Molecular Mechanism Of Berberine On The Regualtion Of VEGF Expression Through The NF-κB Signaling Pathway

Objective: Observing the effect of berberine on the TNFα-induced upregualation ofVEGF and investigating the possible mechanism of NF-κB signal transductionpathway.Methods: MTT was used to assay the effect of berberine on the inhibition of HepG2cells. HepG2cells were stimulated by the indicated concentration（10ng/ml）ofTNFα for0,12h,24h,48h, respectively. To observe the proper time period, theexpression level of VEGF mRNA was examined by Real-time PCR, and theVEGF protein level was assayed by Western blot. After that, HepG2cells werepreincubated with0,10,25,50μmol/Lberberine for24h, respectively, then treatedwith10ng/ml TNFα for24h, the expression level of VEGF mRNA was examinedby Real-time PCR, NF-κB activity was examined by ELSIA, the expression ofVEGF、IκBα、p65were assayed by Western blot.Results:1. The impact of berberine on the cytotoxic effects of HepG2cellsThe effects of berberine on the cytotoxic effects of HepG2cells were examined byMTT, the results were as follows:0μmol/L,10μmol/L,25μmol/L,50μmol/Lberberine can respectively result in the decrease of growth inhibiton of0.82±0.26%,10.54±1.42%,22.68±2.56%,39.16±3.84%, which are revealed indose-dependant manner.2. The effect of TNFα on the mRNA and protein expression level of VEGF inHepG2cells.The mRNA expression level of VEGF was assayed.by Real-time PCR. Comparedwith the control group, the relative mRNA expression quantities of the others. were1.264±0.049%,1.710±0.363%,1、642±0.196%, sperately. The results indicated TNFα induced a increase in VEGF mRNA, the peak at24h（p<0.05vs control）.Also,10ng/ml TNFα-induced a time-dependant increase in VEGF protein level（p<0.05VS control）, which peak at48h.3. The effects of berberine on the TNFα-induced upregration of VEGF expressionHepG2cells were preincubated with0,10,25,50μmol/L berberine for24h,respectively, then treated with10ng/ml TNFα for24h. the mRNAexpression level ofwere assayed VEGF Real-time PCR. Compared with the control group, the relativemRNA expression quantities of the others. were1.710±0.363%、1、587±0.274%、1.486±0.1.911%、1.169±0.085%, sperately, which demonstrated berberineattenduates TNFα-induced upregration of VEGF mRNA expression. Compred withcontrol group, the mRNA expression of TNFα group was elevated（p<0.05VScontrol）, and berberine inhibited the mRNA expression induced by TNFα in aconcentration-dependant manner. Likewise, the effects were also appeared in theprotein expression of VEGF.4. The effect of TNFα on the NF-κB signal transduction pathwayHepG2cells was stimulated by10ng/ml TNFα for0min,30min,1h,2h,respectively. IκBα proteins from cytoplasms were decreased （p<0.05VScontrol）and P65proteins from cell nucleues were increased（p<0.05VS control）,gradually. Meanwhile, NF-κB activity in a time-dependant increase, the peak at2h.5. The effect of berberine on the NF-κB signal transduction pathway induced byTNFαHepG2cells were preincubated with25μmol/L berberine for24h, then treatedwith10ng/ml TNFα for0min，30min，1h,2h, respectively. Compared with the abovegroups, the expression of IκBα protein（p<0.05）from cytoplasms were increased andP65protein from cell nucleues berberine decreased （p<0.05）.We furtherpreincubated with0,10,25,50μmol/L berberine for24h, then treated with TNFα for2h, to investigate the the inhibitory effect of berberine on the NF-κB activity. Theresults indicated that berberine can inhibit the NF-κB activity in dose-dependantmanner.Conclusion: Berberine attenuates TNF-α-induced up-regulation of VEGF expression, thepossible mechanism maybe involve NF-κB pathway. berberine may be a drug forcancer prevention and therapy in hepatocelluar carcinoma.