Molecular Vision 2017; 23:242-250
Received 20 November 2016 | Accepted 12 April 2017 | Published 14 April 2017
1Department of Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, China; 2Chongqing Key Laboratory of Ophthalmology, Chongqing Eye Institute, Chongqing, China
Correspondence to: Xuedong Zhang, Department of Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, No.1 You Yi Road, Yu Zhong District, Chongqing 400016, China; Phone: 0086-23-89012536. FAX: 0086-23-89012536; email: firstname.lastname@example.org
Purpose: To investigate the effect of resolvin D1 (RvD1) on the Nod-like receptor family pyrin domain-containing (NLRP3) inflammasome and the nuclear factor-kappa beta (NF-κB) pathway in streptozotocin (STZ)-induced diabetic retinopathy in rats.
Methods: Ninety-six male rats were divided into four groups: control, STZ, RvD1, and vehicle. The rats with diabetic retinopathy induced by STZ in the RvD1 and vehicle groups were given an intravitreal injection of RvD1 (1,000 ng/kg) or the same dosage of vehicle, respectively. All rats were euthanized 7 days following treatment. Hematoxylin and eosin staining was used to observe the pathological changes in the retinal tissues. The location and expression of the NLRP3 inflammasome components, including NLRP3, caspase-associated recruitment domain (ASC), and caspase-1, in the retinas were detected using immunohistochemistry, real-time PCR, and western blot, respectively. Retinal homogenate of rats were collected for the detection of the downstream molecules interleukin 1 beta (IL-1β) and IL-18 of the NLRP3 inflammasome with enzyme-linked immunosorbent assay kits.
Results: The levels of NLRP3, ASC, cleaved caspase-1, IL-1β, and IL-18 were upregulated in the retinas of the STZ-induced diabetic rats; however, these changes were partially inhibited by the RvD1 treatment. Furthermore, the administration of RvD1 suppressed activation of NF-kB, which was upregulated in STZ-induced diabetic retinopathy.
Conclusions: RvD1 plays a protective role in STZ-induced diabetic retinopathy by inhibiting the level of activation of the NLRP3 inflammasome and associated cytokine production, suggesting targeting of this pathway might be an effective strategy in treatment of diabetic retinopathy.
Diabetic retinopathy (DR) is a common microvascular complication of diabetes, which is regarded as one of the main causes of preventable blindness worldwide . It has been shown that nearly all patients with type 1 diabetes, and more than 60% of patients with type 2 diabetes with a history of diabetes suffer from DR at some point . The number of Americans with DR will triple from 5.5 million in 2005 to 16.0 million in 2050, while the number of vision-threatened patients with DR will increase from 1.2 million in 2005 to 3.4 million in 2050 among patients 40 years old or older. Furthermore, patients with DR aged 65 years old or older will increase to an even greater extent .
The pathogenesis of DR is poorly understood, although many mechanisms have been postulated to contribute to DR, such as activation of the polyol pathway, accumulation of advanced glycation end products (AGEs), the hexosamine pathway, inflammation, and protein kinase C (PKC) activation . Growing evidence has shown that a parainflammatory response, characterized by a chronic low level of inflammation and disorder of immune responses, plays a prominent role in the pathogenesis of DR . Moreover, retinas from animals with diabetes express high levels of proinflammatory cytokines, such as interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), which have been reported to be involved in inflammatory responses in DR . Therefore, in the present study we focused on the role of anti-inflammatory therapies in inhibiting DR.
Pattern-recognition receptors (PRRs) belong to the innate immune system and are responsible for recognizing pathogen-associated molecular patterns (PAMPs) of pathogenic microorganisms, followed by activation of innate and adaptive immune responses. Nod-like receptor family pyrin domain-containing protein (NLRP) is assembled by PRRs and is involved in the development of chronic inflammatory responses . The NLRP3 inflammasome (as the most widely studied inflammasome) comprises NLRP3, apoptosis-associated speck-like protein containing a caspase-associated recruitment domain (ASC), and caspase-1 . NLRP3 interacts with ASC to activate caspase-1, and once activated, the NLRP3 inflammasome induces tissue injury through upregulation of the proinflammatory cytokines IL-18 and IL-1β . A recent study implicated the inappropriate activation of the NLRP3 inflammasome in DR .
