Network-based Pharmacological Analysis And Experiments To Explore The Synergy Antitumor Effect And Mechanism Of Luteolin Combined With Cisplatin On Hela Cell

Objective:Network pharmacology combined with molecular docking were used to determine whether luteolin(LU)is an effective component in Bruceae javanica(BJ)for cervical cancer(CC).In addition,the key targets and potential molecular mechanisms of LU were further verified through in vitro and in vivo experiments,and the mechanism of its synergistic effect to cisplatin(CDDP)was preliminally discussed,providing experimental basis for more reasonable and effective clinical chemotherapy.Methods:1.Drug targets were obtained from TCMSP,and targets of CC were obtained from Gene Cards.The intersection genes were collected.The regulatory network was constructed by Cytoscape.STRING was used to select core targets.The GO and KEGG analyses were done using R 3.6.0 software.Subsequently,molecular docking calculated the binding energy.The LU with the largest negative binding energy was selected as the research object.2.Hela cell lines were cultured in vitro.T he control group,the single LU group,the single CDDP group,and the LU combined CDDP group were set,respectively.The inhibitory rate in each group was determined by CCK8,and the dose concentration with inhibitory rate of about30% was selected for follow-up experiments.Cell morphology was observed under phase contrast microscope at 24 h and 48 h.Cell flow was used to detect the cycle and apoptosis.The invasion and migration ability were also detected.Proteins related were detected by Western blot.3.In vivo studies,the antitumor effects of LU were evaluated using Hela tumor bearing nude mouse models.The weight and volume changes of tumor growth were recorded and the curve was drawn.Tunel staining of the 4 groups was used to analyze tumor apoptosis.Immunohistochemistry was performed on tumor slices of nude mice in 4 groups.Finally,H&E were used to stain organs tissue sections.Liver function indexes,kidney function indexes and blood routine indexes were detected to evaluate the biological safety of LU.4.Image J was used to analyze the image results,SPSS was used to make statistics on each group of data,and Grad Prism was used to draw statistical charts;Data within the group were tested for homogeneity of variance.For data between groups,ANOVA was selected for multiple comparison test and Bonferroni was selected for variance calibration.Results:1.Through the network analysis,a total of 80 corresponding targets were screened out among the 15 components satisfying the ADME.There are 7006 target genes related to cervical cancer,among which 55 drug-cervical cancer intersection genes exist.The M-C-T-P network structure shows that LU is associated with more target genes.2.In vitro CCK 8 results showed that LU inhibited Hela proliferation in a dose-dependent way compared with blank group(P<0.05 or 0.01),and the synergistic effect of LU on CDDP was most obvious at 0.5 μg/ ml.Compared with the control group,LU group and CDDP group: i)The clonogenesis results showed the increase or decrease in clonogenesis ability(P<0.05 or 0.01);ii)In the combination group,G2/M phase increased and the apoptosis of Hela increased(P<0.05 or 0.01);iii)The nuclear contraction or rupture of apoptotic cells in TUNEL staining group was significantly increased(P<0.05 or 0.01);iiii)Flow cytometry showed that the proportion of G2/M phase was significantly increased and that of G0/G1 phase was decreased in the combination group(P<0.05 or 0.01).iiiii)in the combined group,cell migration was inhibited in cell scratch experiment(P<0.05 or 0.01);In Transwell experiment,the invasive ability of combined group was significantly inhibited(P<0.05 or0.01).Compared with the control group,the PI3K/Akt pathway related protein expression levels in LU and CDDP groups were decreased,and the associated histone expression levels were most significantly decreased(P<0.05 or 0.01).3.In the nude mouse tumor transplantation model in vitro,the tumor growth were significantly inhibited in the combination group compared with the control group and the single administration group(P<0.05 or 0.01).H&E staining and TUNEL staining showed that the number of necrotic cell structures and nucleolysis,and the number of green fluorescent apoptotic cells under fluorescence microscope were the most in the combined group compared with others.The combined group had the lowest expression of p-PI3 K,p-AKT,p-ERK,Cyclin B1 and MMP2,and the highest expression of BAX and c-Cas3,which was consistent with the trend of protein expression level of WB in vitro.Biosafety assessment showed that compared with the control group,there were no significant changes in cell and tissue structure in H&E staining of organs in the drug treatment group,same as the blood indexes(P>0.05).Conclusion:1.LU,as one of the active components of BJ,exerts its inhibitory effect on cervical cancer through multi-target and multi-pathway.The main mechanism may be through the PI3K/Akt signaling pathway.2.LU enhanced the down-regulation effect of CDDP on Hela cell proliferation,and luteolin and CDDP had a synergistic relationship.LU combined with CDDP can enhance the apoptosis of cervical cancer Hela cells,which may play a role by affecting the protein expression levels of c-Cas 3,BAX and Bcl-2 genes.Luteolin can enhance the cell cycle G2 arrest of cervical cancer Hela cells induced by CDDP,which may be related to the influence of Cyclin B1 and other cell cycle related proteins.LU combined with CDDP significantly downregulated the protein expressions of MMP 7 and MMP 2 in cervical cancer Hela cells,and synergically inhibited the invasion and metastasis ability of Hela cells.3.LU combined with CDDP can significantly inhibit the growth rate,mass and volume of Hela transplanted tumor in nude mice in vitro.LU can inhibit the PI3K/Akt pathway and down-regulate the expression of ERK,and synergically enhance CDDP in the treatment of cervical cancer without increasing side effects.4.LU is a promising platinum sensitizer.