Based On Data Mining And Network Pharmacology To Explore The Medication Rules And Mechanisms Of Traditional Chinese Medicine In The Treatment Of TON

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Analysis of medication rules of traditional Chinese medicine in treating traumatic optic neuropathy based on data mining technologyObjective:Through collating the clinical research literatures on the treatment of traumatic optic neuropathy(TON)in past 20 years,the data mining technology was used to systematically analyze the traditional Chinese medicine(TCM)data involved in the literature,to explore the rules of Chinese medicine treatment of TON,and to provide reference and guidance for clinical treatment.Methods:The databases such as Wan-Fang Database,China National Knowledge Infrastructure(CNKI)and VIP were searched.Search time was limited from January 2000 to September 2020.The literature was selected according to the inclusion and exclusion criteria and prescriptions were entered into Excel 2016 to analyze the frequency,properties,tastes and effects of Chinese herbs.The software of IBM SPSS25.0 and the software of Modeler 18.0 was used for analyzing cluster analysis and association rules of high frequency herbs.Results:(1)A total of 38 articles were included in this study,and 38 prescriptions were obtained,involving a total of 74 Chinese medicines.The total frequency of all Chinese medicines was 392.The 5 most frequently used Chinese medicines were:Angelica,Ligusticum Chuanxiong,Red Peony,Safflower,Bupleurum.The most commonly used basic prescriptions are Xuefu Zhuyu Decoction and Chufeng Yixing Decoction.(2)In terms of meridian distribution,the frequency of TCM attributed to the liver meridian is the highest,followed by the heart meridian and the spleen meridian;In terms of medicinal properties,warm medicine and cold medicine were used most frequently;in terms of medicinal taste,bitter medicine,sweet medicine and pungent medicine were used most frequently.(3)In terms of the efficacy category of TCM,blood circulation and stasis medicine,and tonic medicine for deficiency were used most frequently.(4)According to the analysis of association rules,when the confidence is 100%,the top three association rules with support level are Ligusticum Chuanxiong-Peach Kernel,Ligusticum Chuanxiong-Peach Kernel-Safflower,and Ligusticum Chuanxiong-Red Peony-Safflower.(5)According to cluster analysis,TCM treating TON can be grouped into four categories:Ligusticum vulgare,Peucedanum,and Fangfeng are grouped into one group;Rehmannia,Radix Paeoniae Rubra,and Angelica are grouped into one group;Safflower,peach kernel,Achyranthes bidentata,and Ligusticum Chuanxiong are grouped into one group;Bupleurum,Fructus Aurantii,and Platycodon grandiflorum are grouped into one category.Conclusions:(1)The top 5 frequency TCM treating TON were angelica,Ligusticum chuanxiong,red peony root,safflower,and bupleurum.In terms of efficacy categories,blood circulation and stasis medicine and tonic medicine for deficiency were mainly used.The top three association rules with support level were Ligusticum Chuanxiong-Peach Kernel,Ligusticum Chuanxiong-Peach Kernel-Safflower,and Ligusticum Chuanxiong-Red Peony-Safflower.All of those reflected the principles of TCM treatment of TON:promoting blood circulation and removing blood stasis,invigorating deficiency and promoting the circulation of qi.(2)The TCM used to treat TON were mostly attributed to the liver and heart meridians,and were mainly bitter and sweet medicines.TCM for ton has both cold and warm properties.All of those were in line with the characteristics of blood-activating and stasis-resolving medicine and tonic medicine.(3)The most commonly used prescriptions for the treatment of TON are Xuefu Zhuyu Decoction and Chufengyi Lose Decoction.Cluster analysis showed the compatibility characteristics for the treatment of TON,which is mainly medicine for promoting blood circulation and removing blood stasis(safflower,peach kernel,chuanxiong,etc.)and invigorating deficiency(white peony,angelica,etc.),supplemented by medicine for expelling wind to relieve superficies(Gaoben,Qianhu,Fangfeng)and Qi-promoting(Bupleurum,Citrus aurantium,Platycodon).PART ? Study on the Mechanisms of Safflower in treating traumatic optic neuropathy Based on Network PharmacologyObjective:To explore the mechanism of safflower in treating Traumatic optic neuropathy(TON)by network pharmacology.Methods:(1)The active ingredients of safflower were screened on TCMSP platform and target information was collected.Uniprot database was used to transform the target proteins into target genes.Cytoscape software was used to construct the screened active ingredients-target gene network model of safflower.(2)The Gene chips of TON were searched from the gene expression database(GEO),and the differentially expressed genes were analyzed by GE02R.Obtain the core target of safflower active ingredients to treat TON by Venn analysis.(3)Protein protein interaction network(PPI)of safflower to the TON gene target was analyzed and constructed by Cytoscape software.Moreover,the core interaction network was extracted according to the BisoGenet and CytoNCA software packages.(4)GO enrichment analysis and KEGG pathway enrichment analysis of intersection genes were performed through DAVID online database.Results:(1)A total of 17 active ingredients and 205 related targets of safflower were obtained in our study.A total of 617 differentially expressed genes of TON in the GSE17117 chip were screened,including 429 up-regulated genes and 188 down-regulated genes.