知识与财富的链接
目的 研究葛根素对人肾癌786-O细胞凋亡的影响及其机制。方法 分别以DMSO(空白对照组)、不同浓度葛根素10、20、40 mg/L和LY294002(PI3K抑制剂)15 mg/L干预对数生长期人肾癌786-O细胞48 h,采用CCK-8法检测细胞增殖抑制率,流式细胞术检测细胞凋亡水平,Western blot法检测磷脂酰肌醇3-激酶(PI3K)、磷酸化PI3K(Tyr317) [p-PI3K(Tyr317)]、蛋白激酶B(Akt)、磷酸化Akt(Ser473)[p-Akt(Ser473)]、糖原合成酶激酶-3β(GSK-3β)、磷酸化GSK-3β (Ser21)[p-GSK-3β(Ser21)]、半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)、激活型Caspase-3(Cleaved Caspase-3)、B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax）表达。结果 与空白对照组比较,葛根素10、20、40 mg/L组和LY294002 15 mg/L组786-O细胞增殖抑制率升高[(17.04±2.92)%、(26.48±4.77)%、(42.79±6.81)%、(35.73±6.12)%比(3.47±0.53)%,P<0.01]、凋亡率升高[(13.72±2.38)%、(30.41±4.05)%、(55.39±6.72)%、(36.08±4.32)%比(2.64±0.51)%,P<0.01];。与空白对照组比较,葛根素20、40 mg/L组和LY294002 15 mg/L组786-O细胞p-PI3K(Tyr317)、p-Akt(Ser473)、p-GSK-3β(Ser21)、Bcl-2表达下调[p-PI3K(Tyr317)：(0.58±0.13)、(0.32±0.07)、(0.38±0.08)比(1.03±0.24);p-Akt(Ser473)：(0.25±0.06)、(0.09±0.03)、(0.11±0.03)比(0.51±0.12);p-GSK-3β(Ser21)：(0.12±0.03)、(0.07±0.02)、(0.09±0.03)比(0.58±0.13);Bcl-2：(0.31±0.08)、(0.18±0.05)、(0.21±0.07)比(0.48±0.12),P均<0.01],Caspase-9、Cleaved Caspase-3、Bax表达上调[Caspase-9：(0.37±0.08)、(0.66±0.13)、(0.58±0.11)比(0.17±0.04);Cleaved Caspase-3：(0.25±0.06)、(0.72±0.16)、(0.43±0.12)比(0.11±0.03);Bax：(0.36±0.08)、(0.62±0.12)、(0.20±0.05)比(0.08±0.02),P均<0.01],PI3K、Akt、GSK-3β磷酸化率降低[PI3K磷酸化率：(0.55±0.15)、(0.29±0.08)、(0.34±0.09)比(0.92±0.28);Akt磷酸化率：(0.24±0.07)、(0.09±0.03)、(0.11±0.04)比(0.53±0.14);GSK-3β磷酸化率：(0.14±0.04)、(0.08±0.03)、(0.10±0.04)比(0.62±0.15),P均<0.01],Bax/Bcl-2比值升高[(1.16±0.27)、(3.41±0.65)、(0.95±0.22)比(0.17±0.04),P<0.01]。与LY294002 15 mg/L组比较,葛根素40 mg/L组786-O细胞增殖抑制率升高[(42.79±6.81)%比(35.73±6.12)%,P<0.05]、凋亡率升高[(55.39±6.72)%比(36.08±4.32)%,P<0.01],Cleaved Caspase-3、Bax表达上调[Cleaved Caspase-3：(0.72±0.16)比(0.43±0.12);Bax：(0.62±0.12)比(0.20±0.05),P均<0.01],Bax/Bcl-2比值升高[(3.41±0.65)比(0.95±0.22),P<0.01],两组间其他指标比较,差异无统计学意义（P>0.05）。结论 葛根素具有诱导人肾癌786-O细胞凋亡的作用,其机制可能与抑制PI3K/Akt/GSK-3β信号通路有关。
Objective To investigate the effect and mechanism of Puerarin on the apoptosis of human renal carcinoma 786-O cells. Methods The DMSO (blank control group), different concentrations of Puerarin (10, 20, 40 mg?/L-1) and LY294002 (PI3K specific inhibitor) 15 mg?