Composing Principles Of Fuzheng Kangai Decoction And Its Mechanism Of Enhancing The Effect Of Gefitinib On Nonsmall Cell Lung Cancer

Objective:Clinical Research:Using the latest data mining algorithms to analyze and summarize the medical records of non-small cell lung cancer with gefitinib treatment from traditional Chinese medicine hospital of Guangdong province,which were trested with Chinese medicine and gefitinib by Professor Wu Wanyin from March 2016 to December 2017.From various perspectives,to analyze and summarize the Wu’s original prescription for the treatment of lung cancer by using traditional Chinese medicine and gefitinib,and excavate the rule of tutor group,which provides the theoretical basis for us to fully learn,inherit and develop the academic thought and experience of Wu.Basic research:1.To construct mi RNAs differentially expressed between gefitinibobtained PC9-GR cell lines,PC9-GR cells and parental cell PC9 cells,and the changes of mi RNA expression after intervention of Fuzheng Kangai Recipe on PC9-GR,so as to screen out Anticancer side reversed gefitinib-resistant mi RNAs.2.To investigate the effect of Fuzheng Kangai Recipe on the proliferation,invasion and migration of PC9 and PC9-GR cell lines from the perspective of mi RNA,and further explore its possible molecular mechanism at the gene and protein level.3.To study the anti tumor effect of FZKA decotion combined with gefitinib and and possible mechanism of FZKA decotion ’s “sensitization” to gefitinib treating NSCLC.Method:Clinical Research:The medical records of non-small cell lung cancer with gefitinib treatment from traditional Chinese medicine hospital of Guangdong province,which were trested with Chinese medicine and gefitinib by Professor Wu Wanyin from March 2016 to December 2017,and the original data were processed.Data collation was established based on Excel database.Based on this,data mining was performed using SPSS Statistics 23 and SPSS Modeler 18,Apriori algorithm was used for association rule analysis,and principal component analysis(Princpal Factor Analysis)was used for factor analysis on non-small cell.The general condition of lung cancer patients and the original prescriptions were analyzed to summarize the characteristics of professors Wu’s treatment of the disease.Basic research:1.Gefitinib-sensitive cell line PC9 was used as the parent,and gefitinib-resistant drug-resistant cell line PC9-GR was constructed by stepwise incremental and intermittent administration.2.The Next Generation Sequencing was used to screen the mi RNA of PC9,C9-GR and GR-FZKA,which were treated by FZKA decotion,and the accuracy and reliability of the sequencing results were verified by the quantitative PCR,and the mi RNA target gene was predicted by the on-line bioinformatics tool,and the dual-luciferase reporter system was used to verify it.3.The inhibitory effect of FZKA decotion,gefitinib single or the two drugs combination on the proliferation of NSCLC cell line PC9 and PC9-GR was detected by CCK-8 assay,and the effect of the combined use of two drugs on two cells was evaluated by the median-effect principle.Plate clone formation assay was used to test the clonality of PC9 and PC9-GR,flow cytometry was used to detect the cell cycle of PC9 and PC9-GR cells and the cell apoptosis under different conditions.The Transwell chamber and scratch test were used to detect the invasion of PC9 and PC9-GR cells combined with a combination of anti cancer,gefitinib,or two drugs.The influence of migration ability was detected by Western blot,and the effects of PC9 and PC9-GR cells on the expression of PTEN and p-AKT and p-Bad protein of mi RNA target gene PTEN were detected by western blot.Results:Clinical Research:1.Of the 1203 prescriptions,41.87% were males and 58.13% were females.Most of the patients were women.The patients are mainly in the 30-yearold or older,among which the youngest is 30 years old,and the oldest is 90 years old.The age range is 50-70 years old(50-60 years old + 60-70 years old),accounting for 59.92% of the total.2.Of the 297 patients included,176 were treated with gefitinib for more than 6 months,accounting for 59.26%,and 135 were