| Glaucoma is an ophthalmic disease with complex pathogenesis,which is often caused by pathological elevated intraocular pressure(IOP),resulting in optic nerve damage and irreversible visual field defect.Epidemiological studies have shown that even when the intraocular pressure is controlled at a low level(≤ 21 mm Hg),some glaucoma patients still have progressive optic nerve damage and progressive narrowing of visual field.Although the existing diagnosis and treatment methods can reduce the intraocular pressure to the target intraocular pressure level,there is still a potential risk of blindness,which poses a huge burden on the society and families.Based on the mechanism characteristics of "multi-target,multi-link and multi-pathway" of traditional Chinese medicine,this paper uses bibliometrics to sort out the general situation and hot spots of traditional Chinese medicine in the diagnosis and treatment of glaucoma in the past 10 years.Then the integrated network pharmacological knowledge is used to explore the possible pharmacological mechanism of Yiqi Mingmu granule,which is the tutor’s clinical experience.Finally,combined with the experimental methods of cell biology,the early prediction mechanism is verified.Thus,a research route of "bibliometrics mining-network pharmacology prediction-cell experiment verification" is constructed to comprehensively clarify the exact mechanism and great advantages of traditional Chinese medicine in the diagnosis and treatment of glaucoma.Part Ⅰ Based on the literature metrology of TCM diagnosis and treatment of glaucoma in recent 10 years research focus and trend analysisObjective:Adopt the method of literature metrology,knowledge visualization mapping for almost 10 years China hownet database about traditional Chinese medicine diagnosis and treatment of glaucoma comb literature system.Methods:Using VOSviewer1.6.14 software and CiteSpace5.8.R3 software as visual knowledge graph drawing tools,we searched the literatures related to the diagnosis and treatment of glaucoma published in the China knowledge Network database from January 1,2012 to December 31,2021.After exporting the required documents in "Refworks" format,the unnecessary documents are initially removed according to the inclusion and exclusion criteria,and then the data are cleaned uniformly and the text format is converted to a degree that can be analyzed and processed by subsequent software.Finally,descriptive statistical analysis and visual map drawing are carried out by using Excel2019,VOSviewer1.6.14 and Cite Space5.8.R3 software respectively.Results:According to the results of descriptive statistical analysis,this study included a total of 404 related articles in the past 10 years,and the number of articles published over the years was very stable,mostly between 35 and 45.Four researchers,Peng Qinghua(24 articles),Peng Jun(17 articles),Li Xiang(16 articles)and Zhang Lixia(11 articles),have published more than 10 articles.The three institutions with the largest number of papers are Hunan University of traditional Chinese Medicine(34articles),Chinese Academy of traditional Chinese Medicine(32 articles)and Chengdu University of traditional Chinese Medicine(29 articles).In terms of the number of articles published in the journal,the number of articles published in the Chinese Journal of traditional Chinese Medicine Ophthalmology(47 articles)is much higher than that of other journals,which is closely related to the professionalism and industry influence of the journal.According to the results of the visual knowledge graph,the cooperative network has formed several main research squares represented by Peng Qinghua,Li Xiang,Zhang Lixia and other researchers,with close cooperation within each team,but dispersed with the rest of the team.The clustering results show that "optic nerve protection" is the key node of research in this field,and clinical or basic research extends around it.TCM treatment mainly includes activating blood circulation and removing blood stasis,soothing the liver and invigorating spleen,regulating qi and so on.The use of proprietary Chinese medicine includes compound Xueshuantong capsule,Qingguangan granule and so on.Acupuncture has also become one of the most important treatment methods in the diagnosis and treatment of glaucoma.It mainly focuses on RGCs apoptosis,which is closely related to autophagy,oxidative stress,optic nerve injury and so on.Conclusions:1.It is necessary to break down barriers between different regions and colleges,strengthen exchanges and cooperation,and further give play to the advantages of traditional Chinese