Study On The Therapeutic Effect Of FTY720 On Diabetic Retinopathy And Its Mechanisms

Background: In the recent years, with the change of people’s diet and lifestyle, the number of patients with diabetic mellitus(DM) is rising year by year and the morbidity of diabetic retinopathy(DR) is growing rapidly, becoming one of the main causes of blindness in working age population.The pathogenesis of DR remains unknown. Inflammatory mechanism has become the research hotspot in recent years and plays an important role in the pathogenesis of DR, contributing to leukostasis and blood retinal barrier(BRB) breakdown. FTY720 is a new immunosuppressant made of the effective components extracted from cordyceps sinensis, and it acts mainly via directly binding to the sphingosine 1 phosphate – receptor(S1PR) expressed on cell surface. Current studies have shown that FTY720 is effective in various inflammatory diseases and cardiovascular diseases, while it remains unknown whether FTY720 has a therapeuty effect on DR. S1P1 and S1P3 are main mediators cooperating in maintenance of endothelial barrier integrity. However, it is unclear whether S1P1/S1P3 is involved in the pathogenesis of DR. Addionally, whether FTY720 can protect BRB in DM rats via directly S1P1 / S1P3 modulation remains unknown and its needs to be explored further.Objects:(1) To explore the therapeutic effect of FTY720 on DR in diabetic rats, including suppression of inflammation and BRB protection;(2) To explore whether retinal S1P1/S1P3 is involved in the pathogenesis of DR;(3) To explore whether FTY720 has the protective effect on BRB in diabetic rats via directly S1P1/S1P3 modulation.Methods:(1) 45 male Wistar rats were randomly divided into normal control group(n=15) and DM group(n=30). Rats in DM group were induced to develop DR by giving an intraperitoneal injection of streptozotocin(STZ, 60mg/kg). Following DM model establishment, body weight and fast blood glucose of all rats were measured at 4w, 8w and 12 w respectively; the rats in DM group were sacrified at 4w and 12 w respectively, and rats in control group were sacrified at 12 w. Hematoxylin and eosin(HE) staining was used to analyze pathological changes in retinas. Evens Blue(EB) angiography and retinal flat mounts were used to analyze retinal vascular permeability. Western blot was used to analyze retinal expression of VEGF.(2) 150 male Wistar rats were induced to develop DM by giving an intraperitoneal injection of STZ(60mg/kg). Following DM model establishment, all diabetic rats were randomly divided into DM group and FTY720 group(DM+FTY720 group). Normal rats were used as controls(control group). There were 50 rats in each group. FTY720 was administered at a dose of 0.3 mg/kg in distilled water by oral gavage daily for 12 weeks. Body weight and fast blood glucose of all the rats were measured at 4 w 8w and 12 w after DM onset. All the rats were sacrificed and used for experiments at 12 weeks after DM model establishment. HE staining was used to analyze pathological changes in retinas. Peripheral blood samples were taken to detect the number of circulating lymphocytes by veterinary hematology system. Western blot was used to analyze the expression of inflammatory cytokines in retinas, including TNF-α、IL-6 and IL-1β. FITC-Concanavalin A(Con A) angiography was used to detect the adherent leukocytes in retinal vessels and all retinal adherent leukocytes were counted under confocal florescence microscopy. The expression and distribution of intercellular cell adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1(VCAM-1) were analyzed by Western blot, immunochemistry and quantitative real time-polymerase chain reaction(RT-PCR).Western blot and RT-PCR were used to analyze the expression of NF-κB(p65) at both protein and m RNA level. Infiltration of inflammatory cells in the retina was detected via immunofluorescence, including CD45 positive total leukocytes, CD11 b positive macrophages and/or activated microglia, and CD3 positive lymphocytes. EB angiography and retinal flat mounts were used to analyze retinal vascular permeability. Additionally, the amount of retinal EB leakage was quantified. Both Western blot and immunochemistry were used to detect the expression and distribution of tight junction proteins in retinas, including ZO-1, Occludin and Claudin-5. Expression of S1P1 and S1P3 was detected via RT-PCR at m RNA level.Results:(1) At each time point, fast blood glucose levels of rats in DM group were significantly increased, while their body weight was significantly decreased compared with control group. HE staining showed disordered and loose arrangement of cells in each retinal layer, and vacuolar degeneration of nucleus and telangiectatic vessels appeared in retinal ganglion cell layer(GCL). EB angiography showed that there was a small amount and local EB leakage at 4w after DM onset, while diffuse leakage appeared at 12 w after DM onset. Retinal expression of VEGF was significantly upregulated in DM group compared with control group, and its expression was significantly higher in DM12 w group than DM4 w group.