半枝莲总黄酮通过PI3K/AKT/mTOR通路诱导肿瘤细胞自噬的体内实验研究

Effect of total flavonoids in Scutellaria barbata in mediating autophagy in tumor cells via PI3K/AKT/mTOR pathway

该研究拟从体内角度研究半枝莲总黄酮(TF-SB)的抗肿瘤作用,并探讨TF-SB对肿瘤细胞自噬的影响及其对PI3K/AKT/mTOR通路的调控作用,为TF-SB的抗肿瘤作用提供实验依据。实验以黑色素瘤荷瘤小鼠模型为研究对象,分为空白对照组、雷帕霉素组(Rap,1.5 mg·kg^(-1))、TF-SB高、中、低剂量组(200,100,50 mg·kg^(-1)),每天给药1次,连续用药11 d,通过测量肿瘤的体积及抑瘤率,来观察TF-SB对黑色素瘤生长的抑制效果;通过TUNEL法检测肿瘤细胞凋亡情况,来进一步验证TF-SB的抗肿瘤活性;通过Western blot法检测LC3-Ⅰ,LC3-Ⅱ的蛋白表达,计算LC3-Ⅱ对LC3-Ⅰ的相对表达量,探讨TF-SB对肿瘤细胞自噬的诱导作用;通过检测PI3K/AKT/mTOR通路标志蛋白的磷酸化水平,研究TF-SB诱导细胞自噬的分子机制。结果表明,TF-SB可有效抑制荷瘤小鼠体内黑色素瘤的生长,减小肿瘤体积、提高抑瘤率,并且可以显著增加肿瘤细胞凋亡指数,增加LC3-Ⅱ/LC3-Ⅰ,抑制p-PI3K,p-AKT及p-mTOR的蛋白表达水平,与对照组比较有显著性差异(P<0.05,P<0.01或P<0.001)。由此可见,半枝莲总黄酮具有抑制体内黑色素瘤生长的作用,其机制可能与通过抑制PI3K/AKT/mTOR通路,诱导肿瘤细胞自噬及凋亡有关。

To investigate the effect of the total flavonoids in Scutellaria barbata（TF-SB） against autophagy in tumor cells in vivo,and further determine whether the mechanism is correlated with the PI3K/AKT/mTOR pathway,which lead to autophagy-induced tumor cell death.Melanoma-bearing mice were prepared and divided into control group,rapamycin group（Rap 1.5 mg·kg^-1）,and high,middle and low-dose TF-SB（200,100,50 mg· kg^-1） groups.The groups were given drugs once a day for successively 11 days.The inhibitory effect of TF-SB on the growth of melanoma was determined by measuring tumor volume and tumor inhibition rate.TUNEL method was used to detect the apoptosis of tumor cells to further verify the antitumor activity of TF-SB.The protein expressions of LC3-Ⅰ and LC3-Ⅱ were detected by Western blot,and the relative expression of LC3-Ⅱ was calculated based on LC3-Ⅱ/LC3-Ⅰ.In addition,the effect of TF-SB on autophagy of tumor cells,the underlying molecular mechanism of TF-SB in inducing autophagy and PI3K/AKT/mTOR pathway marker protein phosphorylation were also studied.According to the results,TF-SB effectively inhibited melanoma growth in mice,reduced tumor volume,increased the tumor inhibition rate,and significantly increased tumor cell apoptosis index and the ratio of LC3-Ⅱ/LC3-I（P〈0.05,P〈0.01 or P〈0.001）.The protein expressions of p-PI3 K,p-AKT and p-mTOR were also suppressed dramatically compared with those in control group（P〈0.05,P〈0.01 or P〈0.001）.In conclusion,the total flavonoids in S.barbata could inhibit the growth of melanoma in vivo by inducing autophagy and apoptosis of tumor cells,which may be correlated with suppression of PI3K/AKT/mTOR pathway.