medicine in the treatment of glaucoma.2.Improve the research on the diagnostic criteria of TCM syndromes,and organically integrate TCM syndrome differentiation with western medicine disease differentiation as far as possible,so as to realize the standardization and standardization of TCM diagnosis and treatment.3.Active components or effective blood-entering components of traditional Chinese medicine are the key to exert pharmacological effects.On the basis of existing studies,we can increase the excavation of drug structure and new functions to make it serve the clinic as much as possible.4.Multi-index evaluation of the therapeutic effect,especially the changes of patients’ psychological status,especially the tracking and evaluation of the long-term effect.5.Exploration of complex pharmacological mechanisms,high-quality evidence-based medicine and real-world research,and multi-efficacy screening of new drugs will be the trend of future development.Part Ⅱ Based on the integration of network pharmacology exploring yiqi bright eye treatment potential mechanism of action of qi deficiency blood stasis type of glaucomaObjective:Integrated network pharmacology was used to explore the potential mechanism of Yiqi Mingmu granule in the treatment of glaucoma with qi deficiency and blood stasis.Methods:Log in to the integrated network pharmacology computing platform of traditional Chinese medicine(TCMIPV2.0),establish the database of chemical composition and candidate targets of Yiqi Mingmu granule,analyze the potential targets of traditional Chinese medicine by GO and KEGG by DAVID6.8,and visually draw the bubble diagram of GO and KEGG by online drawing website Omic Share Tools.The key words "glaucoma" are searched in TCMIPV2.0 ’s disease-related molecular database,and the disease names are selected as "glaucoma"," glaucomatous visual field defect" and " open angle glaucoma" in turn,and the TCM syndrome targets of qi deficiency and blood stasis glaucoma are formed through "syndrome-related molecular mining and functional analysis" in "integrated pharmacological analysis tools collection".Based on STRING database,the target interaction information between Yiqi Mingmu granule and glaucoma with qi deficiency and blood stasis was constructed,and the core chemical components were evaluated by QED,and the multi-dimensional network diagram of "traditional Chinese medicine-component-target-pathway" was drawn with the help of Cytoscape3.7.0.Results:Based on TCMIPV2.0,282 chemical constituents and 815 corresponding targets of Yiqi Mingmu granule were obtained.The enrichment analysis of candidate targets of Yiqi Mingmu granule by GO and KEGG showed that it mainly acts on membrane depolarization,energy metabolism,mitochondria and other related pathways in the process of action potential.Based on the "disease-related molecular database" and "syndrome-related molecular mining and functional analysis" module of TCMIPV2.0,the disease genes obtained by both of them were standardized and deweighted,and571 glaucoma genes related to qi deficiency and blood stasis were obtained.The first 150 nodes are selected as the core target by the arrangement of Degree values from large to small.Among them,there were 113 targets directly acted by Yiqi Mingmu granule and 50 targets for glaucoma with qi deficiency and blood stasis.There were 13 targets between them,which were ALB,PIK3 CA,TLR4,PRKACA,SDHC,NR3C1/GR,ABL1,HNF4 A,SDHB,VCP,RHO,HK1 and ACO2.The GO function analysis and KEGG pathway enrichment analysis of the core targets of Degree showed that the core targets were mainly enriched in Rap1 signal pathway,HIF-1 signal pathway,PI3K-Akt signal pathway and so on.The relationship among the components can be seen from the multi-dimensional network of "traditional Chinese medicine-component-target-pathway" in the treatment of glaucoma of qi deficiency and blood stasis with Yiqi Mingmu granule.Different compounds play a role in the treatment of diseases by acting on different targets such as TNF-α,IL-6,TLR4,NFKB1,AKT,etc.,and then participate in different pathways.Conclusions:1.Yiqi Mingmu granule can give full play to the advantages of traditional Chinese medicine in the diagnosis and treatment of glaucoma with qi deficiency and blood stasis through the way of "multi-component,multi-link,multi-target,multi-pathway".2.Yiqi Mingmu granule may improve the energy metabolism of intracellular microenvironment by regulating the level of local mitochondrial metabolism,mainly by regulating the positive / negative regulation of RNA polymerase Ⅱ promoter transcription,transcription and DNA template,ATP,etc.3.The