Exploration Of The Major Pharmacological Effect And Clinical Positioning Of Hexuemingmu Tablets Against Retinal Degenerative Diseases Based On The Integration Strategy

Purpose:Firstly,taking two common retinal degenerative diseases such as age-related macular degeneration(AMD)and diabetic retinopathy(DR)as examples,the complexity of the two retinal degenerative diseases and their related pathological processes were investigated,effects of Hexuemingmu tablets(HXMM)were predicted;secondly,the overall network and subnetworks of the two diseases were constructed to interfere with the disease by drugs.With quantitative evaluation algorithm of network robustness,the pathological subnetworks of the potential target of HXMM can be evaluated and the major pharmacological effect of HXMM can be predicted;then carried out the experimental verification of retinal cell damaged model to explore the main effect of HXMM and the related active ingredient;finally,in order to provide a reference for the future expansion of HXMM ’s indications,the potential therapeutic effect of other retinal degenerative diseases were explored based on the established efficacy evaluation system of HXMM.This study provides an example for mining the main effects of classical prescriptions of traditional Chinese medicine in complex diseases via an integrated strategy of computational prediction and experimental verification,and a reference for the precise clinical positioning of traditional Chinese medicine.Method:1.Construction of the overall network and the subnetworks of pathological processes in retinal degenerative diseasesIn this study,two disease-related genes of AMD and DR were obtained through the Dis Ge NET database.According to the enrichment analysis results of their biological functions,the similarity clustering of the enriched items was performed,and the items of the two diseases were divided into multiple groups according to the clustering results.For each disease process,the protein interaction network of the overall disease and related pathological processes were established,and data support for the evaluation of the subsequent drug intervention disease network was provided.2.Prediction of multidirectional pharmacological effects of HXMMBased on the rat everted intestinal sac method to prepare HXMM intestinal absorption liquid and identify the intestinal absorption components by liquid chromatography-mass spectrometry technology,the target prediction platform BATMAN-TCM was used to predict the high-confidence targets of intestinal absorption components and based on the DAVID platform,the specific expression of biological function,disease and tissue enrichment analysis were predicted.3.Exploration of the major effects of HXMM on retinal degenerative diseases intervention based on the analysis algorithm of drug intervention disease network disturbanceBased on the previously developed algorithm of drug intervention disease network disturbance,by examining the changes of the disease network topology characteristics before and after drug intervention,the quantitative score of the impact of traditional Chinese medicine targets on the robustness of the disease network can be achieved.The major pharmacological effects of HXMM in intervening retinal degenerative diseases can be explored.4.Pharmacodynamic evaluation of the major effect of HXMM on the intervention of retinal degenerative diseases based on retinal cell damage modelBased on the in vitro experiment of HXMM intestinal absorption liquid,the retinal epithelial cell injured model induced by hydrogen peroxide was designed.To measure the activity of Cell Counting Kit-8(CCK-8),and to measure the Lactate Dehydrogenase(LDH)and Glutathione(GSH)indicators for oxidative stress;Interleukin-6(IL-6)and Tumor Necrosis Factor-α(TNF-α)indicators for inflammation;Vascular Endothelial Growth Factor A(VEGFA)and Vascular Endothelial Growth Factor B(VEGFB)indicators for angiogenesis part.The potential active components of the major effects of HXMM were explored by establishing the association network of "drug-target-pathological processes".5.Prediction of potential indications for other retinal degenerative diseases for HXMMIn this part,based on the analysis algorithm previously of drug intervention disease network disturbance effect,the disease data related to other retinal damages were collected and the disease networks were constructed.A heterogeneous network of "TCM components-disease targets-drug targets" was constructed,potential drug targets were screened out and enriched for analysis,and the possible mechanism of action of HXMM to intervene in diseases was investigated.Result:1.Construction of the overall network and subnetworks of pathological processes in retinal degenerative diseasesThis part of the study detected 685 age-related macular degeneration-related disease targets and 645 diabetic retinopathy-related disease targets based on the database.Combined with the results of biological function enrichment analysis and functional item clustering results,the disease targets were divided into oxidative stress,inflammation,angiogenesis,coagulation,extracellular matrix and other pathological processes.After examining