Effect Of Compound Cinglongyi Capsule And Its Active Component Regiolone On The Expression Of TGF-β1 In HGC-27 Cells

Objective:In this paper,we elaborated the mechanism of action of Compound Qinglongyi Capsule and its active ingredient Regiolone on human gastric cancer cells HGC-27 from the molecular and cellular level,using molecular interaction technology and serum pharmacology technology,with the hospital preparation Compound Qinglongyi Capsule as the research background,which provided the experimental basis and theoretical support for the innovation and development of Chinese medicine anti-tumor drugs.Methods:Based on the Biacore system used to test the specific binding ability of compound Qinglongyi capsule to TGF-β1 protein in vitro,and through serum pharmacology and cellular experiments,further mechanism studies were carried out.Fifteen male SD rats were divided into three groups,i.e.,a blank group,a compound Qinglongyi capsule group,and a cisplatin positive control group,5 rats in each group.Blood was taken from the abdominal aorta and serum was collected.The inhibition effect of compound qinglongyi capsule on gastric cancer cells after administration was observed by cell plate cloning experiment.The cell scratch test was used to observe the migration changes of compound qinglongyi capsule on gastric cancer cells.HE staining was used to observe the morphological changes and apoptosis of the cells after drug administration.The effect of compound qinglongyi capsule on the apoptosis rate of HGC-27 gastric cancer cells was detected by flow cytometry:The protein and mRNA expression levels of TGF-β1,Bax,Bcl-2 and CD 10 before and after treatment were detected by Western-blot and RT-PCR.The specific active components combined with TGF-β1 protein were collected by using the Biacore fishing technology,and the obtained samples were identified and analyzed by UPLC-Q-TOF-MS.The affinity and kinetic analysis of the identified active monomer component Regiolone were again verified by the Biacore system.Besides,the protein and mRNA expression levels of TGF-β1,Bax,Bcl-2 and CD 10 before and after treatment were detected by Western-blot and RT-PCR.Results:The results of Biacore system sensor diagram showed that Compound Qinglongyi Capsule and TGF-β1 protein had good specific binding in vitro,and the response value reached 5369.2RU The cell clone experiment showed that compared with the blank group,the cell clone formation rate of the Compound Qinglongyi Capsule administration group was decreased.The cell scratch test showed that after 48h of observation,the cells in the blank group were almost covered with scratches.The cells in the center of the scratches were not covered in the ci splatin group and the Compound Qinglongyi Capsule administration group.Compared with the blank group,the scratch width in the Compound Qinglongyi Capsule administration group was wider than that in the blank group,and the difference was statistically significant.He staining showed that in the blank control group,there were more living cells,the attachment was relatively firm,the cells were closely connected,and only a very few dead cells.Compared with the blank group,a large number of cells in the cisplatin group and the Compound Qinglongyi Capsule group became round and fell off,and the dead cells were increased,showing a strong cytotoxic effect.Flow cytometry experiment showed that compared with the blank group and ci splatin group,the early and late apoptosis rates of HGC-27 gastric cancer cells were significantly increased after administration of Compound Qinglongyi Capsule,which could significantly promote the apoptosis of HGC-27 gastric cancer cells.The difference was statistically significant.The active ingredient that was compatible with TGF-β1 in Compound Qinglongyi Capsule was fished by Biacore Fishing Technology.After identification and analysis by UPLC-Q-TOF-MS technology,it was compared with the previous database and showed that the combined monomer active ingredient was Regiolone.The affinity kinetic analysis of Biacore showed that the affinity constant(KD)of Regiolone and TGF-β1 was 1.904 X 1 08,confirming the specific binding of Regiolone to TGF-β1 protein and validating the results of the angling technique.The results of Western-blot and Rt-PCR experiments showed that both Compound Qinglongyi Capsule and Regiolone could significantly reduce the protein and gene expressions of TGF-β1,Bcl-2 and CD 10,and significantly increase the protein and gene expression of Bax.Conclusion:In this study,Biacore experiment was used to predict the in vitro specific binding targets of compound Qinglongyi capsule and TGF-β1 protein,which was further verified by pharmacodynamic experiments.The active monomer component of TGF-β1 protein affinity of Compound Qinglongyi Capsule was extracted and identified by Biacore Fishing Technology as Regiolone.Pharmacodynamic experiments verified that both Compound Qinglongyi Capsule and Regiolone can play a role in the treatment of gastric cancer by regulating the expression of anti-tumor related protein factors.This study revealed the material basis and action mechanism of Compound Qinglongyi Capsule in the treatment of gastric cancer.And provides a new method for research and development of new drugs and mechanism research of traditional chinese medicine in the anti-tumor field.