/L-1 were used to interfere with human renal carcinoma 786-O cells in logarithmic growth phase for 48 h. The cell proliferation inhibition rate was detected by CCK-8 method, the cell apoptosis level was detected by flow cytometry, cell cycle-related; the expression of PI3K, p-PI3K(Tyr317), Akt, p-Akt(Ser473), GSK-3β, p-GSK-3β(Ser21), Caspase-9, Cleaved Caspase-3, Bcl-2, Bax. Results Compared with blank control group, the cell proliferation inhibition rate [(17.04±2.92)%, (26.48±4.77)%, (42.79±6.81)%, (35.73±6.12)% vs. (3.47±0.53)%, P<0.01] and apoptosis rate [(13.72±2.38)%, (30.41±4.05)%, (55.39±6.72)%, (36.08±4.32)% vs. (2.64±0.51)%, P<0.01] of 786-O cells in Puerarin 10, 20, 40 mg?/L-1 groups and LY294002 15 mg?/L-1 group were increased. Compared with blank control group, The the expression of p-PI3K(Tyr317), p-Akt(Ser473), p-GSK-3β(Ser21), Bcl-2 of 786-O cells in Puerarin 20, 40 mg/L groups and LY294002 15 mg/L group were down-regulated [p-PI3K(Tyr317): 0.58±0.13, 0.32±0.07, 0.38±0.08 vs. 1.03±0.24; p-Akt(Ser473): 0.25±0.06, 0.09±0.03, 0.11±0.03 vs. 0.51±0.12; p-GSK-3β(Ser21): 0.12±0.03, 0.07±0.02, 0.09±0.03 vs. 0.58±0.13; Bcl-2: 0.31±0.08, 0.18±0.05, 0.21±0.07 vs. 0.48±0.12, all P<0.01]. The expression of Caspase-9, Cleaved Caspase-3, Bax were up-regulated (Caspase-9: 0.37±0.08, 0.66±0.13, 0.58±0.11 vs. 0.17±0.04; Cleaved Caspase-3: 0.25±0.06, 0.72±0.16, 0.43±0.12 vs. 0.11±0.03; Bax: 0.36±0.08, 0.62±0.12, 0.20±0.05 vs. 0.08±0.02, all P<0.01). The phosphorylation rate of PI3K, Akt, GSK-3β were decreased (PI3K phosphorylation rat: 0.55±0.15, 0.29±0.08, 0.34±0.09 vs. 0.92±0.28; Akt phosphorylation rat: 0.24±0.07, 0.09±0.03, 0.11±0.04 vs. 0.53±0.14; GSK-3β phosphorylation rat: 0.14±0.04, 0.08±0.03, 0.10±0.04 vs. 0.62±0.15, all P<0.01) . The ratio of Bax/Bcl-2 was increased (1.16±0.27, 3.41±0.65, 0.95±0.22 vs. 0.17±0.04, P<0.01]. Compared with LY294002 15 mg?/L-1 group, the cell proliferation inhibition rate [(42.79±6.81)% vs. (35.73±6.12)%, P<0.05] and apoptosis rate [(55.39±6.72)% vs. (36.08±4.32)%, P<0.01] in Puerarin 40 mg?/L-1 group was increased. The expression of Cleaved Caspase-3, Bax were up-regulated (Cleaved Caspase-3: 0.72±0.16 vs. 0.43±0.12; Bax: 0.62±0.12 vs. 0.20±0.05, all P<0.01). The ratio of Bax/Bcl-2 was increased (3.41±0.65 vs. 0.95±0.22, P<0.01). While there was no significant difference in other indicators between two groups (P>0.05). Conclusions Puerarin can induce apoptosis of human renal carcinoma 786-O cells, which mechanism may be related to the inhibition of PI3K/Akt/GSK-3β signaling pathway.
