Abstract
Purpose:
To explore the effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on retinal ischemia–reperfusion injury (RIRI) and the protective effect of N‐hydroxy‐N’‐(4‐n‐butyl‐2‐methylphenyl)formamidine (HET0016) on RIRI.
Methods:
Male Sprague–Dawley rats were randomly divided into the normal control group, experimental model group (RIRI group), experimental solvent group (RIRI + solvent group), and experimental treatment group (RIRI + HET0016 group).
Results:
The levels of 20-HETE, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) in the retina of rats at 24 h after reperfusion were measured by enzyme-linked immunosorbent assay. Hematoxylin–eosin staining was used to observe the retinal morphological and thickness changes at 24 h, 48 h, and 7 days after reperfusion. The number and localized expression of matrix metalloproteinase-9–positive cells in the retina of the rats at 24 h after reperfusion and the activation and localized expression of retinal microglia at 48 h after reperfusion were measured using an immunohistochemical method. The nuclear metastasis of nuclear factor kappa-B (NF-κB, p65) cells at 24 h after reperfusion was observed using an immunofluorescence method.
Conclusion:
Overall, 20-HETE might activate microglia to aggravate RIRI by the NF-κB pathway, but HET0016 has significant protective effects for the retina.
Keywords: 20-hydroxyeicosatetraenoic acid, N‐hydroxy‐N’‐(4‐n‐butyl‐2‐methylphenyl)formamidine, nuclear factor kappa-B, retinal ischemia–reperfusion injury
The retina plays a crucial role in the visual pathway by sensing light stimuli and transmitting neural signals.[1] Ischemic retinal diseases account for a significant proportion of ocular diseases, and common causes include hypoperfusion retinopathy induced by inadequate ocular arterial blood supply, ocular ischemic syndrome, central retinal artery occlusion, central retinal vein occlusion, intraocular hypertension, and ophthalmic procedures affecting retinal blood flow.[2] Retinal ischemia–reperfusion injury (RIRI) represents a crucial pathological mechanism underlying diabetic retinopathy (DR), glaucoma, and retinal vascular occlusive diseases, promoting the apoptosis of retinal ganglion cells (RGCs), attributable to retinal dysfunction and visual impairment.[3,4] RIRI management mainly focuses on neuroprotection and treatments against oxidative stress and free radicals, calcium overload, leukocyte actions and inflammatory responses, glutamate‐mediated excitotoxicity, and apoptosis.[5]
Patients with DR have been found to have an increased level of 20‐hydroxyeicosatetraenoic acid (20‐HETE) in the vitreous body.[6] This is a bioactive arachidonic acid generated through cytochrome P450 catalysis. It is reported to play a key role in regulating the myogenic contraction of arteriolar smooth muscles in the kidney, brain, skeletal muscles, and mesentery.[7] Evidence shows that 20‐HETE can activate the nuclear factor kappa-B (NF‐κB) signaling pathway, promote the release of inflammatory cytokines, stimulate microglial proliferation, upregulate matrix metalloproteinase‐9 (MMP‐9) expression, and eventually lead to tissue damage.[8] The 20‐HETE inhibitor N‐hydroxy‐N’‐(4‐n‐butyl‐2‐methylphenyl) formamidine (HET0016) has been found to alleviate inflammatory reactions and tissue edema, inhibit microglial activation, and downregulate MMP‐9 expression through the significant reduction of 20‐HETE.[9]
To date, the secretion level of 20‐HETE and the related mechanism responsible for retinal injury in RIRI remain a research gap. In the current study, a rat model of RIRI is established via the application of high intraocular pressure (IOP) and intravenous injection of HET0016 solution. This study investigates the pathological mechanism of 20‐HETE in RIRI and explores the protective effects of HET0016 on RIRI by using enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), and immunofluorescence (IF) techniques, aiming to provide experimental evidence for the treatment of RIRI.
Methods
Laboratory animals
Male, specific-pathogen-free Sprague–Dawley rats weighing 200 ± 20 g, with clear refraction, round pupils, normal light response, and no abnormalities upon dilated fundus examination, were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The breeding conditions and experimental procedures were in line with the Administrative Regulations on Laboratory Animal Management set out by the State Scientific and Technological Commission. This study obtained approval from the Animal Ethics Committee.
