| Objective:The traditional Chinese medicine theory suggests that Actinidia chinensis Planch Root and Pyrrosia lingua(Thunb.)Farwell(AP)possesses diuretic,blood circulation promoting,and detoxifying properties,making it a common ingredient in folk prescriptions for prostate cancer(PCa)treatment.This study aims to explore the potential synergistic effects of AP in treating PCa by investigating their impact on cell proliferation,migration,apoptosis,as well as analyzing their constituent components.The findings of this research can serve as a valuable reference for further studies and the development of AP as a potential therapy for PCa.Methods:1.Preparation of medicinal materials with AP:The extraction of the Actinidia chinensis Planch Root and Pyrrosia lingua(Thunb.)Farwell in different ratios(1:0,3:1,1:1,1:3,0:1)was carried out using water,sequential water-alcohol and alcohol extraction methods.2.Analysis of the composition of AP:Qualitative analysis of effective monomer components in AP using HPLC and UPLC-Q-Exactive-Orbitrap-MS methods.3.Network pharmacology analysis:The material basis and mechanism of AP in the treatment of prostate cancer were investigated through network pharmacology analysis.4.In vitro antioxidant assay:The antioxidant effects of 15 extracts were preliminary screened by studying the DPPH·and ABTS·~+scavenging abilities of AP.5.Detection of the inhibition rate of PC-3 cells:15 extracts,along with bicalutamide(CDX),were evaluated for their inhibitory rates on PC-3 cells using the MTT assay.Dose-dependent and time-dependent experiments were performed to preliminarily determine the optimal ratio.Combined AP with CDX to detect the synergistic anti-proliferative effect of the combination by MTT assay and determine the optimal combination.6.Cell migration detection:Through cell scratch experiments and inverted phase contrast microscope,observed cell morphology and evaluated the inhibitory effects of the AP,CDX and the combination of them on the migration of PC-3 cells.7.Cell cycle detection:Using a flow cytometer to detect the effects of different drugs on the cell cycle,while simultaneously assessing their initial impact on cell apoptosis.8.Detection of the apoptosis rate of PC-3 cells:TUNEL method was used with fluorescein isothiocyanate(FITC)labeling,and apoptotic cells were observed using laser confocal microscopy.Apoptotic cells were identified as positive cells.9.Detection of PI3K/AKT signaling pathway proteins and Bcl-2 and Bax protein expression in PC-3:Immunofluorescence technology with FITC labeling was used to evaluate the levels of PI3K/AKT signaling pathway proteins and Bcl-2 and Bax proteins in cells from different treatment groups,and to clarify the expression location of the proteins.Results:1.Drug Ingredient Analysis:The 44 components of WEAP-1-0,WEAP-3-1,and WEAP-0-1 were identified by UPLC-Q-Exactive-Orbitrap-MS,including phenolic acids,terpenes,amino acids,coumarins and flavonoids.Among them,compounds such as caffeic acid and betulinic acid had been reported to have anti-tumor effects,which could provide research references for the application of the combination of the AP in the treatment of prostate cancer.2.Network pharmacology analysis:The research results of network pharmacology techniques showed that AP mainly exerts its therapeutic effects on PCa by balancing biological processes such as oxidative stress and reactive oxygen species,regulating pathways such as prostate cancer,the cell cycle and protein glycosylation in cancer,inhibiting cell invasion and migration,and promoting apoptosis.3.In vitro antioxidant experiments:Different combinations of traditional Chinese medicine exhibited DPPH·and ABTS·~+scavenging abilities,with the scavenging effects increasing in a concentration-dependent manner.Both showed a dose-response relationship.The order of the effects of different combination ratios on DPPH·and ABTS·~+scavenging abilities was:AP-1-0>AP-3-1>AP-1-1>AP-1-3>AP-0-1;the order of the effects of different extraction solvents on their scavenging abilities was:water extraction≈water-alcohol extraction>alcohol extraction.4.Detection of the inhibition rate of PC-3 cells:All the extracts showed varying degrees of proliferation inhibition on PC-3 cells,and this effect was concentration-dependent.Combination the best drug extract WEAP-3-1 with CDX showed synergistic effects by reducing drug concentration and increasing the killing effect on tumor cells.5.Cell migration detection:CDX,WEAP-3-1 and combined administration could inhibit PC-3 cell invasion.Compared with the single drug group,the scratch distance of the combination group increased significantly,indicating that the combined treatment group can further inhibit the invasive and migratory abilities of PC-3 cells.6.Cell cycle analysis:Both single and combined treatments led to a reduction in the proportion of cells in the S/G2 phase and caused cell arrest in the G1 phase.The combined treatment particularly accentuated the arrest in the G1 phase.In addition,the proportion of cells in the sub G1 phase significantly increased after both single and combined treatments.By inducing cell arrest in the G1 phase,apoptosis was triggered in PC-3 cells.7.Detection of the apoptosis rate of PC-3:TUNEL experiment results showed a significant increase in the number of green fluorescent cells in the WEAP-3-1 group and the combined administration group,with the combined administration group showing the most significant increase in fluorescent cell numbers.CDX and WEAP-3-1 combined administration could synergistically promote PC-3 cell apoptosis and induce cell death.8.Detection of PI3K/AKT signaling pathway proteins and Bcl-2 and Bax protein expression in PC-3 cells:The results of the immunofluorescence assay showed that the combination treatment of CDX and WEAP-3-1 could downregulated the expression of PI3K/AKT signal pathway proteins,which may inhibit the proliferation of prostate cancer cell line PC-3 by activating PI3K/AKT signal pathway,and upregulated the expression level of Bax protein and downregulated the expression level of Bcl-2 protein,demonstrating a pro-apoptotic effect.Furthermore,the combination treatment was superior to the monotherapy group,and the two drugs acted synergistically to enhance the effect,which may be related to the activation of the Bcl-2/Bax signaling pathway.Conclusion:A total of 44 components were identified from AP using UPLC-Q-Exactive-Orbitrap-MS,and combined network pharmacology research suggested that AP mainly exerted its therapeutic effects on PCa by balancing biological processes such as oxidative stress and reactive oxygen species,regulating pathways such as prostate cancer and the cell cycle,inhibiting cell invasion and migration,and promoting apoptosis.In vitro antioxidant studies demonstrated their ability to scavenge DPPH·and ABTS·~+,highlighting their strong antioxidant properties.The best combined administration combination of WEAP-3-1 and CDX,showed synergistic inhibition of PC-3 cells proliferation and migration,which was better than that of the single drug group.Following combined treatment,the expression level of Bax increased and the expression level of PI3K,AKT,Bcl-2 decreased,suggesting potential activation of the PI3K/AKT and Bcl-2/Bax signaling pathway as a contributing factor. |
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