Based On Bibliometric Analysis,the Research Hotspots Of Malignant Melanoma And Bafulin,and The Molecular Mechanism Of Bufalin Inhibiting The Growth Of Melanoma

Objective: Malignant melanoma(mm)has high malignancy,strong invasiveness and poor prognosis.As a monomer compound with antitumor effect,bufalin has been used by doctors in China for the systemic treatment of advanced tumors.At present,a large number of literatures on bufalin and melanoma are published every year,but there is still a lack of systematic and quantitative research and analysis.Therefore,firstly,this paper systematically analyzes and counts the journal articles related to bufalin and melanoma through quantitative methods,and systematically expounds the research hotspots and current situation of bufalin and melanoma.To provide guidance and reference for scientific research topics and further clinical work.Then,the effects of bufalin on the proliferation,apoptosis,invasion,migration and angiogenesis of malignant melanoma cells and human umbilical vein endothelial cells(HUVECs)were observed and studied by biological cytology experiment.Finally,using the relevant research methods of network pharmacology,cell biology,molecular biology and biochemistry,this paper studies the role and molecular mechanism of bufalin in malignant melanoma,so as to provide a theoretical basis for the clinical application of bufalin in cutaneous melanoma.Methods: Bibliometric analysis: the Chinese literature retrieval strategy is to use CNKI database to collect all the Chinese literature with bufalin and melanoma as the subject words.The title,all authors,source publications,journals,publication time,document type,keywords,research institutions,high-frequency keywords,references and other data of the literature are classified and analyzed.Literature types are not limited to research types,mainly conference literature,journal literature,communication literature and dissertation.The search period is set from January 1,2000 to December 31,2020.Retrieval strategy of English Literature: search all English literature related to bufalin and melanoma included in the web of Science(WOS)database in the format of "bufalin",and download and store the search results in the format of ".txt".The scope of publication is still from January 1,2000 to December 31,2021.All included records include all authors,titles;publication time,literature types,keywords,source publications,keywords and references,and then these data are summarized and analyzed by relevant analysis software.Experimental part: 1.The inhibitory effect of bufalin on the growth of melanoma cells and vascular endothelial cells was detected by CCK-8 cell experiment.2.Plate cloning experiment verified that shibufalin inhibited the proliferation of melanoma cells and vascular endothelial cells.3.The effects of bufalin on apoptosis and cell cycle of melanoma cells were detected by flow cytometry.4.The effect of bufalin on the migration of melanoma cells and vascular endothelial cells was detected by scratch test.5.The invasion and migration ability of melanoma cells and vascular endothelial cells were detected by Transwell method.6.Tubule formation experiment detected that bufalin inhibited the tubular formation of endothelial cells.6.Use bioinformatics,biological database prediction,molecular simulation docking,go,KEGG and PPI analysis to predict the possible targets and signal pathways of bufalin.7.Western blot experiment was used to verify the correctness of the predicted target,so as to clarify the molecular mechanism of bufalin in melanoma cells.Results: Bibliometric analysis: the number of articles published in Chinese has increased year by year,and the number of articles published in English has decreased significantly in recent three years,suggesting that the research tends to be saturated.In English journals on bufalin,the relative influencing factors are not very high,between2-5 points,suggesting that the current research on bufalin is still not very in-depth.Yin Peijie,a scholar from Shanghai Academy of traditional Chinese medicine,has the highest number of articles.The most prominent research institutions are the research teams of Shanghai University of traditional Chinese medicine,Dalian Medical University,Chinese Academy of Sciences and Shanghai Jiaotong University.Internationally,the disciplines of bufalin research are mainly pharmacology,pharmaceutics and oncology.The literature references of bufalin tend to its pharmacology and tumor.The Chinese research focus of bufalin has transitioned from apoptosis and anti-tumor to network pharmacology.High frequency keywords such as apoptosis,metastasis,and autophagy and cell proliferation constitute representative terms in English literature,and they are also hot topics about bufalin.More and more attention has been paid to malignant melanoma.Whether in China or abroad,oncology is the first discipline,followed by dermatology and Venereology.In terms of the number of articles published,the per capita number of articles published abroad is more than 20 times that of Chinese scholars,especially in the United States,which is mainly concentrated in Harvard University,Anderson Cancer Center of the University of Texas and Caitlin cancer center.In the analysis of journal co citation,the relatively high distribution is cancer,nature,cell and New England Journal.These magazines are mainly British and American,indicating that China still needs to be further improved in the field of establishing journals.According to the organization cooperation network map,China’s Sun Yat Sen University,Fudan University,China Medical University and Nanjing University have more international cooperation.The key words in Chinese literature focus on apoptosis,inhibition and cell proliferation.The analysis of English literature keywords found that immunotherapy;navumab and immune checkpoint inhibitors appeared more frequently,followed by prognosis,apoptosis and tumor immunity.The experimental part passed: 1 Bufalin can inhibit the growth of melanoma cells in a dose-dependent and time-dependent manner.The IC50 of bufalin on A375 cells was 20 nmol / L at 24 hours and 17.5 nmol / L at 48 hours;The IC50 of bufalin on A2058 cells was 60 nmol / L at 24 hours and 30 nmol / L at 48 hours.2.With the increase of bufalin