| Background & ObjectiveDiabetic retinopathy(DR) is a microvascular complications in patients with diabetes and is also considered to be a chronic low-grade inflammatory disease. DR is characterized by a breakdown of the blood-retinal barrier(BRB) in the early stages of diabetes. Endothelial cells are the main components in the inner blood-retinal barrier(BRB), and death of retinal endothelial cells is accumulated with the increase of duration of diabetes. Besides endothelial cell death, inflammatory agents also increase vascular permeability through activating inter cellular signals. Interleukin-1β(IL-1β) is upregulation in vitreous, retinas and serum from daibetic patients and rats. IL-1β stimulates enhanced itself synthesis via an autocrine and paracrin manner in endothelial cells, participating in and amplifying inflammatory cascade reaction of retina in diabetes. Forkhead transcription factor(Fox O1) is a transcription factor belonging to Forkhead transcription family, which plays an important role in proliferation regulation, apoptosis induction, oxidative stress suppression, autophagy and differrentiation regulation. Previous studies have demonstrated that the activity of Fox O1 is increased in retinas of type 1 and type 2 diabetic rats, accompanied by increased inflammation response and apoptosis. There might be a possible connection between the increase of Fox O1 bioactivity and inflammation in DR, but the mechanisms of the effect of Fox O1 in endothelial cells are still unclear.It has been reported that IL-1β stimulation of overexpressing the IL-1 receptor results in cytosolic and nuclear Fox O1 protein in primary rat hepotocytes and HEK293 cells. And in macrophages,Fox O1 directly binds to IL-1β promoter and increases its expression. Therefore,it is probable that IL-1β-induced retina endothelial cells overexpression of Fox O1 results in incressed IL-1β expression. Through lentiviral vector –mediated Fox O1 small-interfering RNA(si RNA) inhibiting the expression of Fox O1 in retina endothelial cells and STZ-induced diabetic rats, we investigate the effect and molecular mechanism of Fox O1 in IL-1β-induced autostimulation. MethodsHuman retina microvascular endothelial cells(HRMECs) cultured in glucose of differernt concentrations(5.6, 15, 25 or 35mmol/L) medium for 24 hours, and the expression of Fox O1 and IL-1β in HRMECs were measured by real-time quantitative PCR and Western blotting analysis. HRMECs cultured in recombinant human interleukin-1β(r IL-1β) of differernt concentrations(0, 1, 2.5, 10, 25 or 100 ng/m L) medium for 24 hours, and the expression of Fox O1, IL-1β, Caspase-8 were measured by real-time quantitative PCR(RT-PCR) and Western blotting analysis, and the location of Fox O1 were measured by immunofluorescent staining, and the rate of apoptosis were measured by flow cytometry. HRMECs cultured in IL-1 receptor antagonist(IL-1RA), the inhibitor of MAPKs(U0126,SB203580,SP600125) with or without r IL-1β(10ng/m L), and the protein level of Fox O1、p-ERK、p-P38、p-JNK were assessed by Western blotting. HRMECs cultured in normal medium were transfected with lentiviral vectors Fox O1 small-interfering RNA(LV-si-Fox O1) or empty lentiviral vectors(LVNC). HRMECs with transfection cultured with or without 10ng/m L r IL-1β for 24 hours, and the expression of Fox O1 and IL-1β were measured by real-time PCR and Western blotting analysis.30 rats were randomly selected for diabetes models from 40 SPF male SD rats, the other 10 rats as normal control group(NC group). Diabetic rats were randomly divided into three groups: diabetic group(DM group), diabetes with LV-si-Fox O1 group(LV-si-Fox O1 group), diabetes with LV-NC group(LVNC group). Fox O1-specific small-interfering RNA(si RNA) or empty lentiviral vectors(LVNC)(10μl)was intravitreally injected in diabetic rats before they are were anesthetized using a dissecting microscope, and then eyes were smeared by antibiotic ointment to protect the cornea. Serum glucose and weight were monitored monthly after injection. The rats were anesthetized with