The Mechanism Of Saikosaponin D Killing Glioblastoma Via Endoplasmic Reticulum Stress In Vitro

Objective: To explore the toxic effect and sensitivity analysis of Saikosaponin d(SSD)on Glioblastoma Mutiform(GBM),and explore its molecular mechanism;The synergistic effect of SSD combined with temozolomide(TMZ)on GBM cell lines was clarified,and the sensitivity of TMZ to GBM cells increased by SSD was explored,and its molecular mechanism was further analyzed.It is expected to increase the clinical value of SSD and provide adjuvant therapy strategies for GBM patients.To explore the killing effect and sensitivity analysis of SSD on TMZ in GBM cells.The synergistic effect of SSD combined with TMZ,and the sensitivity of TMZ to GBM cells increased by SSD was explored,and its molecular mechanism was further analyzed.It is expected to increase the clinical value of SSD and provide adjuvant therapy strategies for GBM patients.Methods: CCK-8 method was used to detect the sensitivity of different concentrations of SSD and TMZ to GBM cell lines.The sensitivity and cell vitality of SSD combined with TMZ on GBM cell lines were detected,and the optimal concentration was selected for subsequent experiments.The morphological changes of GBM cell lines before and after administration were observed by light microscope and hematoxylin-eosin(HE)staining.The effects of SSD/TMZ combined treatment on the proliferation of GBM cell lines were detected by cell clonal formation assay.Hoechst33258 fluorescence staining and flow cytometry were used to detect apoptosis of GBM cells.MDC fluorescence staining was used to observe the autophagosomes of GBM cells treated with SSD and TMZ for 48 h.The expression and distribution of apoptotic proteins,endoplasmic reticulum stress proteins and autophagy related proteins in GBM cell lines treated with SSD and TNZ for 48 h were detected by Western blotting Immunocytochemistry(ICC)assay.Results: SSD significantly inhibited the proliferation of RG-2,U87-MG,U251 and LN-428 cells.RG-2,U87-MG and U251 cell lines showed concentration and time dependence,while LN-428 cells showed concentration dependence and sensitivity ranking as U251 cells > U87-MG cells >RG-2 cells > LN-428 cells.Combined administration results showed that SSD significantly enhanced the inhibitory effect of TMZ on GBM cells.Cell cloning and formation experiments also confirmed that combined administration significantly inhibited the proliferation of GBM cell lines.The results of light microscope showed that compared with the control group,the fine adhesion became worse,the floating cells increased,the cell volume decreased,the cytoplasmic protrusion extended,the nuclear shrinkage,and these phenomena were obvious with the increase of the concentration,and the apoptotic bodies were observed in the high concentration group.HE staining showed cytoplasmic elongation in combination administration compared with alone administration.Hoechst 33258 fluorescence staining showed that the number of nuclear bright staining increased with the increase of SSD concentration.GBM cells were detected by flow cytometry with Annexin V/PI double staining,and the number of early and late apoptotic cells increased in the treatment group compared with the control group.MDC fluorescence staining showed that after 48 h of SSD/TMZ combined treatment of GBM cell lines,the cytoplasm of cells in the control group was uniformly stained with yellow-green fluorescence,while the cytoplasm of cells in the drug treatment group showed dense and densely dyed green particles of different sizes,and the number of densely dyed green particles increased with the increase of drug concentration.Western blotting test results showed that compared with the control group,apoptotic proteins Caspase-12,Caspase-9 and Caspase-3 in the dosed group were cleaved,and the expression of cleaved body protein was significantly increased.ICC results showed that the cytoplasm of the dosed group was dark brown compared with the control group.The nucleus of poly ADP-ribose polymerase(PARP)was deeply stained.The expression of endoplasmic reticulum(ER)stress marker protein Glucose regulated protein 78 k D(GRP78)and C/EBP homologous protein(CHOP)was significantly increased.In addition,the protein PKR-like ER kinase(PERK)and Transcription factor-6(ATF6)were activated,and the expressions of autophagy proteins LC3 and Beclin-1 were significantly increased.ICC experiment further confirmed that the expression of autophagy protein was increased in the cytoplasm.Conclusions: SSD inhibited the proliferation of RG-2 cells,U87-MG cells,U251 cells and LN-428 cells,and induced autophagy and apoptosis of GBM cell lines by activating ER stress pathway.Moreover,SSD enhances the sensitivity of GBM cells to TMZ by activating ER stress.This study further expands the application value of SSD,which can be used as a potential adjuvant drug candidate in the treatment of GBM patients,providing new ideas and treatment methods for resistant TMZ in GBM patients.