| Objective:To investigate the role and mechanism of IL-6 inhibitor LMT-28 in prevention of early-stage Diabetic retinopathy(DR)through regulation of gp130/STAT3 signaling pathway.Methods:(1)Building a model of type 1 diabetic mellitus(DM)Sprague-Dawley(SD)rat,as the DM group,and health rats as normal controls(NCs).The body weight,blood glucose and other systemic metabolism and retinal morphological changes were observed after modeling.(2)The inflammatory factors(VEGF,IL-6,ICAM-1,TNF-α),gp130,signal transducer and activator of transcription 3(STAT3),glial fibrillary acidic protein(GFAP),tight junction protein(ZO-1,CLDN5,occludin)in retina of DR rats were observed by pathological and molecular biological methods.(3)The concentrations of IL-6 in serum,aqueous humor and vitreous were detected by ELISA at 2th,4th,6th,8th and 10 th week after DM modeling.(4)Combined with the changes of retinal histological phenotype and molecular phenotype in diabetic rats,we determined the early-stage DR model and observed the expression and function of gp130/STAT3 signaling pathway in early-stage DR.(5)To observe the effect of intravitreal different concentrations of IL-6 inhibitor LMT-28 injection in prevention of early-stage DR.Results:(1)The early-stage model of DR was built,and the findings were as follows:(1)HE staining showed that the thickness of retinal nerve fiber layer in DM group was thinner than that in NCs from the 4th week to the 10 th week after modeling;At 8th and 10 th week after modeling,the cell density of ganglion cell layer in DM group was lower than NCs,and the thickness of retinal neuroepithelial layer was thinner than NCs;The above differences were statistically significant(P < 0.05).(2)Periodic Acid-Schiff staining showed that the ratio of endothelial cells/pericytes and the number of acellular capillary in DM group were significantly increased compared with NCs from 6th to 10 th week after modeling(P < 0.01).Pericytes ghost and hemangiomas were observed in retinal capillaries in DM group at10 th week.(3)Evans blue staining showed: at 4th week after modeling,compared with NCs,there were a small amount of local staining in the retina of DM group;At the 6th and8 th week,the retinal fluorescent leakage in DM group showed cloud like background high fluorescence,and at the 10 th week,the fluorescent leakage showed multiple dot like strong fluorescence.(4)Changes in the transcription level of inflammatory factors in the retina:compared with NCs,VEGF level in the retina of DM group was increased at 2th and8 th week after modeling,but decreased at 6th week;IL-6 level in the retina of DM group was increased at 2th,4th and 10 th week compared with NCs;ICAM-1 and TNF-α levels were increased in the DM group from the 2th week to the 10 th week after modeling compared with NCs;The above differences were statistically significant(P < 0.05).(5)ELISA test showed that IL-6 level in serum of DM group was increased from the 4th week to the 8th week after modeling compared with NCs;From the 2th week to the 10 th week,IL-6 level in aqueous humor of DM group was increased compared with NCs;From the 4th week to 10 th week,IL-6 level in vitreous of DM group was increased compared with NCs;The above differences were statistically significant(P< 0.05).(6)Expression of STAT3 in retina: from the 6th week to 10 th week after modeling,the transcription and translation levels of STAT3 in retina of DM group were increased compared with NCs,the difference was statistically significant(P <0.05).(7)Expression of STAT3 regulatory axis related genes in retina: the transcription level of gp130 in retina of the two groups were the same throughout the course of the disease(P > 0.05).The transcription level of JAK2 in the retina of DM group was increased compared with NCs at 8th and 10 th week,while the negative regulator cell signal transduction inhibitor 3(SOCS3)was increased at 2th,6th and 10 th week,and caspase 7 was significantly increased at 10 th week;The level of SOCS3 protein in the retina of DM group was increased compared with NCs at 2th and 10 th week after modeling,but decreased at 4th and 6 week;The above differences were statistically significant(P < 0.05).(8)Expression of GFAP in retina: at 2th,8th and 10 th week after modeling,the transcription and translation levels of GFAP in the retina of DM group were increased compared with NCs,but decreased at 6th week(P<0.05).Immunohistochemical results showed that the GFAP staining of retina in DM group was significantly stronger than that in NCs at 8th and 10 th week.(9)Expression of tight junctional protein in retina: at 8th week and 10 th week after modeling,the transcription and translation levels of CLDN5 and occludin in retina of DM group were decreased compared with NCs;At 8th week after modeling,the level of ZO-1 m RNA in the retina of DM group was decreased compared with NCs,but increased at 10 th week;From 2th week to 6th week,compared with NCs,ZO-1 protein level in the retina of DM group was decreased,but increased at 8th and10 th week;The above differences were statistically significant(P < 0.05).(10)TUNEL staining showed that apoptosis was observed in the retinal outer nuclear layer in DM group at 4th,8th and 10 th week after modeling,and increased with the progress of the disease.(2)Effect of intravitreal LMT-28 injection on early-stage DR:(1)After intravitreal injection of low concentration(LMT-28 L)and high concentration(LMT-28 H),the STAT3 level in LMT-28 L and LMT-28 H groups was significantly decreased compared with DR group(P < 0.05),but there was no significant difference between the two groups(P > 0.05).(2)After intravitreal LMT-28 solution injection,the occludin level in LMT-28 L and LMT-28 H groups were increased compared with DR group,and LMT-28 H was higher than LMT-28 L(P < 0.05).Compared with DR group,GFAP level was decreased in the two groups,and the level in LMT-28 H was lower than d LMT-28 L(P < 0.05).(3)Retina evans blue staining showed that after intravitreal LMT-28 solution injection,the retinal leakage in LMT-28 H and LMT-28 L groups was decreased compared with DM group,but there were still some vascular leakage.Conclusion:(1)The early-stage DR was formed at 8th week in STZ-induced diabetes rat;(2)IL-6 induced gp130/STAT3 signaling pathway activation,increased retinal vascular leakage in DR,through downregulated the expression of tight junction proteins.(3)LMT-28 had a good effect on early-stage DR through regulation of gp130/STAT3 signaling pathway.and can slow down the progress of early-stage DR. |
PDF Full Text Request