| Aim: To explore the effect and mechanism of Babao Dan(BBD)on epithelial-mesenchymal transition(EMT)of gastric cancer(GC)in vitro and in vivo,and to provide experimental basis for its clinical application.Methods: In vitro study:(1)After treatment with different concentrations of BBD(0,0.25,0.5,0.75 mg/mL),cell scratch assay and Transwell assay were used to detect the cell migration ability;Transwell assay was used to detect the cell invasion ability;adhesion assay was used to detect the cell adhesion ability.(2)After treatment with different concentrations of BBD,Q-PCR assay and Western Blot assay were used to detect the mRNA expression and protein expression of EMT-related genes(E-cadherin,N-cadherin,Vimentin,ZEB1,ZEB2,Twist1,MMP2,MMP9).(3)After treatment with different concentrations of BBD,Western Blot assay was performed to detect the protein expression of TGF-?/Smad pathway-related genes(TGF-?1,Smad2/3,p-Smad2/3).(4)After stimulation with exogenous TGF-?1 factor(10 ng/mL)and treatment with BBD(0.5 mg/mL),Western Blot assay was used to detect the protein expression of TGF-?/Smad pathway-related genes(Smad2/3,p-Smad2/3)and the mRNA expression and protein expression of EMT-related genes(E-cadherin,N-cadherin);Transwell assay was used to detect cell migration ability and invasion ability.In vivo study: The nude mice model of MGC80-3 subcutaneously transplanted tumors was constructed and randomly divided into Control group and BBD group according to tumor volume in nude mice.The BBD group(n=10)was given BBD(0.25g/kg)by gavage,and the Control group(n=10)was given the same volume of saline by gavage for six days a week.The tumor volume and weight of nude mice were measured every other day.After four weeks,the nude mice were sacrificed and the tumors were removed for follow-up experiments:(1)Study the effect of BBD on the growth of gastric cancer xenografts by measuring the volume of transplanted tumors,and study the effect of BBD on the body weight of nude mice by calculating the average body weight of nude mice.(2)Q-PCR assay and Western Blot assay were used to detect the expression of EMT-related and TGF-?/Smad pathway related proteins(E-cadherin,N-cadherin,Vimentin,ZEB1,ZEB2,Twist1,MMP2,MMP9,TGF-?1,Smad2/3,p-Smad2/3).(3)HE assay was used to evaluate the toxicity of BBD on liver and kidney in nude mice.Results: In vitro study:(1)Cell scratch assay and Transwell assay showed that BBD could inhibit cell migration ability(P<0.05 or P<0.01);Transwell assay showed that BBD could inhibit cell invasion ability(P<0.05 or P<0.01);adhesion assay showed that BBD could inhibit cell adhesion ability(P<0.01).(2)Q-PCR assay showed that BBD could up-regulate the mRNA expression of E-cadherin,down-regulate the mRNA expression of N-cadherin and down-regulate the ratio N/E-cadherin(P<0.05 or P<0.01);Western Blot assay showed that BBD could up-regulate the protein expression of E-cadherin(P<0.05 or P<0.01).Meanwhile,The protein expression of N-cadherin,Vimentin,ZEB1,ZEB2,Twist1,MMP2,and MMP9 was down-regulated to different degrees(P<0.05 or P<0.01).BBD had no effect on the protein expression of Vimentin in MGC80-3(P> 0.05).(3)Western Blot assay showed that BBD could down-regulate the protein expression of TGF-?1 and p-Smad2/3(P<0.05 or P<0.01),but the protein expression of Smad2/3 did not change(P>0.05).(4)After stimulation with exogenous TGF-?1 factor,Western Blot assay showed that compared with the Control group,TGF-?1 factor could up-regulate the protein expression of N-cadherin and p-Smad2/3(P<0.05 or P<0.01),but the protein expression of Smad2/3 did not change(P>0.05).Q-PCR assay showed that compared with the Control group TGF-?1 factor could up-regulate the ratio N/E-cadherin mRNA expression(P<0.05 or P<0.01).After treatment with BBD,Western Blot assay showed that compared with the TGF-?1 group BBD could down-regulate the protein expression of N-cadherin and p-Smad2/3(P<0.05 or P<0.01),but the protein expression of Smad2/3 did not change(P>0.05).Q-PCR assay showed that compared with the TGF-?1 group BBD could down-regulate the ratio N/E-cadherin mRNA expression(P<0.01).Transwell assay showed that TGF-?1 could improve cell migration ability and invasion ability(P<0.05 or P<0.01)compared with the control group;however,BBD could reverse this effect(P<0.01).In vivo study:(1)Subcutaneous xenograft tumors in nude mice showed that BBD could inhibit the growth of subcutaneous xenografts in nude mice(P<0.01),but the weight of nude mice did not changed(P>0.05).(2)Q-PCR results showed that the mRNA expression of E-cadherin did not change in the BBD group(P>0.05),whereas the mRNA expression of N-cadherin was down-regulated(P<0.05),and the ratio of N/E-cadherin was down-regulated(P<0.05);Western Blot assay showed that the protein expression of E-cadherin was significantly up-regulated in the BBD group(P<0.01),the protein expression of Vimentin,MMP2,MMP9,and p-Smad2/3 was down-regulated(P<0.05 or P<0.01),while the protein expression of N-cadherin,Twist1,ZEB1,ZEB2,TGF-?1 and Smad2/3 did not change(P>0.05).(3)HE assay showed that BBD had no liver and kidney toxicity in nude miceConclusion: In vitro,BBD can inhibit the migration,invasion and adhesion of gastric cancer cells.In vitro,BBD can inhibit the EMT of gastric cancer cells and the activation of TGF-?/Smad signaling pathway,inhibit the migration and invasion of gastric cancer cells by inhibiting EMT mediated by TGF-?/Smad pathway.BBD can inhibit the proliferation of gastric cancer cells in vivo,inhibit EMT and the activation of TGF-?/Smad signaling pathway. |
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