Effects Of Interleukin-1beta On Glutamine Synthetase In Retinal Müller Cells In Early Diabetic Rats

Diabetes mellitus is a common general disease because of disorder of glycometabolism and attack rate of the disease upgrades year by year. It is estimated by WHO that the patients suffered from this disease will be up to 300 million in 2010．Diabetic retinopathy is the most severe ocular complication of diabetes, which is the leading cause of blindness in a lot of countries. Until now, the mechanism of diabetes mellitus is not understood clearly. Many causes are involved in this disease, including abnormal polyalcohol pathway, advanced glycosylation end products, activation of protein kinase C, oxidative stress, cytokine factors and adhesion moleculers. Many experimental evidences have showed that the pathogenesis of this condition is vascular changes, including vascular permeability, vascular lesions, leukostasis and neovasculation, but microangiopathy is just one aspect of diabetic retinopathy. A number of observations suggest that retinal neurons and glia are also involved in the pathogenesis. Furthermore, they precede the onset of clinically evident vascular injury. A series of studies reported this condition, including loss of color and contrast of sensitivity and abnormal eclectroretinogram in patients before vascular alternations were founded in their retinae. Thus, many evidences have showed that a comprehensive understanding of diabetic retinopathy requires elucidation of the mechanisms by which diabetes affects not only vascular but also nonvascular cells of the retina. Because of their special structures and functions, Müller cells play an important role in diabetic retinopathy.Generally, the mammalian retina contains three types of glial cells. In addition to microglial cells, there are two forms of neuron-supporting macroglial cells, astrocytes and Müller (radial glial) cells. Müller cells, the principal glia of the retina play a number of important roles in normal and disease state. They span the entire depth of the neural retina, radiating from the soma is an inwardly directed process that terminates in an expanded endfoot at the inner border of the retina. Also projecting from the soma is an outwardly directed process that ends in the photoreceptor layer. Microvilli project from this apical process into the subretinal space surrounding the photoreceptors. Endfeet of Müller cells contact and surround blood vessels and secondary processes branching from the main trunk of Müller cells form extensive sheaths that surround neuronal cell bodies, dendrites, and in the optic-fiber layer, the axons of ganglion cells. To some degree, Müller cells are the bridges between the vessels and neurons in retina. In the normal retina, Müller cells(l) regulate extracellular ions concentrations through voltage-gated and transpaort mechanism, (2) regulate the cycle of glutamate by voltage-gated and chemical mechanism, (3) are involved in regulating intraretinal acid-base physiology through bicarbonate ion transporters and carbonic anhydrase-catalyzed CO₂ hydration, (4) provide metabolic support for a series of cells in retina through glucose uptake, glycogen metabolism, aerobic and anaerobic glycolysis, (5) support phtotreceptor oxidative metabolism by producing lactate and (6) regulate the blood flow in retina by chemical mediator. In diabetic retinopathy,(1) Müller cells change the expression and disposition of GFAP and Occludin, which resulting in damage to BRB, (2) the transporter of glutamate and GS in Müller cells are abnormalized, (3) Müller cells involve in PDR because of transformation to fibroblast, (4) functions of retinal vessel are damaged due to abnormal K⁺ ion channel, (5) disturbance of acid-base balance in retina.It was reported that in early diabetic retinopathy, blood-retinal barrier is compromised. Usually, plasma contains 100 to 300μM of glutamate and only 5μM glutamate can be lethal to neurons. A predominant function of Müller cells is to regulate glutamate levels. If extracellular concentration of glutamate exceeds the normal level, retinal neurons can be killed. The glutamate-glutamine cycle in the retina is functioned mainly by glutamate transporter and glutamine synthetase (GS). The transporter of glutamate can transport glutamate into Müller cells and GS can catalyze the amidation of glutamate to glutamine, which removes extra glutamate from the extracellular space efficiently so that the retina can be in healthy condition. However, in diabetes, the ability of regulation of extracellular concentration of glutamate in Müller cells maybe compromised. Ambati and Lieth reported increased glutamate concentrations within the vitreous of patients with diabetes and in diabetic rat retinas. A number