taking medication for longer than 12 months,accounting for 45.45 %.3.The frequency analysis showed that 168 flavors were found in the 1203 prescriptions that were included.Among them,the top fifteen were licorice,Solanum,Taxus,stone,serpent,snakes,Atractylodes,Radix Astragalus,Radix Astragalus,forsythia,Coix root,Rhizoma Curcuma,snake rberry,and corydalis.In this study,Professor Wu Wanyin’s commonly used pair of drugs has 20 combinations,and 8 combinations of multi flavored Chinese medicine combinations,and 13 representative factors have been excavated by factor analysis.3.Factor analysis reveals 13 representative common factors,and analyzes factors in which the cumulative frequency is ranked in the top 10.The first factor is used in fried glutinous rice,glutinous rice glutinous rice,and glutinous rice.Rhizoma Zedoariae and Radix Pseudostellariae have large loads;the second factor has a greater load on Hedysarum diffusa,Hedyotis diffusa,M.rapa,and Pseudostellaria heterophylla;the third factor is Atractylodes macrocephala,heterophylla,Ezhu,Astragalus membranaceus With a larger load;the fourth factor has a larger load on the purple grass,tarsi charcoal and Rhizoma Imperatae;the fifth factor has a larger load in the buffalo horn,silkworm,gastrodia,and yellow peony;sixth Factors in the Lithospermum,Forsythia,Rhizoma Imperatae have a larger load;the seventh factor in the Corydalis,Chuanwu system has a larger load;the eighth factor in Hawthorn,Predicts,Astragalus,Suanzaoren has a larger Load;The ninth factor has larger loads in Solanum nigrum,Radix sophorae,Morus alba,and Glycyrrhiza uralensis;the tenth factor has a larger load on psoralen and pomegranate peel.Basic research:1.Establishing gefitinib drug-resistant cell line PC9-GR.In cell morphology,compared with the parent PC9 cell line,Compared with its parental PC9 cell line,PC9-GR showed little morphological change in cell morphology,but a considerable number of PC9-GR cells grew larger,spindle-shaped,and irregularly shaped,and the proportion of some cells became smaller.Gefitinib inhibited the proliferation of PC9,PC9-GR and A549 cells in a dose-dependent manner.In the same concentration range,the survival curve of PC9 cells decreased rapidly with increasing gefitinib concentrations.PC9-GR cells and A549 cells showed a slowly decreasing trend,and the IC50 s of PC9,PC9-GR and A549 cells were 1.96 μM,19.86 μM and 19.00 μM,respectively.After treatment with 2μM gefitinib for 24 h,flow cytometry results showed that after treatment with 1μM gefitinib for 24 h,compared with PC9 cells,the apoptosis rate of PC9-GR and A549 cells was significantly different(P<0.01);Compared with A549 cells,the difference in apoptosis rate of PC9-GR cells was not statistically significant(P>0.05).The detection results of the cell cycle distribution during the exponential growth phase showed that the distribution of cell cycle in PC9-GR cells was changed to a certain extent compared with PC9 cells: the proportion of cells in G0/G1 phase decreased,and the difference was statistically significant(P< 0.01),the proportion of cells in S phase increased significantly,the difference was statistically significant(P<0.01),and there was no significant change in G2/M phase.2.FZKA decotion inhibits the growth of lung cancer cells.The results of CCK8 showed that FZKA decotion had inhibitory effects on the proliferation of PC9 and PC9-GR cells in a dose-and time-dependent manner.The results of plate cloning experiment,transwell experiment and scratch test showed that FZKA decotion had inhibitory effects on the colony forming ability,invasiveness and migratory ability of PC9 and PC9-GR in a dosedependent manner compared with the control group.With the increase of the concentration of Fuzheng Anti-cancer drugs,the inhibitory effect was increased,and the difference was statistically significant(P<0.05,P<0.01).Flow cytometry results showed that compared with the control group,the proportion of G2/M phase cells in PC9 cells treated with Fuzheng Kangai Recipe decreased,while the proportion of cells in S phase increased,the difference was statistically significant(P< 0.05).In PC9-GR cells,the ratio of G2/M phase cells