Animal model
An animal model of RIRI was established following the method previously proposed by Hartsock et al.[10] Rats in the RIRI group were anesthetized with 0.3% pentobarbital sodium (1.5 mL/100 g) via intraperitoneal injection. Compound tropicamide was used for pupil dilation, followed by the application of proparacaine hydrochloride eye drops. A 30‐G needle was inserted into the anterior chamber for standard infusion without causing corneal penetration or damage to the iris or lens. The other end of the needle was connected to physiological saline and elevated to a distance of 150 cm from the rat’s eyeball, resulting in an IOP of 109.7 mmHg. High perfusion pressure was applied to the anterior chamber for 60 min before needle removal. As the white retina turned orange‐red, tetracycline ointment was applied.
Experimental groups
A total of 112 rats were randomly divided into the normal control (CON) group, model (RIRI) group, solvent treatment (RIRI + solvent) group, and HET0016 treatment (RIRI + HET0016) group, with 28 rats in each group. To build an RIRI model, the RIRI, RIRI + solvent, and RIRI + HET0016 groups were further divided into three subgroups, each according to the following time points of reperfusion: 24 h, 48 h, and 7 days after reperfusion. In the RIRI + HET0016 group, the rats were intravenously injected with HET0016 solution (1 mg/kg) via the tail vein 5–10 min before reperfusion. In the RIRI + solvent group, the rats received an equal volume of 15% hydroxypropyl‐β‐cyclodextrin solution through the tail vein 5–10 min before reperfusion.
Reagents and consumables
HET0016 (#HY-124527, MedChemExpress, USA), hydroxypropyl‐β‐cyclodextrin (#C7070, Solarbio China), NF-κB p65 antibody (#8242, Cell Signaling Technology, USA), IBA1 antibody (#DF6442, Affinity, USA), MMP‐9 antibody (#TA5228, Abmart Medical Technology, China), rat tumor necrosis factor-α (TNF‐α) ELISA kit (#ml002859, Enzyme‐linked Biotechnology, China), and rat 20‐HETE ELISA kit (#ml038319, Enzyme‐linked Biotechnology, China).
Preparation of key reagents
Preparation of 15% hydroxypropyl‐β‐cyclodextrin solution
A total of 15 g of hydroxypropyl‐β‐cyclodextrin powder was weighed using a precision balance. It was dissolved in 0.9% saline solution until no sediment was visible, and then, more saline was added until the volume reached 100 mL.
Preparation of 1 mg/mL HET0016 solution.[11,12,13]
An adequate amount of HET0016 according to body weight (1 mg/kg) was calculated and dissolved in the aforementioned 15% hydroxypropyl‐β‐cyclodextrin solution. The concentration was 1 mg/mL, and the solution was clear and free of sediment.
Preparation of 0.3% phosphate‐buffered saline with Tween 20
Next, 300 μL of Tween 20 was added to 100 mL of 0.1-M phosphate‐buffered saline (PBS) at a pH of 7.4 and mixed thoroughly for future use.
Preparation of 3% bovine serum albumin solution
A total of 3 g of powdered bovine serum albumin was dissolved in 100 mL of 0.3% PBST solution. The solution was stirred until it became clear and transparent.
Outcome measures and assays
Determination of retinal levels of 20‐HETE, TNF‐α and interleukin-1β at 24 h of reperfusion by enzyme-linked immunosorbent assay
Thirty‐two rats meeting the inclusion criteria were randomly divided into the CON group, RIRI group, RIRI + solvent group, and RIRI + HET0016 group, with eight rats in each group. The rats were sacrificed 24 h after reperfusion, and their retinal tissues were harvested and prepared into protein extracts. Standard ELISA was performed to plot a standard curve and determine the equation. Dilutions were calculated using the optical density values of the samples and the equation, and the actual concentration was the diluted concentration multiplied by the dilution ratio.
