Research Into The Anti-hepatoma Effects Of The Total Flavonoids Of Ampelopsis Grossedentata Base On The PI3K/AKT/P53 Signaling Pathway

Objective:To explore the anti-hepatocarcinoma effect of the total Flavonoids of Ampelopsis grossedentata and its possible mechanism.Methods:（1）To study the effect of the total Flavonoids of Ampelopsis grossedentata（TF）on the proliferation of human hepatoma BEL-7404 cells.BEL-7404 cells in logarithmic growth phase are taked as the research object,and the cell concentration is seted to 2.0×10⁴cells/m L.The cell control group and 8 different TF concentration groups(5.0,7.5,11.3,16.9,25.3,38.0,57.0,85.4μg·m L^-1)are seted.Finally,the effect of TF on the proliferation of BEL-7404 cells in vitro is evaluated by MTT assay;（2）The effect of TF on human hepatoma xenografts in nude mice is studied.A nude mouse model of human hepatocarcinoma BEL-7404 cell transplantation is established.The experiment is divided into model group,5-FU group(20 mg·kg^-1)and TF(600,300,150 mg·kg^-1)group.After two weeks of drug intervention,the nude mice are sacrificed.Tumor tissue is isolated and the tumor inhibition rate（IR）and relative tumor proliferation rate（T/C）are calculated.HE staining is used to observe the pathological changes of human liver cancer xenografts in nude mice.（3）The effect of TF on apoptosis of hepatoma cells is detected by TUNEL method;（4）To explore the mechanism of TF against liver cancer from the PI3K/Akt/P53 signal pathway:（4.1）The effects of TF on the expression of pi3k,akt-1,p53,caspase-3,bcl-2 and bax genes in PI3K/Akt/P53 signaling pathway were detected by RT-PCR and q PCR.（4.2）Immunohistochemistry and WB assay were used to detect the effects of TF on the expression of PI3K,AKT-1,P53,CASPASE-3,BCL-2 and Bax proteins in PI3K/Akt/P53 signaling pathway.Results:（1）The results of MTT assay showed that compared with the cell control group,TF at concentrations of 16.9,25.3,36.0,57.0,85.4μg·m L^-1significantly inhibited the proliferation of BEL-7404 cells cultured in vitro（P<0.01）.The concentration of TF at 7.5,11.3μg·m L^-1also inhibited the proliferation of 7404 cells（P<0.05）in a dose-dependent manner,with a half-inhibition rate IC₅₀of 29.15μg·m L^-1.（2）The results of nude mice xenograft tumors showed that compared with the model group,the tumor weight was significantly decreased in the 600 and 300 mg·kg^-1TF groups（P<0.01）,and the 150 mg·kg^-1TF group also reduced the tumor weight（P<0.05）;600,300,150 mg·kg^-1TF group IR were 53.26%,35.94%,26.74%,respectively,T/C was 59.74%,69.66%,84.82%;HE staining pathological examination showed that compared with the model group,the tumor tissues showed different degrees of vacuoles and hepatocarcinoma cell necrosis or apoptosis in the pathological sections of TF 600,300,150 mg·kg^-1.（3）The results of TUNEL assay showed that compared with the model group,the apoptosis of tumor cells in 600,300 mg·kg^-1TF group was significantly increased（P<0.01）.（4）Research results on the mechanism of TF anti-liver cancer:（4.1）The results of RT-PCR and q PCR gene analysis showed that the expression of pi3k,akt-1 and bcl-2 m RNA in the 600 mg·kg^-1TF group was down-regulated compared with the model group（P<0.05 or P<0.01）,while p53,capsase-3,bax m RNA expression was up-regulated（P<0.05 or P<0.01）;300 mg·kg^-1TF group tumor tissue bcl-2 m RNA expression was down-regulated（P<0.05）,while p53,bax m RNA The expression was up-regulated（P<0.05）.（4.2）Immunohistochemical protein analysis results showed that compared with the model group,the expression of PI3K,AKT-1and BCL-2 protein in 600,300 mg·kg^-1TF group was significantly down-regulated（P<0.05 or P<0.01）,while P53,BAX,CASPASE-3 protein expression was significantly up-regulated（P<0.05 or P<0.01）.WB protein analysis results showed that the expression of P53,CASPASE-3 and BAX protein in 600 mg·kg^-1group was significantly up-regulated（P<0.05 or P<0.01）,while compared with the model group,PI3K,AKT-1 and BCL-2protein expression were Significantly down-regulated（P<0.05 or P<0.01）;compared with the model group,the expression of P53 and BAX protein in300mg·kg^-1TF group was up-regulated（P<0.05）,while the expression of BCL-2 protein was down-regulated（P<0.05）.Conclusion（s）:TF has obvious anti-hepatocarcinoma effect,and also inhibit the proliferation of cultured human hepatoma BEL-7404 cells in vitro,which can reduce the weight of human hepatoma BEL-7404 cells and promote the apoptosis of hepatocarcinoma cells.The mechanism may be up-regulate p53,caspase-3,bax related genes and proteins of PI3K/Akt/P53 signaling pathway and down-regulate pi3k,akt-1,bcl-2 related genes and protein expression.