摘要
目的 研究益气解毒方对鼻咽癌细胞增殖、迁移和侵袭的影响,并从转化生长因子 β1(TGF-β1)/SMAD3 信号通路探讨其对增殖、迁移和侵袭的作用机制.方法 (1)将鼻咽癌细胞分为 4 组:溶剂对照组、益气解毒方 0.5 mg·mL-1 组、益气解毒方 1.0 mg·mL-1 组、5-氟尿嘧啶(5-Fluorouracil,5-Fu)2 µg·mL-1 组,采用实时无标记细胞功能分析仪(real time cellular analysis technology,RTCA)监测细胞增殖;创伤愈合实验检测细胞迁移;药物干预 24 h 后,侵袭小室法检测细胞侵袭;Western Blot 法检测细胞 β-catenin、E-cadherin、N-cadherin、TGF-β1、SMAD3 蛋白的表达水平.(2)将鼻咽癌细胞分为 5 组:溶剂对照组、TGF-β1 10 ng·mL-1组、TGF-β1 10 ng·mL-1+益气解毒方 1.0 mg·mL-1 组、益气解毒方 1.0 mg·mL-1 组、LY3200882 10 µmol·L-1 组,采用实时无标记细胞功能分析仪监测细胞增殖;创伤愈合实验检测细胞迁移;药物干预 24 h后,侵袭小室法检测细胞侵袭;Western Blot法检测细胞β-catenin、E-cadherin、N-cadherin、TGF-β1、SMAD3 蛋白的表达水平.(3)随机将裸鼠分为模型组、益气解毒方组和 5-Fu组.皮下部位注射细胞悬液制备鼻咽癌裸鼠移植瘤模型,基本成瘤后,5-Fu组每 2 d腹腔注射 1 次,其余组每天灌胃给药 1 次,连续 3 周.每隔 3 d测量瘤体体积1 次;Western Blot法检测各组组织中β-catenin、E-cadherin、N-cadherin蛋白表达水平.结果 与溶剂对照组比较,益气解毒方(0.5、1.0 mg·mL-1)组的细胞增殖曲线均下降,细胞迁移和侵袭能力均降低(P＜0.05,P＜0.01),且E-cadherin蛋白表达明显升高(P＜0.01),β-catenin、N-cadherin、TGF-β1、SMAD3 蛋白表达均明显降低(P＜0.05,P＜0.01).与模型组比较,益气解毒方组的鼻咽癌移植瘤体积明显减小(P＜0.05),且益气解毒方组移植瘤组织中的β-catenin和N-cadherin蛋白表达明显下调(P＜0.05,P＜0.01),E-cadherin蛋白表达明显上调(P＜0.01).加入TGF-β1 信号通路激活剂和抑制剂后,与益气解毒方 1.0 mg·mL-1 组比较,TGF-β1 10 ng·mL-1+益气解毒方 1.0 mg·mL-1 组的TGF-β1、SMAD3、β-catenin和N-cadherin蛋白表达水平均明显升高(P＜0.05,P＜0.01),且细胞增殖、迁移和侵袭的能力明显增强(P＜0.01).结论 益气解毒方能够通过调控TGF-β1/SMAD3 信号通路抑制鼻咽癌细胞的增殖、迁移和侵袭.
Abstract
Objective To investigate the effect of Yiqi Jiedu Recipe(YQ)on the proliferation,migration,and invasion of nasopharyngeal carcinoma cells,and to explore its mechanisms of action on proliferation,migration,and invasion through the TGF-β1/SMAD3 signaling pathway.Methods(1)The 5-8F cells were divided into four groups:solvent control group,YQ 0.5 mg·mL-1 group,YQ 1.0 mg·mL-1 group,and 5-fluorouracil 2 µg·mL-1 group.Cell proliferation was monitored using real-time cell analysis(RTCA).Wound healing experiment was conducted to assess cell migration.After 24 hours of drug intervention,transwell assay was employed to measure cell invasion.The protein expression levels of β-catenin,E-cadherin,N-cadherin,TGF-β1,and SMAD3 in the cells were evaluated using the Western Blot method.(2)The 5-8F cells were divided into five groups:solvent control group,TGF-β1 10 ng·mL-1 group,TGF-β1 10 ng·mL-1+YQ 1.0 mg·mL-1 group,YQ 1.0 mg·mL-1 group,and LY3200882 10 µmol·L-1 group.Cell proliferation was monitored using RTCA.Wound healing experiment was conducted to assess cell migration.After 24 hours of drug intervention,transwell assay was employed to measure cell invasion.The protein expression levels of β-catenin,E-cadherin,N-cadherin,TGF-β1,and SMAD3 in the cells were evaluated using Western Blot.(3)The nude mice were randomly assigned into the model group,YQ group,and 5-fluorouracil group.Subcutaneous injection of 5-8F cell suspension was performed to establish the xenograft nude mouse model of nasopharyngeal carcinoma.After the tumors reached a certain size,the 5-fluorouracil group received intraperitoneal injection of 5-Fu once every 2 days,while the other groups were orally administered corresponding drugs once a day for three consecutive weeks.Tumor volume was measured every 3 days.Western Blot was conducted to assess the protein expression levels of β-catenin,E-cadherin,and N-cadherin in the tissues of each group.Results Compared with the solvent control group,the proliferation curves of 5-8F cells in the YQ(0.5 mg·mL-1,1.0 mg·mL-1)groups showed a decrease.The migration and invasion abilities of the cells were both reduced(P＜0.05,P＜0.01).Additionally,the expression of E-cadherin protein significantly increased(P＜0.01),while the protein expression of β-catenin,N-cadherin,TGF-β1,and SMAD3 all decreased(P＜0.05,P＜0.01).Compared with the model group,the transplanted tumor volume of nasopharyngeal carcinoma in the YQ group significantly decreased(P＜0.05).Furthermore,the protein expression of β-catenin and N-cadherin in the transplanted tissues of the YQ group was significantly downregulated(P＜0.05,P＜0.01),while the expression of E-cadherin protein was significantly upregulated(P＜0.01).After the addition of the activator and inhibitor of TGF-β1 signaling pathway,compared with the YQ 1.0 mg·mL-1 group,the TGF-β1 10 ng·mL-1+YQ 1.0 mg·mL-1 group showed a significant increase in the expression of TGF-β1,SMAD3,β-catenin,and N-cadherin proteins(P＜0.05,P＜0.01)and obvious enhancement of the abilities of cell proliferation,migration,and invasion(P＜0.01).Conclusion Yiqi Jiedu Recipe can inhibit the proliferation,migration,and invasion by regulating the TGF-β1/SMAD3 signaling pathway.
基金项目
国家自然科学基金项目(81973914)
湖南省教育厅科学研究项目(21B0358)
湖南省教育厅科学研究项目(22A0254)
湖南省中医药管理局资助项目(D2022105)
2021年度湖南中医药大学校级科研基金(2021XJJJ014)
2021年度湖南中医药大学校级科研基金(2021XJJJ008)
湖南省卫健委项目(D202307017740)
NETL
NSTL
万方数据