Preparation of paraffin sections
Eighty rats fulfilling the inclusion criteria were randomly assigned to the CON group (n = 8), RIRI group (n = 24), RIRI + solvent group (n = 24), and RIRI + HET0016 group (n = 24). Furthermore, the RIRI, RIRI + HET0016, and RIRI + solvent groups were divided into 24-h, 48-h, and 7-day reperfusion subgroups (n = 8 in each group). Eyeball specimens were collected to prepare paraffin blocks and sections.
Hematoxylin and eosin staining
Hematoxylin and eosin (HE) staining was performed to observe the retinal morphology at 24 h, 48 h, and 7 days of reperfusion. Photomicrographs were taken by selecting the retinal area 1 mm away from the optic papilla on both sides by using an optical microscope.
IHC
Immunohistochemistry was conducted to visualize the expression and localization of MMP‐9–positive cells in the retina at 24 h of reperfusion, followed by the detection of microglial cell count and location at 48 h of reperfusion. Three random fields were selected for photomicrography.
Immunofluorescence staining
The IF staining was performed to observe the nuclear translocation of p65 in retinal cells at 24 h of reperfusion. Three fields were randomly selected for photomicrography and analysis, with the nucleus blue‐stained and p65 red‐stained.
Statistical analysis
The statistical analysis was performed using SPSS 25.0. All measurement data were presented as mean ± standard deviation (x ± s), and the differences between groups were evaluated using one‐way analysis of variance. When the homogeneity of variance was satisfied, multiple comparisons were conducted using the Student–Newman–Keuls test. In cases of heterogeneity of variance, non‐parametric tests (Mann–Whitney U test) were employed for multiple comparisons. The significance level was set at α =0.05. The software ImageJ was used for counting positive cells and measuring retinal thickness, while another software, GraphPad, was employed to generate statistical graphs.
Results
Determination of retinal levels of 20‐HETE, TNF‐α, and interleukin 1β at 24 h of reperfusion by ELISA
The RIRI and RIRI + solvent groups had significantly higher levels of 20‐HETE, TNF‐α, and interleukin 1β (IL‐1β) in the retina as compared with the CON group (Fig. 1, all P < 0.05). However, the differences were insignificant between the RIRI and RIRI + solvent groups (P = 0.071, 0.962, and 0.624, respectively). In contrast, the RIRI + HET0016 group showed reduced levels of 20‐HETE, TNF‐α, and IL‐1β in comparison with the RIRI and RIRI + solvent groups, and the differences were statistically significant (all P < 0.05). Moreover, the levels of 20‐HETE, TNF‐α, and IL‐1β in the RIRI + HET0016 group were higher than those in the CON group, and the differences were statistically significant (all P < 0.05). These results indicated that HET1006 can significantly inhibit the increase of inflammatory factor secretion caused by RIRI.
Figure 1.
Determination of retinal levels of 20-HETE, TNF-α, and IL-1β by ELISA. Note: A: 20-HETE, B: TNF-α, C: IL-1β; CON group (n = 28): Normal control group, RIRI group (n = 28): Model group, RIRI + solvent group (n = 28): Solvent treatment group, RIRI + HET0016 group (n = 28): HET0016 treatment group; 20-HETE: 20-hydroxyeicosatetraenoic acid. (In Figure 1a, *P < 0.05 compared with the CON group; #P < 0.05 compared with the RIRI group; †P < 0.05 compared with the RIRI + solvent group; ‡P < 0.05 compared with the RIRI + HET0016 group. In Figure 1b, *P < 0.05 compared with the CON group; #P < 0.05 compared with the RIRI group; †P < 0.05 compared with the RIRI + solvent group; ‡P < 0.05 compared with the RIRI + HET0016 group. In Figure 1c, *P < 0.05 compared with the CON group; #P < 0.05 compared with the RIRI group; †P < 0.05 compared with the RIRI + solvent group; ‡P < 0.05 compared with the RIRI + HET0016 group.)
