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

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).

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”.

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

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.

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.

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).

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 MgCl₂ (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.

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.

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.

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).

view in a new window

Table 1. Effects of CCK-8 on blood glucose (mmol/L) and body weight (g) in diabetic cataract rats (mean±SD, n=12)

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. 1 and Table 2).

view in a new window

Table 2. The percentage of opacities of lenses during different stages in three groups

view in a new window

Fig. 1. The rat lenses were examined by slit lamp for clinical signs of cataract after 60 days of STZ injection. A: Clear lens in the control group; B: Severe opacity in the STZ group; C: Mild opacity lens in the CCK-8 group.

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).

view in a new window

Fig. 2. NT antigen in LEC examined by immunofluorescent staining. A: control group; B, C, D: 20, 40 and 60 days in the STZ group; E, F, G: 20, 40 and 60 days in the CCK-8 group.

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.

view in a new window

Fig. 3. A: Detection of NT protein expression in lens of diabetic cataract with western blotting. Lane 1: control group; lanes 2-4: STZ group at 20, 40 and 60 days; lanes 5-7: CCK-8 group at 20, 40 and 60 days. B: Computerised photographic analysis for detection of NT protein in diabetic rat cataract lens with western blotting. *P<0.001 vs control group; ^#P<0.001 vs CCK-8 group.

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).

view in a new window

Fig. 4. RT-PCR detection of iNOS mRNA expression in lens of diabetic cataract with CCK-8. Lane 1: DNA marker; lane 2: control group; lanes 3-5: CCK-8 group at 20, 40 and 60 days; lanes 6-8: STZ group at 20, 40 and 60 days.

view in a new window

Table 3. Comparison of RT-PCR detection of iNOS mRNA expression in lens of diabetic cataract with CCK-8 (mean±SD, n=12)

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).

view in a new window

Fig. 5. Gene array expression. Red colour shows upregulated genes; green colour shows downregulated genes; black colour shows no changes. As time passed, STZ group showed upregulation iNOS mRNA (Nos1 and Nos2, red colour), while CCK-8 group showed downregulation iNOS mRNA (green colour).

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 colleagues³⁵reported 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.^36-37We 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.

REFERENCES

1. Virag L, Szabo E, Gergely P, Szabo C. Peroxynitrite induced cytotoxicity: mechanism and opportunities for intervention. Toxicol Lett 2003; 113-124:140-141.

2. Szabo C. Multiple pathways of peroxynitrite cytotoxicity. Toxicol Lett 2003; 105-112: 140-141.

3. Miyamoto S, Martinez GR, Martins AP, Medeiros MH, Di Mascio P. Direct evidence of single molecular oxygen [O₂(1Deltag)] production in the reaction of linoleic acid hydroperoxide with peroxynitrite. J Am Chem Soc 2003; 125: 4510-4517.

4. Gow AJ, Thom SR, Ischiropoulos H. Nitric oxide and peroxynitrite-mediated pulmonary cell death. Am J Physiol 1998; 274: 112-118.

5. Hao LN, Ling YL, He SZ, Mao QY, Liang JQ. Damage role of peroxynitrite in the formation of diabetic rat cataract and antagonism of puerarin. Chin J Ophthalmol(Chin) 2004; 114:311-317.

6. Hao LN, Ling YL, Gu ZY, Huang XL, He SZ. Changes of inducible nitric oxide synthase mRNA in the antagonism of puerarin to peroxynitrite induced rat diabetic cataract. Chin J Ophthalmol(Chin) 2004; 144:368-375.

7. Hao LN, Mao QY, Ling YL, He SZ. Gene expression and biomedical changes during the formation of rat diabetic cataract. Chin Ophthalmic Res(Chin) 2005; 23: 297-300.

8. Bradford MM. A refined and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254.

9. Reno C, Boykim R, Martinez ML, David A. Temporal alteration in mRNA levels for proteinases and inhibitors and their potential regulators in the healing medial collateral ligament. Biochem Biophysi Res Commun 1998;252:757-763.

10. Kojima M, Sasaki K. Reinvestigation of streptozotocin induced diabetic cataract as a standard experimental model. Nippon Ganka Gakkai Zasshi 1993;97:324-332.

11. Shibata S, Natori Y, Nishihara T, Tomisaka K, Matsumoto K, Sansawa H, et al. Antioxidant and anti-cataract effects of chlorella on rats with streptozotocin induced diabetes. J Nutr Sci Vitaminol 2003;l49:334-339.

12. Ettl A, Daxer A, Gottinger W, Schmid E. Inhibition of experimental diabetic cataract by topical administration of RS-verapamil hydrochloride. Br J Ophthalmol 2004; 88: 44-47.

13. Swamy-Mruthinti S, Shaw SM, Zhao HR, Green K, Abraham EC. Evidence of a glycemic threshold for the development of cataracts in diabetic rats. Curr Eye Res 1999; 18: 423-429.

14. Follansbee MH, Beyer KH Jr, Vesell ES. Studies on pyrazinoylguanidine 6. Prevention of cataracts in STZ-diabetic rats. Pharmacology 1997; 54: 256-260.

15. Ugazio G, Bosia S, Burdino E, Grignolo F. Amelioration of diabetes and cataract by Na₃VO₄ plus U-83836E in streptozotocin treated rats. Res Commun Mol Pathol Pharmacol 1994; 85: 313-328.

