| Background Retinal ischemia-reperfusion injury(RIRI)is a common pathological process,which leads to the death of retinal ganglion cells(RGCs)and degeneration,derangement of the retinal structure and dysfunction of the retina seriously,that can result in irreversible visual impairment and have a poor prognosis in many ophthalmic diseases.The pathogenesis of RIRI is very complicated and the mechanisms of neuronal degeneration have not yet been completely elucidated,but it has been recognized that various factors such as oxygen free radical generation,overload of intracellular calcium and microvascular injury combined effects.In addition,inflammatory factor-mediated immune inflammation and recruitment of leukocytes into the retina are particularly involved.Many studies have confirmed that there is a close relationship between RIRI and inflammatory factors.In the subacute phase of RIRI,the inflammation related genes are activated,thereby resulting in the massive releases of a variety of inflammatory cytokines,such as TNF-?,IL-1,IL-6 and other inflammatory mediators.At the same time,a variety of cytokines have a protective effect in the RIR injury.Interleukin-33(IL-33)is a recently identified multifunctional cytokine in 2005 and participates in diverse processes,including infectious diseases,allergic diseases,autoimmune disorders,cardiovascular disease and neoplastic when combined with its specific ceceptor,ST2.IL-33 is also known to function as an inflammatory cytokine to induce Th2 cytokine production.Some previous studies have demonstrated that IL-33 is a "double-edged sword" which has both proinflammatory and protective effects on the body,mainly depending on the type of disease and the immune environment.Studies have indicated that IL-33 has a protective effect on the body in the tissues and organs of ischemia-reperfusion injury.The effect may be related to IL-33 induces immuno-shift of Th cells from Thl to Th2 type response,suppress the expression of inflammatory cytokine and neutrophils activation related to infiltration,inhibits the apoptosis.However,the role and mechanism of IL-33 in RIRI have not been studied.The present study was designed to determine whether IL-33 is a protective factor in RIRI and the underlying mechanism was investigated.In recent years,with the deepening study of retinal ischemic diseases,some previous studies have demonstrated inflammatory response and apoptosis play an important role in RIRI.IL-1? and TNF-? are the most important proinflammatory factors and the release of inflammatory mediators and leukocyte infiltration amplifies inflammatory lesions in RIRI.Apoptosis is one of the major forms of injury and lead to sustained tissue damage and loss of function,especially in RGCs.Apoptosis is a special way of cell death and regulated by many genes and proteins.Bcl protein family has an important regulatory role in the process of cell apoptosis,in which Bcl-2 is the most important anti apoptotic protein,while Bax has a role in promoting apoptosis.NF-?B is a multifunctional nuclear transcription factor and a key regulator in inflammation and apoptosis in all cells.The activation of NF-?B can result in the expression of related inflammatory factors such as TNF-?,IL-1? and may mediate apoptosis in retinal ganglion cells,which is closely related to RIR injury.Matrix metalloproteinase 9(MMP-9)can degrade the extracellular matrix,destroy the blood retinal barrier,promote the occurrence of retinal edema,and participate in the inflammatory response after RIRI.A growing body of evidence has suggested that the protection of ischemic retinal neurons can be achieved by interfering with inflammatory responses and apoptotic pathways in RIRI.Therefore,in this study,we established the rat RIRI models to investigate the dynamic expression and distribution of IL-33 in the retinas,and preliminarily reveal the possible significance of IL-33 in RIRI.But it is not clear whether it is effective in promoting inflammation or inhibiting inflammation.Besides,the rats were randomized to receive an intravitreous injection of 2?g recombinant IL-33(rIL-33)or PBS at 1 hour before ischemia respectively,to observe the effects of exogenous IL-33 on RIRI in rats.Through the observation of retinal function,histopathology,inflammatory factors and apoptosis proteins,the possible mechanism of IL-33 on retinal ischemia-reperfusion injury in rats was preliminary discussed.And it can provide theoretical evidence for further therapy of retinal ischemia-reperfusion injury.Prat 