Resolvin D1 (RvD1) is derived from the w-3-polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) . The therapeutic promise of RvD1 in chronic inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and asthma, has been recognized . However, the anti-inflammatory role of RvD1 in DR has not been elucidated. Therefore, we performed this study to investigate the effect and the possible mechanism of RvD1 in the retina and whether the NLRP3 inflammasome is a target for RvD1 in DR.
Male Sprague–Dawley rats (200–250 g, 8 weeks old) were obtained from the Animal Care Committee of Chongqing Medical University (Chongqing, China), were raised in a specific pathogen-free room, and received a standard laboratory rat diet and water. Diabetes was induced with a single intraperitoneal injection of freshly prepared streptozotocin (STZ) solution in citrate buffer (pH 4.5) at 60 mg/kg in rats. The rats were considered to be diabetic with non-fasting blood glucose 250 mg/dl or greater after 1 week. Diabetic rats were raised for 12 weeks with a regular diet and unlimited water. All animal protocols were approved by the Ethics Committee of Chongqing Medical University and in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.
Ninety-six rats were included in this study and randomly assigned to four groups: the control (n = 26), STZ (n = 26), RvD1 (n = 22), and vehicle group (n = 22). All animals, except those in the control group, were STZ-treated rats. The rats in the RvD1 group were given an intravitreal injection of RvD1 (1000 ng/kg) using sterile syringes fitted with a 30-gauge needle containing 5 μl of reconstituted RvD1 solution. Rats in the vehicle group were given an intravitreal injection of the same amount of saline and ethanol. All animals were euthanized 7 days after the respective treatment. . The rats were deeply anaesthetized by an intraperitoneal injection of 10% chloral hydrate (300 mg/kg).
The blood–retinal barrier (BRB) permeability was performed using the Evans blue (EB) technique. The rats were deeply anaesthetized with an intraperitoneal injection of 10% chloral hydrate (300 mg/kg) and then injected with EB dye solution dissolved in low concentration saline at a concentration of 30 mg/ml through the tail vein, at a dosage of 45 mg/kg. After 120 min, the rats were perfused with PBS (1X; 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH7.4) followed by 1% paraformaldehyde via the left ventricle. After dissecting and drying, the weight of the retina was measured. Each retina was incubated in 200 μl formamide overnight at 70 °C and centrifuged at 20,000 ×g for 60 min. Supernatant retinal extracts were tested spectrophotometrically at 620 nm and 740 nm.
The eyeballs were harvested and fixed via immersion in 4% paraformaldehyde for 24 h. The 10% buffered formalin-fixed retina tissue was embedded in paraffin and sectioned at 5 μm. Following staining with hematoxylin and eosin (H&E), the paraffin sections were observed and photographed with a fluorescence microscope (. Leica, Bannockburn, IL, DM6000).
The sections were washed four times for 5 min in PBS following deparaffinization and antigen retrieval. The slides were then blocked with goat serum for 30 min. All sections were subsequently incubated with anti-NLRP3 antibody (1:200 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), anti-ASC antibody (1:200 dilution, Santa Cruz Biotechnology), and anti-caspase-1 antibody (1:200 dilution, Santa Cruz Biotechnology) at 4 °C overnight, followed by incubation with goat anti-rabbit biotinylated antibody (ZSGB-Bio, Beijing, China) at room temperature. Staining was developed with diaminobenzidine (DAB) and counterstained with hematoxylin. Sections incubated in PBS were used as negative controls. Three sections per eye were selected, with four rats in each group. Retinal images were obtained at the same distance from the optic nerve. Images of the immunohistochemically stained tissue sections were collected using an Olympus BX60 microscope (Olympus Optical Co Ltd, Tokyo, Japan).
