| ObjectiveThis research is one part of National Natural Science Foundation of China“Study on the method of discovering active substance in anti-liver cancer of Oroxylum based on microfluidic cell biological chip technology”?No.81874342?.This topic is based on the research on the extraction and purification of effective components of Orozylum indicum?L.?Vent.,fingerprint,content determination and spectral efficiency relationship of Total flavonoids of anti-tumor components of Orozylum indicum using modern analytical instruments and methods,such as HPLC,defining its pharmacodynamic basis for anti-hepatoma effect.DEN-induced rat liver cancer model and microfluidic chip technology were employed to verify the anti-hepatoma effects of total flavonoids and Oroxin B in vivo and in vitro.From the perspectives of metabolomics,genomics,and related genes and proteins,this thesis explained the mechanism of anti-hepatoma effect of Orozylum indicum,provided experimental and theoretical basis for the clinical application and further development of traditional Chinese medicine Orozylum indicum as anti-hepatoma drug.Methods1.With the HPLC method,study on fingerprint of Orozylum indicum water and ethanol extract,and determination of the content of the Oroxin B;Based on the fingerprint,with gray correlation analysis and Person correlation analysis,study on the spectral effect relationship of total flavonoids in Orozylum indicum.2.Based on the Microfluidic chip technology,using the Hoechst 33342/PI method,cell apoptotic and necrosis rate as examining index,carry out the study of total flavonoids and Oroxin B on the influence of Human hepatoma cell HepG2 proliferation.Cell relative mobility rate as examining index,carry out the study of total flavonoids and Oroxin B on the influence of Human hepatoma cell HepG2 migration.3.Establishment of rat liver cancer model by DEN chemical induction,study on the pharmacodynamics of total flavonoids and Oroxin B in vivo against liver tumors by organ index,liver pathological section,serum cytokine content,AFP/ALT/AST level,etc.4.By HPLC-QTOF-MS technique,and use the MPP 12.6 software analysing the plasma samples of DEN hepatocarcinoma rats which intervene by total flavonoids and Oroxin B,and investigate the changes in endogenous metabolites,finding potential biomarkers and metabolic pathways,illuminate the mechanism of hepatocellular carcinoma treatment in vivo of total flavonoids and Oroxin B from metabolomics aspects.5.Using Illumina Sequencer and Agilent Microarray Scanner,study on the effects of total flavonoids and Oroxin B on the expression of transcriptome?mRNA?and non-transcriptome?microRNA?in liver tissues of DEN hepatocarcinoma rats,finding differentially expressed mRNA and microRNA,perform GO analysis and metabolic pathway analysis,interpretation of the mechanism of anti-hepatoma of total flavonoids and Oroxin B from genomics aspects.6.Using PT-PCR and Western Blot method,detecting the related mRNA and protein expression in liver tissues of DEN hepatocarcinoma rats after total flavonoids and Oroxin B treatmnet.Results1.Fingerprints of water and ethanol extracts from Orozylum indicum were established.The results of the determination of the Oroxin B in water extracts from Orozylum indicum were shown to be 13.98,12.94,20.34,16.83,15.78,15.37,18.44,15.78 mg·g-1,respectively;The results of the determination of the Oroxin B in ethanol extracts from Orozylum indicum were shown to be 17.65,26.45,30.99,27.62,23.46,22.62,30.42,24.03 mg·g-1,respectively;The results of gray correlation were as follows:Oroxin B>Chrysin-7-O-?-D-glucuronide>Oroxin A>Baicalein>Chrysin;The results of Person correlation analysis was as follows:Oroxin B>Baicalein>Oroxin A>Chrysin>Chrysin-7-O-?-D-glucuronide.2.The microfluidic chip for drug screening research was designed and fabricated.The in vitro pharmacodynamic experiment was carried out by using the chip.The results showed that the total flavonoids and Oroxin B of Orozylum indicum had significant inhibitory effects on human hepatoma HepG2 cells,the highest rates were 78.42%and 76.69%respectively.The microfluidic chip for cell migration research was designed and fabricated.The in vitro migration experiment was carried out with the chip.The results showed that the total flavonoids and Oroxin B could inhibit the migration of human hepatoma HepG2 cells,the highest rates were 16.58%and 17.54%.3.The rat model of hepatocellular carcinoma induced by DEN was established.The results of in vivo pharmacodynamic study based on DEN hepatocarcinoma rats showed that the liver tissue of the blank group was rosy and smooth,and the liver tissue of the model group was densely covered with nodular nodules.The diameter of the tumor was more than 1mm,and the liver tissue of the total flavonoids and Oroxin B of the group had different