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The Expression of Opticin in Vitreous Body and Retina of Diabetes Mellitus Rats

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The Expression of Opticin in Vitreous Body and Retina of Diabetes Mellitus Rats Asian Biomedicine Vol. 6 No. 3 June 2012; 439 - 444 DOI: 10.5372/1905-7415.0603.074 Brief communication (Original) The expression of opticin in vitreous body and retina of diabetes mellitus rats Hong Luo, Xiaoping Qiu, Yancheng Xu Department of ophthalmology, Zhongnan Hosipital, Wuhan University, Wuhan 430077, China Background: Diabetic retinopathy is a common complication of diabetes mellitus. Opcitin is a glycoprotein present in the vitreous body. Its role in diabetic retinopathy needs to be further defined. Objective: Investigate and compare the mRNA and protein levels of opticin in vitreous body and retina in normal and diabetic rats. Methods: Twenty-four male Sprague-Dawley (SD) rats were randomly divided into two groups (12/group), a streptozocin-induced diabetes (STZ) group and a control group. In the STZ group, 1% sterile STZ solution was injected into the rats intraperitonally (60mg/kg). An equal volume of sodium citrate buffer solution was administrated in the rats from the control group. The rats were sacrificed one month after various treatments. The eye bodies of three rats from each group were removed and fixed with 4% paraformaldehydeion for the following pathological analysis. Meanwhile, the vitreous bodies and retina of the other nine rats from each group were removed for the real-time PCR, immunohistochemistry, and western blot assays. Results: The mRNA level of opticin in the vitreous body of diabetes mellitus (DM) rats was 5.66% of that of the control ones (p <0.01). The expression of opticin mRNA in retina of DM rats was 9.28% of that of the control ones (p <0.01). In addition, opticin protein was expressed in the vitreous body and retina of the normal rats, whereas it was negative in the DM ones. Conclusions: The opticin expression in vitreous body and retina of diabetes rats was significantly decreased or even disappeared, which may suggest a key role of opticin in the development of diabetic retinopathy. Keywords: Diabetic retinopathy, opticin, rat Diabetic retinopathy (DR) is one of the most It was confirmed that Opticin could significantly common complications of diabetes mellitus (DM), and suppress vascular endothelial growth factor opticin- it is the fourth cause for the blindness followed A (VEGF-A) and fibroblast growth factor-2 (FGF-2) by cataract, corneal disease, and glaucoma. With induced proliferation, metastasis, and budding of the increasing prevalence of DM, the number of DR bovine vascular endothelial cells [5]. It might be due patients is increasing, which can pose a significant to vascular disappearance in the vitreous body in the burden to the society. development of eye or some proliferative diseases such Opticin, a new glycoprotein found by Reardon as proliferative diabetic retinopathy (PDR) [2]. et al. [1], belongs to the third category of leucine-rich In the present study we produced streptozotocin repeats (LRR) proteoglycan family. It can stably exist (STZ) induced diabetic rat model. Then we assayed in water solution with a dimmer of approximately the mRNA and protein expressions of opticin in the 90kDa. Opticin is mainly present in the vitreous body vitreous body and retina. Based on these, we tried to and its mRNA can be determined in various tissues. clarify the possible role of opticin in the development Thus, Opticin plays an important role in maintaining of PDR. vitreous body stability [2], linking the vitreous body [3] and the retina, and inhibiting angiogenesis [4-6]. Materials and methods Main reagents Correspondence to: Hong Luo, Department of ophthalmology, STZ and anti-Opticin antibody were purchased Zhongnan Hosipital, Wuhan University, Wuhan 430077, China. from Santa Cruz Biotech Co., Ltd. (CA, USA). First E-mail: [email protected] strand cDNA synthesis kit Fermentas and PCR kit 440 H. Luo, et al. K0171 were obtained from MBI Biotech Co., Ltd. kit (Toyobo, Japan). Then the following primers (USA). were used: Opticin (150-bp product), forward (5’-GCCACTTAATTTGCATTTCGC-3’) and reverse Establishment of animal model and grouping (5’-ATCCCTTTCTTTCAT GGTTCTCC-3’); β-actin Twenty-four SPF Sprague-Dawley (SD) rats (149-bp product), forward (52 -GAAAAGATGACC weighing 200 to 250g were provided by the CAGATCATG-32 ) and reverse (52 -ATCTTCATGA Experimental Center of Chinese Center for Disease GGTAGTCCGTC-32 ). The primers were synthesized Control and Prevention (Beijing, China). All the by Sangon Biological Engineering Technology & experiments were conducted in accordance with the Services Co., Ltd. (Shanghai, China). national guidelines for the care and use of laboratory 25 μl of standard reaction system included 12.5 μl animals. This study was approved by the Ethnic of Real time PCR Master Mix SYBR GreenI, 0.5 μl Committee of Hubei Province. of primer forward (10 μmol/l), 0.5 μl of