycemia plays a central role in the development of diabetic retinopathy; prolonged exposure of the cells to high glucose is shown to cause both acute and reversible changes in cellular metabolism, and long term irreversible changes in stable macromolecules (Madsen-Bouterse and Kowluru 2008).

Hesperidin is a kind of ubiquitous plant component and has a variety of biological effects. Moreover, it has also been reported that hesperidin reduced by around 10–30% the synthesis of NO by macrophages when inducible NO synthase was already expressed with LPS/IFN-gamma for 24 h (Rao et al. 2008). Sakata et al. (2003) indicated hesperidin as a COX-2 and iNOS inhibitor, might be related to the anti-inflammatory and anti-tumorigenic efficacies. Treatment with hesperidin suppressed production of PGE2, nitrogen dioxide (NO2), and expression of iNOS protein (Sakata et al. 2003).

In this study, hesperidin was isolated from Pericarpium Citri Reticulatae of Newhall Citrus reticulata Blanco and its inhibitory activity on iNOS induction in RPE cells treated by high glucose was determined. MTT assay discovered that the cell viability in high glucose model group was obvious lower than in the normal control group and the difference had statistical significance, which indicated high glucose had resulted in the injury of RPE cells; however, the cell viability in the experimental groups, which had been pretreated by hesperidin, was higher than in the high glucose model group. It was found that hesperidin potently inhibits high- glucose-inducible NO production by suppressing iNOS expression in RPE cells. It showed that the injury of RPE cells induced by high glucose might be associated with the activation of iNOS and mass production of NO. Further studies will have to elucidate the molecular mechanisms of protection of hesperidin on RPE cells.

References

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Hong J, Yuan Z, Shuai J et al (2007) High glucose induce production of iNOS by human retinal pigment epithelium cells through activation of the p38 signal pathway. Acta Universitatis Medicinalis Nanjing (Natural Science) 27(9):970–973

Izumi N, Nagaoka T, Mori F et al (2006) Relation between plasma nitric oxide levels and diabetic retinopathy. Jpn J Ophthalmol 50(5):465–468

Kanaze FI, Gabrieli C, Kokkalou E et al (2003) Simultaneous reversed-phase high-performance liquid chromatographic method for the determination of diosmin, hesperidin and naringin in

different citrus fruit juices and pharmaceutical formulations. J Pharm Biomed Anal 33(2): 243–249

Leal EC, Manivannan A, Hosoya K et al (2007) Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy. Invest Ophthalmol Vis Sci 48(11):5257–5265

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Toda N, Nakanishi-Toda M (2007) Nitric oxide: ocular blood flow, glaucoma, and diabetic retinopathy. Prog Retin Eye Res 26(3):205–238

Tommasini S, Calabrò ML, Stancanelli R et al (2005) The inclusion complexes of hesperetin and its 7-rhamnoglucoside with (2-hydroxypropyl)-beta-cyclodextrin. J Pharm Biomed Anal 39 (3–4):572–580

Chapter 23

Profiling MicroRNAs Differentially Expressed

in Rabbit Retina

Naihong Yan, Ke Ma, Jia Ma, Wei Chen, Yun Wang, Guiqun Cao, Dominic Man-Kit Lam, and Xuyang Liu

Abstract MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression at the post-transcriptional level. Recent studies indicate that miRNAs may constitute a major mechanism underlying mammal’s retinal development. The overall objective of this study is to compare and contrast retinal miRNAs expression between newborn and adult rabbits, and to identify some of the genes possibly associated with retinal development. Retinas were isolated from 3-day-old and 2- month-old rabbits. A miRNA microarray designed to detect 924 miRNAs was used to determine the expression profile of miRNAs from newborn and adult rabbits. The expression of twenty-eight miRNAs was found to differ significantly between newborn and adult rabbit retina. Among these, 17 appear to be up-regulated and the other 11 miRNAs down-regulated, suggesting a role of differential miRNA expression in retinal development. Computer prediction tools indicate that some of the target genes might be directly associated with signal pathways relevant to visual development.

23.1 Introduction

MicroRNAs (miRNAs) are an abundant class of non-coding RNAs (typically 19 23 nucleotides) playing an important role in regulation of post-transcriptional gene expression. The first discovered miRNA, lin-4, is involved in developmental timing in the nematode Caenorhabditis elegans (Lee et al. 1993). After that, thousands of miRNAs have since been identified in various organisms through random cloning and sequencing or computational prediction. Recent advances have led to a more detailed understanding of miRNA biogenesis and function. Primary miRNA transcripts are processed by the RNaseIII nuclease Drosha, into pre-miRNAs. The

X. Liu (B)

Department of Ophthalmology, Torsten Wiesel Research Institute, West China Hospital, Sichuan University, Chengdu, China

e-mail: xliu1213@yahoo.com.cn

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_23,C Springer Science+Business Media, LLC 2010