Animals
Male Sprague Dawley (SD) rats were purchased from the Guangdong animal experimental center, Hospital of Guangzhou University of Traditional Chinese Medicine. Rats were housed in pathogen-free microisolator cages with access to food and water ad libitum with a 12:12 hour light–dark cycle and constant temperature and humidity.
Grouping and drug administration
Table 1. Drug administration
Group
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Detail
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Normal
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intraperitoneal injection of citrate buffer
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DM
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intraperitoneal injection of streptozotocin (STZ)
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hUCMSCs
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intraperitoneal injection of STZ + intravitreal injection of hUCMSCs
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tBHQ
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intraperitoneal injection of STZ + Oral tBHQ
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tBHQ-hUCMSCs
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intraperitoneal injection of STZ + Oral tBHQ + intravitreal injection of hUCMSCs
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ATRA-hUCMSCs
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intraperitoneal injection of STZ + Oral ATRA + intravitreal injection of hUCMSCs
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All of the eight-week-old male rats were fixed and received an intraperitoneal injection of 60mg/kg STZ (Sigma, Code: S0130) immediately after dissolving it in 0.1M citrate buffer pH 4.5, or citrate buffer only (Normal rats). One week later, 1mL/100g peanut oil was administered to the Normal and DM groups orally once per day. Ten weeks later, in the hUCMSCs, tBHQ-hUCMSCs and ATRA-hUCMSCs groups, hUCMSCs were injected into the vitreous. tBHQ and ATRA were dissolved in peanut oil at concentrations of 15mg/mL and 0.1mg/mL respectively. One week after modeling, 15mg/100g of tBHQ per day was administered orally to the tBHQ and tBHQ-hUCMSCs groups, and 0.1mg/100g of ATRA per day was administered orally to the ATRA-hUCMSCs group.
Blood glucose quantification
Blood samples were collected from the tail vein of non-fasted alert rats, and glucose levels were determined with the glucometer system Accu-Chek Performa from Roche Diagnostic (Mannheim, Germany).
Glycated hemoglobin and plasma insulin quantification
After the rats were anesthetized, blood was collected from the abdominal aorta. The percentage of HbA1c was measured using the DCA2000 Analyzer (Bayer). The insulin concentrations were assessed using a mouse insulin ultrasensitive ELISA kit (Mercodia, Code: 10-1250-10).
Ex vivo expansion and characterization of hUCMSCs
The hUCMSCs were purchased from Vcanbio Cell & Gene Engineer Corp (Tianjin, China). The seed cells of P2 generation were resuscitated in a water bath at 37℃. The cells were carefully blown and resuscitated with complete culture medium. The formula of the medium was as follows: 89% DMEM-F12 (Gibco, Code: 11330), 10% FBS (Biosharp, Code: 04-001-1ACS), 1% penicillin and streptomycin (Beyotime Biotechnology, Code: C0222). The solution was centrifuged, the supernatant discarded, and the medium was re-suspended, blown and well shaken, then syringed into an aseptic culture bottle, which was labeled and placed in a 5% CO2, saturated humidity incubator at a constant 37 ℃ temperature to maintain the culture. When the cell density reached about 80%, the culture medium was discarded, the cells rinsed twice with normal saline, TrypLE (Gibco, Code: 125603-029) digestive juice added to digest the cells for two minutes in the cell incubator. After this period 10mL saline (Shijiazhuang No.4 Pharmoceutical, Code: H13023201) was added to end digestion and the solution was again centrifuged. After discarding the supernatant, the medium was returned to the centrifuge tube, and the cells were gently blown for uniform distribution in the culture medium. The cell suspension was syringed into the culture flask and placed into the cell incubator to maintain culture.
Once the cells were cultured to P5 generation, they were characterized. The digested cells fell away and were diluted to a density of 1 × 105/μL, incubated with FITC-CD34 (Abcam, Code: ab131589), FITC-CD45 (Abcam, Code: ab27287) and FITC-CD90 (Abcam, Code: ab11155) for 1 hour and washed again with buffered saline. The residual first antibody was removed, FITC-IgG (Abcam, Code: ab6854) secondary antibody diluent was added, and the solution was incubated at room temperature without light for one hour, then washed again with buffered saline to prevent unbound secondary antibodies from affecting the experimental results. The cells were transferred for detection of immunofluorescent markers by flow cytometry.
Intravitreal administration of hUCMSCs
Rats were anesthetized using intraperitoneal injection of 3% pentobarbital sodium. Pupils were dilated with tropicamide acetate (Dirui, Code: 20103127) before surgery, and 5μL hUCMSCs cell suspension was administered using a microsyringe after topical anesthesia. The conjunctival sac was rinsed with normal saline, then the syringe needle was inserted normal to the scleral surface at the 1-3 o'clock position at the limbus. The syringe was pressed slowly to inject the cell diluent into the vitreous cavity, kept in place for a few seconds, then was slowly withdrawn and the ocular surface at this location was gently pressed using a cotton swab for several seconds to prevent the liquid from escaping.
