The Mechanism Of Guiyuan Tiluo Yangxue Decoction In Promoting The Survival Of Retinal Ganglion Cells Based

BackgroundNon-arteritic anterior ischemic optic neuropathy(NAION)is the most common optic nerve infarction disease second only to glaucoma in the middle-aged and elderly population over 50 years of age,similar to "optic nerve stroke",which seriously endangers visual function and even causes blindness.The exact pathogenesis of NAION remains unclear and may be related to hemodynamic abnormalities in the optic nerve head area,but the location of associated vascular lesions and the mechanism of ischemia remain controversial.The short posterior ciliary arteries(SPCAs)are the main source of blood supply to the optic nerve head,but also supply the choroid around the anterior part of the lamina cribrosa of optic nerve.The shape between the anterior optic nerve of the lamina cribrosa and the peripapillary choroid is like a"watershed".If this area is in a hypoperfusion state,it is easy to cause local ischemic injury,induce optic nerve axonal degeneration,and secondary retinal ganglion cells(RGCs)apoptosis.Epidemiological studies have shown that the annual prevalence of NAION in the Chinese adult population is 1 in 16,000,and it is estimated that there are about 30,000 new patients each year.With the increasing aging of the population in China,the prevalence of NAION is also increasing.At the same time,NAION is a difficult and hot topic in the field of neuro-ophthalmology,and it is urgent to seek effective strategies to protect the optic nerve.Currently,there is no specific drug for NAION.Drug therapy mainly includes glucocorticoids,neurotrophic agents,vasodilators,etc.Non-drug therapy mainly includes optic nerve sheath decompression,hyperbaric oxygen,enhanced external counterpulsation,etc..However,the actual effects of these treatments are not ideal.In recent years,traditional Chinese medicine has played an important role in protecting the optic nerve,with good clinical benefits,which has been generally recognized by the majority of peers.A large number of basic studies have shown that Chinese herbal compounds have the overall regulatory characteristics of multiple components,multiple targets,and multiple pathways,which can promote RGCs survival and alleviate optic nerve axonal degeneration."Inner Canon of Huangdi" said:"qi desertion before unclear vision while ability to see after presence of blood in eyes".It shows that there is a close relationship between qi and blood and eyes.Feng Jun,chief physician,combined with his clinical experience for more than 30 years,believed that NAION was caused by local "blood desertion" of the optic nerve in the anterior part of the lamina cribrosa.Its essence of pathogenesis is the blood stasis of collaterals and the blood deficiency of collateral channels,resulting in malnutrition of eye tie.At the same time,he proposed the method of dredging collaterals and nourishing blood.According to this theory,he formed the empirical formula-Guiyuan Tiluo Yangxue decoction(GTY),which can dredge ocular collaterals and nourish ocular blood and eye tie.Previous studies have preliminarily confirmed that GTY can improve the visual function of patients with NAION.At the same time,it can inhibit RGCs apoptosis and reduce optic nerve axonal degeneration,and also has an optic nerve protective effect.However,its mechanism of protecting RGCs is still not clear.In this study,we established a rat model of nonarteritic anterior ischemic optic neuropathy(rNAION)by using photodynamic therapy.From the aspect of histopathology,the macroscopic and ultrastructural characteristics of optic nerve and retina were observed.In this study,we established a rat model of nonarteritic anterior ischemic optic neuropathy(rNAION)by using photodynamic therapy.From the aspect of histopathology,the macroscopic and ultrastructural characteristics of optic nerve and retina were observed.From the perspective of molecular biology,it is deeply explored whether GTY inhibits optic nerve axonal degeneration and promotes RGCs survival by activating the PI3K/Akt pathway,and then positively or negatively regulating the expression of downstream substrates,such as Akt,CREB,GSK3? and caspase3.In this way,the scientific theoretical connotation of "eyes obtain blood and there is vision" is revealed.Part ? Establishment of a rat model of nonarteritic anterior ischemic optic neuropathy by