Prediction And Validation Analysis Of Lycii Fructus On Improving Age-Related Macular Degeneration By Network Pharmacology

Age-related macular degeneration(AMD)is the third leading cause of blindness in the world,which mainly affects the vision of the elderly,leading to serious vision loss or blindness.Modern pharmacological studies have shown that AMD is mainly related to oxidative stress,inflammatory response and RPE autophagy dysfunction.Traditional Chinese medicine(TCM)believes that AMD is mostly caused by deficiency of liver and kidney.As a traditional Chinese medicine commonly used to improve the eyesight,Lycii Fructus has been clearly recorded in "Medicine Theory" as early as it can replenish the deficiency of vital energy,easy color,improve the eyesight,peace of mind and longevity.Modern studies have shown that Lycii Fructus has the activities of anti-oxidation,anti-inflammation,regulating immunity,protecting liver and improving eyesight.According to the association analysis of AMD common Chinese medicine on TCM auxiliary platform,Lycii Fructus is the important medicine for TCM clinical treatment of AMD.Through literature research,it is found that the recent and modern studies on the improvement of AMD by Lycii Fructus are mostly focused on water extract,alcohol extract and crude polysaccharide of Lycii Fructus,while there are few studies on other parts of Lycii Fructus.This paper aims to systematically screen and evaluate the activity of different parts of Lycii Fructus to improve AMD based on chemical composition by network pharmacological prediction and experiments.By deeply revealing the material basis of the efficacy of Chinese wolfberry in improving vision,in order to achieve the purpose of promoting the transformation of Chinese wolfberry products,and at the same time provide reference and thinking for the research and development of clinical AMD drugs.Chapter one,Literature researchSection 1,Research progress on AMD pathological mechanismAs a degenerative disease,early pathological changes of AMD mainly involve the presence of medium sized drusen in Bruch’s membrane,the accumulation of lipofuscin in retinal pigment epithelial cells and the loss of choroidal capillary endothelial cells.Two clinical manifestations are typical for advanced AMD:choroidal neovascularization and geographic atrophy,which mainly affects retinal pigment epithelial cells and Photoreceptor cells,resulting in central vision impairment or blindness.The pathogenesis of AMD mainly involves oxidative stress,inflammatory response,and abnormalities in autophagy,complement,lipid metabolism,extracellular matrix,angiogenesis and other pathways,and there is much overlap between pathological mechanisms,especially between early and dry AMD.Many molecular mechanisms related to aging,including oxidative stress,inflammation,impaired protein homeostasis,mitochondrial dysfunction/DNA damage,have been studied in AMD,but most of them focused on RPE cells,and the molecular biological mechanism of choroidal microvascular changes in AMD has not been fully elucidated.Section 2,Advances in clinical drug research and application of AMDAccording to different stages of the disease,AMD can be divided into early and late stage.There is currently no therapeutic drugs for dry AMD,and ARDES antioxidants can be used to delay the progression from mid-stage AMD to late-stage dry AMD.In recent years,clinical studies on dry AMD include:Complement pathway inhibitor(APL-2Ⅱ、Eculizumab、LFG316、ARC1905、Lampalizumab);Visual cycle inhibitors(Fenretinide、ACU-4429);Amyloid beta antibody(RN6G、GSK93377687);MA09-HRPE cell stem cell transplantation;AAVCAGSCD59 gene therapy,However,the above methods did not achieve satisfactory clinical effect.The first-line treatment for wet AMD is intravitreal injection of anti-VEGF drugs,including Pegaptanib,ranibizumab,Bevacizumab,Brolucizumab and Conbercept.Other clinical studies in wet AMD include:PEDF inhibitors(E10030);Tie-2 activator(AKB-9778、Faricimab);Genetic product RGX-314.Through the analysis of the pathological mechanism of AMD,it can be seen that AMD is a multi-factor disease,and the intervention aimed at a certain pathologic pathway may not achieve a good therapeutic effect,and multi-factor combination drug is expected to become the future research direction for the treatment of dry AMD.Studies have shown that the long-term inhibition of retinal angiogenesis caused by VEGF treatment will aggravate the progression of geographic atrophy and lead to visual loss.Therefore,new wet AMD targets still need to be explored.Looking into the future treatment of AMD,preventing the occurrence of disease,enhancing retinal function while controlling disease,multi-factor combined drug use and exploring more convenient ways of drug administration are worthy consideration.Section3,Research progress of Lycii Fructus in improvement of AMDTraditional medicine believes that deficiency of liver and kidney is the main pathogenesis of AMD.As a traditional Chinese medicine for improving eyesight,Lycii Fructus has the effect of nourishing liver and kidney,nourishing essence and filling marrow.Modern pharmacological studies have shown that Lycium barbarum polysaccharide can improve AMD by improving oxidative stress,inhibiting inflammatory response,reducing the accumulation of lipofuscin,inhibiting Bruch membrane thickening and protecting RPE cells.In addition,zeaxanthin and lutein in Lycii Fructus can protect RPE cells by down-regulating the expression of MMP2 and TIMP-2 protease.Betaine can down-regulate the expression of VEGF in retina and inhibit the formation of CNV.In conclusion,modern pharmacological