of studies have reported the dysfunction of the glutamate transporter in retinal Müller cells in diabetes, but studies about the influence of GS on glial glutamate metabolism in the condition is not intensive. Consequently, our study is focused on GS. GS is a glial-specific enzyme, which is the key enzyme to control the recycling of the neurotransmitter glutamate and prevent the glutamate accumulation. Glutamate, released by glutaminergic neurons, is taken up by glial cells and converted by GS to the nontoxic amino acid glutamine. Glutamine is then released by the glial cells and returns to the neurons, where it is hydrolyzed by glutaminase to form glutamate again. In diabetic retinopathy, the recycle is abnormal. Lieth and LaNoue found down-regulation of GS in diabetic rat retinas. The molecular mechanisms that modulate GS expression in diabetes are not yet known.Recent studies have suggested that the pathogenesis of diabetic retinopathy is similar to a chronic inflammatory disease. Some studies reported that in the vitreous fluid of the patients with proliferative diabetic retinopathy and in the retinae of diabetic rats, increased levels of cytokines, including interleukin-1(?) (IL-1(?)), IL-6 and IL-8 were detected. A current study reported that IL-1(?) can accelerate apoptosis of retinal capillary via action of NF-kB, and the process is exacerbated in high glucose conditions. In diabetes, the expression of IL-1(?) dramatically upregulates in the glial cells and endothelial cells, the abnormality is similar to those observed in retinal ischaemia reperfusion. It suggests that IL-1(?) may play an important role in the pathogenesis of diabetic retinopathy. Especially, some researchers consider IL-1(?) may play a primary role in Müller cells in diabetic retinopathy.The purpose of this project was to investigate the role of IL-1(?) on the GS in retinal Müller cells in normal and high glucose conditions or in experimentally diabetic rats and the mechanism of changes of expression of GS in retinal Müller cells induced by IL-1(?).Partâ… Changes of expressions of GS, c-Jun and IL-1(?) in retinas in early diabeticratsSectionâ… [Abstract] Objective To investigate the changes of expressions of GS, c-Jun, and IL-1(?) in retinas in early diabetic rats. Methods Diabetic rats were induced with streptozotocin injection and divided into four groups at random (control, 1month, 2month or 3month group). The expressions of GS, IL-1(?) and c-Jun in retinas in experimental and control groups were analyzed by immunohistochemistry and western blot. Expressions of their mRNAs were detected by Realtime-RT-PCR. Results The expressions of GS and GSmRNA were not changed in retinas in control group or in 1m and 2m groups, but they decreased obviously in 3m group(P<0．01) . The expressions of c-Jun ,IL-1(?) and their mRNAs in retina in control group were lower, but in retinas of diabetic rats they increased significantly .especially in 3 month group(P<0．01) . Conclusion In retinas in early diabetic rats, it was founded that expressions of GS decreased and expressions of c-Jun and IL-1(?) increased. The possible mechanism may be activation of c-Jun by IL-1(?).Sectionâ…¡[Abstract] Objective To investigate the changes of expressions of GS, c-Jun and IL-1(?) in retinal Müller cells in high glucose conditions. Methods Cultured Müller cells were divided into control group (5mMol/l glucose) or experimental group (25mMol/l glucose ) at random. After incubation for 24h, the expressions of GS, IL-1(?) and c-Jun in retinal Müller cells in experimental and control groups were analyzed by immunohistochemistry and western blot. Expressions of their mRNAs were detected by Realtime-RT-PCR. Results From the data, it was founded that in high glucose conditions, expressions of GS and GSmRNA were down-regulated and expressions of of c-Jun, IL-1(?) and their mRNAs were up-regulated signicantly. Conclusion High glucose may result in decrease in GS expression and increase in c-Jun and IL-1(?) expressions in Müller cells. It is suggested that IL-1(?) may down-regulate expression of GS by activating c-Jun.Partâ…¡Effect of IL-1βon the expression of GS in retinal Müller cells andpossible mechanism[Abstract] Objective To investigate the change of expression of GS by IL-1(?) in retina Müller cells in early diabetic rats and its possible mechanism. Methods (1) After injection of different doses of IL-1(?)(0, 100, 500, 1000ng/ml) into the vitreous cavities of 48 rats (each group has 12 rats) for 24 h, the expressions of GS , c-Jun and their mRNAs in retina were analyzed by indirect immunofluorescence, western blot or Realtime-RT-PCR respectively. (2) Cultured Müller cells were divided into control group (5mMol/l glucose) or experimental group (25mMol/l glucose ) at random. After incubation with different doses of IL-1(?) (0, 0．