(2)11 core targets for safflower treatment of TON were screened out by Venn analysis.(3)A total of 14 core target proteins were screened out through PPI analysis.(4)The GO enrichment analysis results showed that these targets that were mainly concentrated in extracellular space,cytosol and apical part of cell.The biological processes of these targets included the negative regulation of apoptotic process,response to organic substance,positive regulation of apoptotic process,etc.Related molecular functions were nuclear hormone receptor binding and protein complex binding.(5)The enrichment analysis results of KEGG transition showed that the possible pathways of safflower for treating TON were mainly TNF signaling pathway,pathways in cancer,estrogen signaling pathway and Toll-like receptor signaling pathway.Conclusion:Based on the network pharmacological analysis of safflower in treating TON,we deduce that safflower may regulate TNF and other signaling pathways by regulating CASP3,MMP9 and other targets,thereby intervening in the apoptosis of RGCs to play a neuroprotective role.Part ? Effect of safflower injection on TNF apoptosis signaling pathway of RGC-5 cells induced by L-glutamateObjective:The experiment is to investigate the molecular mechanism of safflower injection in inhibiting the apoptosis of retinal gaglion cells after optic nerve injury in vitro.Methods:(1)Our experiment used staurosporine to induce RGC-5 cell line neuronal differentiation.CCK-8 was used to screen the optimal sodium L-glutamate concentration and injury time of RGC-5 cells,and safflower injection required for intervention.(2)RGC-5 cells induced by staurosporine were labeled as 5 experimental groups:Group A(Cell control group),Group B(Glutamate model group),Group C(safflower injection group),Group D(Ac-DEVD-CHO group),Group E(safflower injection and Ac-DEVD-CHO group).Group A was added with DMEM-H medium,and Groups B-E were added with the selected concentration of sodium L-glutamate medium.After incubation according to the selected injury time,Group A was added with DMEM-H medium,Group B was added with the same concentration of sodium L-glutamate medium,and Group C was added with safflower injection with the selected concentration,Group D was added with Ac-DEVD-CHO(10?M),Group E was added with safflower injection with the selected concentration and Ac-DEVD-CHO(10?M),respectively.All 5 groups were tested after 24-hour incubation.(3)Experimental index detection:The expression of Brn3a in RGC-5 was detected by immunofluorescence.CCK-8 and flow cytometry were used to detect the survival and apoptosis rate of RGC-5 cells in each experimental group,respectively.The protein levels of TNFR1,FADD,Caspase-8 and Caspase-3 were detected by Western-blot analysis.(5)Expression of proteins in TNFR1/FADD/caspase8/Caspase3 apoptosis signaling pathway:Results:(1)The neuronal morphological changes of RGC-5 could be induced with the concentration of 0.5 ?mol/L staurosporine medium for 1 hour.The apoptosis rate of staurosporine-differented RGC-5 cells could reach 43.98%,which was the closest to IC50,with a concentration of 8 mmol/L sodium L-glutamate interfering for 18 h.60%safflower injection interfered with RGC-5 cells for 24h,the inhibition rate of staurosporine-differented RGC-5 could reach 41.34%,which was the closest to IC50.Finally,the concentration of 0.5 ?mol/L STSN was selected to induce RGC-5 cells for 1h to promote differentiation and 10 mmol/L sodium L-glutamate was used to intervene for 18 h to establish the RGC-5 injury model.70%safflower injection was selected as the intervention concentration.(2)Immunofluorescence results showed that RGC-5 cells can express Brn3a,which is a specific transcription factor expressed in retinal ganglion cells in rats and mouse.(3)The results of CCK-8 showed that compared with Group A,the cell survival rate of Group B was 68.35%,while the rates of Group C to E were 77.51%,81.13%and 86.88%respectively.In addition,the survival rate of Group C,Group D and Group E was were significantly higher than that of Group B(P<0.01).Flow cytometry showed that the RGC-5 cells apoptosis rates in each group were:3.43%in Group A,25.75%in Group B,15.88%in Group C,14.21%in Group D,11.56%in Group E.Compared with group B,the apoptosis rates of group C,D and E were significantly lower(P<0.01).Compared with group B,the protein expression levels of TNFR1,FADD,Caspase-8 and Caspase-3 in groups C,D,and E were significantly reduced,and the differences were statistically significant(P<0.05).There was no significant statistical difference in the protein expression levels of TNFR1,FADD and caspase-8 between group C and group D(P>0.05),indicating that safflower injection and inhibitor had the same effect in regulating the protein expression levels of TNFR1,FADD and caspase-8.In the expression level of Caspase-3,there was a significant statistical difference between group C and D(P<0.05),indicating that the specific Caspase-3 inhibitor(Ac-DEVD-CHO)was more effective in reducing the level of Caspase-3 than safflower injection.In addition,the protein levels of Caspase-8 and Caspase-3 in Group E were significantly lower than those in group C and D(P<0.05),which indicated that safflower injection combined with Ac-DEVD-CHO could synergistically inhibit RGC-5 apoptosis.Conclusions:(1)Safflower injection can effectively inhibit the apoptosis of glutamate injured RGC-5 and improve its survival rate.(2)70%Safflower injection can exert an obvious anti-apoptotic effect,which may be achieved by inhibiting the expression levels of TNFR1,FADD,Caspase-8 and Caspase-3,and regulating the TNF apoptotic signal pathway.