Photodynamic therapyPurpose:To evaluate the stability,feasibility and repeatability of rNAION established by photodynamic therapy,which provides an important research tool for subsequent experiments.Methods:The rNAION model was induced by injecting a solution of rose bengal(RB)into the tail vein,followed by continuous irradiation of the optic disc using a 532 nm wavelength laser.On the 1st and 28th day after the completion of modeling,color fundus photography(CFP)was taken to observe the morphological characteristics of the retina and optic nerve in rats.On the first day,fundus fluorescein angiography(FFA)was performed by tail vein injection of 10%fluorescein sodium injection.Then we dynamically took FFA images of the posterior pole of the fundus of rats centered on the optic disc to observe the fluorescence filling and leakage in the retina,choroidal vessels,and optic disc region of rats.On 22nd day,RGCs were retrogradely labeled with 3%fluorogold(FG)via the superior colliculus,and panretinal preparations were performed to quantitatively analyze RGCs survival.Meanwhile,the histopathological features of the optic nerve and retina of rats were observed by hematoxylin-eosin(HE)staining.Results:1 On the first day after the animal model was established,the CFP of rats with rNAION showed blurred optic disc borders and slightly lighter color.It indicates papilledema of the optic nerve,and the border of the "double whorled ring" encircling the optic disc is blurred.On the 28th day,the CFP of rNAION showed a more pronounced stereoscopic "depression"sensation in the optic disc,which may be optic atrophy.2 In the early stage,FFA showed retarded fluorescence filling of retinal and choroidal vessels in rNAION,and massive fluorescence accumulation at the optic disc.In the late stage,the fluorescence of retinal and choroidal vessels gradually subsided with blood flow.A large amount of fluorescence leakage and accumulation were still observed at the optic disc,which was evident in the middle and upper two-thirds.3 H&E staining showed that the local diffuse proliferation,invasion and migration of glial cell nuclei in the optic nerve of rNAION were observed at high magnification.The structure of local nerve fiber bundles in the optic nerve was destroyed,and there was abnormal proliferation of glia.The optic nerve head was deeply depressed,suggesting significant optic atrophy.A large amount of RGCs loss was observed in the retinal nerve fiber layer of rNAION.The retinal nerve fiber layer of rNAION became thinner and atrophied,but no obvious abnormality was observed in the other retinal layers.4 Under the fluorescence microscope,the overall distribution of RGCs in the model group was significantly sparse.It can be seen that the loss of RGCs around the optic disc is relatively mild,and the damage of RGCs in the mid-periphery of the retina gradually worsens.It showed quadrant incremental damage characteristics.Compared with the normal group,the density of RGCs in the experimental eyes of rats in the model group decreased significantly,and the difference was statistically significant(P<0.01).Compared with the model group,the survival rate of RGCs in the normal group of rats was 2.1 times higher than that in the model group,and the difference was statistically significant(P<0.01).Conclusion:1 Photodynamic therapy can successfully induce rNAION model,which is a mature,stable and reliable way to establish animal models.Its morphological characteristics of retina and optic nerve are in good agreement with human NAION.2 Selective loss of RGCs and axonal degeneration of the optic nerve are the main pathological outcomes of rNAION.3 Retrograde labeling of RGCs by application of fluoro-gold on the surface of superior colliculus is the gold standard for evaluating the rNAION model successfully induced by photodynamic therapy.Part ? Pathomorphological changes of Guiyuan Tiluo Yangxue decoction in promoting the survival of retinal ganglion cellsPurpose:On the one hand,to observe the effect of Guiyuan Tiluo Yangxue decoction on the macroscopic and ultrastructural characteristics of the retina and optic nerve in rNAION from the perspective of histopathology,on the other hand,to quantitatively analyze the survival of RGCs in different groups.Its purpose is to elucidate the pathophysiological basis of "eyes obtain blood and there is vision".Methods:The rNAION was induced by photodynamic method,and