Total mRNA was extracted from the retinal samples with TRIzol Reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol. Quantitative PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Foster City, CA). PCR primers employed were as follows: NLRP3, 5′-CCA GGG CTC TGT TCA TTG-3′ (forward) and 5′-CCT TGG CTT TCA CTT CG-3′ (reverse); ASC, 5′-CCC ATA GAC CTC ACT GAT AAA C-3′ (forward) and 5′-AGA GCA TCC AGC AAA CCA-3′ (reverse); caspase-1, 5′-AGG AGG GAA TAT GTG GG-3′ (forward) and 5′-AAC CTT GGG CTT GTC TT-3′ (reverse); IL-1β, 5′-CCT TGT GCA AGT GTC TGA AG-3′ (forward) and 5′-GGG CTT GGA AGC AAT CCT TA-3′ (reverse); IL-18, 5′-CGC AGT AAT ACG GAG CAT AAA TGA C-3′ (forward) and 5′-GGT AGA CAT CCT TCC ATC CTT CAC-3′ (reverse); and β-actin, 5′-AGG GAA ATC GTG CGT GAC-3′ (forward) and 5′-CGC TCA TTG CCG ATA GTG-3′ (reverse). Cycling conditions were 30 s of polymerase activation at 95 °C, followed by 40 cycles at 95 °C for 5 s and at 60 °C for 34 s. The data were analyzed using the 2-ΔΔCT method.
Retinas were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors or a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Haimen, China) and centrifuged at 12,000 ×g for 20 min at 4 °C. Protein samples were loaded on a Tris glycine gel, separated with 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred onto polyvinylidene difluoride membranes. The membranes were then blocked with 5% skim milk for 2 h at 37 °C and incubated overnight at 4 °C with the following antibodies: anti-NLRP3 antibody (Santa Cruz Biotechnology) at 1:200 dilution, anti-ASC antibody (Santa Cruz Biotechnology) at 1:200 dilution, anti-caspase-1 antibody (Proteintech, Wuhan, China) at 1:500 dilution, antibodies to IκBα (Santa Cruz Biotechnology) at 1:500 dilution, nuclear factor-kappa B (NF-κB) p65 (Santa Cruz Biotechnology) at 1:200 dilution, and p-NF-kB p65 (Abcam, Cambridge, UK) at 1:700 dilution. After being washed with Tris-buffered saline with Tween 20 (TBS-T), the membranes were then incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (Sigma, St. Louis, MO) for 1 h at 1:5,000 dilution at room temperature. The immunoreactive bands were visualized using an enhanced chemiluminescence method and quantified with densitometry analysis (Quantity One, Hercules, CA). GAPDH was used as a loading control.
Retinas were isolated and homogenized in 150 μl of RIPA lysis buffer, supplemented with 1% protease inhibitors followed by centrifugation at 15,000 ×g for 25 min at 4 °C. The levels of IL-1β and IL-18 were measured in the extracted supernatant using commercial kits. The kits used for the measurement of cytokines were as follows: IL-1β (eBioscience, Santa Clara, CA, Ref: BMS630, Lot: 87,225,015) and IL-18 (Novex, Invitrogen, Camarillo, CA, Ref: KRC2341, Lot: 130,401/A).
Data were expressed as mean ± standard deviation (SD). Statistical comparisons among the groups were performed using one-way ANOVA (ANOVA) followed by the Student–Newman–Keuls (SNK) test for multiple comparisons. A p value of less than 0.05 was considered statistically significant. Statistical calculations were performed using SPSS Software 19.0 (SPSS, Inc., Chicago, IL).
H&E staining showed a homogenous surface of the retina with the ganglion cells arranged in a monolayer, and regular arrangement of cells at the inner and outer nuclear layers in the control group (Figure 1A). Twelve weeks after the STZ treatment, we observed disruption of the retinal structures, and the inner and outer nuclear layers appeared to be disordered (Figure 1B,C). The rats in the RvD1 group showed improvements in the distribution of the inner and outer nuclear layers compared with the STZ group (Figure 1D). In addition, as shown in Figure 1E, the permeability of the BRB in the STZ group was statistically significantly increased 2.4-fold compared with the controls (p<0.01).
