| ObjectiveTo investigate the proliferation of live cancer Bel-7402 cells effected by Gexiazhuyutang typical Chinese traditional medicine named To investigate the effect of the named Ge-xiazhuyutang on Bel-7402 cells and rats liver cancer tissue, about phosphatase and tensin homologue deleted from chromosome 10(PTEN ) protein and the PI3K/Akt signal transduction pathway about signal factor: phosphoinositide-3- kinase(PI3K) and the phosphorylation of serine threonine kinase(P-Akt) , we try to clarify the mechanisms that mechanisms of Gexia- zhuyutang resisting liver cancer.Methods1 .In vitro study:20 male Wistars rats were at random divided into four group(five per group):control regular groups, cisplaintin groups, Gexiazhuyutang groups, combiming treatment groups, cisplaintin was confected into 0.300mg/ml liquid, and Gexiazhuyutang was steamed and filtrated and confected into 0.740g/ml drug liquid, which was fed to rats, control regular groups was fed with saline water, then prepared drug serum and normal rat serum. Bel-7402 cells cultured and passaged were divided into four groups that were treated with serum of cisplaintin, serum of Gexiazhuyutang, serum of combiming treatment and normal rat serum respectively.Cell proliferation that were treated with serum of cisplaintin, serum of Gexiazhuyutang, serum of combiming treatment and normal rat serum respectively was detected by by the methylthiazolyl tetrazolium(MTT) assay. And calculated cell restrained rat(%).The expression of PTEN protein, PI3K and the phosphorylation of Akt at different time was detected by Western blotting analysis.2.In vivo study:Animals grouping,replicating model,treatment, indexes testing Rats were divided into 5 groups: normal control group; liver cancer model control group; cisplaintin treatment group; Gexiazhuyutang treatment group; combining treatment group. In each group, there are 7 rats which are all male. The method of replicating animal models refers to that of Wu Shijiu and Wang Jinbo. Ascites with walker-256 tumor cell lines was injected into subcutaneous tissues of rats. After solid carcinoma grew, circumjacent tissue of the tumor was cut into pieces about 2 mm~3. They were paunched to expose the liver after being hocused. Tumor pieces were planted under the liver envelop by ophthalmic forceps. Asorbable gelatin sponge filled the wound to keep tumor pieces from emerge and to avoid bleeding. Then abdominal cavity was closed. Each rat was injected penicillin for 3 days, 2 lakh units every day. Rats of liver cancer model control group; Living in environment of high temperature and high damp. Rats of normal control group were not operated, fed with general fodder, living in general environment. Rats of normal control group and Rats of liver cancer model control group were not treated. Rats of cisplaintin treatment group were treated with Gexiazhuyutang for 15 days,0.740g/ml qd, IP. Rats of combining treatment group were treated with cisplaintin for 7 times, 0.3mg/ml,one time every other day. Rats of combining treatment group were treated with Gexiazhuyutang and cisplaintin by the same dose and usage as cisplaintin treatment group and Gexiazhuyutang. 20 days later, all rats were put to death.The expression of PTEN protein, PI3K and the phosphorylation of Akt with tissue of normal control group and liver cancer model control group and cisplaintin treatment group and Gexiazhuyutang treatment group and combining treatment group. In each group at different time was detected by Western blotting analysis.Results1.Effect of Baoganning dosage to the proliferation of Bel-7402 cells at 48h by the ONE-way: F=1834.977,P=0.000,LSD was applied between every two groups. Drug serum of cisplaintin groups, Gexiazhuyutang groups, combiming treatment groups significantly inhibit Bel-7402 proliferation compared with normal group(P=0.000, P＜0.01), Compared with cisplaintin groups, Gexiazhuyutang groups had difference (P=0.048, P＜0.05) and combiming treatment groups significantly inhibit Bel-7402 cells proliferation(P=0.000, P＜0.01).There was significant difference was observed between Gexiazhuyutang groups and combiming treatment groups (P=0.000, P＜0.01).2.Effect of Baoganning dosage to the proliferation of Bel-7402 cells at 72h by the ONE-way: F=3737.239,P=0.000,LSD was applied