degrees of improvement compared with the model group;Total flavonoids and Oroxin B of Orozylum indicum can reduce the spleen index,increase the thymus index of rats to different degrees,it also can reduce the levels of AFP,ALT,AST,TNF-?,IL-12 and increase the level of IL-2 in serum.4.According to the results of HPLC-QTOF-MS,the changes of endogenous metabolites in the plasma of rats after the intervention of total flavonoids and Oroxin B of Orozylum indicum were analyzed by related databases.The results showed that 285 metabolites were changed after the intervention of total flavonoids,of which 41 were up-regulated and 244were down-regulated,such as Arachidonic acid,Prostaglandins,N-arachidonoyl L-serine,etc.The changes are mainly related to Tryptophan metaboilsh,Bile acid synthesis,Arachidonic acid metaboilsh,Linoleic acid metabolism,Serine metabolism and other pathways;A total201 metabolites were changed after the intervention of Oroxin B,of which 34 were up-regulated and 167 were down-regulated.The changes are mainly related to such as Arachidonic acid,Prostaglandins,PA(16:0/18:1?11Z?,Prostaglandin E2?PGE2?,etc.The changes are mainly related to Amino acid metabolism,Energy metabolism,Bile acid synthesis,Arachidonic acid metabolism,Calcium signaling pathway,Phospholipase signaling pathway,MAPK signaling pathway,etc.5.Illumina transcriptome assay was used to detect the effect of total flavonoids from Orozylum indicum on the expression of mRNA in liver tissues of DEN hepatocarcinoma rats.The results showed that there were 1491 genes were changed,739 of which were up-regulated and 752 genes were down-regulated.The genes which altered expression after administration were subjected to correlation pathway annotation and enrichment analysis.The results showed that the differentially expressed genes are mainly related to the Arachidonic acid metabolism,Wnt signaling pathway,Angiogenesis Angiogenesis pathway,PI3 kinase pathway,TCA cycle and respiratory electron transport,etc.;Agilent Microarray Scanner was used to detect the effect of total flavonoids and Oroxin B on the expression of microRNA in liver tissues of DEN hepatocarcinoma rats.The results showed that 82 miRNAs changed after the intervention of total flavonoids,and 23 of them were up-regulated,59 The miRNAs were down-regulated;89 miRNAs were changed after the intervention of Oroxin B,36 miRNAs were up-regulated and 53 miRNAs were down-regulated.There were 36 miRNAs regulated by total flavonoids and Oroxin B,29 down-regulated and 7 up-regulated.Differentially expressed miRNAs may be involved in signaling pathways such as MAPK signaling pathway,NF-kappa B signaling pathway,TGF-beta signaling pathway,PI3K-Akt signaling pathway,and TNF-?signaling pathway,etc.6.The results of RT-PCR and Western blot showed that the total flavonoids and Oroxin B of Orozylum indicum could reduce the expression of PI3K and p38 mRNA,and increase the expression of Akt,PTEN,Caspase 9,and Caspase3 mRNA;The total flavonoids and Oroxin B of Orozylum indicum could reduce the expression of PI3K,p38,p-p38,TGF-?and p-Akt proteins,and increase the expression of PTEN,Caspase 9,and Caspase 3 proteins.Conclusion1.The highest content of the Oroxin B in Orozylum indicum water or ethanol extracts of different origins was in Zhangjiajie,Hunan Province.Among the monomer components of total flavonoids,the most relevant effect on anti-hepatoma was Oroxin B.2.Total flavonoids and Oroxin B of Orozylum indicum have a good inhibitory effect on human hepatoma HepG2 cells proliferation and migration in vitro.3.Total flavonoids and Oroxin B of Orozylum indicum have a good anti-hepatoma effect in vivo,showing unique advantages in improving the quality of life and prolonging the life cycle of patients with hepatocellular carcinoma.4.Total flavonoids and Oroxin B of Orozylum indicum have certain regulatory effects on metabolites in DEN hepatocarcinoma rats,which may be related to Amino acid metabolism,Energy metabolism,Bile acid synthesis,Arachidonic acid metabolism,Calcium signaling pathway,Phospholipase signaling pathway,MAPK signaling pathway and other related ways,etc.5.Total flavonoids and Oroxin B of Orozylum indicum have a certain regulatory effect on transcriptome and non-transcriptome genes in DEN hepatocarcinoma rats,possibly with Arachidonic acid metabolism,Wnt signaling pathway,Angiogenesis pathway,PI3 kinase pathway,TGF-beta signaling pathway,PI3K-Akt signaling pathway,TNF-?signaling pathway and other metabolic or signaling pathways.6.Total flavonoids and Oroxin B of Orozylum indicum can promote the apoptosis,and inhibit the proliferation and migration of tumor cells,which may be related to the regulation of PI3K/Akt signaling pathway,p38 MAPK signaling pathway and TGF-?signaling pathway. |
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