Immunohistochemical results showed that NLRP3, ASC, and caspase-1 were specifically located in the ganglion cell layer and the inner and outer nuclear layers (Figure 2A). The number of cells expressing NLRP3, ASC, and caspase-1 was remarkably increased in the STZ group (p<0.001) but decreased in the RvD1 group compared with the STZ group (Figure 2B; p<0.01). Compared with the control group, the mRNA levels of NLRP3, ASC, and caspase-1 were statistically significantly increased in the STZ group (p<0.05), which were inhibited by RvD1 administration as seen in Figure 2C (p<0.05).
In addition, as shown in Figure 2D–G intravitreal RvD1 administration statistically significantly inhibited the protein expression of NLRP3, ASC, and cleaved caspase-1 compared with the STZ group (p<0.05). Nevertheless, we did not observe any statistically significant difference between the STZ and vehicle groups.
To investigate the changes in the downstream molecules mature IL-1β and IL-18 of NLRP3 inflammasome activation, we detected the levels with enzyme-linked immunosorbent assay (ELISA). As shown in Figure 3A,B, the treatment with STZ statistically significantly increased the expression of IL-1β and IL-18 (both p<0.001), while treatment with RvD1 showed a statistically significant reduction in the protein levels of IL-1β and IL-18 compared with the STZ group (p<0.01 and p<0.001, respectively). Meanwhile, consistent with the ELISA findings, the mRNA levels of IL-1β and IL-18 were decreased in the RvD1 group (Figure 3C,D).
Given the importance of NF-κB in the transcription of the cytokines in the NLRP3 inflammasome, we used western blotting to analyze protein expression. As seen in Figure 4, the levels of NF-κB phosphorylation (Figure 4A,B) and degradation of IκBα (Figure 4C,D) in the rats with STZ-induced DR were markedly increased (both p<0.05), whereas RvD1 suppressed the expression of NF-κB phosphorylation and decreased IκBα (both p<0.05).
In this study, we observed pathological morphology of the retina in STZ-induced retinopathy and the breakdown of the BRB, which contributes to the development of DR, resulting in hemorrhage, edema, and retinal detachment . In addition, we found increased expression of the NLRP3 inflammasomes, including NLRP3, ASC, caspase-1, and its downstream inflammatory molecules, as well as the activation of NF-κB in diabetic retinas. In the present study, we also demonstrated that the upregulation of the NLRP3 inflammasome and the NF-κB pathway in diabetic rats was attenuated after treatment with RvD1. These findings indicate that RvD1 may inhibit the NLRP3 inflammasome and the NF-κB signaling pathway and may contribute to attenuation of the early stage of DR.
The disruption of the BRB occurs in the early stage of STZ-treated retinopathy . Diabetic-induced endothelial dysfunction, leukostasis, and inflammation play an important role in the pathogenesis of DR, accounting for the initial change in BRB damage . IL-1β increases vascular permeability via NF-κB activation, leukocyte adhesion, and retinal capillary cell apoptosis . In the present study, the permeability of the BRB in the STZ group was statistically significantly increased after 3 months, which may be accounted for by the actions of IL-1β and IL-18. In addition, we observed disruption of the inner and outer retinal nuclear layers in diabetic rats.
Hyperglycemia and other stresses lead to a series of inflammatory mediators in diabetes, which trigger parainflammatory responses that might cause abnormal leukocyte–endothelial interactions and, ultimately, retinal microvascular damage . Chronic inflammation is one of the key triggers in the pathogenesis of DR, and a reduction in inflammation has been reported to alleviate the development and progression of DR . For this reason, we aimed to prevent chronic inflammation in a model of DR. Studies have reported that NLRP plays an important role in the development of chronic inflammatory responses through IL-1β and IL-18 secretion. Activation of the NLRP3 inflammasome has been demonstrated in a wide variety of diseases, including asbestosis, Alzheimer disease, atherosclerosis, and gout [19-21]. An emerging role of the NLRP3 inflammasome in the development of diabetic nephropathy suggested the involvement of NLNRP3 in renal inflammation . In addition, Shi et al.  observed that the NLRP3 inflammasome could be activated in ARPE-19 cells under high glucose stress in vitro. In this study, we also found that the NLRP3 inflammasome pathway was involved in the development of DR in a type 1 diabetic rat model, which further confirmed the role of the NLRP3 inflammasome in DR.
