| Objective: Diffuse large B cell lymphoma(DLBCL)is one of the most common and malignant adult lymphoid tissue tumors in China.DLBCL has the characteristics of rapid invasive growth,high degree of malignancy and poor prognosis.Therefore,new drugs or treatment strategies are urgently needed to improve the survival rate or delay the recurrence of DLBCL.Bufalin(BF)is a monomer isolated from toad venom.It has been reported that it has antitumor effect on many kinds of cancer.However,there are few reports about the effect of bufalin on the growth of dlbc,and its mechanism has not been fully elucidated.Therefore,through bibliometric and bioinformatics analysis of bufalin related Chinese and English literature,this paper makes clear the current research status of bufalin,analyzes the research hotspots,and makes a comprehensive and systematic evaluation of the current research literature of bufalin,so as to provide guidance for the selection of the topic.Then we elucidated the role and mechanism of bufalin in DLBCL by means of bioinformatics,molecular biology,biochemistry and other methods,so as to provide a theoretical basis for the clinical application of bufalin.Methods: The first part: bibliometric analysis of bufalin related literature.1.Data resources and search strategy: Chinese Literature: search all the Chinese literature about bufalin as the subject word in CNKI database,the time period is from January 1,2000 to December 31,2020.Using the correlation analysis function of the database,this paper analyzes the current Chinese research status of bufalin.English Literature: from January1,2000 to December 31,2019,Pub Med database was searched for all the related literatures about bufalin with the search formula of "bufalin".The search results were stored in ".XML" format,and the literatures were screened and quality controlled.2.Data extraction and matrix setting of English Literature: after downloading the data,the co-occurrence matrix is created by BICOMB software.3.Double clustering analysis of high-frequency Keywords: gcluto software is used to conduct double clustering analysis on the high-frequency keywords.4.The establishment of strategy analysis map: draw the theme map according to the centrality and density of the theme words,and generate the strategy map by using graphpad prism 7 software.The x-axis represents the centrality or external cohesion index,and the y-axis represents the internal cohesion index or density.5.Transwell experiment showed that bufalin could inhibit the migration of dlbc cells.6.Bioinformatics analysis suggested that bufalin might regulate the signaling pathway of NFATc1,and molecular docking suggested that bufalin could interact with NFATc1.7.Relevant databases confirmed that NFATc1 was differentially expressed in DLBCL lymphoma and was associated with poor prognosis.Immunofluorescence showed that NFATc1 was mainly expressed in the nucleus.8.After si RNA knockout NFATc1,cell proliferation decreased and apoptosis increased.Bioinformatics predicted that NFATc1 might affect c-myc,and WB test confirmed that the expression of c-myc decreased after NFATc1 knockout.9.The distribution of intracellular calcium was inhibited by bufalin.Therefore,the mechanism of bufalin in DLBCL may play an anti-tumor role through Ca2+ / NFATc1 / cmyc signaling pathway.10.In vivo tumor test showed that bufalin could significantly reduce tumor growth in mice.Immunohistochemistry showed that the expression of NFATc1,cmyc and Ki-67 was decreased.The second part: Objective to investigate the effect and mechanism of bufalin in DLBCL.1.The growth inhibition of bufalin on DLBCL cells was detected by CCK-8 cell proliferation test.2.DAPI staining and flow cytometry were used to detect the effect of bufalin on DLBCL cell apoptosis.3.To observe the effect of bufalin on DLBCL cell cycle by flow cytometry.4.Transwell method was used to detect the migration ability of DLBCL cells treated with bufalin.5.Using bioinformatics and molecular docking to predict the target of bufalin in DLBCL.6.The molecular mechanism of bufalin in DLBCL was elucidated by immunofluorescence assay,Western blot,immunohistochemistry,intracellular calcium fluorescence detection,si RNA transfection,real-time quantitative PCR and animal experiments.Results: The first part: From 2000 to 2020,a total of 147 Chinese articles related to bufalin were published,which reached the peak in 2019.Every year,the effect of bufalin on tumor proliferation was studied,suggesting that the main effect of bufalin may be to inhibit cell proliferation.In the subject analysis,it is mainly distributed in traditional Chinese medicine,tumor and urology.Among the literatures about bufalin published by relevant institutions,Shanghai University of traditional Chinese medicine,Second Military Medical University and Shandong University of traditional Chinese Medicine published more.Among the journals published,the top ones are the Journal of naval medicine of the Chinese people's Liberation Army,Chinese herbal medicine,Chinese Journal of traditional Chinese medicine,modern oncology,Chinese patent medicine,and Journal of Shanghai University of traditional Chinese medicine.In terms of related sub themes,the effects of bufalin were