concentration,the colony formation of melanoma cells decreased significantly.3.In the apoptosis experiment,in the blank control group,the number of apoptotic cells was less than 5%,while in the dosing group,the number of apoptotic cells reached 10-20% at10 nm and 30-40% at 20 nm.4 At 10 nm and 20 nm,the G2 M phase cells of melanoma cells increased significantly,and this process was dose-dependent.5.In melanoma cell lines A375 and A2058,after treatment with bufalin,the scratch widened and the migration ability of melanoma cell lines decreased with the passage of time.The migration ability of cells in the scratch group was stronger than that in the control group.6.After treatment with bufalin,fewer cells were transferred to the lower chamber of Transwell,but more cells were in the control group,suggesting that bufalin can inhibit the migration of melanoma cells in a concentration dependent manner.7.In Transwell chamber invasion experiment,when the concentration of bufalin was 5nm and 10 nm,the invasion ability of melanoma cells decreased significantly and the number of cells decreased significantly.8.Bufalin inhibited the activity of HUVECs in a dose-dependent manner at different time points.The IC50 was 40 nm at 24 hours and 20 nm at 48 hours.9.Plate cloning experiment further confirmed the inhibitory effect of bufalin on the growth of HUVECs.Compared with the control group,the number of clone formation of HUVECs in the experimental group added with bufalin(5nm,10nm)was significantly reduced.10.The scratch width becomes wider in the scratch test.Especially at the concentration of 10 nm,the width was significantly larger than that of the blank control group.11.In Transwell experiment,with the increase of bufalin concentration,fewer cells migrated to the lower surface of Transwell chamber,which was statistically significant after software analysis,which further confirmed that bufalin could inhibit the migration of HUVECs cell lines.12.After HUVECs cells were treated with bufalin solution at 0,5 and 10 nm in turn,compared with the control group,the number of vascular network formed by HUVECs changed little at the concentration of 5 nm,but with the increase of concentration,the number of vascular network decreased significantly and the tubular structure was damaged.13.The possible targets of bufalin were obtained from pharmmapper database.The genes of melanoma and angiogenesis were obtained from malacards database and geneclip database.Through the intersection of the four,57 key targets of bufalin that may affect angiogenesis and melanoma growth were obtained.These 57 genes were identified as bufalin related target genes.13.The analysis of go results shows that stimulation response,biological regulation,cell information transmission and biofilm process are the main biological processes of bufalin related genes,which are mainly reflected in regulating protein metabolism,cell generation and nitrogen acid metabolism.In terms of molecular function,the main activities of these genes include protein binding,ion binding and regulation of transferase activity,mainly the combination of protein and enzyme molecules.These genes are related to various cell components,including cytoplasm,nucleus and cell membrane,mainly in the form of protein binding.14.KEGG analysis enriched 20 bufalin related pathways,among which PI3K-Akt pathway is closely related to malignant melanoma.15.PPI protein interaction analysis showed that MAPK,AKT1 and p53 were the central genes with the most interaction.16.Molecular docking simulates the molecular interaction between bufalin and AKT1,and bufalin forms a hydrogen bond with AKT1 at the 53 rd site of asparagine(ASN 53).17.After treatment with bufalin,there was no significant change in the level of PI3 K protein in melanoma cells and vascular endothelial cells,but the level of p-Akt protein decreased significantly,indicating that bufalin will play a role by inhibiting the level of Akt protein phosphorylation.18.After adding PI3 K protein inhibitor sc-79,the expression level of p-Akt protein increased,and the increased effect will be reversed by bufalin.The recovery experiment proved that bufalin inhibited melanoma growth and angiogenesis through PI3K-Akt signal pathway.Conclusions: Bibliometric analysis: at present,the research heat of bufalin has decreased and tends to be saturated.From the cluster analysis of high-frequency words,the hot content has transitioned from cell proliferation and apoptosis to network pharmacology and drug detection.In the research of bufalin,China is in the leading and core position,but it is not closely connected with other countries and the degree of cross-national institutional cooperation is low.Journal analysis shows that the research on bufalin needs to be in-depth,and the exploration on the action mechanism and treatment of bufalin is the key direction of current research.On the research of melanoma,the number of papers published in both China and abroad has increased year by year,but the per capita number of papers published abroad is more than 20 times that of Chinese scholars.In the field of melanoma research,the level of scientific research abroad is significantly stronger than that in China.From the perspective of discipline distribution,the research on malignant melanoma in China is clinical and abroad is mechanism.Key words such as apoptosis,immunosuppression and biomarkers appear frequently,indicating that the research scope of melanoma is wide,involving clinical characteristics,pathogenesis,immunotherapy and other aspects.Among them,immunoepidemic treatment and tumor microenvironment are the hot research direction of malignant melanoma.Experimental part: 1 Bufalin can inhibit the proliferation,migration and invasion of melanoma cells.2.Bufalin can promote the apoptosis of melanoma cells and block the cell cycle in G2 M phase.3.Bufalin can inhibit the proliferation,migration and invasion of vascular endothelial cells.4.Bufalin can inhibit the formation of HUVECs tubules.5.Bufalin can inhibit the growth and angiogenesis of melanoma by affecting the phosphorylation level of active protein Akt in PI3K-Akt signaling pathway.The specific mechanism may be that bufalin forms a hydrogen bond with AKT1 at the 53 rd site of asparagine,thus affecting the phosphorylation level of Akt protein.