ABSTRACT
ObjectiveTo investigate the effect of Biejiajian Wan （BJJW） on transforming growth factor-β1 （TGF-β1）-induced epithelial-mesenchymal transition （EMT） of HepG2 cells， and explore its mechanism against EMT of hepatocellular carcinoma cells. MethodHepG2 cells were randomly divided into a blank group， a TGF-β1 model group （10 μg·L-1 TGF-β1）， a low-dose BJJW group （10 μg·L-1 TGF-β1+0.55 g·kg-1 BJJW）， a medium-dose BJJW group （10 μg·L-1 TGF-β1+1.1 g·kg-1 BJJW）， a high-dose BJJW group （10 μg·L-1 TGF-β1+2.2 g·kg-1 BJJW）， and a sorafenib group （10 μg·L-1 TGF-β1+0.03 g·kg-1 sorafenib）. The EMT model was induced by 10 μg·L-1 TGF-β1 in HepG2 cells. After treatment with corresponding medicated serum， cell counting kit -8 （CCK-8） assay was used to detect cell proliferation. Cell migration ability was detected by the Transwell assay and wound healing assay. The protein expression related to EMT and nuclear factor-kappa B （NF-κB） signaling pathway was detected by cell immunofluorescence assay and Western blot. ResultCompared with the blank group 4 days later， the TGF-β1 model group showed fusiform and loose cells with widened gap and antennae reaching out， decreased protein expression of E-cadherin （P<0.05）， and increased protein expression of N-cadherin and vimentin （P<0.05）， which indicated that the EMT model was properly induced in HepG2 cells by TGF-β1 stimulation for 4 days. After 48 hours of treatment with the corresponding medicated serum， each medication group showed inhibited proliferation of HepG2 cells that had undergone EMT， especially the low- and high-dose BJJW groups （P<0.01）， and the medium-dose BJJW group showed increased E-cadherin protein expression （P<0.05） and decreased p-p65， N-cadherin， and vimentin protein expression （P<0.05）， as compared with the TGF-β1 model group. As revealed by the transwell assay and wound healing assay， TGF-β1 enhanced the migration ability of HepG2 cells （P<0.05， P<0.01） compared with the results in the blank group， compared with the TGF-β1 model group， the medication groups showed inhibited migration ability of HepG2 cells （P<0.05， P<0.01）. Compared with the blank group， the TGF-β1 model group promoted the expression of p65 and Snail into the nucleus. Compared with the TGF-β1 model group， the medication groups inhibited the expression of p65 and Snail into the nucleus. ConclusionBJJW may inhibit the EMT， proliferation， and migration of HepG2 cells induced by TGF-β1 by suppressing the NF-κB signaling pathway to exert an anti-hepatocellular carcinoma effect.
ABSTRACT
Objective:To study the effect of Biejiajian Wan on the epithelial-mesenchymal transition （EMT） of rat hepatic oval cells induced by transforming growth factor- β1（TGF-β1）， in order to explore its mechanism in reversing EMT. Method:WB-F344 cells were divided into five groups： blank group， TGF-β1 model group （10 μg·L-1TGF-β1）， low-dose group （10 μg·L-1TGF-β1+0.55 g·kg-1Biejiajian Wan）， medium-dose group （10 μg·L-1TGF-β1+1.1 g·kg-1Biejiajian Wan）， high-dose group （10 μg·L-1TGF-β1+2.2 g·kg-1Biejiajian Wan）. Except blank group， TGF-β1 was used to induce WB-F344 cells in all of the remaining groups to construct an EMT model. After the cells were treated with low， medium and high doses of Biejiajian Wan serum， the changes of migration ability of WB-F344 cells were detected by cell scratching test. The expressions of E-cadherin， N-cadherin and Vimentin were detected by Western blot. Real-time PCR was used to detect the changes in the expression of β-catenin mRNA. The expression of β-catenin was detected by cell immunofluorescence assay. Result:Compared with normal WB-F344 cells， the intercellular space of WB-F344 cells became loose from tight， and the morphology changed from cobblestone to fibroblast after TGF-β1 induced WB-F344 cells for 4 days， and the expression of E-cadherin protein decreased， while the expression of N-cadherin protein increased （P<0.01）， indicating that the EMT model of WB-F344 cells was successfully built. Compared with the blank group， the migration ability of WB-F344 cells in TGF-β1 model group was enhanced （P<0.01）， compared with TGF-β1 model group， Biejiajian Wan could significantly inhibit the migration ability of WB-F344 cells； specifically， the low-dose group had no statistical significance， and the medium and high-dose groups had statistical significance （P<0.05）. Western blot results showed that compared with the blank group， the expression of E-cadherin decreased， whereas those of N-cadherin and Vimentin increased in the TGF-β1 model group （P<0.01）， compared with TGF-β1 model group， E-cadherin protein expression was increased in the low， medium and high-dose groups， while the expressions of N-cadherin and Vimentin was decreased； specifically， the low-dose groups had no statistical significance， and the medium and high dose groups had statistical significance （P<0.05，P<0.01）. Real-time PCR results showed that compared with the blank group， the mRNA expression of β-catenin in the TGF-β1 model group was increased （P<0.05）， whereas compared with TGF-β1 model group， the mRNA expression of β-catenin in the low， medium and high-dose groups of Biejiajian Wan was reduced （P<0.01）. The results of cellular immunofluorescence showed that compared with the blank group， the fluorescence expression of β-catenin in the cell nucleus was enhanced in the TGF-β1 model group； and compared with the TGF-β1 model group， the expression of β -catenin in the cell nucleus of the low， medium and high-dose groups of Biejiajian Wan decreased， and the inhibitory effect of Biejiajian Wan on β-catenin in the cell nucleus was positively correlated with its concentration. Conclusion:Biejiajian Wan may reverse the EMT process that TGF-β1 induced WB-F344 cells， and inhibit the migration of WB-F344 cells by inhibiting Wnt/β-catenin signaling pathway.