primer reverse μ μ μ The animals were raised in different cages (2/ (10 mol/l), 1 l of cDNA, and 10.5 l of ddH2O. The cage) and subjected to a 12-hour light/dark cycle. The following reactions were performed for 40 cycles. The animal room was maintained at 20±2°C and 30 to 60% annealing temperature was maintained at 55°C; the relative humidity. The rats were given free access rest of the conditions included denaturation at 94°C to standard food and water. The rats were randomly for five seconds followed by extension at 72°C for 10 divided into STZ injection group and a control group seconds. The data were analyzed using IQ5 software (12/group). In the STZ injection group, 1% sterile STZ of Gene express module (Bio-Rad, USA). Three solution was injected into the rats intraperitonally replicate reactions were performed and values were (60mg/kg). An equal volume of 0.1mol/l sodium normalized to the housekeeping gene GAPDH. CT dihydrogen citrate buffer (6mg/kg) was injected into values were determined by using the 7500 System SDS the rats from the control group. Software (version 1.2.3; Applied Biosystems, USA). Blood and urine sample were collected 72 hours Expression ratios were finally calculated in accordance after the injections. The diabetic model was confirmed with 2-ΔΔCT method. to be successfully established when the blood glucose was equal to or more than 16.7mmol/l and urine Immunohistochemistry analysis glucose was positive. The blood glucose was The eyeballs were fixed in 4% paraformaldehyde measured using a ONE TOUCH blood glucose meter for 24 hours. Then they were processed using degrease, or standard glucose testing strips. In this part of the rehydration in graded alcohols, and further processed experiment, the weight and blood glucose were using the streptavidin immunoperoxidase method. In monitored every 2 weeks. After 1 month, the rats were brief, the sections were submitted for antigen retrieval sacrificed under an anesthesia by an intraperitonal at 92 to 95°C for 10 minutes in citrate buffer (0.01mol/ injection of 10% ketamine (0.1 ml/100g). The eyeballs l, pH6.0). Subsequently, the slides were incubated were removed from three rats in each group. The in 10% normal serum for 30 minutes, followed by vitreous body and retina were collected from the incubation overnight at 4°C with the diluted rabbit anti- residual nine rats in each group. The eyeballs were rat Opticin polyclonal antibody (Santa Cruz, USA) treated with 4% paraformaldehyde fixation for the antibody (1:100). The slides were then incubated with following assay. The vitreous body and retina were biotinylated anti-rabbit immunoglobulins for 30 minutes stored in liquid nitrogen for later use. at 37°C, followed by streptavidin peroxidase complex for 15 minutes at 37°C. PBS (pH7.4) was selected as Quantitative PCR assay of Opticin mRNA in a negative control. vitreous body and retina Vitreous body and retina were homogenized in a Western blot assay glass homogenizer and then 1.5 ml Trizol (Invitrogen, The total tissue protein was extracted using a USA) was added. Total RNA was extracted using an total protein extract kit in accordance with the RNeasy Mini Kit (Qiagen, USA) in accordance with manufacturer’s instructions. The concentration of the the manufacturer’s instructions. total protein was quantified by Bradford method. After DNase I treatment, 2 μg of RNA was Thirty micrograms of the proteins were separated reversely transcribed using a ReverTra Ace-TM on 12% sodium dodecyl sulfate polyacrylamide gel Vol. 6 No. 3 Opticin and diabetic retinopathy 441 June 2012 electrophoresis (SDS-PAGE), and then the proteins Results were transferred onto a sheet of polyvinylidene The expressions of opticin mRNA is down- fluoride (PVDF) transfer membrane. The transfer regulated in vitreous body and retina in the membrane was semi-dry at 300 mA for 30 minutes. diabetes rats Then, the membrane was blocked by 5% skim milk The mRNA levels of opticin in the vitreous body for four hours. The membrane was washed three times and retina of DM rats was significantly reduced with tris buffered saline (TBS) for five minutes each. compared to control. The opticin in the vitreous body Subsequently, rabbit anti-rat opticin polyclonal and retina of DM rats was only 5.66% and 9.28% antibody (Santa Cruz Biotechnology, USA) antibody of the control respectively (p <0.01) as shown in (1:200) was added and incubated at 4°C overnight. Figure 1. Then HRP-conjugated goat anti- rabbit IgG (1:3000) (Zhongshan Golden Bridge Biotechnology Co., Beijing, The expression and location of opticin protein in China) was added and incubated at room temperature vitreous body and retina in the diabetes rats for another two hours. The membrane was stained Western blot result revealed that opticin protein by enhanced chemiluminescence (ECL) reagent clearly appeared at a site of about 45 kDa. However, (Pierce, USA) and imaged on X-ray film (Fuji film, there was no strap in the vitreous body and retina of Japan) by autoradiography.
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