then SD rats were randomly divided into normal group,model group,mecobalamin group,low-dose Chinese medicine group,and medium-dose Chinese medicine group according to random number table.Except that the normal group was not given any treatment,the model group was fed with distilled water,while the group with drug intervention were correspondingly given mecobalamin solution and different doses of traditional Chinese medicine once daily for 4 weeks.At the end of the intervention,the histopathological features of the retina and optic nerve of rats in different groups were qualitatively analyzed by HE staining.The survival of RGCs was quantitatively analyzed by FG retrograde labeling through the superior colliculus.Transmission electron microscopy(TEM)was used to observe the ultrastructural characteristics of the retinal ganglion cells and optic nerve of rats in different groups.Results:1 HE staining showed that compared with the model group,mecobalamin group and low-dose TCM group,the nuclear loss of RGCs in the retinal nerve fiber layer of rats in the medium-dose and high-dose TCM groups was reduced at high magnification.The retinal nerve fiber layer thinning and atrophy were alleviated in the medium and high dose TCM groups,and no significant abnormalities were observed in the internal structures of the other retinal layers.There was local diffuse proliferation and invasion of glial cells in the optic nerve of the medium-dose and high-dose TCM groups,along with nuclear migration and disarrangement.The optic nerve fiber bundles were abnormally invaded and the optic nerve head was slightly depressed.However,except for the normal gr oup,the degree of optic nerve degeneration was milder than that of the other groups.2 Compared with the model group,the RGCs density of the TCM intervention groups at different doses showed a relatively increasing trend with increasing drug concentration.Among them,the medium and high dose TCM groups had a significant effect,and this difference was statistically significant(P<0.05 or P<0.01).There was a significant difference in the survival rate of RGCs between the groups(P<0.01).Compared with the model group,the survival rate of RGCs in each drug intervention group was more than 50%,except for the low-dose TCM group.The survival rate of RGCs in the medium-dose and high-dose TCM group was significantly higher than that in the model group,mecobalamin group,and low-dose TCM group,all of which were statistically different(P<0.01).3 The results of transmission electron microscopy(TEM)revealed that the cell capsule of RGCs in the normal group was clear and intact under ultra-high magnification and generally oval.The organelles such as chromatin,mitochondria,endoplasmic reticulum and nucleus in retinal ganglion cells were abundant and structurally intact.The nuclei of retinal ganglion cells were oval,the nuclear capsule was clear and intact,and the chromatin distribution in the nucleus was uniform.In the rNAION group,RGCs had vacuolated intracellular chromatin with uneven distribution,swollen mitochondria,and swollen and deformed endoplasmic reticulum.The retinal ganglion cell nuclear capsule was indistinct,irregular,pyknotic,and the chromatin in the nucleus was sparse and unevenly distributed.The medium and high dose TCM groups could significantly inhibit intracellular subcellular organelle destruction in RGCs.4 TEM was used to observe the ultrastructural characteristics of the optic nerve in different groups.Its results revealed that the optic nerve in the normal group was composed of axons with different sizes and regular arrangement,and the axons were separated by connective tissue.In the rNAION group,most of the axonal myelin sheaths were disintegrated and thinned,axons were swollen,atrophic and degenerated,and axons were sparsely distributed and disorganized.Medium and high doses of Chinese herbal medicine could significantly inhibit myelin disintegration and thinning of optic nerve axons and reduce axonal atrophy and degeneration.Conclusion1 Guiyuan Tiluo Yangxue decoction can inhibit apoptosis of RGCs and alleviate optic nerve degeneration,which is to improve the density and survival rate of RGCs.It has the effect of protecting optic nerve.Thus,the pathophysiological basis of "eyes obtain blood and there is vision" is revealed.2 With the increase of drug dose,the effect of Guiyuan Tiluo Yangxue decoction on promoting the survival