芦根多糖诱导自噬和凋亡抑制非小细胞肺癌A549细胞增殖

Inhibition of Rhizoma Reed Polysaccharide on the Proliferation of Non-small Cell Lung Cancer Cells A549 by Inducing Autophagy and Apoptosis

目的探讨芦根多糖抑制非小细胞肺癌生长、增殖的作用及相关分子机制。方法建立A549细胞体外模型,加入芦根多糖干预后,CCK-8法检测细胞活性,流式细胞仪检测A549细胞凋亡情况,免疫印迹法(Western blotting)检测A549细胞的LC3-Ⅱ/Ⅰ、AKT、p-AKT、PI3K、p-PI3K蛋白表达;建立体内移植瘤小鼠模型,加入芦根多糖作用,并以顺铂为阳性对照,观察芦根多糖对荷瘤小鼠肿瘤生长影响,免疫组化法检测移植瘤小鼠A549细胞肿瘤组织凋亡蛋白Bax、Bcl-2、Caspase-3蛋白表达。结果100μg·mL^-1的芦根多糖在48及72 h时显著抑制A549细胞增殖(P<0.05);芦根多糖可使A549细胞凋亡的比例显著升高(P<0.05),A549细胞增殖的LC3-Ⅱ/Ⅰ、p-AKT/AKT、p-PI3K/PI3K比值显著升高(P<0.05),而AKT、PI3K蛋白表达水平无明显变化。裸鼠移植A549细胞7 d后,腋下开始生长肿瘤块,第13天时小鼠肿瘤体积达到90 mm 3,模型成功率90.0%;给药第9天开始,顺铂组及芦根多糖组肿瘤生长曲线平缓,肿瘤体积明显低于对照组(P<0.05)。给药第18天时,与对照组比较,顺铂和芦根多糖对小鼠肿瘤的抑制率分别为39.1%,35.9%(P<0.05),瘤细胞的凋亡蛋白Bax、Casepes-3表达显著升高(P<0.05),Bcl-2蛋白表达显著降低(P<0.05)。结论芦根多糖可能通过激活自噬和促进凋亡抑制A549细胞增殖,可作为一种潜在的非小细胞肺癌的治疗药物。

Objective To investigate the role and related molecular mechanisms of Rhizoma reed polysaccharide in inhibiting the proliferation of non-small cell lung cancer(NSCLC).Methods A549 cell model in vitro was established.CCK-8 method was used to detect cell activity,and flow cytometry was used to detect apoptosis of A549 cells.Western blotting was used to detect LC3-Ⅱ/Ⅰ,AKT,p-AKT,PI3K,and p-PI3K protein expressions in A549 cells.Tumor model in mice was established,and the efficacy of Rhizoma reed polysaccharide on the growth of tumors in mice was observed and compared with the positive control cisplatin.The expressions of apoptotic proteins Bax,Bcl-2,and Caspase-3 in A549 cells were detected by immunohistochemistry.Results The proliferation of A549 cells was significantly inhibited by 100μg·mL^-1 of Rhizoma reed polysaccharide at 48 and 72 h(P<0.05);Rhizoma reed polysaccharide could significantly increase the apoptotic rate of A549 cells(P<0.05),and the LC3-Ⅱ/Ⅰ,p-AKT/AKT,p-PI3K/PI3K ratio of A549 cells increased significantly(P<0.05),while the expression levels of AKT and PI3K protein did not change significantly(P<0.05).Seven days after transplantation of A549 cells in nude mice,tumor mass began to grow in axilla.On the 13th day,the volume of tumors reached 90 mm 3,and the success rate of the modeling was 90.0%.From the 9th day of administration,the growth curves of tumors in the cisplatin group and the Rhizoma reed polysaccharide group were smooth,and the volumes of tumors were significantly lower than those in the control group(P<0.05).On the 18th day of administration,compared with the control group,the inhibition rates of cisplatin and Rhizoma reed Polysaccharide were 39.1%and 35.9%respectively(P<0.05).The expressions of apoptotic proteins Bax and Casepes-3 were significantly increased(P<0.05),while the expression of Bcl-2 was significantly decreased(P<0.05).Conclusion Rhizoma reed polysaccharide may inhibit the proliferation of A549 cells by activating autophagy and promoting apoptosis,and thus can be used as a potential therapeutic drug for non-small cell lung cancer.