HE staining–based morphological changes in the retina at 24 h, 48 h, and 7 days of reperfusion
The retina consists of 10 layers, and three distinct layers can be observed under light microscopy, as follows: the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer. HE staining revealed that the retinas in the CON group had well-organized layers and clear structural integrity. At 24 h and 48 h post modeling, the retinas in the RIRI and RIRI + solvent groups showed disrupted morphology, loose cell arrangement, vacuoles, nuclear condensation, and signs of swelling and thickening. Compared with the RIRI and RIRI + solvent groups, the RIRI + HET0016 group showed reduced pathological changes in the retinas, with a more orderly cell arrangement and lessened swelling. At 7 days post modeling, the RIRI and RIRI + solvent groups exhibited reduced retinal thickness, nuclear dissolution, and decreased RGCs. In contrast, the RIRI + HET0016 group experienced an increase in retinal thickness as well as in the number of RGCs [Fig. 2].
Figure 2.
HE staining of the retina at 7 days of reperfusion (100×). Note: CON group (n = 28): Normal control group, RIRI group (n = 28): Model group, RIRI + solvent group (n = 28): Solvent treatment group, RIRI + HET0016 group (n = 28): HET0016 treatment group
IHC detection of MMP‐9–positive cell expression and localization in the retina at 24 h of reperfusion
The CON group showed minimal expression of MMP‐9–positive cells, while the RIRI and RIRI + solvent groups exhibited increased expression of MMP‐9 in all layers of the retina, especially in the GCL [Fig. 3]. The MMP‐9–positive cell expression of the RIRI and RIRI + solvent groups was significantly different from that of the CON group (P < 0.05). However, no significant difference in MMP‐9–positive cell expression was observed between the RIRI and RIRI + solvent groups (P = 0.897). Compared with the RIRI and RIRI + solvent groups, the RIRI + HET0016 group showed a significant reduction in MMP‐9–positive cells (P < 0.05). Compared with the CON group, the RIRI + HET0016 group had a significantly higher MMP‐9–positive cell count (P < 0.05).
Figure 3.
IHC detection of MMP-9-positive cell count. (a) Representative images of MMP-9 immunohistochemistry (400×), (b) Quantitative results of MMP-9 immunohistochemistry. Note: CON group (n = 28): Normal control group, RIRI group (n = 28): Model group, RIRI + solvent group (n = 28): Solvent treatment group, RIRI + HET0016 group (n = 28): HET0016 treatment group. IHC staining was conducted to determine MMP-9-positive cell count in the retina, where positive cells appeared dark brown with blue-stained nuclei. *P < 0.05 compared with the CON group; #P < 0.05 compared with the RIRI group; †P < 0.05 compared with the RIRI + solvent group; ‡P < 0.05 compared with the RIRI + HET0016 group
IHC detection of microglial activation status and localization at 48 h of reperfusion
Microglial cell expression was detected using its specific marker IBA1. A small number of activated microglial cells were observed in the CON group, while significantly increased microglial activation was noted in the RIRI and RIRI + solvent groups, characterized by enlarged cell bodies typically distributed in the GCL and inner plexiform layer (Fig. 4, P < 0.05). The RIRI and RIRI + solvent groups showed no significant difference in microglial activation (P = 0.713). Compared with the RIRI and RIRI + solvent groups, the RIRI + HET0016 group experienced a significant reduction in activated microglial cells (P < 0.05). Compared with the CON group, the RIRI + HET0016 group had a significantly higher number of activated microglial cells (P < 0.05).
Figure 4.
IHC detection of IBA1-positive cell count. (a) Representative images of IBA1 immunohistochemistry (400×), (b) Quantitative results of IBA1 immunohistochemistry. Note: CON group (n = 28): Normal control group, RIRI group (n = 28): Model group, RIRI + solvent group (n = 28): Solvent treatment group, RIRI + HET0016 group (n = 28): HET0016 treatment group. *P < 0.05 compared with the CON group; #P < 0.05 compared with the RIRI group; †P < 0.05 compared with the RIRI + solvent group; ‡P < 0.05 compared with the RIRI + HET0016 group
IF staining of p65 localization at 24 h of reperfusion
The IF results of p65 are shown in Fig. 5. In the CON group, p65 was mostly expressed in the cytoplasm but rarely in the nucleus. Compared with the CON group, the RIRI and RIRI + solvent groups showed significantly increased nuclear expression of p65, primarily in the GCL and INL (P < 0.05, respectively), but the difference between the RIRI group and the RIRI + solvent group lacked statistical significance (P = 0.603). In the RIRI + HET0016 group, the nuclear translocation of p65 was significantly reduced compared with that in the RIRI and RIRI + solvent groups (P < 0.05).
