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The antagonism of cholecystokinin octapeptide-8 to the peroxynitrite oxidation on a diabetic cataractal rat model

HAO, Li-na; LING, Yi-qun; MAO, Qi-yan; LING, Yi-ling; HE, Shou-zhi

Section Editor(s): SHEN, Xi-bin; GUO, Li-shao

Original article
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Background Cataracts is considered be formed because of an abnormal glucose metabolic pathway or oxidative stress. We explored the damaging role of ONOO- and antagonism of cholecystokinin octapeptide-8 (CCK-8) in diabetic cataractal rat lenses.

Methods A diabetic cataractal animal model was established by peritoneal injection of streptozotocine (STZ). Thirty-six normal SD rats were taken as control group; seventy-two were given STZ (45 mg/kg) and then divided into STZ group and CCK-8 group (peritoneal injection CCK-8). STZ induced diabetic rats were treated with CCK-8 for 60 days. Lenses were examined with slit lamp at 20, 40 and 60 days. Immunofluorescent staining and Western blot analysis were used for determining nitrotyrosine (NT, a marker for ONOO-). RT-PCR and gene array analysis were used for determining the expression of inducible nitric oxide synthetase mRNA (iNOS mRNA) in lens epithelium (LEC).

Results STZ group rats developed lens opacity by 20 days that reached a high level by 60 days after STZ injection. CCK-8 group rats delayed the cataract formation. There was no distinct expression of NT and iNOS mRNA in control group. In STZ group, there were distinct expression of NT and upregulation of iNOS mRNA; however, CCK-8 group showed weak expression of NT and downregulation of iNOS mRNA.

Conclusions NT, which may be a new form of oxidative stress, was expressed in diabetic rat LEC although CCK-8 could reverse NT damage in LEC. The results suggested that CCK-8 might be a useful therapeutic agent against diabetic cataract. The antagonizing mechanism of CCK-8 may be related to direct antagonism of ONOO- as well as its inhibition of the expression of iNOS mRNA for production of NO and therefore decrease in the formation of ONOO-.

Edited by

Department of Pathophysiology, Hebei Medical University, Shijiazhuang 050011, China (Hao LN, Ling YQ and Ling YL)

Life Science College, Beijing University, Beijing 100866, China (Mao QY)

Department of Ophthalmology, PLA General Hospital, Beijing 100866, China (He SZ)

Correspondence to: Dr. Ling Yi-ling, Department of Pathophysiology, Hebei Medical University, Shijiazhuang 050011, China (Tel: 86-311-8626572000. Fax: 86-311-6077634. Email: lingyiling@tom.com)

(Received December 25, 2005)

The current theory is that cataracts are formed because of an abnormal glucose metabolic pathway or oxidative stress. Oxidative stress is defined as the generation of oxidising species that exceed the antioxidant defences. Traditional reactive species include oxygen, hydrogen peroxide (H2O2), nitric oxide (NO) and superoxide anion (O2). Recent studies have focused on peroxynitrite (ONOO-). It is formed by rapid reaction of NO and O2, and may be an important mediator of cytotoxicity in oxidative process.1-4

We have reported that lens oxidation may occur by the ONOO- pathway which is antagonized by puerarin.5-7 In this study we show that ONOO- was produced in the epithelium of diabetic cataractal lenses and that production was attenuated by cholecystokinin octapeptide-8 (CCK-8).

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METHODS

Animals

Pathogen free, male, Sprague Dawley (SD) rats (5-6 weeks old) were maintained and treated in accordance with The Association for Research in Vision and Ophthalmology (ARVO) Resolution “Statement for the Use of Animals in Ophthalmic and Vision Research”.

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Antibodies and reagents

Monoclonal mouse antiNT antibody, goat antimouse fluorescent isothiocyanate (FITC) antibody, streptozotocine (STZ) and CCK-8 were purchased from Sigma Company, USA.