chloral hydrate at the end of twelve weeks, and the eyes were immediately removed and fixed in 4% paraformaldehyde for HE stain and immunohistochemistry, and in 4% pre-cooling glutaraldehyde for electron microscopic examination. The isolation of whole retina from freshed eyes was preserved in liquid nitrogen rapidly for detecting the expression of Fox O1 and IL-1β by real-time PCRand Western blotting analysis. Results1. High glucose promoted the expression of Fox O1 and IL-1β in HRMECsThe protein and m RNA level of Fox O1 and IL-1β were increased with increasing glucose concentration especially when the concentration was 35 mmol/L(P<0.05).2. r IL-1β-induced early apoptosis of HRMECs is concentration-dependent, but independent of Caspase-8The early apoptosis rate of normal cells was 0.5%. HRMECs cultured in virous concentration of r IL-1β(0, 1, 2.5, 10, 25 or 100 ng/m L), and the early apoptosis rate were 5.3 %, 6.6%, 8.1%,8.3%, 12.6% respectively. Different concentrations of r IL-1β did not affect the expression level of Caspase-8(P>0.05).3. Different concentration of r IL-1β stimulated upregulation of expression of Fox O1 and IL-1βCompared with normal group, the m RNA and protein levels of Fox O1 and IL-1β were elevated with increasing concentration of r IL-1β(all P<0.05). The expression of Fox O1 in the cytoplasm and the nucleus which was detected by immunofluorescent staining increases with increasing concentration of r IL-1β, and the expression was significantly increased(P<0.05), especially when the concentration of r IL-1β was 25 and 100 mmol/L.4. IL-1 receptor antagonist(IL-1RA) and the inhibitors of MAPKs(U0126, SB203580, SP600125) reduced r IL-1β-induced expression of Fox O1 in HRMECs.IL-1RA can reduced r IL-1β-induced expression of Fox O1 and IL-1β. MAPKs(p-ERK、p-P38、p-JNK) were activated by r IL-1β in HRMECs, and IL-1RA can blocked the phosphorylation of MAPKs. Compared with normal group, the protein expression of Fox O1 was inhibited by the inhibitor of MAPKs.5. The expression of Fox O1 and IL-1β were attenuated in transfected cells of LV si-Fox O1 with r IL-1β stimulation.Transfection efficiency of HRMECs as primary cells were detected by flow cytometry for transfected group and control transfected group, and the results were 68.1% and 58.5% respectively. Compared with normal group, the protein and m RNA level of Fox O1 and IL-1β were significantly increased in IL-1β group and LV-NC+r IL-1β group(P<0.001); Compared with r IL-1β group, Fox O1 was slightly reduced in LV si-Fox O1+IL-1β group(P<0.05), but the level of IL-1β was significantly reduced in LV si-Fox O1+IL-1β group(P<0.001).6. The expression of Fox O1 and IL-1β were decreased in the retinal of animal with transfection of lentiviral vector.Compared with NC group, the expression of Fox O1 and IL-1β were significantly increased in DM group(P<0.05), while the expression were insignificance in the LV-si-Fox O1 group(P>0.05). The result of immunohistochemistry showed that the immunostaining of Fox O1 and IL-1β were significantly strengthened in the DM group compared with NC group, while they were slightly strengthened in the LV-si-Fox O1 group. Immunostaining of Fox O1 was mainly showed in the retina of outer plexiform layer, inner nuclear layer, inner plexiform layer and ganglion cell layer, and it was in the retina of full layer for the expression of IL-1β. Electron microscopy showed that the morphology of mitochondria in retina endothelial cells and lumen were normal in NC group, while the mitochondria were swollen and lumen was narrowing in DM group and LVNC group. The mitochondria were slightly swollen and lumen was not narrowing in LV si group. Conclusions1. In HRMECs, IL-1β can activated and phosphorylated MAPKs through IL-1 receptor, then increased the expression of Fox O1 and IL-1β.mitochondrial swelling2. The expression of IL-1β were reduced in the retina of diabetic rats with transfection resulting in down regulation of Fox O1,accompanied by improvement of mitochondrial swelling in endothelial cell. |
PDF Full Text Request