enrichment results showed that the prescription was closely related to HIF-1 signal pathway and PI3K-Akt signal pathway.It reduced the optic nerve damage of glaucoma by reducing the level of oxidative stress and down-regulating the expression of inflammatory factors such as TNF-α,IL-6 and TLR4.Part Ⅲ Protective effect of drug-containing serum of Yiqi Mingmu granule on RGC-5 cells injured by hypoxiaObjective:To evaluate the protective effect of drug-containing serum of Yiqi Mingmu granule on RGC-5 cell model induced by cobalt chloride.Methods:The cell model of RGC-5 hypoxia injury induced by CoCl2 solution was established.The rats were divided into blank serum group and Yiqi Mingmu granule containing serum group.The rats were fed with the same volume of normal saline or Yiqi Mingjie granule decoction,and the blood was separated to prepare drug-containing serum.The cultured cells were divided into five groups: normal group,control group and high and low drug-containing serum dose group.Different volume fractions of drug-containing serum were used to intervene and detect the required indexes.It includes: CCK-8 method to detect the optimal concentration and time of modeling,the effects of different concentrations of drug-containing serum on the viability of normal RGC-5 and hypoxia-injured RGC-5 cells;flow cytometry to detect the effect of drug-containing serum on hypoxia-induced apoptosis of RGC-5cells induced by CoCl2;effect of biological transmission electron microscope on ultrastructure of hypoxia-induced RGC-5 cells induced by CoCl2.The effects of colorimetry and WST-1 on the GSH-Px activity and SOD level of hypoxic RGC-5cells induced by CoCl2;the effect of drug-containing serum on the expression of apoptosis-related factors m RNA induced by CoCl2 in hypoxic RGC-5 cells;chart drawing and data analysis were processed by Graph Pad Prism9 software.Results:1.Compared with the normal group(0 umol/L CoCl2),the lethal rate of RGC-5cells was 50% after incubated with 400 umol/L CoCl2 solution for 24 hours,so 400μmol/L CoCl2 solution was selected and cultured for 24 hours as the condition for the establishment of hypoxic injury model of RGC-5 cells.2.1.25%,2.5% and 5% drug-containing serum could promote the growth of normal RGC-5 cells in a dose-dependent manner.After co-incubating with 400umol/L CoCl2 for 24 hours,2.5% and 5% drug-containing serum groups could promote the growth of hypoxic cells induced by CoCl2.3.The results of flow cytometry showed that compared with the hypoxia model group,the apoptosis rate was significantly decreased after co-incubation with 1.25%,2.5% and 5% drug-containing serum.4.The results of biological transmission electron microscope showed that compared with the normal group,the hypoxia model group showed nuclear swelling,structural disorder,atrophy or even deformity,mitochondrial swelling,crest rupture and final vacuole-like changes,messy cytoplasmic distribution,nucleolar pyknosis or disappearance.After the intervention of Yiqi Mingmu granule containing serum,compared with the model group,the cell morphology was more regular,a small amount of mitochondrial vacuolar changes were found,the mitochondrial crest was clearly visible,the nucleolus was round and a small part of chromatin condensed.5.Compared with the normal group,the activities of SOD and GSH-Px in cells decreased after the intervention of CoCl2 solution(P < 0.01).Compared with the model group,the activities of SOD and GSH-Px increased in different concentrations of Yiqi Mingmu granule containing serum for 24 hours.6.Compared with the blank serum group,the expression of HIF-1 α,caspase-3and bax increased and the expression of Bcl-2 decreased after intervention with CoCl2 solution.Compared with the hypoxia model group,2.5% and 5% drug-containing serum could increase the expression of Bcl-2 and decrease the expression of HIF-1 α,caspase-3 and bax(P<0.05,0.01).Results:1.The drug-containing serum of Yiqi Mingmu granule can prevent the apoptosis of RGC-5 cells induced by CoCl2.2.In terms of ultrastructure,the drug-containing serum can significantly improve the nuclear swelling and structural disorder,although there are a small amount of mitochondrial vacuole-like changes,but the mitochondrial crest is clearly visible,the nucleolus is round and a small part of chromatin condenses.3,protect RGC-5 cells from oxidative stress by increasing the activities of SOD and GSH-Px.4.By increasing the expression of Bcl-2 and reducing the expression of HIF-1 α,caspase-3 and bax,the cell injury under hypoxia was alleviated. |
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