mainly studied on apoptosis,toxicology,inhibition and cell cycle.Matrix analysis of the subject words and sub subject words showed that apoptosis,anti-tumor effect,mechanism of action and cell proliferation ranked top.The results of matrix analysis also suggest that bufalin has been reported in pancreatic cancer,breast cancer,prostate cancer,liver cancer,colon cancer and osteosarcoma.There is a report in Burkitt lymphoma,but not in DLBCL.In English literature search,474 papers met the above criteria.The main research fields of bufalin related literatures are pharmacology,oncology,chemistry,biochemistry and molecular biology.China has made the largest contribution to the related research of bufalin,accounting for more than 70% of the published papers.The United States and Japan ranked second and third in the number of papers,respectively.More than 5 articles about bufalin have been published in 21 journals,including "oncology letters","event based comprehensive and alternative medicine" and "International Journal of Oncology".There is a report about bufalin in lymphoma in English literature.3.A total of 50high-frequency keywords are enriched.By double cluster analysis,the keywords were divided into five categories: "drug therapy in related tumors","relevant descriptive information of bufalin","mechanisms of drug effects","pharmaceutical information of bufalin" and "metabolism related researches on bufalin".Among these five categories,the mechanism and treatment of bufalin will be the main research direction in the future.4.Analysis of key genes of bufalin.CASP3,Jun,VEGFA,AKT1,mapk8,MMP9,mapk3,mapk1,Fos and MMP2 were the most frequently interacted genes.The KEGG pathway analysis showed that there were 20 most important bufalin related pathways.Go analysis showed that biological regulation,metabolic process and stimulation response were the main biological processes of bufalin related genes.In terms of molecular functions,the important functions of these genes include protein binding,ion binding,nucleic acid binding and nucleotide binding.The second part: 1.Bufalin inhibited the growth of DLBCL cells in a dose and time-dependent manner.The 24 h IC50 of bufalin on su-dhl-10 cells was 5 nmol / L,48 h IC50 was 3.7 nmol / L;the 24 h IC50 of bufalin on su-dhl-6 cells was 20 nmol / L,48 h IC50 was 7 nmol / L.2.DAPI staining showed that with the increase of bufalin concentration,DLBCL cells showed morphological changes of apoptosis,such as chromatin condensation,nuclear fragmentation and apoptotic body formation.3.Flow cytometry also showed that bufalin promoted the apoptosis of DLBCL cells.The expression of Bcl-2 and Mcl-1 decreased in a dose-dependent manner,while the expression of PARP,Bax and c-caspase3 increased significantly.4.Cell cycle arrest was detected by flow cytometry.The results showed that with the increase of drug concentration,the number of cells in G2 M phase of DLBCL cells increased in a dose-dependent manner.5.Transwell experiment showed that bufalin could inhibit the migration of dlbc cells.6.Bioinformatics analysis suggested that bufalin might regulate the signaling pathway of NFATc1,and molecular docking suggested that bufalin could interact with NFATc1.7.Relevant databases confirmed that NFATc1 was differentially expressed in DLBCL lymphoma and was associated with poor prognosis.Immunofluorescence showed that NFATc1 was mainly expressed in the nucleus.8.After si RNA knockout NFATc1,cell proliferation decreased and apoptosis increased.Bioinformatics predicted that NFATc1 might affect c-myc,and WB test confirmed that the expression of c-myc decreased after NFATc1 knockout.9.The distribution of intracellular calcium was inhibited by bufalin.Therefore,the mechanism of bufalin in DLBCL may play an anti-tumor role through Ca2 + / NFATc1 / cmyc signaling pathway.10.In vivo tumor test showed that bufalin could significantly reduce tumor growth in mice.Immunohistochemistry showed that the expression of NFATc1,cmyc and Ki-67 was decreased.Conclusions: 1.This study is the first time to combine text mining with bioinformatics to comprehensively analyze the related literature of bufalin.The results show that the exploration of the mechanism and treatment of bufalin is the focus of attention.Bufalin has anti-tumor effect in a variety of tumors,and the most reported is the effect of inducing apoptosis,among which the apoptosis related gene Caspase3 is the most fully studied.Bufalin can inhibit the proliferation,invasion and metastasis of cancer cells through PI3 K / Akt,hedgehog,mapk-jnk,Wnt / ?-Catenin,TGF-? / Smad,integrin signaling pathway and NF-k B.The statistical analysis of the research status of bufalin and bufalin in recent 20 years provides us with a few suggestions for the research direction of bufalin.2.The results showed that bufalin could effectively inhibit the growth of DLBCL cancer cells in vitro and in vivo.The possible mechanism is to induce cell apoptosis and arrest cell cycle in G2 / m by regulating Ca2 + / NFATc1 / cmyc pathway and inhibit cell proliferation.Our results suggest that bufalin can be used as a potential therapeutic drug to improve the survival rate of DLBCL patients. |
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