Healthy male Sprague-Dawley rats (n=64, aged 8-10weeks, weighed between
200 and 250 g) were used for all experiments. All rats were purchased from the
Experimental Animal Center of Chongqing Medical University, where rats were
housed in cages on a 12-h light-dark schedule with food and water ad libitum. All procedures were performed in accordance to the Chongqing Medical University’s Animal Care and Use Committee Guidelines.
Induction of type 2 diabetes mellitus
After one week of adaptive feeding, 40 rats were randomly divided into two groups: Normal group and Diabetic group. Rats in the Normal group were fed with normal chow diet, while those in the DM group with a high-fat and high-glucose diet. Four weeks later, streptozotocin (STZ; Sigma-Aldrich) was prepared in acetate buffer and administered (30 mg/kg) via intraperitoneal injections for 5 consecutive days at 7–9 weeks of age. Normal group mice were administered an intraperitoneal injection of
acetate buffer (pH 4.5). After injection, blood glucose level greater than 16.7mmol/L was taken as the successful establishment of type 2 DM model. After successful diabetes induction, rats were treated according to the study design. At the end of the treatment, animals were sacrificed with overdose pentobarbital. Rat eyeballs were enucleated and immediately put into 40 g/L paraformaldehyde for hematoxylin and eosin (H-E) staining or immunohistochemistry. Retinas were isolated carefully and snap frozen in liquid nitrogen for western blot or enzyme-linked immunosorbent assay (ELISA).
Intravitreal treatment of Cs-A/HMGB-1
This study comprised of two experimental parts. First, rats in the Normal and DM group were deeply anesthetized by pentobarbital injection, and topical ocular anesthesia was achieved with 0.4% benoxinate hydrochloride of eye drop. After pupils were dilated satisfactorily, rats were injected with Cs-A (42 ng/2 μL Dimethyl sulfoxide (DMSO)) intravitreally in the left eye with a 30-gauge micro-injector under a dissecting microscope. For the control, 2μL DMSO was injected into the right eye of the same rat. According to the treatment, rat eyes could be categorized into four groups: Normal, Normal+Cs-A, DM and DM+Cs-A group. Rats with operational complications such as vitreous hemorrhage, retinal detachment or death were excluded from our study. Forty-eight hours after administration of Cs-A, rats were anesthetized and sacrificed for further experiments.
The second part of the experiment was that twenty-four normal healthy rats
(Sprague-Dawley, non-diabetic, 200 - 250 g) were subjected to intravitreal injections
into the left eyes using the method described above. Rats were divided into three
groups according to the injection substances: Normal control group (n=8) received
5μL sterile phosphate buffer saline (PBS); Normal+HMGB-1 group (n=8) received
sterilized solution of recombinant HMGB-1 (5 ng/5 μL PBS; R&D Systems,
Minneapolis, MN); and Normal+HMGB-1+Cs-A group (n=8) received a combination
of HMGB-1(5 ng/5 μL PBS) and Cs-A (42 ng/2 μL DMSO).
H-E staining of the retinal tissues
Rat eyeballs were dehydrated using graded ethanol and embedded in paraffn. Sections of five-micron thickness were cut and transferred to triethoxysilane-coated slides.
The tissues were stained with hematoxylin–eosin and examined for morphometry. Images were taken through an Olympus BX60 microscope (Olympus Optical Co Ltd, Tokyo, Japan).
Immunohistochemistry and Western blot
For immunohistochemistry, the paraffin-embedded retinal sections (5μm) were
dewaxed and dehydrated. Endogenous peroxidase was quenched with 3% H2O2
blocker for 10 minutes at room temperature. Then, the sections were incubated with
anti-HMGB-1 antibody (1:500, Epitomics, Cambridge, U.K.) at 4 ℃ overnight. After
extensive washing with PBS, the sections were incubated with horseradish peroxidase
-streptavidin immunoglobulin (1:500, Abcam, Cambridge, UK) for 30 min, developed with diaminobenzidine for 3 min and counterstained with hematoxylin. Quantitative image analysis was performed with Image-Pro Plus 6.0 software (Media Cybernetics Inc, Bethesda, Maryland MD, USA). Ten fields per retina were randomly selected and the densitometry mean values of HMGB-1 immunostaining were determined.
For western blot, protein extracts were collected with a tissue lysis buffer (PBS containing 10 mM EDTA, 1% Triton X-100 and the protease inhibitor cocktail) and the concentrations were determined using a bicinchoninic acid protein assay kit (Bio-Rad, Richmond, CA). Protein samples were separated on polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1h. Primary antibodies were incubated at 4°C overnight at the following dilutions: anti-HMGB-1 antibody (1:500, Epitomics, Cambridge, U.K.), anti- IL-1β antibody (1:500,Epitomics, Cambridge, U.K), anti-TNF-α antibody (1:500, Abcam, Cambridge, U.K) or anti-actin antibody (1:1000, Sigma, St. Louis, MO, USA), and then incubated with secondary horseradish peroxidase-conjugated antibody (1:1000, Abcam, Cambridge, U.K.) for 1h at 37℃. The immunocomplexes were visualized by the ECL chemiluminescence method. Subsequently, semi-quantitative analysis was performed using the quantity one software (Bio-Rad, Richmond, CA). The amount of target proteins was quantified relative to the level of β-actin.
ELISA Assay of IL-1β and TNF-α
At the end of the treatment, each retina was dissected and homogenized in 100 μL
of lysis buffer supplemented with protease inhibitors (Beyotime). Samples were
centrifuged at a speed of 12,000 rpm for 10 min at 4 °C and the supernatants were
collected. After protein concentrations were assessed with the Bio-Rad method
(Bio-Rad, Richmond, CA), samples were subjected to corresponding ELISA kits
(R&D Systems, Minneapolis, MN) for the determination of IL-1β and TNF-α levels
according to the manufacturer’s instructions. The absorbance at 450nm was read on
an automated plate reader (Spectra Max Gemini UVmax; Molecular Devices,
Sunnyvale, CA). All measurements were performed in triplicate and the tissue sample
concentration was calculated from a stand curve and corrected for protein
RNA extraction and RT-PCR
The retinas were separated from eyeballs. Total RNA was extracted from mouse retinal tissues using a RNAiso kit (Invitrogen, Paisley, UK) according to the manufacturer’s instructions. Reverse transcription was carried out on the extracted total RNA by a reverse transcription kit (Mbi, Glen Burnie, MD, USA) to obtain cDNA, and the operation steps were carried out according to the reverse transcription kit manufacturer’s instructions. Amplifcation was carried out by SYBR Green kit (Roche Diagnostics, Basel, Switzerland), and IL-1β primer, TNF-α primer and GAPDH primer were synthesized and designed by Shanghai Sangon Biological Engineering Technology & Services Corporation. More details are shown in Table 1.
The PCR amplifcation conditions were as follows: pre-incubation for 5 min at 94°C, 35 cycles of amplification (94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec). Both IL-1β and TNF-α were normalized to GAPDH expression using the 2-ΔΔCt method.
Table 1 Primer sequence
The data were expressed as mean ± standard deviation (SD). Data were analyzed
using the one-way analysis of variance (ANOVA) followed by Bonferroni's post
hoc test. All statistical analyses were performed with GraphPad Prism software
(version 4.0, GraphPad Software, San Diego, Calif.). P value less than 0.05 was
considered statistically significant.