Figure 5.
IF staining of p65 nuclear translocation (a) Representative images of p65 immunofluorescence (400×), (b) Quantitative results of p65 immunofluorescence. Note: CON group (n = 28): Normal control group, RIRI group (n = 28): Model group, RIRI + solvent group (n = 28): Solvent treatment group, RIRI + HET0016 group (n = 28): HET0016 treatment group. *P < 0.05 compared with the CON group; #P < 0.05 compared with the RIRI group; †P < 0.05 compared with the RIRI + solvent group; ‡P < 0.05 compared with the RIRI + HET0016 group
Discussion
RIRI can induce RGC apoptosis and eventually lead to physiological dysfunction of the retina.[14] Palmhof et al.[15] reported that damage to RGCs occurs during the early phase of reperfusion. RIRI has been found to play a role in the NF‐κB signaling pathway,[16] apoptotic signaling pathway,[17] and autophagy signaling pathway.[18] Despite the current knowledge of these complex signaling pathways and related pathological mechanisms, there is a lack of effective protection from reperfusion injury. Studies have shown that the 20‐HETE level is elevated in mouse brain tissue after cerebral ischemia–reperfusion,[19] in the cerebral cortex of mice with traumatic brain injury, in the plasma of patients with ischemic diseases,[20] and in the vitreous body of patients with DR.[21] Like 20‐HETE, epoxyeicosatrienoic acids can also be generated in the retinal circulation and are reported to be associated with retinal angiogenesis under hypoxic conditions.[22] Nevertheless, very little is known about the relationship between 20‐HETE and RIRI.
In this study, the retinal level of 20‐HETE at 24 h of reperfusion was significantly elevated in the RIRI group and the RIRI + solvent group compared with the CON group. Notably, early intervention with HET0016 resulted in a reduction in the retinal level of 20‐HETE in the RIRI model. It is believed that the use of HET0016 could reverse the RIRI‐induced increase in 20‐HETE secretion and thereby reduce inflammatory responses and tissue edema. Interleukin 1β is an important mediator of immune response in an inflammatory environment. Evidence has shown that increased IOP could activate retinal inflammatory responses, stimulate the release of IL‐1β and other pro‐inflammatory molecules, and lead to RIRI. In this study, the retinal levels of 20‐HETE and the inflammatory cytokines TNF‐α and IL‐1β were significantly reduced in the RIRI + HET0016 group as compared with the other groups. The study by Zhang et al.[2] investigated the effect of curcumin on the retinal expression of IL‐1β in rats with RIRI and discovered that RIRI can be mitigated through IL‐1β modulation.