of RGCs gradually increased in a dose-dependent manner.3 Guiyuan Tiluo Yangxue decoction can inhibit the destruction of subcellular organelle structure of RGCs and optic nerve axonal degeneration.Part ? Mechanism of Guiyuan Tiluo Yangxue decoction in Promoting the Survival of Retinal Ganglion Cells via modulating PI3K/Akt PathwayPurpose:From the perspective of molecular biology,to investigate whether Guiyuan Tiluo Yangxue decoction promotes the survival of RGCs and inhibits optic nerve axonal degeneration via activating the PI3K/Akt pathway,and then positively or negatively regulating the expression of downstream substrates,such as Akt,CREB,GSK 3? and caspase3.At the same time,it further reveals the molecular effect mechanism of "qi desertion before unclear vision while ability to see after presence of blood in eyes".Methods:The rat model of non-arteritic anterior ischemic optic neuropathy was established by photodynamic therapy.Then,SD rats were randomly divided into normal group,model group,mecobalamin group,low-dose Chinese medicine group,medium-dose Chinese medicine group,and high-dose Chinese medicine group according to the random number table.On the second day after the establishment of the animal model,except that the normal group did not receive any treatment,the model group was fed with distilled water,and the drug intervention group was fed with mecobalamin solution and different doses of Guiyuan Tiluo Yangxue decoction,respectively,once daily for 4 weeks of continuous intervention.The gene expressions of PI3K mRNA,Akt mRNA,CREB mRNA,GSK 3? mRNA and Caspase3 mRNA in the retina of rats in different groups were detected by RT-qPCR.Meanwhile,Western blot was used to detect the protein expression of PI3K,Akt,p-Akt(ser473),CREB,p-CREB(ser133),GSK3?,p-GSK3?(ser9)and caspase3 in the retina of rats in different groups.Results:1 Results of RT-PCR revealed that the expression levels of PI3K mRNA,Akt mRNA,CREB mRNA and GSK 3? mRNA in the retina of the medium and high-dose TCM groups were increased to varying degrees compared with the model group,and this difference was statistically significant(P<0.05 or P<0.01).PI3K mRNA,Akt mRNA and GSK 3? mRNA in the retina of the mecobalamin group showed an up-regulation trend,and the difference was statistically significant(P<0.05).Compared with the model group,the caspase 3 mRNA expression levels of the retinas in the medium-dose group and high-dose TCM group were significantly decreased,and this difference was statistically significant(P<0.05 or P<0.01).2 Results of Western blot revealed that the protein expression of PI3K in the retina of the medium-dose group and high-dose TCM groups showed an up-regulation trend compared with the model group,and this difference was statistically significant(P<0.05).Compared with the model group,different doses of Guiyuan Tiluo Yangxue decoction could up-regulate the relative protein expression of p-Akt/Akt,p-CREB/CREB,and p-GSK3?/GSK3? in the retina in a dose-dependent manner,and the differences were statistically significant(P<0.05 or P<0.01).mecobalamin could up-regulate the relative protein expression of p-Akt/Akt,p-CREB/CREB,and p-GSK3?/GSK3?,and the differences were statistically significant(P<0.05 orP<0.01).Compared with the model group,different doses of Guiyuan Tiluo Yangxue decoction could down-regulate the protein expression of caspase3 in a dose-dependent manner,and the differences were statistically significant(P<0.05 or P<0.01).Conclusion:1 Guiyuan Tiluo Yangxue decoction is equivalent to exogenous compound neurotrophic agents and may activate the PI3K/Akt pathway.It can up-regulate p-Akt expression,and activated Akt can promote the protein phosphorylation of downstream targets CREB and GSK3?,which in turn increases the activity of CREB and promotes GSK3? inactivation.It can inhibit the protein expression level of caspase 3 in retina.It can prevent RGCs apoptosis,reduce optic nerve axonal degeneration,and play a role in protecting the optic nerve.2 Guiyuan Tiluo Yangxue decoction can promote the survival of RGCs and inhibit optic nerve axonal degeneration.Its mechanism may be related to the activation of the PI3K/Akt pathway.Then,it up-regulated the expression of Akt mRNA,CREB mRNA and GSK3? mRNA in the retina,but also down-regulated the expression of caspase3 mRNA in the retina.It confirms the scientific theoretical basis of "eyes obtain blood and there is vision".