Protection Of Signal Transduction Pathways Regulated By AG On Retina Of Rat With Chronic Elevation Of IOP

IntroductionNowadays the main treatment for glaucoma is to lower the intraocular pressure (IOP)by way of medicine or surgery;however,simply lowering the IOP is not enough to prevent the progressive damage to the optic nerve.Other pathomechanisms can also cause the damage to the retinal ganglia cells and optic nerve.All factors that cause the apoptosis of RGC such as toxicity of Glutamate,interruption of nutrition factors, abnormality of blood vessels,activation of glial cell and toxicity of NO etc,are related to the pathological changes of glaucoma.However,the mechanisms of signal pathways of all those toxic factors and regulation of apoptosis gene are not clear.Therefore,it is important to make sure how the signal pathway works and the regulation of apoptosis gene from different levels.Ischemia-reperfusion model is received as an important model to study the retinal damage and RGC apoptosis;however it is not so commonly used in chronic intraocular hypertension.In the process of chronic intraocular hypertension,the retinal nerve fibre layer is thinning and the apoptotic retinal ganglia cells are increasing as the time goes, accompanied with the abnormal activation or inhibition of signal transduction pathways and abnormal expression of apoptosis related genes.Thus the assessment of the regulation of various signal transduction pathways and apoptosis related genes can be a reliable indicator to evaluate nerve protectants.Signal transduction pathway and apoptosis related gene is a hot topic is today's molecular biology study.It has been found that there are several transcription factor and signal transduction pathway related to cell growth,proliferation,and apoptosis; they are PI-3K(phosphatidylinositol-3-kinase)/AKT pathway,NF-κB(transcription factor)pathway and the regulation of cell apoptosis related gene Caspase-9(Cysteine proteinase)and so forth.PI-3K also known as phosphatidylinositol-3-kinase is a special kinase to catalyze the reaction of phosphatidylinositol.It has been found in recent decade that PI-3K is an ester type second messenger related to cell transduction.Some study presumed that PI-3K and its downstream molecule AKT are involved in the regulation of the activity of NF-κB(16);moreover,PI-3K-AKT pathway can regulate cell survival by inhibiting the activation of apoptotic promoter,such as Caspase-9.NF-κB also named as transcription factor,is a transduced element wide exists in various kinds of cells in human body.As a multiple direction regulatory protein it can mediate cell survival signal,prevent cell apoptosis.In various study,it has been proved that the actuating signal of cell anti-apoptosis effect is mediated by all kinds of survival factors receptor kinases;they stimulate cascade signal transduction pathway centered by NF-κB,which precipitates the activation of BAL-2 family and the apoptosis of Caspase-9,which are all NF-κB regulated genes.Caspase is a family of cysteine proteinase.It has been classified into two groups according to primary structure and the length of structure domain on N end:(1) Promoters:function as the initiating factor of provocation and regulation of apoptosis, including Caspase-8,9,10,and so on;(2)effectors:mainly cause apoptosis directly by enzymolysis of the substrate protein,including Caspase-3,6,7,and so on.As cited above,we have known that PI-3K can up-regulate NF-κB and down-regulate Caspase-9 in the complicated signal transduction pathway.AG is a safe effective induced nitricoxide synthase(INOS)inhibitor,which is extensively applied in investigation of myocardial ischemia and cerebral anoxia but is seldom reported in investigation of glaucoma.It is reported by Neufield and his coworkers that in the mouse model of chronic intraocular hypertension,AG is protecting optic nerve;however,the mechanism of protection function of retina in chronic intraocular hypertension is still being approached,as well as whether it is mediating anti-apoptosis through all kinds of signal transduction pathways.On account of above presumption,this experiment aimed at studying normal and intraocular hypertension retina tissue at the level of gene and protein to find out after using AG,the change of transcription factor PI-3K,NF-κB and apoptosis related gene Caspase-9 as well as the protein expression.Thus approach the relationship between chronic intraocular hypertension and transcription factor PI-3K,NF-κB and apoptosis related gene Caspase-9,and the affection of transcription factor PI-3K,NF-κB and apoptosis related gene Caspase-9 in AG mediated anti-RGCs-apoptosis regulation and reveal the mechanism of optic nerve injury and protection signal transduction in glaucoma to some degree.We hope the findings be a certain guideline for the clinical work to find a best optic nerve protective agent which can produce a marked effect in multiple signal pathways and supply a new treatment target and thought.Materials and MethodsThis experiment includes three parts:PartⅠ:The affection of AG to the expression of Caspase-9 in chronic intraocular hypertension of mouse model.Using immunohistochemistry staining, RT-PCR and Western blotting respectively to detect the variation of the Caspase-9 expression in normal mouse retina(group A),chronic intraocular hypertension(group B)and chronic intraocular hypertension treated by AG(group