香叶木苷对替莫唑胺作用于大鼠神经胶质瘤细胞C6效果的影响

Impact of diosmin on the effect of temozolomide on Glioma cells C6 of rat

目的探究香叶木苷对替莫唑胺作用于大鼠神经胶质瘤细胞C6效果的影响。方法将大鼠神经胶质瘤细胞C6分为4组:空白对照组、替莫唑胺组(200μmol·L^-1)、香叶木苷组(20μmol·L^-1)和联合组(替莫唑胺+香叶木苷,浓度同上)。以CCK-8检测细胞增殖倍数,以Transwell检测细胞侵袭,以Annexin V-EGFP/PI双染法检测细胞凋亡,以蛋白质印迹法检测E钙黏蛋白(E-cad)、N钙黏蛋白(N-cad)、波形蛋白(Vimentin)、核因子κB p65(NF-κB p65)和蛋白激酶B(Akt)通路表达(灰度值)。结果空白对照组、香叶木苷组、替莫唑胺组和联合组在第3天的细胞增殖倍数分别为(4.88±0.34),(4.22±0.40),(3.51±0.30)和(2.73±0.22)倍;这4组的克隆形成率分别为(65.5±12.1)%,(52.7±9.3)%,(26.2±8.0)%和(12.0±5.2)%;这4组的细胞凋亡率分别为(2.51±0.88)%,(5.62±1.80)%,(16.73±3.86)%和(31.45±5.22)%;这4组的细胞细胞侵袭数分别为(154±37),(108±26),(36±9)和(9±5)个;这4组的E-cad表达分别为0.03±0.01,0.12±0.04,0.24±0.04和0.45±0.06;这4组的N-cad表达分别为0.96±0.15,0.56±0.08,0.28±0.06,0.06±0.02;这4组的Vimentin表达分别为0.38±0.07,0.20±0.05,0.08±0.04,0.01±0.01;这4组的p-Akt/Akt比例为分别为0.37±0.04,0.26±0.06,0.16±0.04,0.06±0.02;这4组的p-P65/P65比率为0.30±0.05,0.20±0.04,0.13±0.03,0.03±0.01。上述指标:香叶木苷组、替莫唑胺组和联合组与空白对照组比较,差异均有统计学意义(均P<0.05);联合组与替莫唑胺组比较,差异均有统计学意义(均P<0.05)。结论香叶木苷可能通过NF-κB p65、Akt通路增强替莫唑胺对大鼠神经胶质瘤细胞C6增殖、侵袭和上皮间质转化的抑制作用。

Objective To investigate the impact of diosmin on the effect of temozolomide(TMZ) on rat Glioma C6 cells. Methods Rat Glioma C6 cells were divided into four groups:blank control group,TMZ group(200 μmol·L^-1),diosmin group(20 μmol·L^-1) and joint group(temozolomide+ diosmin,the concentration is the same as above). Cell proliferation multiple was detected by CCK-8. Cell invasion was detected by Transwell. Apoptosis was detected by Annexin V-EGFP/PI double staining method. Expression(gray value) of the E-cadherin(E-cad),N-cadherin(N-cad),Vimentin,nuclear factor κB p65(NF-κB p65) and protein kinase B(Akt) pathway proteins were detected by Western blot. Results The cell proliferation multiple in blank control group,diosmin group,TMZ group,and joint group were(4. 88 ± 0. 34),(4. 22 ± 0. 40),(3. 51 ± 0. 30),and(2. 73 ± 0. 22) times;the colony formation rates in the four groups were respectively(65. 5 ± 12. 1) %,(52. 7 ± 9. 3) %,(26. 2 ± 8. 0) %,(12. 0 ± 5. 2) %;the apoptosis rates in the four groups were(2. 51 ± 0. 88) %,(5. 62 ± 1. 80) %,(16. 73 ± 3. 86) %,(31. 45 ± 5. 22) %;the number of cell invasion in the four groups were(154 ± 37),(108 ± 26),(36 ± 9),(9 ± 5);expression of the E-cad protein in the four groups were 0. 03 ± 0. 01,0. 12 ± 0. 04,0. 24 ± 0. 04,0. 45 ± 0. 06;expression of the N-cad protein in the four groups were 0. 96 ± 0. 15,0. 56 ± 0. 08,0. 28 ± 0. 06,0. 06 ± 0. 02;expression of the Vimentin in the four groups were 0. 38 ± 0. 07,0. 20 ± 0. 05,0. 08 ± 0. 04,0. 01 ± 0. 01;the p-Akt/Akt ratio in the four groups were 0. 37 ± 0. 04,0. 26 ± 0. 06,0. 16 ± 0. 04,0. 06 ± 0. 02;the p-P65/P65 ratio in the four groups were 0. 30 ± 0. 05,0. 20 ± 0. 04,0. 13 ± 0. 03,0. 03 ± 0. 01.Comparison between diosmin group,TMZ group,joint group and blank control group,the difference of the above indicators were statistically significant(all P < 0. 05);comparison between joint group and TMZ group,the difference of the above indicators were statistically significant(all P < 0. 05). Conclusion Diosmin may enhance the inhibitory effect of temozolomide on the proliferation,invasion and epithelial-mesenchymal transformation of rat Glioma C6 cells through the NF-kappa B p65 and Akt pathways.