芪玉三龙汤调节肺癌组织核转录蛋白表达抑制荷瘤小鼠瘤体生长的作用机制研究

Study on mechanism of Qiyusanlong decoction inhibiting tumor growth in tumor-bearing mice by regulating the expression of nuclear transcription protein in lung cancer tissue

目的 观察芪玉三龙汤对肺癌荷瘤小鼠瘤体的抑瘤效应及对Wnt/β-连环蛋白（Wnt/β-catenin）信号通路中糖原合成酶激酶3β（GSK3β）、磷酸化GSK3β（p-GSK3β）、β-catenin蛋白表达的影响.方法 采用Lewis肺癌细胞株培养移植法建立肺癌荷瘤小鼠模型,按随机数字表法分为模型组、化疗组和芪玉三龙汤低、中、高剂量组以及联合组,每组8只.制模第2天开始,芪玉三龙汤低、中、高剂量组小鼠分别按20.12、40.24、80.48 g·kg-1·d-1灌胃中药,化疗组每周腹腔注射0.4 mL顺铂,联合组给予高剂量中药灌胃和顺铂腹腔注射联合用药,模型组给予等量生理盐水,连续给药21 d.实验结束后处死小鼠取肺组织,称取瘤质量,计算抑瘤率;用蛋白质免疫印迹试验（Western Blot）检测肿瘤组织GSK3β、p-GSK3β、β-catenin的蛋白表达水平.结果 与模型组比较,各药物组瘤质量、GSK3β、β-catenin的蛋白表达水平均明显降低,抑瘤率、p-GSK3β的蛋白表达水平明显升高,联合组瘤质量、抑瘤率的变化较芪玉三龙汤低、中、高剂量组更显著 〔瘤质量（g）：1.48±0.71比4.53±1.34、4.27±0.62、3.45±1.05,抑瘤率：73.23％比13.51％、18.32％、33.86％,均P〈0.01〕,联合组瘤质量、抑瘤率与化疗组相当〔瘤质量（g）：1.48±0.71比1.49±0.68,抑瘤率：73.23％比73.01％,均P〉0.05〕;而联合组GSK3β、p-GSK3β的蛋白表达水平变化也较芪玉三龙汤低、中、高剂量组和化疗组更显著〔GSK3β/β-肌动蛋白（β-actin）：0.58±0.03比1.02±0.02、1.06±0.04、0.96±0.04、0.78±0.05,p-GSK3β/β-actin：1.93±0.05比1.40±0.09、1.41±0.06、1.60±0.06、1.79±0.02,均P〈0.05〕,但对β-catenin蛋白表达的影响以芪玉三龙汤高剂量组的降低程度较化疗组和芪玉三龙汤低中剂量组及联合组更显著（β-catenin/β-actin：0.16±0.01比0.80±0.05、1.33±0.04、0.74±0.05、0.73±0.02）.结论 芪玉三龙汤对肺癌移植瘤具有温和的抑制作用,高剂量抑制肿瘤生长作用较优,且量效关系明显.芪玉三龙汤和化疗药联用具有协同效应,对肺癌具有一定的抑制作用.

Objective To observe the anti-tumor effect of Qiyusanlong decoction on lung cancer tumor-bearing mice and its influence on the protein expressions of glycogen synthase kinase 3 β （GSK3 β ）, phosphorylation GSK3 β （p-GSK3β ） and β -eatenin in Wnt/β -catenin signaling pathway. Methods The model of tumor-bearing mice was established by cultivating and transplanting Lewis lung cancer cell lines, which were divided into six groups： the model group, the chemotherapy group, the groups with low, medium, high dose of Qiyusanlong decoction respectively and the combination group by random number table, with 8 mice in each group. The next day after modeling, the mice in low, medium, high dose of Qiyusanlong decoction groups were given 20.12, 40.24, 80.48 g·kg^-1· d^-1 by garage respectively, each mouse in the chemotherapy group was given 0.4 mL cisplatin by intra-peritoneal injection once a week, the mice in the combination group were given high dose of Qiyusanlong decoction by gavage as well as cisplatin by intra-peritoneal injection, and the mice in the model group were given the same amount of normal saline; the course of management in all groups lasted for 21 days. The mice were executed at the end of experiment, the lung tissue was taken, the weight of tumor mass was measured, and the tumor-inhibiting rate was calculated; the protein immune Western Blot test was adopted to detect the protein expression levels of GSK3 β, p-GSK3 β, and β -catenin in lung tumor tissue. Results Compared with the model group, the tumor mass and levels of protein expressions of GSK3 β and β -catenin were obviously decreased in various treatment groups, the tumor-inhibiting rate and the protein expression of p-GSK3 β were obviously increased, and the degrees of changes of tumor mass and tumor-inhibiting rate in the combination group were more significant than those in the groups with low, medium, high dose of Qiyusanlong decoction [tumor mass （g）： 1.48 ± 0.71 vs. 4.53 ± 1.34, 4.27 ±0.62, 3.45 ±1.05, tumor-inhibiting rate： 73.23% vs. 13.51%, 18.32%, 33.86%, all P 〈 0.01]; thetumor mass and tumor-inhibiting rate in the combination group were almost the same as those in the chemotherapy group [tumor mass （g）： 1.48±0.71 vs. 1.49±0.68, tumor-inhibiting rate： 73.23% vs. 73,01%, both P 〉 0.05]; the degrees of protein expression changes of GSK3β and p-GSK313 in the combination group were more significant than those in the low, medium, high dose Qiyusanlong decoction groups as well as the chemotherapy group （GSK3 β / β -actin： 0.58 ± 0.03 vs. 1.02 ± 0.02, 1.06 ± 0.04, 0.96 ± 0.04, 0.78± 0.05; p-GSK3β/ β -actin： 1.93 ± 0.05 vs. 1.40 ± 0.09, 1.41± 0.06, 1.60± 0.06, 1.79 ±0.02, all P 〈 0.05）. But the effect on β -catenin protein expression was more significant in high dose of Qiyusanlong decoction group than those in chemotherapy group and low and middle dose of Qiyusanlong decoction groups and combination group （ β -catenin/β -actin： 0.16 ± 0.01 vs. 0.80 ± 0.05, 1.33 ± 0.04, 0.74± 0.05, 0.73 ± 0.02）. Conclusions Qiyusanlong decoction has mild inhibitory effect on xenograft of lung cancer, and its high dose has a better inhibitory effect, showing obvious dose-effect relationship. Qiyusanlong decoction in combination with chemotherapy has synergistic inhibitory effect on lung cancer.