between every two groups. Drug serum of cisplaintin groups, Gexiazhuyutang groups, combiming treatment groups significantly inhibit Bel-7402 proliferation compared with normal group(P=0.000, P＜0.01), Compared with cisplaintin groups, Gexiazhuyutang groups had difference (P=0.020, P＜0.05) and combiming treatment groups significantlyinhibit Bel-7402 cells proliferation(P=0.000,P＜0.01).There was significant difference was observed between Gexiazhuyutang groups and combiming treatment groups(P=0.005, P＜0.01).3.Compared to the blank control, the serum with Gexiazhuyutang ingredients decreased the level of PTEN at 5min, maximized at 30min, returned at 3h,at 6h PTEN resume to the blank control level. Compared to the blank control, Gexiazhuyutang ingredients, cisplaintin ingredients, combiming treatment ingredients significantly inhibit the expression of PTEN at 3h,but the treatment of combiming treatment ingredients was the best well, Gexiazhuyutang ingredients take second place, the last was cisplaintin ingredients.4. Compared to the blank control, the serum with Gexiazhuyutang ingredients decreased the level of PI3K at 5min, minimized at 10min, returned at 30min,at 60min PI3K resume to the blank control level. Compared to the blank control, Gexiazhuyutang ingredients, cisplaintin ingredients, combiming treatment ingredients significantly inhibit the expression of PI3K at 10min,but the treatment of combiming treatment ingredients was the best well, Gexiazhuyutang ingredients take second place, the last was cisplaintin ingredients.5. Compared to the blank control, the serum with Gexiazhuyutang ingredients decreased the level of P-Akt at 10min, minimized at 30min, at 60min P-Akt resume to the blank control level. Compared to the blank control, Gexiazhuyutang ingredients, cisplaintin ingredients, combiming treatment ingredients significantly inhibit the expression of P-Akt at 30min,but the treatment of combiming treatment ingredients was the best well, Gexiazhuyutang ingredients take second place, the last was cisplaintin ingredients.6.Compared to the blank control, liver cancer model control group, Gexiazhuyutang group, cisplaintin group, combiming treatment group significantly promoted the expression of PTEN protein; Compared to the liver cancer model control group, Gexiazhuyutang group, cisplaintin group, combiming treatment group more promoted the expression of PTEN protein; Compared to the cisplaintin group, Gexiazhuyutang group, and combiming treatment group more promoted the expression of PTEN protein, but combiming treatment group more recreased the expression of PTEN.7.Compared to the blank control, liver cancer model control group, Gexiazhuyutang group, cisplaintin group, combiming treatment group significantly decreased the expression of PI3K; Compared to the liver cancer model control group, Gexiazhuyutang group, cisplaintin group, combiming treatment group more decreased the expression of PI3K; Compared to the cisplaintin group, Gexiazhuyutang group, and combiming treatment group more decreased the expression of PI3K, but combiming treatment group more recreased the expression of PI3K.8.Compared to the blank control, liver cancer model control group, Gexiazhuyutang group, cisplaintin group, combiming treatment group significantly decreased the expression of P-Akt ; Compared to the liver cancer model control group, Gexiazhuyutang group, cisplaintin group, combiming treatment group more decreased the expression of P-Akt ; Compared to the cisplaintin group, Gexiazhuyutang group, and combiming treatment group more decreased the expression of P-Akt, but combiming treatment group more recreased the expression of P-Akt.Conclusion1.Gexiazhuyutang could restrain the proliferation of live cancer Bel-7402 cells.2.The one of mechanisms of the Gexiazhuyutang risiting liver cancer may be Baoganning's up-regulation of PTEN.3. Gexiazhuyutang could inhibit expression of PI3K,Akt ,which may be the one of main mechanisms of Gexiazhuyutang's down-regulation PI3K,P-Akt expression.4.Proved that PTEN protein and the PI3K/Akt signal transduction pathway about signal factor: PI3K and the phosphorylation of P-Akt expression may repel each other.5.Gexiazhuyutang and cisplaintin may help to effect each other.
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