养阴化瘀解毒方通过PI3K/Akt信号通路调控低氧环境下结肠癌细胞的自噬和凋亡

Yangyin Huayu Jiedu Preseription Regulates Autophagy and Apoptosis of Colon Cancer Cells in Hypoxic Environment Through PI3K/Akt Signaling Pathway

目的:观察不同氧浓度环境对结肠癌细胞增殖、自噬的影响,并基于磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路探讨养阴化瘀解毒方在低氧环境下对结肠癌细胞自噬及凋亡的影响。方法:HCT-116细胞分为常氧组、1%低氧组、5%低氧组,通过细胞增殖与毒性检测(MTS)法检测细胞存活率,单丹磺酰尸胺(MDC)染色观察不同氧浓度下细胞自噬荧光表达情况;在5%低氧微环境下用养阴化瘀解毒方干预HCT-116细胞,分为正常组、空白血清组、养阴化瘀解毒方组(养阴化瘀解毒方含药血清5%、15%、25%);通过MTS法计算各组细胞的抑制率;单丹磺酰尸胺(MDC)染色检测各组自噬细胞率的变化;利用膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)/碘化丙锭(PI)流式细胞术检测各组细胞凋亡率;蛋白免疫印迹法(Western blot)检测不同组自噬蛋白微管相关蛋白1轻链3(LC3Ⅱ/Ⅰ)、酵母Atg6同源物(Beclin-1)、泛素结合蛋白(p62)和凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2/腺病毒E1B相互作用蛋白3(BNIP-3)、Bcl-2关联X蛋白(Bax)、活化的胱天蛋白酶-3(cleaved Caspase-3)及缺氧诱导因子-1α(HIF-1α)和通路蛋白磷脂酰肌醇3-激酶(PI3K)、磷酸化PI3K(p-PI3K)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)表达情况。结果:与常氧组比较,1%、5%低氧组细胞存活率均明显上升,以5%低氧组细胞存活率提升幅度最大(P<0.01);与常氧组比较,1%、5%低氧组细胞自噬荧光强度均明显增强,且以5%低氧环境下MDC染色自噬荧光表达最强;低氧环境下,与空白血清组比较,养阴化瘀解毒方组(15%、25%)细胞抑制率明显提高、自噬细胞率降低、细胞凋亡率升高(P<0.01);养阴化瘀解毒方组(15%、25%)Beclin-1蛋白表达降低(P<0.05);与养阴化瘀解毒方组(5%)比较,养阴化瘀解毒方组(25%)Beclin-1蛋白表达降低(P<0.05);与空白血清组比较,养阴化瘀解毒方组(5%、15%、25%)LC3Ⅱ/Ⅰ蛋白表达均降低(P<0.05,P<0.01);养阴化瘀解毒方组(15%、25%)p62蛋白表达升高(P<0.01);与养阴化瘀解毒方低剂量组比较,养阴化瘀解毒方组(25%)p62蛋白表达升高(P<0.05);与空白血清组比较,养阴化瘀解毒方组(25%)促凋亡蛋白BNIP-3、Bax表达升高,抑凋亡蛋白Bcl-2表达降低(P<0.01);与养阴化瘀解毒方组(5%)比较,养阴化瘀解毒方组(25%)Bax蛋白表达上升(P<0.05);与空白血清组比较,养阴化瘀解毒方组(15%、25%)低氧因子HIF-1α表达降低(P<0.01);养阴化瘀解毒方组(25%)p-PI3K/PI3K、p-Akt/Akt表达上升(P<0.05,P<0.01);与养阴化瘀解毒方中剂量比较,养阴化瘀解毒方组(25%)p-Akt/Akt表达上升(P<0.05)。结论:低氧微环境能明显促进结肠癌细胞自噬及增殖,发挥细胞保护作用,养阴化瘀解毒方在5%低氧环境下能明显抑制结肠癌细胞自噬和增殖,并促进细胞凋亡,其机制可能与上调PI3K/Akt信号通路有关。

Objective:To investigate the effect of different oxygen concentration on the proliferation and autophagy of colon cancer cells and to explore the effect of Yangyin Huayu Jiedu Preseription(YHJP)on autophagy and apoptosis of colon cancer cells under hypoxia based on phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)signaling pathway.Method:HCT-116 cells were divided into normoxia group,1%O2 group,and 5%O2 group.Cell viability was detected by cell proliferation assay(MTS),and autophagy was observed based on monodansylcadaverine(MDC)staining.HCT-116 cells were treated with YHJP in 5%O2 microenvironment.The cells were divided into normal group,blank serum group,and low-,medium-,highdose YHJP groups(5%,15%,25%serum containing YHJP).Cell inhibition rate in each group was calculated by MTS,and changes in the rate of autophagy were detected based on MDC staining.Annexin V-fluorescein isothiocyanate(FITC)/propidium iodide(PI)was employed to detect the apoptosis rate of each group.Western blotting was applied to measure the expression of autophagy proteins microtubule-associated protein 1 light chain 3(LC3Ⅱ/Ⅰ),yeast Atg6 homolog(Beclin-1),ubiquitin-binding scaffold protein p62(p62),apoptosis-related proteins B-cell lymphoma-2(Bcl-2),Bcl-2/adenovirus E1B interacting protein 3(BNIP-3),and Bcl-2 associated X protein(Bax),cleaved cysteine-aspartic acid protease-3(Caspase-3),hypoxia-inducible factor-1α(HIF-1α)and pathway proteins PI3K,phosphorylated(p)-PI3K,Akt,and p-Akt.Result:Cell survival rates of the 1%O2 and 5%O2 groups were increased compared with that in the normoxia group,particularly the 5%O2 group(P<0.01).The fluorescence intensity for autophagy in 1%O2 and 5%O2 groups was significantly increased compared with that in the normoxia group,especially the 5%O2 group.In the presence of 5%O2,compared with the blank serum group,medium-dose and high-dose YHJP groups showed high cell inhibition rate,low autophagy rate,high apoptosis rate(P<0.01),and low expression of Beclin-1 protein(P<0.05).Compared with low-dose YHJP group,high-dose YHJP group demonstrated low expression of Beclin-1 protein(P<0.05).Compared with the blank serum group,the three YHJP groups had low expression of LC3Ⅱ/Ⅰprotein(P<0.05,P<0.01).Compared with the blank serum group,medium-dose and high-dose YHJP groups showed high expression of p62 protein(P<0.01).Compared with low-dose YHJP group,high-dose YHJP group showed high expression of p62 protein(P<0.05).Compared with the blank serum group,high-dose YHJP increased the expression of BNIP-3 and Bax and decreased the expression of Bcl-2(P<0.01).The expression of Bax protein in the high-dose YHJP group was increased compared with that in the low-dose YHJP group(P<0.05).The expression of HIF-1αin the medium-dose and high-dose YHJP groups was decreased(P<0.01)and the expression of p-PI3K/PI3K and p-Akt/Akt in the high-dose YHJP group was increased(P<0.05,P<0.01)compared with that in the blank serum group.The expression of p-Akt/Akt was higher in the high-dose YHJP group than in the medium-dose YHJP(P<0.05).Conclusion:Hypoxic microenvironment can significantly promote autophagy and proliferation of colon cancer cells.YHJP can significantly inhibit autophagy and proliferation and promote apoptosis of colon cancer cells in 5%O2 environment by up-regulating the PI3K/Akt signaling pathway.