1,1, 5, 10ng/ml) for 24h, the expressions of GS and c-Jun in retinal Müller cells in experimental and control groups were analyzed by indirect immunofluorescence and western blot. Expressions of their mRNAs were detected by Realtime-RT-PCR too. Results (1) After injection of different doses of IL-1(?) into the vitreous cavities of rats for 24 h, it was found that expressions of GS in 500ng/mlIL-1(?) and 1000ng/mlIL-1(?) groups decreased significantly compared with their expressions in control group. In 100ng/mlIL-1(?) group, expression of c-Jun increased significantly and expressions of c-Jun could be up-regulated in a dose-dependent manner by IL-1(?). (2) After being treated with different doses of IL-1(?) for 24h, expressions of GS was down-regulated significantly and expressions of c-Jun was up-regulated significantly in a dose-dependent manner from the dose of lng/ml IL-1(?), especially in high glucose conditions. Conclusions After being injected with IL-1(?), the down-regulation of GS was founded, which may cause elevation of glutamate so that the ganglion cells were damaged. The possible mechanism may be activation of c-Jun by IL-1(?). Partâ…¢To study the effect of IL-1ra against IL-1βon the expression of GS inretinal Müller cells[Abstract] Objective To study the effect of IL-1ra against IL-1βon the expression of GS in retinal Müller cells and possible interventional method. Methods (1) After injection of different doses of IL-1ra (0, 500, 1000ng/ml) with 1000ng/ml IL-1(?) into the vitreous cavities of 27 rats (each group has 9 rats) for 24 h, the expressions of GS , c-Jun and their mRNAs in retinas were analyzed by indirect immunofluorescence, western blot or Realtime-RT-PCR respectively. (2) Cultured Müller cells were put into 25mMol/l glucose conditions, 100ng/mlIL-1ra was added into experimental group and nothing was added in control group. Also another two groups were set up, 100ng/mlIL-1ra with 10ng/mlIL-1(?) were added into one group, in another group only 10ng/mlIL-1(?) was added. After incubation for 24h, the expressions of GS and c-Jun in retinal Müller cells were analyzed by indirect immunofluorescence and western blot. Expressions of their mRNAs were detected by Realtime-RT-PCR too. Results (1) After injection of different doses of IL-1ra with 1000ng/ml IL-1(?) into the vitreous cavities for 24 h, it was founded that in 0ng/ml or 500ng/ml IL-1ra group, expression of GS or c-Jun was not changed obviously. But in 1000ng/ml IL-1ra group, it was detected that expression of GS increased or c-Jun decreased significantly. (2) After being treated by IL-1ra for 24h in high glucose conditions, it was founded expression of GS was up-regulated and expression of c-Jun was down-regulated significantly respectively in experimental group. After being treated by IL-1(?) with or without IL-1ra for 24h in high glucose conditions, it was detected that IL-1ra was against the effect of IL-1(?) on expressions of GS and c-Jun. Conclusions The possible mechanism that IL-1(?) down-regulates GS expression by activating c-Jun was investigated again. It is suggested that IL-1ra can be against the effect of IL-1(?) on GS expression. Partâ…£Effects of IL-1βon the proliferation and apoptosis of retinal Müller cellsin high glucose conditions and their possible mechanisms[Abstract] Objective To study the effects of IL-1βon the proliferation and apoptosis of retinal Müller cells in high glucose conditions and their possible mechanisms. Methods Cultured Müller cells were divided into control group (5mMol/l glucose) or experimental group (25mMol/l glucose ) at random. After incubation with different doses of IL-1(?) (0, 0．1,1, 5, 10ng/ml) for 24h, the proliferation of Müller cells was determined with MTT method and apoptosis of Müller cells was analyzed by flow cytometry after Annexin V-fluorescein isothiocyanate/Propidium idoium(Annexin V-FITC/PI) staining. Expressions of pSTAT3 induced by IL-1βin Müller cells were analyzed by immunohistochemistry and western blot respectively. Results Proliferation of retinal Müller cells increased in two groups with increasing of concentration of IL-1(?), especially in high glucose condition. Apoptosis of retinal Müller cells in two groups was not changed obviously with increasing of concentration of IL-1(?). Expressions of pSTAT3 induced by IL-1βin Müller cells increased significantly from the concentration of 1ng/ml IL-1βin a dose-dependent manner. Conclusions It is suggested that in mimic diabetes situation IL-1βmay accelerate proliferation of retinal Müller cells. Accelerating proliferation of Müller cells by activation of pSTAT3 may be possible mechanism. At the same time, apoptosis of Müller cells induced by IL-1βis not founded.