A Study On The Effects Of Celastrus Orbiculatus Extracts On Apoptosis Of Human Gliomablastoma U251 Cell

Glioblastoma is the most common primary neurological tumor.The current treatment for glioblastoma is still based on traditional surgery,and adjuvant chemo-radiotherapy.However,as glioblastoma s grow invasively,it is difficult to separate the tumor tissue from the normal and remove them all.Thus,glioblastoma s would get a resurgence easily after operation and as a result glioblastoma patients got a low survival rate.Regular anti-glioblastoma drugs inhibit proliferation and induce apoptosis mainly via cytotoxic effect to control the tumor.Meanwhile its side effect is also great which would do damage to the normal tissue,and easy to form drug resistance,which would affect the therapeutic efficacy and life quality.As a result,there is an urgent need of new anti-glioblastoma drugs.Celastrus orbiculatus is a genus of deciduous woody vines of the genus euonymus.It is also known as Guoshanlong,Dananshe and et al.Celastrus orbiculatus was first recorded in Zhiwu Mingshi Tukao as herbal medicine,which could cure many disease including traumatic injury,waist and leg pain.Our previous studies found that C.orbiculatus ethyl acetate extract(COE)had a significant intervention on inhibiting the proliferation,invasion and metastasis of glioblastoma.While its impact on apoptosis of glioblastoma cells is still unknown.In this subject,COE-induced apoptosis of glioblastoma U251 cells and its related molecular mechanisms was studied for the first time.Different concentrations of COE were used to study the effects of the drug on the apoptosis of human glioblastoma U251 cells and the possible molecular mechanisms involved.And the result could lay a foundation for the development and further clinical application in the treatment of malignant glioblastoma.Different molecular biology methods was used in this subject to study the effects of COE on apoptosis of human glioblastoma cell lines,elucidating the molecular mechanisms of its effects,and providing mechanism support for Chinese medicine against brain tumors,and the theoretical basis for the development of anti-glioblastoma Chinese medicine with independent property rights.Part?Effect of Celastrus Orbiculatus Extracts on Glioblastoma U251 cell Objective:To study the effects of different concentrations of COE on the proliferation and apoptosis of human glioblastoma cells.Methods:Human glioblastoma U251 cells in logarithmic growth phase were randomly divided into control group and treatment group(the treatment group was given COE with the concentration of 10,20,40,80,160,320 ?g/mL,respectively).Cell viability was measured at three time points(24 h,48 h,and 72 h)by MTT assay.According to MTT results,cytotoxicity was ruled out.COE at low,medium and high concentrations(40,80,120 ?g/mL)was selected as the subsequent experimental concentration.Negative control group,positive control drugs(temozolomide,TMZ,40 ?M)and COE interfering with U251 cells for 24 h,and the AnnexinV/FITC-PI dual-label flow cytometry test was performed to detect apoptosis,JC-1 detected changes in mitochondrial membrane potential.Results:Proliferation of U251 cells was inhibited in a time-and concentration-dependent manner.According to the inhibitory rate,the calculated IC50 of COE in 24 h was 127.06 ?g/mL.After treated with different concentrations of COE for 24 hours,the apoptosis rate of U251 cells increased and the mitochondrial membrane potential decreased gradually with the increase of COE concentration.Conclusion:COE can inhibit the proliferation of human glioblastoma U251 cells and promote the apoptosis of U251 cells through the mitochondrial pathway.Part II The Molecular Mechanism of Celastrus Orbiculatus Extract Promoting Apoptosis of Human Glioblastoma U251 Cell via Mitochondrial Pathway and Microfilament RemodelingObjective:Transmission electron microscopy was used to observe the ultrastructural changes in glioblastoma U251 cell after COE intervention,and further investigating the effects of COE on apoptosis-related mitochondrial pathway and microfilament remodeling.Methods:The ultrastructure of human glioblastoma U251 cell was observed by transmission electron microscopy after the treatment of COE(80 ?g/mL)for 24 h.Western blot was used to detect the expression of mitochondrial apoptosis-related protein in U251 cells after the treatment of Different concentrationsof COE(40,80,120 ?g/mL)for 24 h.Immunofluorescence staining was used to observe the change of microfilament skeleton of glioblastoma cell after COE intervention.Western blot was used to detect the expression level of apoptosis cytoskeleton-related proteins in U251 after COE intervention for 24 h.Results:After being treatet with COE(80 ?g/mL),cells swelled,the number of mitochondria increased,the volume became smaller,the number of mitochondrial ridges decreased,and the reticular formation collapsed.The arrangement of microfilaments was disordered.The results of Western blot showed that the protein expression of Bcl2L12 and Bcl-2 was down-regulated and the expression of Bax,Cyt-C and Caspase-3 were up-regulated after COE treatment.Immunofluorescence experiments showed that the distribution of microfilament was changed to the surroundings instead of the center after COE and Lat A treatment.Western blot results showed that the down-regulation of PI3K,Akt and Bcl-2 and the up-regulation of Caspase-3 were related to the application of COE and Lat A.The above changes were more pronounced in the COE combined with the Lat A group.Conclusion:COE can induce apoptosis of U251 cells via mitochondrial pathway;COE can affect the remodeling of microfilament skeleton of glioblastoma cell,leading to apoptosis of U251 cells.Part ? The Effect of Celastrus Orbiculatus Extract on Human Glioblastoma Xenograft and Molecular MechanismsObjective:To observe the effect of COE on the anti-glioblastoma of U251 tumor-bearing nude mice.Methods:2 × 106 logarithmic growth phase human glioblastoma U251 cells were implanted subcutaneously in the nude mice and were divided into 3 groups immediately after three weeks of injection,including negative control group,COE treatment group and positive control group.The COE group and the positive control group were treated with COE and TMZ 40 mg/kg/d by gavage.The control group was given equal volume of normal saline.The body weight of nude mice and the length of the subcutaneous tumors were measured every three days.In vivo fluorescence imaging was performed every 7 days.After 21 days,PBS was perfused to isolate subcutaneous tumors.The tumors were weighed and photographed.The expression of mitochondrial apoptosis-related proteins and microfilament skeleton-related proteins in tumor tissues was detected by immunohistochemistry.Results:The tumor volume and tumor weight of the COE and TMZ intervention groups were both lower than those of the control group,and the inhibitory effect of COE was more obvious.The results of immunohistochemistry showed that the expression levels of Caspase-3,Bax were up-regulated and the expression of Bcl-2,PI3K,and p-Akt were down-regulated.Conclusion:COE can significantly inhibit the growth of subcutaneous xenograft glioblastoma tissue,and the inhibitory effect is more obvious than TMZ.The mechanism may be related to the activation of mitochondrial apoptosis and the remodeling of tumor cell microfilaments.