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Groups and animal model

Animals were divided into three groups: control group, STZ group and CCK-8 group, each containing 36 animals. SD rats in STZ group and CCK-8 group were injected peritoneally with STZ (45 mg/kg) to establish animal model. The control group received the same amount of saline. Three days after injection, CCK-8 group received peritoneal injection of CCK-8 100 μg/kg at 9 am each day. Concentrations of CCK-8 from 25 μg/kg to 100 μg/kg for peritoneal injection were tested before experiment to determine optimum dose. The blood glucose and body weight were estimated at 20, 40 and 60 days and animals lenses were examined by slit lamp, on the same day after STZ injection, for clinical signs of cataract and graded on 0-5 scale7 then enucleated for further study.

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Immunofluorescent staining

The rats were sacrificed and one eye was enucleated immediately in each group at 20, 40 and 60 days. There were nine eyes in this experiment. The lenses were dissected via posterior incision under dissecting microscope and then fixed in 70% ethanol for 24 hours. The lenses were screened with PBS until they became milky then the suspension were centrifuged 1000 rpm for 4 minutes. The sediment was mixed with PBS, filtered, centrifuged again, then suspended with PBS. Using indirect immunofluorescent labelling antibody technique, NT (1:400) was added to the suspension and reacted in dark for 30 minutes at room temperature; goat antimouse FITC labelling antibody was added for another 30 minutes under the same condition. The suspension was examined under fluorescent microscope.

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Western blotting

The rats were sacrificed and one eye was enucleated immediately in each group at 20, 40 and 60 days. There were nine eyes in this experiment. Lenses were prepared as described, homogenized and solubilized in ice cold PBS containing protease inhibitors, phenylmethylsulfonyl fluoride (1 μg/ml), aprotinin (1 μg/ml), leupeptin (1 μg/ml), pepstatin A (1 μg/ml) and EDTA (1 mmol/l). The homogenate was centrifuged at 15 000 rpm at 4°C for 10 minutes. The protein content of the supernatants was determined by the Bradford method.8 After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% linear slab gel under reducing conditions, separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane using a semidry electrophoretic transfer cell (Trans-blot; Bio-Rad, USA). Blot was stained at room temperature with a 1:600 dilution of monoclonal mouse, antiNT antibody overnight at 4°C. After washing and incubation with horseradish peroxidase conjugated secondary antibody (1:1000 dilution), blot was developed using the enhanced chemiluminescence Western blot analysis detection system (ECL Plus; Amersham Pharmacia Biotech, USA).

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RT-PCR analysis

The rats were sacrificed and one eye was enucleated immediately in each three group at 20, 40 and 60 days. There were nine eyes in this experiment. Equal amounts of the total RNA were used to detect the mRNA levels of iNOS by RT-PCR (GeneAmp RNA-PCR kit; Applied Biosystems, USA). Total RNA was extracted from the rat lenses in three groups, according to the kit manufacturer’s specifications. The sense and antisense oligonucleotide primers for rat iNOS 9 were synthesized by Biological Engineering Corporation.

The primer sequences are: iNOS (262 bp) sense primer 1: 5'-CGCCCTTCCGCAGTTCT-3'; sense primer 2: 5′-TCCAGGAGGACATGCAGCAC-3′. β-actin (420 bp) sense primer 1: 5′-GAGACCT TCAACACCCAGCC-3′; sense primer 2: 5′-GCGGG GCATCGGAACCGCTCA-3′. In addition, 4 μg of RNA in a total volume of 20 μl (pH 8.3) were for synthesizing the cDNA. RT-PCR was first performed at 24°C for 10 minutes, then at 42°C for 15 minutes. The reaction mixture was heated at 99°C for 5 minutes, and the RT product was mixed with DNA polymerase (AmpliTaq; Applied Biosystems, USA) and the sense primer in a buffer containing 20 mmol Tris HCl, 50 mmol KCl, 2.0 mmol MgCI2 (pH 8.3), and 50 mmol of each dNTP in a 100 μl volume. The mixture was then amplified by PCR: an initial denaturing at 94°C for 2 minutes, 29 cycles of 45 seconds at 55°C then extension of the primer at 72°C for 10 minutes. All reactions were adjusted for iNOS expression. The negative controls were omission of RNA template or reverse transcriptase from the reaction mixture. PCR products were analyzed on 2% agarose gel.

