The Mechanism Of Schizandrin Inhibiting Proliferation And Migration Of Neuroblastoma SH-SY5Y Cells

BackgroundsNeuroblastoma(NB)is a neuroendocrine tumor that can originate from any nerve ridge of the sympathetic nervous system,it mostly located on the adrenal gland,while it can also be found in the neck,chest,abdomen and pelvic nerve tissue.Nearly half of NB occurs within 2 years old,some NB can subside spontaneously or transform into benign tumors while most are difficult to be cured with poor prognosis.At present,surgery,chemotherapy and radiotherapy are still the main treatment methods for neuroblastoma.However,due to the serious side effects of most treatments,it is urgent to search new drugs which are more safe and more effective.It is reported that schisandra chinensis has important anti-cancer functions.Schisandrin,the main ingredient of lignans,has been reported many times for its anti-cancer effect,but the specific mechanism is not yet clear.Because schizandrin is a small fat-soluble substance,maybe it can cross the blood-brain barrier,which can be used as a neurotherapeutic drug.In this experiment,schizandrin was applied to neuroblastoma SH-SY5 Y cells for the first time to explore whether it could inhibit the proliferation and invasion of SH-SY5 Y cells and to disscuss its possible mechanism,the results may provide a new target for NB clinical treatment.Objective To explore the possible mechanism of schizandrin inhibiting proliferation and migration of SH-SY5 Y cells so as to provide new theoretical support for the clinical treatment of NB.Methods1.Schisandrin with different concentrations was added to SH-SY5 Y cells for different time,respectively.MTT was used to detect cell activity of differene groups,so as to determine the appropriate drug action time and concentration of Schisandrin.2.SH-SY5 Y cells were treated with Schisandrin at different concentrations for a certain amount of time.Apoptosis was detected by DAPI staining and flow cytometry.According to the experimental results,the expression levels of mRNA and protein of Bcl-2,Bcl-xl,Bax and p-AMPK genes were detected by qRT-RCR and Western Blot assay.3.SH-SY5 Y cells were treated with Schisandrin at different concentrations for a certain amount of time,and the cell cycle was detected by flow cytometry.According to the experimental results,the expression levels of mRNA and protein of cyclin D1,CDK4 and P21 genes were detected by qRT-RCR and Western Blot assay.4.SH-SY5 Y cells were treated with different concentrations of Schisandrin for a certain amount of time,and the migration ability was detected by scratch test.At the same time,schisandrin was added to SH-SY5 Y cells with the concentrations of 200 cells/mL,then SH-SY5 Y cells were cultured for many days.According to this phenomenon,the levels of mRNA and proteins of MMP9,HIF-1a and VEGF in each group were detected by qRT-RCR and Western Blot assay,respectively.Results1.SH-SY5 Y cells treated with different concentrations of Schisandrin were cultured for different time respectively,the results of MTT showed that compared with the control group cells,the inhibition ratio of the group treated with Schisandrin is markedly increased with the increase of drug concentration and drug actin time,which was positively correlated with the time and dose of the drug.2.The results of DAPI staining and flow cytometry showed that the number of apoptosis in the group treated with schisandrin was higher than that in control group.The results of qRT-RCR showed that compared with the control group,the mRNA level of Bcl-2 and Bcl-xl decreased.The results of Western Blot showed that the protein level of Bcl-2 decreased,while the protein level of Bax and p-AMPK increased.3.Flow cytometry was used to detect cell cycle in each treatment group.The results showed that,compared with the control group,the proportion of cells in G2/M phase increased and the proportion of G1 phase decreased with the increase of drug concentration.The results of qRT-RCR and Western Blot showed that compared with the control group,the mRNA and protein levels of cyclinD1 and CDK4 decreased,negatively correlated with the dose of Schisandrin.But the protein expression of P21 increased,positive correlation with the drug dosage.4.The results of scratch test showed that compared with the control group,the migration ability of cells treated with schisandrin was significantly decreased.Colony cloning assay showed that the number and size of cell clones treated with schisandrin were significantly lower than those in the control group,negatively correlated with the drug concentration.The results of qRT-PCR and Western Blot showed that the levels of mRNA and protein of MMP9,HIF-1? and VEGF were lower than those of the control group.It was negatively correlated with the dose of Schisandrin.Conclusions1.Schizandrin inhibited the proliferation and invasion of human neuroblastoma SH-SY5 Y cells in a time-and dose-dependent manner.2.Schisandrin may block the cell cycle by affecting the expression of cyclinD1,CDK4 and P21 or by inducing apoptosis of SH-SY5 Y cells by regulating the expression of Bcl-2 family genes,so as to play an anti-proliferation role.3.Schisandrin inhibits the migration of SH-SY5 Y cells by inhibiting the expression of MMP9,HIF-1? and VEGF genes.