基于PI3K/Akt信号通路探讨乌梅丸含药血清对胰腺癌细胞增殖、侵袭、迁移和凋亡的影响  

Effect of Wumeiwan-medicated Serum on Proliferation,Invasion,Migration,Apoptosis of Pancreatic Cancer Cells Based on PI3K/Akt Signaling Pathway

作　　者：王熙 张莹雯[1,3] WANG Xi;ZHANG Ying-wen(Hubei University of Chinese Medicine,Wuhan 430061,China;Wuhan Hospital of Traditional Chinese Medicine,Wuhan 430014,China;Zhongnan Hospital of Wuhan University,Wuhan 430071,China)

机构地区：[1]湖北中医药大学,武汉430061 [2]武汉市中医医院,武汉430014 [3]武汉大学中南医院,武汉430071

出　　处：《中国实验方剂学杂志》2022年第6期34-42,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：湖北省卫健委中医科研项目(ZY2019M072);武汉市卫健委面上重点科研项目(WZ21A09);武汉市卫健委中青年医学骨干人才培养工程(武卫通[2020]55号)。

摘　　要：目的:观察乌梅丸含药血清对人胰腺癌SW1990细胞增殖、侵袭、迁移和凋亡的影响,同时探讨其可能的作用机制。方法:制备乌梅丸含药血清,体外培养胰腺癌细胞株SW1990,采用细胞增殖与活性检测(CCK-8)法筛选乌梅丸含药血清最佳作用时间用于后续实验;将SW1990细胞分为空白组和乌梅丸低、中、高组(含药血清2%、4%、8%),利用平板克隆实验、细胞划痕实验和细胞侵袭实验分别检测其克隆形成、迁移和侵袭能力;流式细胞术检测乌梅丸含药血清对胰腺癌SW1900细胞凋亡的影响;通过蛋白免疫印迹法(Western blot)检测SW1990细胞中凋亡相关蛋白,B细胞淋巴瘤-2(Bcl-2),Bcl-2相关X蛋白(Bax),细胞色素C(Cyt C),活化的半胱氨酸天冬氨酸蛋白水解酶-3(cleaved Caspase-3)、cleaved Caspase-9,磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)通路中PI3K,磷酸化(p)-PI3K,Akt,磷酸化(p)-Akt蛋白表达水平。结果:与空白组比较,作用72 h,乌梅丸低、中、高组吸光度A显著降低(P<0.01),与乌梅丸低组比较,乌梅丸中、高组A均显著降低(P<0.01),与乌梅丸中组比较,乌梅丸高组A显著降低(P<0.01),说明乌梅丸作用72 h可呈剂量依赖性抑制SW1990细胞增殖能力,药物最佳作用时间为72 h;与空白组比较,乌梅丸低、中、高组SW1990细胞侵袭能力显著减弱(P<0.01),且呈浓度依赖性;与空白组比较,乌梅丸低、中、高组细胞克隆形成能力、迁移能力均有下降(P<0.05,P<0.01),且呈浓度依赖性;与空白组比较,乌梅丸低、中、高组细胞的总凋亡率显著升高(P<0.01),乌梅丸诱导凋亡作用随给药剂量增加而增强;与空白组比较,乌梅丸低、中、高组Bcl-2蛋白表达显著降低(P<0.01),cleaved Caspase-3,cleaved Caspase-9,Cyt C,Bax蛋白表达升高(P<0.05,P<0.01),且呈现出一定的量效关系;与空白组比较,乌梅丸低、中、高组p-PI3K,p-Akt蛋白表达均有减少(P<0.05,P<0.01),且随着剂量的增加,蛋白表达逐渐降低,pObjective: To observe the effects of Wumeiwan-medicated serum on the proliferation,invasion, migration, and apoptosis of human pancreatic cancer SW1990 cells and explore the underlying mechanism. Method: The Wumeiwan-medicated serum was prepared and the pancreatic cancer SW1990 cell line was cultured in vitro. The optimal time of Wumeiwan-medicated serum was selected for subsequent experiments by cell counting kit-8(CCK-8). SW1990 cells were divided into a control group and low-(2%),medium-(4%),and high-dose(8%)Wumeiwan-medicated serum groups. The colony-forming,migration,and invasion abilities were detected by clonogenic assay,wound healing assay,and transwell migration assay. Flow cytometry was used to detect the effect of Wumeiwan-medicated serum on the apoptosis of pancreatic cancer SW1900 cells. Western blot was used to detect the expression levels of apoptosis-related proteins,such as B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),cytochrome C(Cyt C),cleaved cysteinyl aspartatespecific protease-3(cleaved Caspase-3),cleaved cysteinyl aspartate-specific protease-9(cleaved Caspase-9),as well as phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(Akt),and phosphorylated Akt(p-Akt)in PI3K/Akt pathway in SW1990 cells. Result: Compared with blank group,Wumeiwan groups showed decreased absorbance(A)72 h after drug intervention(P<0.01). Compared with the low-dose group,the medium-and high-dose groups showed decreased A(P<0.01). Compared with the mediumdose group,the high-dose group showed decreased A(P<0.01). It indicates that Wumeiwan can inhibit SW1990 cell proliferation in a dose-dependent manner after 72 h,and the optimal action time is 72 h. Compared with the blank group,the Wumeiwan groups showed weakened invasion of SW1990 cells(P<0.01),reduced colonyforming and migration abilities(P<0.05,P<0.01)in a dose-dependent manner,and increased total apoptosis rates(P<0.01). The inducing effect of Wumeiwan on apoptosis increased with the increase in dosage. Compared with the blan

关 键 词：乌梅丸含药血清 胰腺癌 增殖 侵袭 转移 磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路 

分 类 号：R22[医药卫生—中医基础理论] R242[医药卫生—中医学]