(2) At each time point, fast blood glucose levels of rats in DM group were significantly increased, while their body weight was significantly decreased compared with control group. HE staining showed thinning of retinal thickness, disordered arrangement of cells in each retinal layer, unclear boundary of each retinal layer, cell nucleus swelling and telangiectatic vessels in DM group; while after treatment with FTY720, all the changes were alleviated; Analysis of periphery blood samples showed the number of circulating lymphocytes in DM+FTY720 group was significantly decreased,compared with DM group. Expression of inflammatory cytokines was significantly unregulated in DM group compared with control group, including TNF-α、IL-6 and IL-1β. However, their expression were significantly decreased in DM+FTY720 group, compared with DM group. FITC-Con A angiography showed that there was a large amount of adherent leukocytes in retinal vessels of diabetic rats and mainly at the position of the vessular furcation, while nearly no adherent leukocytes could be seen in DM+FTY720 group. The result of adherent leukocytes counting showed that the number of adherent leukocytes in retinal vessels was significantly decreased in DM+FTY720 group, compared with DM group. Immunochemical staining showed that ICAM-1 and VCAM-1 positive staining was abound in DM group and mainly in GCL and nerve fiber layer(NFL), while both positive staining was very little in DM+FTY720 group. Western blot and RT-PCR analyses demonstrated that expression of ICAM-1 and VCAM-1 in DM group was significantly higher than control group at both protein and m RNA levels, while their expression was significantly decreased in DM+FTY720 group compared with DM group. Western blot and RT-PCR analyses demonstrated that expression of NF-κB(p65) was significantly higher in DM group than control group at both protein and m RNA levels, while its expression was significantly decreased in DM+FTY720 group when compared with DM group. Immunofluorescent staining demonstrated there were many retinal CD45 positive leukocytes in DM group, including CD11 b positive macrophages/activated microglia. However, both positive cells were very few in DM+FTY720 group. Nearly no CD3 positive lymphocytes could be seen in all three groups. In control group, retinal vascular structure was clear and no leakage could be seen. There was diffuse leakage and vascular tortuosity in retinas of DM group, while only local retinal leakage could be seen in DM+FTY720 group. Quantification of retinal EB leakage showed retinal vascular leakage was significantly increased in DM group compared with control group, while it was significantly lower in DM+FTY720 group than DM group. Immunochemical staining showed that there were abundantly ZO-1, Occludin and Claudin-5 positive staining in retinas of control group, mainly in GCL, NFL, outer plexiform layer(OPL) and inner nuclear layer(INL); only a few retinal positive staining could be seen in DM group; retinal positive staining of these tight junction proteins in DM+FTY720 group was relatively higher than DM group. Western blot analysis demonstrated that retinal expression of ZO-1, Occludin and Claudin-5 was significantly decreased in DM group in comparison with control group, while their expression was significantly higher in DM+FTY720 group than DM group. RT-PCR analysis showed that there was abound expression of S1P1 and S1P3 in retinas of control rats at m RNA level. However, their expression was significantly decreased in DM group, which was reversed by FTY720 treatment..Conclusions:(1) FTY720 has a therapeutic effect on DR in diabetic rats, through suppressing retinal inflammation and protecting BRB;(2) Downregulation of S1P1/S1P3 is involved in the BRB breakdown in diabetic rats;(3) FTY720 protects BRB through two mechanisms, one is inflammatiory supression and the other one is upregulation of S1P1/S1P3 expression.