| Background:Retinal ischemia-reperfusion injury (RIRI) is a common pathological process and can result in vision loss. It primarily occurs due to reduction in the intraocular pressure as a consequence of ocular hypertension, thrombolytic treatment of retinal vascular occlusion, diabetic retinopathy or other ophthalmic operations able to affect the retinal blood flow. Although its mechanism has not yet been fully elucidated, inflammatory factor-mediated immune inflammation is particularly involved, while classic mechanisms such as oxygen free radical generation, calcium overload and change in nitric oxide concentration also have roles. In the subacute phase of RIRI (several hours to several days after RIRI), the expression of inflammation related genes is activated, thereby resulting in the increased production of interleukin-8 (IL-8), IL-6, tumor necrosis factor-α (TNF-α), interferon regulatory factor (IRF), nuclear factor-kappa B (NF-κB) and other inflammatory mediators. These inflammatory mediators, through a series of cascade reactions, may induce leukocyte rolling, adhesion, accumulation and infiltration to the lesions. The infiltrating leukocytes further release numerous cytokines and chemokines, causing blood-retinal barrier destruction and ultimately resulting in retinal neuronal cell death.T helper cells (Th) are traditionally divided into two cell subgroups:Thi (mainly producing interferon y and IL-2) and TI12 (mainly producing IL-4,-5, and-13). Recently, a new type of CD4+ effector cell, T helper cell 17 (Th17), is identified. Th17 cells mainly produce IL-17, which plays a key role in the inflammation, infection and defense response. Subsequent studies reveal that IL-17 is not only produced by Th17 cells but secreted by epithelial cells, neuroglial cells, endothelial cells, NK cells, γδT cells, eosinophils, neutrophils and other cells. Moreover, the production and activity of IL-17 can be regulated by other cytokines. For example, IL-23, a cytokine produced by dendritic cells, microgliocytes, phagocytes and other antigen-presenting cells can not only promote the differentiation of Th cells into Th17 cells but stimulate T cells to secrete IL-17. It has been reported that IL-23, as an upstream positive regulatory factor of IL-17, can participate in the IL-17-mediated pathophysiological process. In rat cerebral ischemia-reperfusion injury model, the IL-23 and IL-17 expressions significantly increased in the brain. Moreover, brain injury in this model could be attenuated by IL-23 antibody or IL-17 antibody or in gene knockout IL-17- or IL-23p19- animals. Similarly, IL-23/IL-17 related ischemia-reperfusion injury is also confirmed in the heart, liver, and lung. Therefore, IL-23 and IL-17 can aggravate tissue damage, and has now become a new focus in studies on ischemia-reperfusion injury. The retina as a part of brain tissue, we doubt whether the pathway of IL-23/IL-17 participated RIRI. This question is one part content of our study.Curcumin is a natural product extracted from turmeric (Curcumalonga) and has been used as an herb for centuries. Because of its anti-inflammatory, anti-oxidant, anti-cell proliferation and anti-apoptotic properties (among its other biological activities), curcumin has been widely used in studies on systemic diseases (e.g, diabetes, cardiovascular disease, multiple sclerosis, Alzheimer’s disease, and cancers) as well as ocular diseases, including diabetic retinopathy, retinitis pigmentosa, and glaucoma. Numerous studies have shown that curcumin may exert a neuroprotective effect on the retina, acting mainly as an inhibitor of retinal ganglion cell apoptosis, retinal pigment cell proliferation and vascular degeneration.This study was undertaken to explore whether the IL-23/IL-17 pathway participated in the pathological process of RIRI and to investigate the effect of curcumin on the IL-23 and IL-17 expressions in the retina of Sprague-Dawley (SD) rats following RIRI, thus providing a new theoretical basis for the treatment of RIRI with curcumin.The first part Objective:To observe the expression of interleukin-23 (IL-23) and interleukin-17 (IL-17) in rat retina after injury by ischemia-reperfusion, and to explore the role in RIRI. Methods A total of 75 SD rats were divided into normal control group (NCG) and model group (MG). The rats in NCG were not treated. The rats in MG were induced