1.The expression and significance of IL-33 in ischemia-reperfusion injury of retina in ratsObjective Through the establishment of RIRI model,to detecte the dynamic expression and localization of IL-33 in the retinal tissue of rats,and to investigatethe possible role of IL-33 in RIRI.Methods 60 SD rats were randomly divided into six groups using digital table method:the normal control group(NCG)and model group(MG),and MG group was divided into five points in time of 6 hours,12 hours,24 hours,72 hours and 144 hours,and there were 10 rats in each point.Retinal ischemia reperfusion injury was induced by anterior chamber perfusion method of raising the intraocular pressure in the right eye.Optical microscopy was used to observe the retinal structure at each time point after Hematoxylin and eosin(H-E)staining.Immunohistochemistry staining was used to detect the expression and localization of IL-33 protein in the retina.The level of IL-33,IL-? and TNF-? at each time point in retina was detect by using Enzyme linked immunosorbent assay(ELISA)method and the correlation between the several kinds of cytokines was analyzed.Results The RIRI model was successfully established.HE staining showed that the retinal structure and erch layer were normal,neat,and there were very few inflammatory cell infiltration.Pathological changes such as retinal edema,loosely arranged cells and disorganized we structure re found in MG at each time point.Nerve fiber layer and INL is particularly evident,accompanied by many inflammatory cells infiltration.Immunohistochemical staining showed that IL-33 in the retina of NCG was low expression,but was significantly enhanced in MG.The positive signal mainly expressed in the nucleus and cytoplasm in retinal ganglion cell layer(GCL)and INL.ELISA assay results showed that the expression level of IL-33 in MG at 6h,12h,24h,72h,144h in retina were increased,and the differences were statistically significant compared with the NCG group(P<0.01).The expression of IL-33 peaked at 24h and was positively correlated with the levels of IL-1? and TNF-?(r=0.845,0.895;P=0.034,0.016).Conclusion The expression of IL-33 in rats retinal tissues with RIRI increased significantly,and the expression level was positively correlated with other inflammatory factors,suggesting that IL-33 may play an important role in RIRI.Part 2.Effect of IL-33 preconditioning on retinal ischemia-reperfusion injury in ratsObjective To observe the effect of IL-33 preconditioning on retinal ischemia-reperfusion injury in rats.Methods 60 SD rats were randomly divided into four groups using digital table method:(1)normal control group(NCG),(2)RIR injury model group(MG),(3)IL-33 pretreatment group(IL-33),(4)PBS group(PBS),(n=15 per group).Rats in IL-33 group and PBS group received an intravitreous injection of 2?g recombinant IL-33(Peprotech,America)or an equal amounts of PBS with microinjector at 1 hour before RIR injury modeling respectively.All the animals were taken right eye for modeling.After reperfusion for 24h,the following tests were performed:(1)Electroretinogram(ERG)detection,(2)HE pathological staining to observe the retinal tissue structure changes,infiltration of inflammatory cell number,the thickness of retina and the number of RGCs under the light microscope;(3)Terminal deoxynucleotidyl transferase dUTP Nick marker enzyme mediated(Tunel)method to detect the cell apoptosis in retinas.Results ERG results showed that compared with the NCG group,the b wave amplitude of MG group were significantly decreased(t=5.765,P=0.000).It indicated that ischemia-reperfusion can obviously damage the physiological function of retinal inner layer cells.While in IL-33 group,b wave amplitude were significantly higher than those in MG group and PBS group(t=2.420,3.024;P=0.022,0.005)and b wave ratio were significantly higher than those in MG group and PBS group(t=9.144,9.597;P=0.000,0.000).HE staining results showed eduction of the retina edema,more neatly cells arranged and decreased inflammatory cell infiltration could be found in IL-33 group.The number of inflammatory cell had significant difference compared with MG and PBS group(t=3.697,4.715;P=0.0017,0.0002).The retinal ganglion cell count in IL-33 group had significant difference(t=2.807,3.188;P=0.012,0.005)compared with MG and PBS groups.Tunel proved that the apoptotic cells in IL-33 pretreatment group showed low positive expression,and the apoptotic index was significantly lower than that in group MG and group PBS,the difference was statistically