the overlap,the specific multi-processes subnetworks were constructed.2.Prediction of multidirectional pharmacological effects of HXMMIn this part of the study,based on the identified components of the drug-containing intestinal absorption liquid,854 potential drug targets of HXMM were obtained.The results of the overall biological function enrichment analysis showed that the potential targets were mainly enriched in blood circulation,lipid biosynthesis,oxidative stress response,angiogenesis,etc.In addition,the results of disease enrichment analysis showed that the potential targets of HXMM were mainly enriched in diseases such as vascular diseases and vision-related diseases;the results of tissue enrichment analysis showed that the potential targets of HXMM were in the retina and lens.3.Exploration of the major effects of HXMM on retinal degenerative diseases intervention based on the analysis algorithm of drug intervention disease network disturbanceIn this part,based on the analysis algorithm of drug intervention disease network disturbance effect,a traditional Chinese medicine efficacy prediction system was established,and the disturbance coefficient of HXMM affecting the robustness of the disease network was used to mine the major pathological processes subnetwork of HXMM intervening in diseases.Included in FDA-approved drug,it is regarded as a multi-target drug,and its perturbation coefficient on the disease network is used as a reference to make better understanding.It was found that the potential target of HXMM can reduce the oxidative stress,inflammation,angiogenesis and other subnetworks.Therefore,it can be speculated that HXMM could have a relatively strong intervention effect on oxidative stress,inflammation,angiogenesis in retinal degenerative diseases.4.Pharmacodynamic evaluation of the major effect of HXMM on the intervention of retinal degenerative diseases based on retinal cell damage modelThe results of in vitro experiments showed that the survival rate of retinal epithelial cells was significantly reduced after hydrogen peroxide treatment,the oxidative stress index LDH was significantly increased,and the GSH level was decreased;the contents of inflammatory factors IL-6 and TNF-α were increased;vascular endothelial growth factor VEGFA,VEGFB expression were increased.Compared with the model group(only treated with hydrogen peroxide),the group pretreated with HXMM intestinal absorption solution had improved cell survival rate after hydrogen peroxide treatment;while GSH level increased,LDH level decreased,suggesting that the level of oxidative stress decreased;IL-6 and TNF-α level decreased,indicating its anti-inflammatory effect;the decreased expression of vascular endothelial growth factors VEGFA and VEGFB suggested its anti-angiogenesis effect.In conclusion,HXMM could reduce the damage of hydrogen peroxide to retinal epithelial cells by intervening in oxidative stress,inflammatory response and angiogenesis.By establishing the processes between the active ingredients and the major effects,13 active ingredients related to the major effect related to HXMM were obtained.5.Prediction of potential indications for other retinal injury diseases of HXMMBased on the analysis algorithm of drug intervention disease network disturbance effect,the potential targets of HXMM were found.The disease network disturbance coefficient was higher for four indications such as pterygium,keratitis,ocular hypertension,and corneal neovascularization.It is suggested that HXMM has a potential therapeutic effect on the above four indications.Conclusion:This study focuses on the large and complex networks of age-related macular degeneration and diabetic retinopathy and the subnetworks of related pathological processes.Using the analysis algorithm of drug intervention disease network disturbance effect,it was found that HXMM have an impact on the overall disease network and pathological processes.The perturbation coefficients of the networks were different,among which the perturbation coefficients for the oxidative stress,inflammation and angiogenesis subnetworks were higher,suggesting that the major processes of the intervention in retinal degenerative diseases were oxidative stress,inflammation and angiogenesis.Compared with the traditional network pharmacology method,the algorithm preliminarily realizes the quantitative evaluation of the intervention effect of drugs on disease at the molecular level.The results of in vitro cell experiments were consistent with the findings of computational predictions,and HXMM can effectively reduce retinal damage caused by Acute Retinal Pigment Epithelial(ARPE)-19 cells treated with hydrogen oxide by producing antioxidant,anti-inflammatory,and antiangiogenesis activities.Subsequently,with the help of the analysis algorithm of drug intervention disease network disturbance effect,it can be proposed that there is a potential therapeutic effect of HXMM on the indications of pterygium,keratitis,ocular hypertension,corneal neovascularization,etc.It is beneficial to modern medical indications of traditional Chinese medicine compounds and the precise application of intervention in complex diseases provide new strategies.