studies on the improvement of AMD of Lycii Fructus are mostly focused on Lycium barbarum polysaccharide,while there are few studies on other ingredients of Lycii Fructus.Chapter two,Prediction and validation analysis of improvement of age-related macular degeneration by Lycii Fructus based on network pharmacologySection 1,Pharmacological prediction of Lycii Fructus in improving age-related macular degenerationThe results showed that 88 active components in Lycii Fructus could improve the AMD by improving VEGF,IL-8,IL-6,TNF,MMP9 and other 80 targets,participating in biological processes such as oxidative stress and inflammatory response,and affecting the signaling pathways such as AGE-RAGE,IL-17,TNF and so on.Flavonoids,carotenoids and aromatic acids in different compounds of Lycii Fructus can improve oxidative stress and inflammatory response.In addition,flavonoids also participate in improving retinal blood circulation;Carotenoids are involved in improving visual development and nerve signal transduction.Aromatic acids are involved in extracellular matrix remodeling.Section 2,Validation analysis and activity screening of Lycii Fructus in improving age-related macular degenerationOn the basis of combing the pathological mechanism of AMD,combined with the prediction results of network pharmacology,the targets of SOD,HO-1,Nrf2,IL-6,IL-1β,TNF-α,oxidative stress and inflammatory reaction biological events were selected to verify the effect of Lycii Fructus on improving AMD.Meanwhile,the anti-inflammatory and antioxidant activities of different parts of Lycii Fructus were screened and evaluated.1.Preparation of samples from different parts of Lycii FructusThe water extract of Lycii Fructus,95%ethanol extract after water extraction,polysaccharide fraction,Pigment fraction,Water elution fraction,30%ethanol elution fraction,70%ethanol elution fraction,and 95%ethanol elution fraction.A total of 8 samples were obtained.The content of total polysaccharide in polysaccharide fraction was 28.317%,betaine in water extraction was 7.432%,total flavonoids in 70%ethanol-eluted fraction was 17.196%,zeaxanthin was 0.318%,β-carotene was 0.998%,and zeaxanthin dipalmitate was 9.671%in pigment fraction.2.Validation and screening of anti-inflammatory activity of different parts of Lycii Fructus based on BV2 cellsThrough the establishment of BV2 cells induced by lipopolysaccharide inflammatory damage model,different parts of Lycii Fructus anti-inflammatory activity screening results show:The water extract of Lycii Fructus,95%ethanol extract after water extraction,polysaccharide fraction,Pigment fraction,Water elution fraction,30%ethanol elution fraction,70%ethanol elution fraction,and 95%ethanol elution fraction could down-regulate mRNA levels of IL-6,IL-1β and TNF-α in BV2 cells to different degrees(P<0.05,0.01,0.001).This study confirmed the prediction results of network pharmacology:Lycii Fructus can improve AMD by regulating IL-6,IL-1β,TNF-α targets,inhibiting the inflammatory response process.3.Validation and screening of antioxidant activity of different parts of Lycii Fructus based on ARPE-19 cellsBased on the oxidative stress injury model of ARPE-19 cells induced by sodium iodate,the results showed that 70%ethanol fraction and 95%ethanol fraction of Lycii Fructus could significantly increase the survival rate of ARPE-19 cells induced by sodium iodate(P<0.001,0.01,0.05),and the effect of 95%ethanol fraction was better than that of 70%ethanol fraction.It was found that 95%ethanol fraction of Lycii Fructus could increase the activity of SOD in ARPE-19 cells.GSH content,reduce the content MDA in cells,promote the entry of Nrf2 protein into the nucleus,and up-regulate the expression of antioxidant enzyme HO-1.Moreover 95%ethanol parts can inhibit the expression of LC3B II and up-regulate the expression of p62,regulate the autophagy function of ARPE-19 cells.This experiment confirmed the predicted results of network pharmacology:Lycii Fructus can improve AMD by regulating SOD and HO-1 targets and affecting the oxidative stress state of the body.Chapter three,Effect of Lycium Fructus Extract on Retinal Degradation of AMD Mice Induced by Compound Hydroquinone DietSection 1,Effect of Lycii Fructus on Retinal Damage in AMD MiceThe water extract and alcohol extract of Lycii Fructus for 4 months could significantly improve the inner retinal layer disorder and partially restore the ganglion layer thickness of model group mice.The levels of IL-6 and TNF-α in serum and the levels of IL-6,IL-1β and TNF-α in retina of model group were decreased.Section 2,Effect of Lycii Fructus on Retinal Damage in AMD Mice Based on Serum MetabolomicsBased on serum metabolomics analysis of the mechanism of Lycii Fructus improving retinal injury in AMD mice,the results showed that the plasma metabolic profile of model group appeared significant difference with the youth group.Thirteen different metabolites were identified,involving the metabolism of Taurine and hypotaurine metabolism,sphingolipid metabolism,retinol metabolism,unsaturated fatty acids metabolism and primary bile acid metabolism.The water extract and alcohol extract of Lycii Fructus could adjust the plasma metabolic profile of the model group to the youth group,and the water extract of Lycii Fructus could adjust the levels of phosphatidylcholine,phosphatidylethanolamine and docosahexaenoic acid,and improve the metabolism of Sphingolipid and unsaturated fatty acid.The alcohol extract of Lycii Fructus can affect the Taurine and hypotaurine metabolism and sphingolipid by regulating the levels of taurine、phosphatidylcholine and phosphatidylethanolamine.