with normal saline by anterior chamber perfusion creating a RIRI model. Optical microscopy was used to observe the retinal structure after hematoxylin and eosin (H-E) staining. Immunohistochemistry staining was used to detect specific lesions expressing IL-23 and IL-17. Both levels of retina were quantified by Western-blot and Enzyme-linked immunosorbent assay (ELISA) in 12, 24,72,144 hours after RIRI. Results The retinal structure of NCG was normal, Pathological changes such as retinal edema, disorganized structure, empty spaces and loosely packed cells and inflammatory cell’s infiltration were found in MG. Immunohistochemistry staining results showed that the expression of IL-23 and IL-17 were very few in NCG, but were increased significantly in MG. The positive signal mainly located in ganglion cell layer (GCL) and inner nuclear layer (INL) of retina. Western-blot test analysis revealed that IL-23 and IL-17 expressions were increased significantly after RIRI, IL-23 was the highest at 24h, while IL-17 was the highest at 72h respectivly after RIRI. ELISA assay showed that the expression of IL-23, IL-17 in MG were increased, and had a significant statistical difference compared to NCG (P<0.01). Conclusions The expression level of IL-23 and IL-17 was increased significantly in the retina after RIRI, which maybe suggests that both aggravate inflammatory response and participate in RIRI, leading to retinal damage.The second part Objective:This study aimed to investigate the effect of curcumin on the retinal structure and the expressions of IL-23 and IL-17 in the rat retina after RIRI. Methods A total of 150 SD were randomly divided into MG, low-dose curcumin group (LDCG) and high-dose curcumin group (HDCG) (n=50 per group). RIRI was generated by anterior chamber perfusion of normal saline to the right eye. The left eye served as NCG. Rats in LDCG and HDCG received an intraperitoneal injection of 20 mg/kg/d and 100 mg/kg/d curcumin respectively, at 30 min before RIRI and once daily after RIRI. Retinal structure and inflammation were evaluated after H-E staining. Expression of IL-17 and IL-23 protein was determined by immunohistochemical method. The number of cell apoptosis in retina was examined by the TUNEL method. Western-blot and ELISA assay were used to measure the level of both expressions at 12,24,72, and 144h after RIRI. Results The retinal structure of NCG was normal. Retinal edema, disorganized retinal structure, empty spaces or loosely packed cells and inflammatory cell infiltration were observed in MG and LDCG groups, whereas the morphological changes in HDCG group were improved as compared to MG and LDCG groups. Immunohistochemistry showed that IL-23 and IL-17 were few in NCG, but increased significantly in MG and LDCG groups. The TUNEL method showed there had no brown nuclear cells in NCG, but MG retina had more apoptotic cells, mainly in the GCL and INL. LDCG and HDCG had a few apoptoic cells, the apoptosis index (AI) was deceased according to MG (vs MG, P<0.01). Western-blot assay and ELISA also showed that IL-23 and IL-17 expressions increased significantly after RIRI (vs NCG, P<0.01). Moreover, the IL-23 expression reached a peak at 24h, whereas IL-17 expression peaked at 72h after RIRI. Curcumin reduced IL-23 and IL-17 expressions significantly in a dose-dependent manner (vs MG, P<0.01). Conclusion The IL-23 and IL-17 expressions increase after RIRI and curcumin significantly reduces retinal IL-23 and IL-17 expressions at each time point in a dose-dependent manner. Then curcumin could inhibit leukocytes infiltration, down retinal neuronal cell AI and improve the retinal pathologic changes.Abvoal all, Our study results not only showed that the IL-23/IL-17 pathway participated in the pathology of RIRI but revealed that IL-23 reached its peak at 24h, whereas IL-17 peaked at 72h after RIRI.This results also showed that IL-23 acts immediately after injury, whereas IL-17 play an important role in the delayed phase of the injury. By blocking IL-23/IL-17 pathway, there will provide a better therapeutic time window for treating RIRI. Furthmore, curcumin may attenuate the retinal inflammation, reduce the retinal cell AI and inhibit the IL-23 and IL-17 expressions following RIRI. The anti-inflammation of curcumin shows new evidence for the treatment of RIRI. |
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