Nuclear factor kappa-B is a nuclear transcription factor ubiquitously present in various cells and is involved in diverse inflammatory and immune responses.[23,24] The NF‐κB activator 20‐HETE can stimulate IκB phosphorylation and nuclear translocation of p65 by activating IKK.[25,26] The activation of NF‐κB leads to increased synthesis of IL‐1β and TNF‐α, and these inflammatory cytokines, in turn, activate NF‐κB, creating a positive feedback loop that exacerbates reperfusion injury.[27] Matrix metalloproteinases are a class of endopeptidases that require metal (zinc, calcium) ions as cofactors for activity. Matrix metalloproteinase‐2, MMP‐9, and MMP‐14 have been extensively studied compared with other members of the MMP family.[8] MMP-9 is normally expressed at low levels. However, it can be expressed abnormally or overexpressed under conditions of ischemia, hypoxia, inflammation, and oxidative stress, which can result in neuronal degeneration and necrosis.[9] Nuclear factor kappa-B can promote MMP‐9 expression in various cells.[28] According to the IF staining analysis, p65 was mainly expressed in the cytoplasm in the CON group. After 24 h of modeling, the RIRI and RIRI + solvent groups showed increased nuclear expression of p65. In the RIRI + HET0016 group, the nuclear expression of p65 decreased following early intervention with HET0016. In other words, HET0016 can inhibit the 20‐HETE–induced nuclear translocation of p65 and suppress the activation of the NF‐κB pathway. The IHC staining results showed minimal expression of MMP‐9 in the CON group, increased MMP‐9 expression in the RIRI and RIRI + solvent groups at 24 h of modeling, and a significant reduction in MMP‐9–positive cells in the RIRI + HET0016 group after early intervention with HET0016 as compared with the RIRI and RIRI + solvent groups. It was inferred that HET0016 could reduce the secretion of MMP‐9 by inhibiting the synthesis of 20‐HETE, thereby providing some protection for the blood–retinal barrier. Wang et al.[29] also demonstrated the protective effect and mechanism of 20‐HETE in RIRI‐induced retinal injury by downregulating MMP‐9 expression.
Microglial cells are resident immune cells in the central nervous system and the retina.[30] In the resting state, microglial cells exhibit a highly branched morphology, secrete neurotrophic factors, and serve as scavengers to maintain normal brain and retinal function.[30,31] In the retina of healthy adults, microglial cells can be found around RGC bodies as well as in the outer and inner plexiform layers, where they are responsible for local environment monitoring, metabolic byproduct elimination, cell debris ingestion, and regulation of intercellular communication.[31,32] Proliferation and morphological changes of microglial cells can be ascribed to focal injury, reperfusion injury, aging, and cellular degeneration.[33,34,35] Activated microglial cells produce a high level of pro‐inflammatory cytokines, such as TNF‐α and IL‐1β. Retinal and other neuroinflammatory conditions are characterized by microglial activation and the release of pro‐inflammatory cytokines. In this study, at 24 h of modeling, both the RIRI group and RIRI + solvent group showed increased secretion of 20‐HETE, TNF‐α, and IL‐1β, while the early intervention with HET0016 induced reductions in the levels of 20‐HETE, TNF‐α, and IL‐1β. These findings suggested that 20‐HETE can stimulate the secretion of inflammatory cytokines TNF‐α and IL‐1β by retinal cells in RIRI, leading to aggravated inflammatory responses. At 48 h of reperfusion, the RIRI and RIRI + solvent groups showed significant microglial proliferation, whereas early intervention with HET0016 induced a substantial decrease in microglial cells. These results suggest that HET0016 can inhibit 20‐HETE–induced microglial cell proliferation in RIRI.
In conclusion, the retinal level of 20‐HETE was found to be elevated 24 h after the establishment of a rat model of RIRI, and the 20‐HETE inhibitor HET0016 could alleviate reperfusion injury through inhibition of the NF‐κB signaling pathway, reduced release of related inflammatory cytokines, downregulation of MMP‐9 expression, and suppression of microglial activation. These findings can provide a research basis for the treatment of RIRI; offer a new approach for preserving vision in patients with glaucoma, ischemic optic neuropathy, and DR; and supply new clues for further investigations into the underlying mechanisms of RIRI.
Conclusion
20-HETE can activate microglia through NF-κB pathway to participate in retinal ischemia-reperfusion injury in rats. HET0016 treatment can reduce the pathological changes of the retina.
Author contributions
All authors contributed to data analysis, drafting, and revising the article and have agreed on the journal to which the article will be submitted, gave final approval of the version to be published, and agreed to be accountable for all aspects of the work.
Data availability statement
All data generated or analyzed during this study are included in this published article.
Financial support and sponsorship
This study was supported by the Joint Construction Project of Henan Medical Science and Technology Research Project-Mechanism of 20-HETE controlling NF-κB pathway in RIRI.
Project number: LHGJ20190423
Conflicts of interest
There are no conflicts of interest.
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Associated Data
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Data Availability Statement
All data generated or analyzed during this study are included in this published article.