HE staining
Rats were euthanized by cervical dislocation and their eyes were enucleated and fixed in 4% paraformaldehyde (Biosharp, Code: BL539A). The eyes were immersed in 50%, 70%, 85% and 95% ethanol for 1 hour each, completely immersed in anhydrous ethanol Ⅰ, Ⅱ and Ⅲ for 1 hour for dehydration, then in xylene Ⅰ and Ⅱ for 30 minutes. After heating to dissolve the paraffin the transparent eyeball was soaked in the paraffin for 1 hour. Serial sections were made along the axis parallel to the optic disc and cornea, about 4pm. The slices were dyed in the following order: Xylene Ⅰ, then Ⅱ, 10 minutes each; a 1:1 mixture of xylene and anhydrous ethanol 5 minutes; 100%, 95%, 85%, 75% ethanol, 5 minutes each; distilled water, 2 minutes; hematoxylin staining, 8 minutes; distilled water, 2 minutes; 1% hydrochloric acid alcohol, 0.5 minute; tap water, 1 minute; 5% ammonia, 1 minute; eosin, 2 minutes; distilled water, 2 minutes; 75%, 85%, 95%, 100% ethanol, 5 minutes each; Xylene Ⅰ and Ⅱ, 5 minutes each. A neutral resin sealing film was applied, and the sections were observed and photographed under a light microscope.
Quantification of retinal ganglion cells (RGCs)
The number of RGCs in the ganglion cell layer was quantified using a PM-10AD microscope (Olympus, Japan). The labeled cells from temporal lobe to serrated nostril were counted on five consecutive sections. The samples were blindly analyzed by two independent observers. The data are expressed as the number of RGCs per 100 μm retinal length.
Quantification of blood inflammatory factors
The serum was diluted five times with sample dilution. According to the ELISA kit instructions (Mbbiology, Yancheng, China; IL-6 Code: MM-0190R2; TNF-α Code: MM-0180R2; iNOS Code: MM-0454M2; IFN-γ, Code: MM-0198R2), the optical density (OD) value was detected by enzyme labeling instrument at 450nm wavelength. The concentrations of IL-6, IL-10, TNF- α, iNOS and IFN- γ were calculated.
Quantification of antioxidant capacity
Quantity of SOD in rats’ blood was determined using the xanthine oxidase method, MDA using the thiobarbituric acid method[21] and GSH-Px using chemical colorimetry[22].
Western Blot Analysis
Target proteins were detected via western blotting. VEGF, Nrf2, HO-1, NF-κBp65 and cyclooxygenase-2 (COX-2) were targeted. β-actin was used as a reference protein. Each protein was repeated at least three times. The bands were analyzed by densitometry using the image analysis system (Image J, National Institute of Health, Bethesda, USA). 30~60 μg of proteins were transferred to a polyvinylidene fluoride membrane (BIO-RAD, Code: 162-0177) via immunoblotting after the electrophoresis using the Bio-Rad Mini-Protean Tetra electrophoresis “wettransfer” system (Bio-Rad, USA). Anti-VEGF (1:2000, Abcam, Code: ab47154), anti-Nrf2 (1:1000, Abcam, Code: ab89443), anti-HO-1 (1:1000, Abcam, Code: ab13248), anti-NF-κBp65 (1:2000, Cell Signaling Technology, Code: 3033S), anti-COX-2 (1:2000, Abcam, Code: ab188184) and anti-β-actin (1:5000, Abcam, Code: ab8226) were used as primary antibodies. Blotting was performed at least three times to confirm the reproducibility of the results. Bands were analyzed densitometrically using an image analysis system.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Retinal RNA was extracted using a One Step PrimeScript™ Ⅲ RT-qPCR Mix RNA extraction kit (Takara, Code: RR600S). The purity and concentration of RNA were determined using a spectrophotometer (Nano Photometer NP80, IMPLEN, Germany). ReverTra Ace qPCR RT Master Mix with gDNA remover kit (TOYOBO, 96600) was used for reverse transcription and synthesis of RNA. The mRNA expression of IL-1β, TNF-α, Nrf2, HO-1 were quantitatively detected using a SYBR Green Realtime PCR Master Mix kit (TOYOBO, 0722-841400). The mRNA expression was normalized to β-actin. The sequence of forward and reverse primers of RNA are shown in Table 2. Each group consisted of three samples, each in triplicate. Relative gene expression between groups was calculated using the 2-△△CT method.
Table 2 qRT-PCR Forward/Reverse(F/R) primers sequences
Primers
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Sequences 5′-3′
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β-actin
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5′-CCTCTATGCCAACACAGTGC-3′
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5′-CATCGTACTCCTGCTTGCTG-3′
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Nrf2
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5′-TCCCATTTGTAGATGACCATGAG-3′
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5′-CCATGTCCTGCTCTATGCTG-3′
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HO-1
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5′-ACAGAGGAACACAAAGACCAG-3′
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5′-GTGTCTGGGATGAGCTAGTG-3′
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IL-1β
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5′-TCCAGGATGAGGACATGAGCAC-3′
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5′-GAACGTCACACACCAGCAGGTTA-3′
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TNF-α
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5′-CAGGCGGTGCCTATGTCTCA-3′
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5′-GGCTACAGGCTTGTCACTCGAA-3′
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Statistical analyses
SPSS version 20.0 was used for statistical analysis. A Shapiro-Wilk test was used to check normality, mean ± standard deviation (SD) was used to describe normally distributed measurement data, and parametric tests were used for statistical analysis. A one-way ANOVA test was used to compare the means among the three groups and Levene Statistic was used to test the homogeneity of variance. If the variance was uniform, Bonferroni correction was used for pairwise comparison between groups. If the variance was uneven, Dunnett T3 test was used. P values < 0.05 were considered statistically significant.