| Objective To explore the effect of Gecko crude peptides(GCPs)on proliferation,apoptosis and migration of(SH-SY5Y)neuroblastoma cells and its molecular mechanisms.To investigate the effect of vitamin C combined with GCPs on SH-SY5 Y cells.The effect of some polypeptide monomers on SH-SY5 Y cells was preliminarily studied.Methods The effect of Gecko crude peptides(GCPs)on proliferation,apoptosis and migration of(SH-SY5Y)neuroblastoma cells.MTT method was used to screen out the appropriate GCPs concentration to SH-SY5 Y cells.Firstly,SH-SY5 Y cells were divided into the control group and six other groups with different concentrations(0.08,0.12,0.16,0.20,0.24,0.32 mg/m L)GCPs for 24 h,48 h,72 h.The morphological changes of SH-SY5 Y cells were observed by inverted microscope.Wound healing assay was performed to observe the influence of GCPs on the migration of SH-SY5 Y cells.The migration of SH-SY5 Y cells were detected by transwell migration assay.Hoechst 33258 fluorescence staining was used to detect the effects of GCPs on nuclear morphology of SH-SY5 Y cells.Flow cytometry was used to detect the apoptotic rate of SH-SY5 Y cells which were treated with GCPs.The expression of caspase-3 and caspase-9 were detected by Western blot assay;PEPCK enzyme activity was detected after GCP treatment in SH-SY5 Y cells.To verify whether PEPCK overexpression could affect SH-SY5 Y cells proliferation.Firstly,overexpressed PEPCK plasmid were transfected into SH-SY5 Y cells,and then the growth status of the cells were observed.Secondly,the nuclear changes of SH-SY5 Y cells were detected by Hoechst 33258 fluorescence staining.Finally,wound healing assay was performed to observe the influence on SH-SY5 Y cells.Effects of GCPs combined with Vitamin C on the proliferation,migration and apoptosis of SH-SY5 Y cells: For each group of logarithmic growth cells,a solution of GCPs at a concentration of 0.15 mg/m L was added.Firstly,SH-SY5 Y cells were divided into the control group and eight other groups with different concentrations(0.005,0.01,0.05,0.10,0.15,0.2,0.25,0.3 mmol/L)Vitamin C for 24 h,48 h.Then cell proliferation was assessed using MTT.The morphological changes of SH-SY5 Y cells were observed by inverted microscope.Wound healing assay was performed to observe the influence of Vitamin C on the migration of SH-SY5 Y cells.Flow cytometry was used to detect the apoptotic rate of SH-SY5 Y cells which were treated with Vitamin C.MTT assay was used to detect the activity of monomer peptides on SH-SY5 Y cells.Results After treatment of GCPs for 24 h,48 h,72 h,the 50% inhibitory dose(IC50)values were 0.24 mg/m L,0.21 mg/m L,0.18 mg/m L,respectively.Compared with the blank control group,the healing ability were inhibited at GCPs low,medium and high doses of SH-SY5 Y cells(P < 0.05);Compared with the control group,GCPs could significantly inhibit the ability of SH-SY5 Y cells to penetrate the filtration membrane.The higher the concentration,the more obvious the inhibition.Under the observation of fluorescence microscope,the blue fluorescence of the blank control group was uniform,round and in good shape.In the GCPs group,the blue fluorescence distribution were uneven,the nucleus were reduced and irregular,and there were stronger fluorescence,and the apoptostic morphology were more obvious.Compared with the blank control group,the higher the concentration of GCPs,the higher the apoptosis rate of SH-SY5 Y cells,P < 0.05;The protein expression of caspase-3 and caspase-9 were upregulated after treatment of GCPs.After treatment of GCPs on SHSY5 Y cells,the PEPCK enzyme activity was increased.When overexpressed PEPCK plasmid were transfected into SH-SY5 Y cells,apoptosis rate were increased,scratch healing ability were weakened,and the nucleus appeared senescence.After treatment of Vitamin C for 24 h,48 h,the 50% inhibitory dose(IC50)values were 0.1242 mmol/L and 0.1213 mmol/L,respectively.Vitamin C can enhance the effect of GCPs on inhibiting the proliferation of SH-SY5 Y cells.And the inhibitory effect is more significant with the increase of time and Vitamin C concentration.SHSY5 Y cells in the blank group grew adherent to the wall,with uniform size,long protuberances,clear and complete contour,good growth status and uniform cytoplasm.GCPs(0.1 mg/m L)group cells had a slight swelling of the cells body,growth and aggregation,and a clear cell boundary.In the Vitamin C(1 mmol/m L)group,the cells body was slightly swollen and the protuberance was shortened.GCPs(0.1 mg/m L)+Vitamin C(1 mmol/m L)cells body swelling was severe,protrusions were shortened significantly.And cells became round and bright,and cell fragments appeared.Compared with the blank control group,GCPs(0.1 mg/m L)group and Vitamin C(0.1mmol/m L)group showed reduced scratch healing ability,but there were no significant difference(P > 0.05).Compared with the blank control group,the scratch healing ability of GCPs(0.1 mg/m L)+ Vitamin C(0.11 mmol/m L)group was significantly reduced(P < 0.05).After Hoechst 33258 fluorescence staining,the fluorescence size and distribution of cells nuclei in the blank control group were uniform,circular and in good shape.However,the distribution of cell nucleus blue fluorescence in the GCPs(0.1 mg/m L)treatment group was uniform,and the cell nucleus fluorescence was basically the same as that in the blank control group.The treated cells in the Vitamin C(1 mmol/m L)group had slightly shrunken nuclei compared with those in the control group.In the GCPs(0.1 mg/m L)+ Vitamin C(1 mmol/m L)group,the fluorescence intensity increased,the nucleus significantly shrunk,the fluorescence distribution was uneven,and strong fluorophore appeared.When the monomer peptide acted on SH-SY5 Y cells for 24 h,there were 7monomer peptides that had a pro-proliferation effect on SH-SY5 Y cells when the concentration reached 0.1 mg/m L.In a certain concentration range,the proliferation rate increased with the increase of the concentration.There were two monomer peptides that showed weak inhibition of proliferation.When the monomer peptide on SH-SY5 Y cells for 48 h,only two monomer peptides can promote the proliferation of SH-SY5 Y cells.The other 7 showed inhibitory effects on proliferation,among which the inhibitory effects of monomer STPS and monomer EQLQ were significant.Nine gecko polypeptide monomers had different effects on the proliferation of SH-SY5 Y cells.The effect of the same monomer on the SH-SY5 Y cells is different with the different action time.Conclusion GCPs can inhibit proliferation and migration of SH-SY5 Y cells,which mechanism may be associated with the caspase pathway and PEPCK gene enzyme activity.Overexpression of PEPCK gene can inhibit the proliferation and migration of SH-SY5 Y cells and induce their apoptosis,which may be related to the caspase pathway and energy metabolism.Vitamin C enhance the ability of GCPs to inhibit the proliferation and migration of SH-SY5 Y cells and increase the apoptosis rate of SH-SY5 Y cells.Vitamin C can reduce the concentration of GCPs used.Nine gecko polypeptide monomers had different effects on the proliferation of SH-SY5 Y cells.The effect of the same monomer on the SH-SY5 Y cells is different with the different action time. |
