Protective Effect And Mechanism Of Icariin On X-ray Induced Neural Stem Cells Injury

Objective:To explore the protective effect and mechanism of icariin on X-ray induced neural stem cells injury,so as to provide a new idea for exploring potential anti-radiation agent.Methods:NE-4C neural stem cells were cultured in vitro and irradiated with different doses of X-rays（0,1,2.5,5,10 Gy）,the cell survival rate was detected by CCK-8 method after 12 h,24 h and 48 h respectively.After the cells were treated with different concentrations of icariin（0,2.5,5,10,20,40μM）for 48 hours,the cells survival rate was measured by CCK-8 method.Pretreated with different concentrations of icariin（0,2.5,5,10,20,40μM）for 30 minutes,the cells were irradiated with 5 Gy X-rays to establish the radiation model.CCK-8 assay was used to detect the cell survival rate after 48 h.The cells were divided into 5 groups:control group（Con group）,irradiated group（IR group）,and low,medium and high concentration icariin treated before irradiating.The effects of Icariin and X-ray on migration ability of NE-4C cells were detected by cell scratch test.Hoechst 33258 staining was used to observe cell apoptosis.Western blot assay was used to detect the expressions of apoptosis-related proteins Bax and Bcl-2,gamma-H2AX,autophagy related proteins LC3 II/LC3 I,Beclin-1 and oxidative stress-related proteins Nrf2、Keap1、HO-1、NQO1.Results:（1）The results of CCK-8 assay showed that 48 h after irradiation,different radiation doses of X-ray significantly decreased the survival rate of NE-4C cells,and showed a dose-dependent effect.48 h after irradiation of 5 Gy X-ray,the survival rate of NE-4C cells was about 51%.The survival rate of NE-4C cells did not change significantly with different concentrations of icariin.After icariin and X-ray treatment,different concentrations of icariin could significantly increase the cell survival rate,and it was concentration-dependent in a certain concentration range（2.5,5,10μM）.（2）The cell scratch experiment showed that X-ray irradiation could significantly inhibit the migration of NE-4C cells when compared to the control group,and icariin treatment could significantly promote the migration.（3）Hoechst 33258staining showed that compared with the control group,the blue fluorescence in the IR group was enhanced and brightened,showing obvious apoptotic characteristics.After icariin treatment,the blue fluorescence of cell granules was decreased to different degrees.（4）WB results showed that compared with the control group,the protein expressions of Bax/Bcl-2,gamma-H₂AX,LC3 II/LC3I,Beclin-1,NQO1 and HO-1 in IR group were increased,while the expression level of Keap1 was decreased.Icariin can reduce the expressions of Bax/Bcl-2,gamma-H₂AX and Beclin-1,and increase the protein content of Keap1,HO-1 and NQO1.Conclusion:（1）X-ray irradiation can cause damage to the proliferation and migration of NE-4C neural stem cells.（2）Icariin can improve the proliferation and migration of NE-4C cells induced by radiation.（3）The neuroprotective effect of icariin on radiation-induced neural stem cell injury may be related to the decrease of apoptosis and DNA damage by down-regulating the expression levels of Bax/Bcl-2,Beclin-1 and gamma-H2AX,and up-regulating the expression levels of HO-1 and NQO1 proteins.