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Gene arrays

The rats were sacrificed and one eye was enucleated immediately in each group at 20, 40 and 60 days. There were nine eyes in this experiment. Lenses used in arrays were dissected free of any contaminating tissue and homogenized in trizol reagent. RNA extraction was carried out according to the manufacture’s protocol. Concentration and RNA quality were assessed via spectrophotometry and formaldehyde gel electrophoresis. Amplified mRNA was then labelled with Cy3 or Cy5 (Random Primer DNA Labelling Kit, Bao-Boiscience Engineering Corporation, China). Successfully labelled control and experimental targets with 12 housekeeping genes and 12 synthesized 70 mer oligo DNA as positive and negative controls were then combined and prepared for hybridization. Array slides were obtained from Qiagen Corporation, USA and incubated in prehybridization for 1 hour at 42°C. Targets were dried via vacuum centrifugation then resuspended in 50 μl hybridization solution with 1μl Cot 1 DNA and 1μl poly A oligonucleotide as blocking agents, heated to 95°C for 5 minutes and then added to the face of one slide. The printed face of the second slide of the pair was then placed face to face with the first using the same probe. Slide pairs were then placed on a level plastic cover above some 1 × SSC moistened tissue in a slide box. The slide box was sealed and placed floating in a water bath and hybridized for 24-48 hours at 42°C. Following hybridization, slides were washed twice in wash solution for 20 minutes, dipped in nuclease free water then spray dried. Finally, the backs of the slides were cleaned with ddH2O, wiped with 100% ethanol, then wiped dry and scanned by Scan Array Express Scanner (Packard Bioscience Corporation, USA).

RT-PCR array confirmations were the same as above RT-PCR analysis. Gene Pix Pro 4.0 photo soft ware (Axon Instruments Corporation, USA) was used for clustering analysis. Two folds higher divergence were regarded as divergent expression gene.

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Statistical analysis

SPSS v10.0 was used for statistical analysis. The results are expressed as means ± SD. Statistical significance was determined by single factor analysis of variance (ANOVA) followed by the Fisher post hoc test for multiple comparisons. A P value less than 0.001 was considered significant.

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RESULTS

Animal model

The STZ group exhibited typical diabetic symptoms compared to the control and the CCK-8 groups. There was also a remarkable glucose concentration and body difference among three groups during 20, 40 and 60 experiment days (Table 1).

Table 1

Table 1

Slit lamp examination showed the lenses were clear in the control group during the entire experimental period. At 20 days, lenses in the STZ group became opaque (Table 2); at 40 days, opacity developed in the CCK-8 and STZ groups. At 60 days, opacity developed in the CCK-8 and STZ groups (Fig. 1and Table 2).

Fig. 1.

Fig. 1.

Table 2

Table 2

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Immunofluorescent staining

NT negative antigen was visible as a faint green colour in the nucleus and cytoplasm. NT positive antigen appeared as an orange yellow colour under the fluorescent microscope. A faint green colour was visible in the nucleus and cytoplasm of the control group. The STZ group colour changed from green yellow to orange yellow colour during the period of 20 to 60 days. In comparison, CCK-8 group colour ranged from faint green to yellow colour during the period of 20 to 40 days of experiment, then changed to green yellow at 60 days (Fig. 2).

Fig. 2.

Fig. 2.

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Western blotting

Using Western blot analysis, a faint expression of NT could be seen in the control group. A gradual to strong expression of NT was observed at different stages of the experiment in STZ group. But expression of NT in CCK-8 group changed gradually from faint to strong during the period of 20 to 40 days, then turn to weak at 60 days (Fig. 3A). Computerised photographic analysis indicated that there were significant differences among three groups (P< 0.001, Fig. 3B); The trial was repeated twice with the same result).