increased compared with the control group,and the difference was statistically significant(P<0.05).Compared with the control group,the proportion of early apoptotic cells increased gradually with increasing concentration of Fuzheng Anti-cancer drugs.3.FZKA decotion combined with gefitinib inhibit lung cancer growth.CCK8 experiment,plate cloning experiment,Transwell experiment,scratch test,flow cytometry experiment,etc.showed that FZKA decotion alone drug group,Gefitinib single drug group and combined drug administration group were able to PC9,PC9-The proliferation ability,colony formation ability,invasiveness,migration ability,and anti-apoptosis ability of GR were all inhibited,and the combined administration group was stronger than the Fuzheng Kangai Fang alone group and the gefitinib single drug group,and the difference was statistically significant.Significance(P<0.01);Compared with gefitinib alone group,the combined administration has better cytostatic effect(P<0.01).4.High-throughput sequencing results showed that hsa-mi R-141-3p was differentially expressed in PC9,PC9-GR and GR-FZKA.The q PCR verification results showed that compared with PC9,PC9-GR,the expression was upregulated,and the difference was statistically significant(P<0.01).The expression of hsa-mi R-141-3p that had been up-regulated in PC9-GR cells after treatment with Fuzheng Anti-cancer was down-regulated,and the difference was statistically significant.Significance(P<0.05).CCK8 proliferation inhibition experiments and plate cloning experiments showed that hsa-mi R-141-3p reduced the sensitivity of lung cancer cells to gefitinib.Target Scan mi RNA and RNAhybid software predict that PTEN is the target gene for hsa-mi R-141-3p.5.FZKA decotion regulates the effect of gefitinib on Lung Cancer by Regulating hsa-mi R-141-3p q RT-PCR results showed that compared with the blank group,the expression of hsa-mi R-141-3p was gradually down-regulated with increasing concentration of Fuzheng anticancer drug,showing a dosedependent,statistically significant difference(P<0.01).Western Blot results showed that compared with the blank group,Fuzheng anticancer drug concentration-dependent upregulation of PTEN protein expression in PC9 and PC9-GR cells,time-dependent down-regulation of p-Akt(Ser473)protein expression,time-dependent up-regulation p-Bad(Ser112)protein expression and protein expression differences were statistically significant(P<0.05,P<0.01).The results of combined administration showed that compared with the control group,Fuzheng Kangaifang alone drug group,gefitinib single drug group and combined administration group could up-regulate the expression of PTEN protein,and the effect of combined administration group was better than Fuzheng Kang There was a statistically significant difference between the monotherapy group and the gefitinib group(P<0.05).Compared with the gefitinib group,the combined treatment group had better ability to inhibit cell migration and the difference was statistically significant.Significance(P<0.05).Conclusions:1.Prof.Wu Wanyin advocates that the pathogenesis of lung cancer is mainly due to deficiency of lung and spleen qi,phlegm and blood stasis.The type of syndrome is mainly qi deficiency and phlegm,and the treatment advocates the combination of syndrome differentiation and disease differentiation.Gold to Fuzheng,heat to dissolve phlegm with anti-cancer,Kang-Ai Kang-Ai prescription,lung cancer hemoptysis,can be added Zizhu grass,lobster charcoal,Rhizoma Imperatae;lung rash itching,can add Lithospermum,Forsythia,white Chine root;lung cancer chest pain,can add Corydalis,system Chuanwu;poor lung cancer,can add hawthorn;lung cancer insomnia,can add Suanzaoren;lung cancer diarrhea,can add psoralen,pomegranate skin.2.FZKA decotion can down-regulate the expression of hsa-mi R-141-3p,and it can synergistically inhibit the proliferation of PC9 and PC9-GR cells in vitro in combination with gefitinib,and through the PI3K/AKT pathway.Up-regulates the expression of tumor suppressor gene PTEN and simultaneously inhibits the activation of AKT phosphorylation,thereby promoting pro-apoptotic protein Bad phosphorylation induces apoptosis.