The Study On Therapeutic Basis And Effective Mechanism Of Coptis Rhizoma In Treatment Of Lung Adenocarcinoma

Objective: This study aimed to find out the potential targets and mechanisms of Oxyepiberberine（OPB）isolated from Coptis Rhizoma（Huanglian,HL）in the treatment of Lung adenocarcinoma（LUAD）,using network pharmacology and biological experimental.Methods: HL was divided into four parts by systematic solvent extraction.Using human lung adenocarcinoma cells（A549）as subjects,the effective antiLUAD parts of HL were screened by MTT method.High performance liquid chromatography（HPLC）method were analyzed to screen of the active compounds of effective anti-LUAD parts,then to A549 cells and BEAS-2B cells as the subjects,the key compounds with the best anti-tumor effect and the least toxic and side effects on normal cells were screened out through MTT assays.Then,through network pharmacology and molecular docking technology,the relevant targets of effective substances in HLfor the treatment of LUAD were searched,and the C-T-P network diagram and PPI network diagram were established to screen the key targets of effective substances against LUAD.Further analysis of docking energy and residual sites between effective compound and key targets through molecular docking technology.After analyzing the biological functions of the core targets,the effects of effective compound on proliferation,migration and apoptosis of A549 cells were investigated by MTT,plate cloning,scratches assay and Hoechst 33342 assay.Western Blot was used to investigate the regulation of effective compound on the expression of apoptosis-related proteins,migration-related proteins and core target proteins in A549 cells,and to elucidate the mechanism of effective compound against LUAD in vitro.Finally,tumor-bearing mice were used to verify the anti-LUAD activity and the regulation of effective compound on related proteins.BALB/c mice were subcutaneously injected with LLC lung cancer cells to establish the LLC lung cancer subcutaneous solid tumor mouse model.Dasatinib was used as positive control drug to observe the effects of effective compound on the growth status,tumor growth and body weight change of mice.Meanwhile,hematoxylin-eosin（H＆E）staining was used to examine the changes of tumor tissues in each group.Immunohistochemical experiments were performed to investigate the regulatory effects of effective compound on apoptosis-related proteins,migration-related proteins and key target proteins screened by network pharmacology,and further elucidate the mechanism of effective compound against LUAD in vivo at the cellular protein level.Results: MTT assay showed that the n-butanol part of HL was an effective part against LUAD.The results of HPLC showed that n-butanol of HL mainly consisted of seven effective alkaloids.They are Jatrorrhizine,Columbamine,Epiberberine,Coptisine,Palmatine,Berberine and oxyberberine,respectively.MTT assay results of these 7 compounds on A549 cells showed that OPB inhibited the proliferation of tumor cells and had the least toxic and side effects on normal cells.Therefore,OPB was identified as the key compound against LUAD in HL.The results of C-T-P and PPI network diagram constructed by network pharmacology showed that PIK3 CD,SRC,MAPK1,BRAF and MET were the key targets of effective substances in HL against LUAD.The results of molecular docking experiments showed that the docking energy of OPB and SRC was the highest,suggesting that SRC might be the core target of effective substance of HL against LUAD.In vitro MTT results showed that OPB significantly inhibited the proliferation of A549 in a dose-dependent manner.The survival rate of A549 cells was 50% when OPB concentration reached 16 μM 48 h after administration.The results of plate cloning experiment showed that OPB reduced the number of A549 cell clones in a dose-dependent manner.Hoechst 33342 staining showed that OPB promoted apoptosis of A549 cells in a dose-dependent manner.The results of Would healing experiment showed that OPB could inhibit the migration of A549 cells.Western blot analysis showed that OPB significantly down-regulated the expression of Bcl-2,MMP2 and MMP9 proteins（P < 0.05 or P < 0.001）,significantly upregulated Cleaved-Caspase3 protein expression（P < 0.05 or P < 0.001）,which further verified the effect of OPB on promoting apoptosis and inhibiting migration of A549 cells.Meanwhile,OPB significantly inhibited the protein expression levels of SRC,FAK,Ras,Raf,MEK and P-ERK（P < 0.05 or P< 0.01 or P < 0.001）,which verified the results of network pharmacology and confirmed that OPB inhibited the proliferation and migration of tumor cells through SRC-dominated signaling pathway and promoted the apoptosis of tumor cells.In the whole tumor-bearing mice experiment,the growth condition of the op B-treated mice was significantly better than that of the model group and the positive drug Dasatinib group.Compared with Dasatinib,there was no significant change in the body weight of mice in the OPB group alone,while the body weight of mice in the positive group decreased,suggesting that the side effects of OPB group on mice were less than that of Dasatinib group.OPB high-dose group,low-dose group and Dasatinib group significantly inhibited tumor growth and significantly reduced the trend of tumor volume growth in mice.The OPB high-dose,low-dose,and Dasatinib groups significantly reduced the final tumor weight（P < 0.01）.H＆E staining showed that the density of tumor cells decreased and the nuclei were destroyed in OPB and Dasatinib groups.Finally,immunohistochemical results showed that high and low doses of OPB and Dasatinib significantly regulated apoptosis,migration and SRCrelated pathway protein expression in tumor cells,which was consistent with network pharmacology and in vitro cell experiments.Conclusion: 1.The effective part of HL against LUAD is n-butanol,and the most effective compound against LUAD is OPB.2.OPB can dosedependently inhibit the proliferation,cloning and migration A549 in vitro,promote the apoptosis of A549 cell,and have no obvious toxicity to human normal lung epithelial cells.3.OPB can significantly inhibit tumor growth in vivo,and has no side effects on mice.4.OPB inhibits the proliferation and migration of LUAD cells and promotes the apoptosis of LUAD cells through the SRC-FAK-Ras-Raf-MEK-p-ERK pathway,thus inhibiting the progression of LUAD.This study elucidated the mechanism of the effective substance of HL against LUAD in bioinformatics and in vivo and in vitro.