Study On The Inhibitory Effect And Mechanism Of Xihuang Pill And Its Medicinal Ingredients On Stemness Of Glioma

Background and PurposeGlioblastoma is the most common malignant tumor of the central nervous system in clinical practice.Due to the characteristics of extensive infiltration and growth of gliomas,it is difficult to radically remove them by surgery.Therefore,it is necessary to combine radiotherapy and chemotherapy,targeted or Immunotherapy and other comprehensive treatment methods.However,the recurrence and mortality rates of glioblastoma patients remain high.With the deepening of research,a large amount of evidence suggests that glioma stem cells may be the main factor causing the occurrence,invasion,recurrence and drug resistance of gliomas,and they are the key bottleneck restricting the efficacy and prognosis of glioma treatment.Xihuang pill(XHP)is a commonly used adjuvant drug for cancer patients in my country.It can inhibit tumor cell proliferation,induce tumor cell apoptosis,and resist tumor invasion and migration.However,its specific mechanism of action has not been fully elucidated.This subject aims to explore and verify the molecular mechanism of XHP’s inhibitory effect on glioblastoma stemness,and provide experimental data and a theoretical basis for the clinical application and drug development of XHP in the treatment of glioma.MethodsIn this study,UPLC-QTOF-MS was used to identify the main chemical components of XHP.Using network pharmacology and bioinformatics methods,the molecular network targeting GSCs of the active ingredients in XHP was constructed.Cell viability,self-renewal ability,apoptosis and expression of GSCs markers were detected by CCK-8 assay,3D-tumorsphere formation assay and flow cytometry,respectively.GEPIA was used to evaluate the relationship between GSCs markers(CD 133 and SOX2)and key proteins of EGFR/Akt/mTOR signaling pathway,and verified by western blot.A stable Akt-overexpressed GBM cell line was constructed using lentivirus to evaluate the regulation of Akt signaling in glioma stem cells.The active components in XHP were screened using molecular docking,Lipinski’s rule,and CCK-8 methods.The regulating effect and mechanism of the screened active monomer on the stemness of GSCs were verified using FACS and Western blotting.Futher,the effect of XHP on the growth of glioblastoma was analyzed by a subcutaneous transplantation model of BALB/c-nu nude mice,and pathological changes in tumor tissues were detected by hematoxylin-eosin staining and TUNEL staining.The expression of GSCs markers in tumor tissue was identified by Western blot and immunofluorescence.ResultsThrough bioinformatics analysis,5 active compounds and 55 targets that XHP acts on glioma stem cells were obtained.In vitro experiments showed that besides inducing apoptosis of GBM 3D-tumorspheres,XHP could also reduce the number of 3D-tumorspheres,the proportion of CD133+cells and the expression levels of GSCs markers(CD 133 and SOX2).In addition,XHP can attenuate the expression of CD 133,EGFR,p-Akt,p-mTOR and SOX2 in GBM spheres,and significantly inhibit the expression of p-Akt and SOX2 in stable Akt-overexpressed GBM cells.Among the five active ingredients,quercetin had a stronger inhibitory effect on GSC stemness.Further in vivo experiments suggested that XHP could reduce the expression of GSCs markers(CD133 and SOX2),and delay the progression of subcutaneously transplanted glioblastoma in BALB/c-nu nude mice.ConclusionComputational predictions and experimental verification demonstrated that XHP could suppress the stemness of GBM spheres,restrict their capacity for self-renewal and induce apoptosis by down-regulating SOX2 through the CD133/EGFR/Akt/mTOR signaling cascade both in vitro and in vivo,thereby restricting the progression of subcutaneously transplanted glioblastoma in BALB/cnu nude mice.Quercetin may be the active ingredient in XHP that actually reduces GBM stemness.