七方胃痛颗粒对过表达14-3-3ζ人胃腺癌AGS细胞上皮间质转化的抑制作用及机制研究

Inhibitory effect and mechanism of Qifang Weitong granule on epithelial-mesenchymal transition in human gastric adenocarcinoma AGS cells overexpressing 14-3-3ζ

目的研究七方胃痛颗粒对过表达14-3-3ζ人胃腺癌AGS细胞上皮间质转化的抑制作用及相关机制。方法取体外培养的人胃腺癌AGS细胞,经转化生长因子β1(TGF-β1)诱导发生上皮间质转化后进行实验。实验分为6组:空白EMT组(正常DMEM培养基培养)、14-3-3ζ过表达组(慢病毒转染过表达的EMT细胞加正常DMEM培养基培养)、TGF-β/Smad通路抑制剂组(培养基中加入LY2109761抑制剂培养)、中药血清组(加10%七方胃痛颗粒含药血清培养)、14-3-3ζ过表达TGF-β/Smad通路抑制剂组(慢病毒过表达EMT细胞加LY2109761抑制剂培养)、14-3-3ζ过表达中药血清组(慢病毒过表达EMT细胞加10%七方胃痛颗粒含药血清培养)。镜下观察各组细胞的形态,采用细胞划痕实验与Transwell细胞迁移法检测各组细胞的迁移与侵袭能力,采用免疫荧光技术、蛋白质印迹技术检测TGF-β/Smad信号通路下游关键蛋白Smad2与p-Smad2/3的表达情况。结果14-3-3ζ过表达组梭形间充质样细胞数量多,细胞黏附能力下降,细胞间排列松散,且出现丝状伪足;中药血清组和TGF-β/Smad通路抑制剂组细胞间的连接较为紧密,伪足相对减少;14-3-3ζ过表达中药血清组和14-3-3ζ过表达TGF-β/Smad通路抑制剂组细胞间的细胞间的连接更紧密。14-3-3ζ过表达组的细胞迁移速率明显快于空白EMT组(P<0.05),细胞侵袭数量明显多于空白EMT组(P<0.05),Smad2、p-Smad2/3蛋白表达量明显高于空白EMT组(P<0.05);中药血清组、14-3-3ζ过表达中药血清组、TGF-β/Smad通路抑制剂组、14-3-3ζ过表达TGF-β/Smad通路抑制剂组细胞迁移速率明显慢于14-3-3ζ过表达组和空白EMT组(P均<0.05),细胞侵袭数量明显少于14-3-3ζ过表达组和空白EMT组(P均<0.05),Smad2、p-Smad2/3蛋白表达量明显低于14-3-3ζ过表达组和空白EMT组(P均<0.05)。结论七方胃痛颗粒能抑制14-3-3ζ过表达AGS细胞上皮间质转化的侵袭、迁移能力,其抗癌作用的发挥很可能与抑制14-3-3ζ/TGF-β/Smad信号通路相关。

Objective It is to study the inhibitory effect and related mechanism of Qifang Weitong granule on epithelial-mesenchymal transition in human gastric adenocarcinoma AGS cells overexpressing 14-3-3ζ.Methods The human gastric adenocarcinoma AGS cells cultured in vitro were harvested and subjected to epithelial-mesenchymal transition(EMT)induced by transforming growth factorβ1(TGF-β1).The experiment was divided into 6 groups:blank EMT group(cultured in normal DMEM medium),14-3-3ζoverexpression group(cultured in normal DMEM medium with overexpressed EMT cells transfected by lentivirus),TGF-β/Smad pathway inhibitor group(cultured in the medium with LY2109761 inhibitor),and traditional Chinese medicine serum group(cultured in the medium containing 10%Qifang Weitong granule),14-3-3ζoverexpression TGF-β/Smad pathway inhibitor group(cultured with lentivirus overexpression EMT cells plus LY2109761 inhibitor),14-3-3ζoverexpression traditional Chinese medicine serum group(cultured with lentivirus overexpression EMT cells plus the serum containing 10%Qifang Weitong granules).The morphology of the cells in each group was observed under a microscope,and the migration and invasion abilities of the cells in each group were detected by cell scratch assay and Transwell cell migration assay.The expression of key downstream proteins Smad2 and p-Smad2/3 in TGF-β/Smad signaling pathway was detected by immunofluorescence and western blotting.Results There were a lot of spindle-shaped mesenchymal-like cells in the 14-3-3ζoverexpression group,with decreased cell adhesion and loose intercellular arrangement,and filopodia appeared;the connection between cells was tighter,and the pseudopodia was relatively reduced in the traditional Chinese medicine serum group and TGF-β/Smad pathway inhibitor group;the connection between cells was more tighter in the 14-3-3ζoverexpression Chinese medicine serum group and the 14-3-3ζoverexpression TGF-β/Smad pathway inhibitor group.The migration rate of cells in the 14-3-3ζoverexpression group was significantly faster than that in the blank EMT group(P<0.05),the number of invasion cell was significantly higher than that in the blank EMT group(P<0.05),and the protein expressions of Smad2 and p-Smad2/3 was significantly higher than that in the blank EMT group(P<0.05);The cell migration rates were significantly slower(all P<0.05),the numbers ofinvasion cell were significantly less(all P<0.05),and the expression levels of Smad2 and p-Smad2/3 proteins were significantly lower in the traditional Chinese medicine serum group,TGF-β/Smad pathway inhibitor group,14-3-3ζoverexpression TGF-β/Smad pathway inhibitor group,14-3-3ζoverexpression traditional Chinese medicine serum group than those in the 14-3-3ζoverexpression group and the blank EMT group(all P<0.05).Conclusion Qifang Weitong Granules can inhibit the invasion and migration of 14-3-3ζoverexpressing AGS cells in epithelial-mesenchymal transition,and its anti-cancer effect may be related to the inhibition of 14-3-3ζ/TGF-β/Smad signaling pathway.