significant(t=4.560,5.225;P=0.0002,0.0001).The data revealed that IL-33 pretreatment maintained better retinal function and structure,inhibited leukocytes infiltration and reduced apoptosis of retinal ganglion cells.Conclusion IL-33 preconditioning can protect retinal function and tissue structure and inhibit the apoptosis of retinal ganglion cells after ischemia-reperfusion injury in rats.Part 3.Preliminary study of the mechanism of IL-33 precon-ditioning on retinal ischemia-reperfusion injury in ratsObjective To discuss the underlying mechanism of IL-33 pretreatment on retina protection in RIRI in rats,and to provide a new way and theoretical basis for the clinical treatment of RIR injury.Methods 100 SD rats were randomly divided into normal control group(NCG),RIR injury model group(MG),IL-33 pretreatment group(IL-33),and PBS group(PBS),(n=25 per group).The RIR injury model was established by acute elevated intraocular pressure to the right eye.Rats in IL-33 group received an intravitreous injection of 2?g recombinant IL-33(rIL-33)at 1 hour before ischemia respectively.The rats were sacrificed at 24 hours after reperfusion and the eyeballs were obtained to detect the following tests:(1)Immunohistochemistry was used to observe the expression of Bcl-2 and Bax;(2)The protein level of Bcl-2,Bax and NF-?B p65 in the retina was detected by Western blotting;(3)The mRNA level of Bcl-2,Bax and MMP-9 in the retina was detected by Real-time PCR;(4)The level of IL-10 and TNF-? in retina was detect by using ELISA method.(5)MMP-9 activity was detected using Gelatin Zymography assay.Results Immunohistochemistry confirmed that pretreatment with IL-33 could induce the expression of Bcl-2(t=2.334,2.388;P=0.0479,0.0440)and inhibit the expression of Bax(t=4.794,5.190;P=0.0014,0.0008)than those in MG and PBS group.Western blot also showed that IL-33 upregulated the expression of Bcl-2(t=3.339,2.686;P=0.0102,0.0277)and decreased the expression of Bax(t=4.010,4.406;P=0.0039,0.0023)and NF-?B p65(t=3.404,3.721;P=0.0093,0.0059)compared with MG and PBS group.Real-time PCR indicated that the expression of Bcl-2 mRNA in IL-33 group was significantly increased(t=9.195,9.887;P=0.000,0.000),while the expression of Bax mRAN(t=5.911,5.793;P=0.0004,0.0004)and MMP-9 mRNA(t=5.175,6.152;P=0.0008,0.0003)decreased significantly,as compared to the MG and PBS group.Gelatin zymography assay showed that IL-33 pretreatment inhibited the activity of MMP-9(t=5.781,7.023;P=0.0004,0.0001)compared with MG and PBS group.ELISA demonstrated that IL-33 could inhibit the release of IL-1?(t=10.349,P=0.000)and TNF-?(t=7.200,P=0.0001).Conclusion IL-33 can prevent apoptosis,attenuate NF-?B p65 presence in the retina and thereby inhibited the activation of NF-?B,inhibit the release of IL-1? and TNF-?,and reduce the expression and activity of MMP-9.These findings suggest that IL-33 may be a new promising agent which can attenuate the RIR injury by reducing inflammatory cell infiltration and preventing apoptosis.Above all,our study showed a new cytokine,IL-33,participated in the pathology of RIRI first time through the establishment of SD rat model of RIRI in the first part of this paper.We revealed that IL-33 reached its peak at 24h and positively correlated with other inflammatory cytokines,suggesting that IL-33 may play a an important role in retinal ischemia reperfusion inflammatory responses after injury,involved in the pathological process of RIRI.IL-33 is an "early warning molecule",which can be secreted by damaged or necrotic epithelia and endothelial cells when stimulated by inflammatory factors and physicochemical factors.But the precise role of IL-33 in the pathogenesis of RIRI is not clear,so the second part of this thesis revealed that IL-33 can protect the function and structure of RIR injury by IL-33 pretreatment before modeling.In the third part,we further explore the possible mechanism,and find that IL-33 pretreatment can reduce the release of inflammatory factors,regulate the expression of related apoptotic proteins and reduce the apoptosis of retinal neurons.IL-33 may be an anti-inflammatory factor and an inhibitor of apoptosis in the rat model of RIRI and contribute to the development of RIRI combined with proinflammatory factor.There is a dynamic balance between IL-33 and proinflammatory factors.However,the specific transmission pathway of IL-33 and its signaling pathway IL-33/ST2 in retinal ischemia-reperfusion injury should be further studied. |
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