16. Ruf JC, Ciavatti M, Gustafsson T, Renaud S. Effect of D-myo-inositol on platelet function and composition and on cataract development in streptozotocin induced diabetic rats. Biochem Med Metab Biol 1992; 48: 46-55.

17. Yuen EC, Gunther EC, Bothwell M. Nitric oxide activation of TrkB through peroxynitrite. Neuroreport 2000;9: 3593-3597.

18. Ornek K, Karel F, Buyukbingol Z. May nitric oxide molecule have a role in the pathogenesis of human cataract? Exp Eye Res 2003;76:23-27.

19. Pacher P, Szabo C. Role of peroxynitrite in the pathogenesis of cardiovascular complications of diabetes. Curr Opin Pharmaco 2006; 6:136-141.

20. Jawerbaum A, Higa R, White V, Capobianco E, Pustovrh C, Sinner D, et al. Peroxynitrites and impaired modulation of nitric oxide concentrations in embryos from diabetic rats during early organogenesis. Reproduction 2005; 130: 695-703.

21. Hao LN, Wang QL, Ling YL, Meng XY, Zhang XJ, Liang JQ, et al. Effect of cholecystokinin octaptide on diabetic cataract of rats. Basic Clin Med 2005; 25: 646-652.

22. Li SJ, Cong B, Yan YL, Yao YX, Ma CL, Ling YL. Cholecystokinin octapeptide inhibits the in vitro expression of CD14 in rat pulmonary interstitial macrophages induced by lipopolysaccharide. Chin Med J 2002; 115: 276-279.[PubMed]

23. Konturek PC, Brzozowski T, Sulekova Z, Brzozowska I, Duda A, Meixner H, et al. Role of leptin in ulcer healing. Eur J Pharmacol 2001; 414: 87-97.

24. Gow AJ, Thom SR, Ischiropoulos H. Nitric oxide and peroxynitrite-mediated pulmonary cell death. AM J Physiol 1998; 274: 112-118.

25. Olson LP, Bartberger MD, Houk KN. Peroxynitrate and peroxynitrite: a complete basis set investigation of similarities and differences between these NOx species. J Am Chem Soc 2003; 125: 3999-4006.

26. Cuzzocrea S, Zingarelli B, Caputi AP. Peroxynitrate- mediated DNA strand breakage activates poly(ADP-ribose) synthetase and causes cellular energy depletion in a nonseptic shock model induced by zymosan in the rat. Shock 1998; 9: 336-340.

27. Shibuki H, Katai N, Yodoi J, Uchida K, Yoshimura N. Lipid peroxidation and peroxynitrite in retinal ischaemia-reperfusion injury. Invest Ophthalmol Vis Sci 2000; 41:3607-3614.

28. Beauregard C, Brandt PC, Chiou GC. Induction of nitric oxide synthase and over-production of nitric oxide by interleukin-1beta in cultured lacrimal gland acinar cells. Exp Eye Res 2003; 77:109-114.

29. Webb T, Gulley S, Pruitt F, Esdaile AR, Sharma SK, Cox JE, et al. Cholecystokinin-8 increases Fos-like immunoreactivity in myenteric neurons of the duodenum and jejunum more after intraperitoneal than after intravenous injection. Neurosci Lett 2005; 389:157-162.

30. Hashimoto H, Onaka T, Kawasaki M, Chen L, Mera T, Soya A, et al. Effects of cholecystokinin (CCK)-8 on hypothalamic oxytocin-secreting neurons in rats lacking CCK-A receptor. Auton Neurosci 2005; 121:16-25.

31. Tagen MB, Beinfeld MC. Recombinant prohormone convertase 1 and 2 cleave purified pro cholecystokinin (CCK) and a synthetic peptide containing CCK 8 Gly Arg Arg and the carboxyl-terminal flanking peptide. Peptides 2005; 26: 2530-2535.

32. Sayegh AI, Reeve JR Jr, Lampley ST, Hart B, Gulley S, Esdaile AR, et al. Role for the enteric nervous system in the regulation of satiety via cholecystokinin-8. J Am Vet Med Assoc 2005; 226:1809-1816.

33. Eisen S, Phillips RJ, Geary N, Baronowsky EA, Powley TL, Smith GP. Inhibitory effects on intake of cholecystokinin-8 and cholecystokinin-33 in rats with hepatic proper or common hepatic branch vagal innervation. Am J Physiol Regul Integr Comp Physiol 2005; 289:R456-R462.

34. Tirassa P, Costa N, Aloe L. CCK-8 prevents the development of kindling and regulates the GABA and NPY expression in the hippocampus of pentylenetetrazole (PTZ)-treated adult rats. Neurophamacology 2005; 48: 732-742.

35. Kuntz E, Pinget M, Damge P. Cholecystokinin octapeptide: a potential growth factor for pancreatic beta cells in diabetic rats. JOP 2004; 5: 464-475.

36. Ahren B, Holst JJ, Efendic S. Antidiabetogenic action of cholecystokinin-8 in type 2 diabetes. J Clin Endocrinol Metab 2000; 85:1043-1048.

37. Risset J, Julien S, Laine J. Localization of cholecystokinin receptor subtypes in the endocine pancreas. Histochem Cytochem 2003; 51: 1501-1513.