Fig. 3. A:

Fig. 3. A:

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RT-PCR analysis

There was no expression of iNOS mRNA in the control group. There was distinct upregulation of iNOS mRNA in the STZ group with time, but expression of iNOS mRNA in the CCK-8 group appeared to be gradually upregulated during the period of 20 to 40 days of experiment, then downregulated by 60 days (Fig. 4). Computerised photographic analysis indicated that there were significant differences among the three groups (P<0.001, Table 2).

Fig. 4.

Fig. 4.

Table 3

Table 3

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Gene arrays

With CCK-8 treatment, expression of iNOS mRNA appeared gradually upregulated during the period of 20 to 40 days of experiment, then downregulated by 60 days (Fig. 5).

Fig. 5.

Fig. 5.

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DISCUSSION

We established diabetic cataract animal model of rats in vivo to elucidate the mechanism of oxidative stress in the process of cataract formation and to develop therapy to inhibit cataract formation.10-16

Using immunofluorescent staining and Western blotting analysis, we verified that ONOO- was produced during the formation of diabetic cataract. LEC are the most metabolically active region in lens. The restorative inhibition of lens depends on the antioxidant enzymes produced in the LEC.

Using RT-PCR gene array technique, we verified that inducible nitric oxide synthetase (iNOS) might contribute to oxidative stress by helping to produce more powerful oxidants such as ONO-. iNOS is the major enzyme involved in the production of NO, which is a signalling molecule in several pathways.17 Under pathological conditions in the lenses of humans, rats and rabbits,18 upregulation of iNOS mRNA in LEC leads to overproduction of iNOS and NO as well as increasing O2. Increased NO and O2 produce ONOO- a strong oxidant. The changes in NO, iNOS and ONOO- during diabetic cataract formation were not clear. Our studies found that NT increased in the LEC of diabetic rats. Peroxynitrite generation has been implicated in the induction of apoptosis seen in diseases such as diabetes.19

Recent studies also reported that high glucose and peroxynitrite are associated with tyrosine nitration, inactivation of prostacyclin synthase, thromboxane/prostaglandin H2 receptor mediated apoptosis and adhesion molecule expression in cultured human aortic endothelial cells.20 We found that CCK-8 in LEC21 and other tissues reduced the oxidation induced by ONOO- and/or strengthened the antioxidant system.22 CCK-8 could have also inhibited the expression of iNOS mRNA thus decreasing the formation of ONOO-,23 Expression of a small amount of NT in the control provides physiological evidence for the existence of ONOO-. When exogenous NO and ONOO- reacted with ox pulmonary endothelium, only ONOO- could induce cell apoptosis.24 ONOO- affects the cell’s oxidising and repair systems, ionic channels, proteinase, nitroproteins and inhibits respiration in mitochondria, leading to cell apoptosis and apoptosis mediated by mitochondria.25,26 During retinal ischaemia and reperfusion, NO and ONOO- are produced in eyes.27 We also found that ONOO- induced apoptosis in LEC.5 Therefore, iNOS induced overproduction of NO, later combined with O2 to form ONOO- as well as other oxidants.28

CCK-8, which has many physiological functions, is distributed in the stomach, intestine and central nervous system.29-34 Kuntz E and his colleagues35 reported that CCK-8 could improve blood glucose concentrations in type 1 diabetic rats, which correlated with an increase in beta cell mass. This study found that the damaging affect of ONOO and iNOS could be inhibited by CCK-8 which may make it a useful therapeutic agent for diabetic cataracts.36-37 We found that up to 40 days, the effect of CCK-8 was not apparent, while after 40 days its effect was dramatic. The reason for this may be related to time required for the concentration of CCK-8 to reach a critical level.

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    Keywords:

    diabetes mellitus; lens; oxidative; cholecystokinin octapeptide-8

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