RvD1, derived from the essential omega-3 fatty acid docosahexaenoic acid (DHA), has been shown to have a protective role in several diseases, including hepatic ischemia/reperfusion injury , adjuvant-induced arthritis , peritonitis , and uveitis . However, whether RvD1 can regulate the NLRP3 inflammasome and prevent the progression of DR is unclear. Because o-3-polyunsaturated fatty acids have been reported to protect against retinopathy and reduce angiogenesis , we speculate that RvD1 may exert its anti-inflammatory effects by targeting the NLRP3 inflammasome. Our results showed that RvD1 inhibited the expression of the NLRP3 inflammasome and its downstream signaling pathway in diabetic retinas, which promoted the resolution of inflammation in DR. Therefore, we concluded that RvD1 might be a potent inhibitor of the NLRP3 inflammasome signaling pathway, which could maintain potent anti-inflammatory properties in a diabetic rat model of chronic inflammation.
Caspases, categorized as either proinflammatory or proapoptotic factors, can lead to inflammation or cell death. Caspase-1, activated by the NLRP3 inflammasome, mediates cytokines such as IL-1β . Activated caspase-1 processes pro-IL-1β to a mature form and subsequently induces proinflammatory cytokine release and inflammation . IL-1β is a potent proinflammatory mediator, and maturation and release are mediated by inflammasome activation. Secretion of the inflammatory cytokines IL-1β and IL-18 play a pivotal role in the development of DR and are critically regulated by the NLPR3 inflammasome [31,32]. Consistent with previous studies, the present data revealed that levels of the inflammatory cytokines IL-1β and IL-18 were elevated in the STZ-induced DR model but decreased with the administration of RvD1. Collectively, RvD1 downregulated the expression of IL-1β and IL-18, most likely through the inhibition of the NLRP3 inflammasome signaling pathway.
NF-κB, a ubiquitously expressed transcription factor that regulates many inflammatory cytokines, has been shown to regulate DR. Under physiologic conditions, NF-κB is silenced by its inhibitor, IkB. Persistent activation of NF-κB initiates an increase in inflammatory cytokines during the inflammatory response that regulates cell proliferation, invasion, apoptosis, and metastasis . It is known that the activation of NF-κB is involved in the NLRP3 inflammasome . Our results showed enhanced phosphorylation of NF-κB and degradation of IκBα and increased inflammatory cytokine production in STZ-treated rats retinas compared with the control group. In accordance with previous studies showing that RvD1 regulates the phosphorylation of NF-κB , the present results revealed that RvD1 treatment inactivated the NF-κB-related signaling pathway via inhibition of NF-κB phosphorylation and blockade of the degradation of IκBα, suggesting an inhibitory effect of RvD1 on NF-κB activity and subsequent inflammatory responses in DR.
In conclusion, we found a suppressive action of RvD1 on the activation of NLRP3 inflammasome-associated component proteins, including NLRP3, ASC, caspase-1, and NF-κB activation. We also found an inhibitory role of RvD1 relative to the production of IL-1β and IL-18. The present study results indicate that the NLRP3 inflammasome contributes to the pathogenesis of STZ-induced DR, and RvD1 may be an alternative therapeutic agent for DR.
This study was supported by funds from National Natural Science Foundation of China (81,371,043), the special fund of Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003) and National Key Clinical Specialties Construction Program of China. The authors would like to express their thanks to M r. Peizeng Yang and Mrs. Chaokui Wang for their kind advice.