C)on the 3rd day,7th day,14th day,21st day,28th day.PartⅡ:The affection of AG to the expression of NF-κB in chronic intraocular hypertension of mouse model.Using immunohistochemistry staining,RT-PCR and Western blotting respectively to detect the variation of the NF-κB expression in normal mouse retina(group A),chronic intraocular hypertension(group B)and chronic intraocular hypertension treated by AG(group C)on the 3rd day,7th day,14th day, 21st day,28th day.PartⅢ:The affection of AG to the expression of PI-3K in chronic intraocular hypertension of mouse model.Using immunohistochemistry staining,RT-PCR and Western blotting respectively to detect the variation of the apoptosis related gene PI-3K expression in normal mouse retina(group A),chronic intraocular hypertension(group B)and chronic intraocular hypertension treated by AG(group C)on the 3rd day,7th day,14th day,21st day,28th day.Results1,Expression of Caspse-9 affected by AG on retina of rats with chronic IOP elevation(1)Group A(control group):There are 10 layers in mouse retina,which is similar to human being.Immunohistochemistry staining shows that the expression of Caspase-9 merely exists in ganglion cell layer,0-1/HP.There's only trace quantity expression detected by RT-PCR and Western blotting.(2)Group B:The RGCs and nerve fiber layer are thinner than control group after modeling on each time point except the 3rd day(P＜0.05 or 0.01). Immunohistochemistry staining shows that the expression of Caspase-9 locates mainly in the nuclei in ganglion cell layer and inner nuclear layer,the number of positive nuclei increased and the color of staining was darker as time went by and both reached the peak during the 7th day and the 14th day.RT-PCR and Western blotting show that the expression of Caspase-9 is stronger than group A on each time point except the 3rd day(P＜0.05 or 0.01).(3)Group C:The retinal ganglion cell and nerve fiber layer are thinner than control group on each time point,but thicker than group B(P＜0.05).Immunohistochemistry staining shows that the expression intensity of Caspase-9 in ganglion cell layer and inner nuclear layer is between group A and B(P＜0.05).RT-PCR and Western blotting show that the expression time course of Caspase-9 is similar to group B,however the peak appeared during the 7th and 14th day,but was less than group B(P＜0.05).2,Expression of NF-κB affected by AG on retina of rats with chronic IOP elevation(1)Group A:Histology examination is similar to group A in PartⅠ,which shows trace quantity expression of NF-κB protein in ganglion cell layer and inner nuclear layer.Western blotting shows no significant expression.(3)Group B:The retinal ganglion cell and nerve fiber layer are thinner than control group on each time point(P＜0.05).Immunohistochemistry staining shows that the positive expression of NF-κB reached peak during the 7th and the 14th day(P＜0.05). Western blotting shows that the expression ofNF-κB is stronger than group A on each time point(P＜0.05).(3)Group C:The retinal ganglion cell and nerve fiber layer are thinner than control group on each time point,but thicker than group B(P＜0.05).Immunohistochemistry staining shows that the expression time course ofNF-κB protein is similar to group B, but the intensity is stronger(P＜0.05).RT-PCR and Western blotting show that the expression ofNF-κB protein are stronger than group B on each time point(P＜0.05).3,Expression of PI-3K affected by AG on retina of rats with chronic IOP elevation(1)Group A:Histology examination is similar to group A in PartⅠ,which shows trace quantity expression of PI-3K protein.RT-PCR and Western blotting show weak positive expression.(2)Group B:The retinal ganglion cell and nerve fiber layer are thinner than control group on each time point(P＜0.05).Immunohistochemistry staining shows that the expression intensity of PI-3K protein cells accentuated as time went by(P＜0.05 or 0.01),and reached peak the 7th day after modeling,then attenuated ever since.RT-PCR and Western blotting show the positive expression time course of PI-3K meets the result of immunohistochemistry staining.(3)Group C:The retinal ganglion cell and nerve fiber layer are thinner than control group on each time point,but thicker than group B(P＜0.05).Immunohistochemistry staining,RT-PCR and Western blotting show that on the same point the expression intensity of PI-3K is stronger than group B significantly(P＜0.05 or 0.01),reached peak on the 7th day after modeling.Conclusion1,Some apoptosis promoter(such as Caspase-9)and apoptosis inhibitor(such as NF-κB,PI-3K)may act at the same time under the condition of chronic intraocular hypertension.2,Under the condition of chronic intraocular hypertension,AG protects the retina by way of down regulating the expression of apoptosis related gene Caspase-9.3,Under the condition of chronic intraocular hypertension,AG protects the retina by way of up regulating the expression of nuclear factor NF-κB.4,Under the condition of chronic intraocular hypertension,AG protects the retina by way of up regulating the expression of PI-3K.On the whole,the signal transduction pathways are more than complicated,which leaves so many things to be approached.This investigation shows that AG may play a part on multiple points of signal transduction pathway to protect the retina under the condition of chronic intraocular hypertension.The specific detail mechanism is still in need of further research later.