基于糖原合成酶激酶3β/核因子-κB通路探讨参芪抑瘤方联合顺铂对H22肝癌荷瘤小鼠的抑瘤作用

Exploring the tumor suppression effect of Shenqi Yiliu decoction combined with cisplatin on H22 hepatocellular carcinoma tumor-bearing mice based on glycogen synthase kinase 3β/nuclear factor-kB pathway

目的基于糖原合成酶激酶3β(GSK3β)/核因子-κB(NF-κB)通路探讨参芪抑瘤方联合顺铂对H22肝癌荷瘤小鼠的抑瘤作用。方法皮下接种H22瘤株建立小鼠H22实体移植瘤模型,待造模成功后随机分为模型组、对照组和低、中、高剂量实验组,每组10只;另取10只正常小鼠作为空白组。空白组和模型组均给予0.9%NaCl;对照组腹腔注射2.5 mg·kg^(-1)顺铂,每周2次;低、中、高剂量实验组灌胃给予13.52、27.03和54.06 g·kg^(-1)·d^(-1)参芪抑瘤方煎液+腹腔注射2.5 mg·kg^(-1)顺铂,每周2次。6组小鼠均连续治疗13 d,处死小鼠,计算肿瘤体积;用蛋白质印迹法检测瘤组织中GSK3β/NF-κB通路相关蛋白的表达水平;用反转录酶-聚合酶链反应检测GSK3β/NF-κB通路相关基因和细胞增殖抗原标记物(Ki67)和细胞增殖核抗原(PCNA)mRNA相对表达量。结果中、高剂量实验组和对照组、模型组的第13天肿瘤体积分别为(2014.80±885.60)、(1324.75±873.69)、(2459.45±663.30)和(3355.50±631.72)mm3,p-GSK3β蛋白相对表达水平分别为1.05±0.16、0.86±0.14、1.20±0.21和1.66±0.12,p-NF-κB(p65)蛋白相对表达水平分别为0.45±0.11、0.38±0.10、0.55±0.11和0.74±0.05,GSK3βmRNA相对表达量分别为0.22±0.13、0.08±0.03、0.50±0.07和1.00±0.00,NF-κB mRNA相对表达量分别为0.22±0.04、0.10±0.09、0.45±0.06和1.00±0.00,Ki67 mRNA相对表达量分别为0.48±0.08、0.10±0.03、0.68±0.11和1.00±0.00,PCNA mRNA相对表达量分别为0.46±0.08、0.20±0.02、0.61±0.16和1.00±0.00。对照组和中、高剂量实验组的上述指标与模型组比较,差异均有统计学意义(均P<0.05)。结论参芪抑瘤方对H22肝癌小鼠有显著的抑瘤作用,能显著降低Ki67和PCNA的表达,其机制可能是通过抑制GSK-3β/NF-κB信号通路响应。

Objective To explore the anti-tumor effect of Shenqi Yiliu decoction combined with cisplatin on H22 hepatoma-bearing mice based on glycogen synthase kinase 3β(GSK3β)//nuclear factor-kB(NF-kB)pathway.Methods H22 tumor strain was subcutaneously inoculated to establish H22 solid transplanted tumor model in mice.After the model was successfully established,the mice were randomly divided into model group,control group and experimental-L,-M,-H groups,10 mice in each group,the other 10 normal mice were taken as blank group.The blank and model groups were given 0.9%NaCl;the control group was given 2.5 mg·kg^(-1)cisplatin by intraperitoneal injection twice a week;the experimental-L,-M,-H groups were given 13.52,27.03,54.06 g·kg1.d^(-1)Shenqi Yiliu decoction by gavage+2.5 mg kg^(-1)cisplatin by intraperitoneal injection,twice a week.After 13 days of continuous treatment,the mice were killed and the tumor volume was calculated.Western blot was used to detect the protein content of GSK3β/NF-kB pathway in tumor tissues.Reverse transcriptase-polymerase chain reaction was used to detect the relative expressions of CSK3β/NF-kB pathway related genes and cell proliferation antigen marker(Ki67)and cell proliferation nuclear antigen(PCNA)mRNA in each intervention group.Results On the 13th day,the tumor volumes of the experimental-M,-H groups and control group,model group were(2014.80±885.60),(1324.75±873.69),(2459.45±663.30)and(3355.50±631.72)mm^(3);the expression levels of p-GSK3βprotein were 1.05±0.16,0.86±0.14,1.20±0.21 and 1.66±0.12;the expression levels of p-NF-kB(p65)protein were 0.45±0.11,0.38±0.10,0.55±0.11 and 0.74±0.05;GSK3βmRNA were 0.22±0.13,0.08±0.03,0.50±0.07 and 1.00±0.00;NF-kB mRNA were 0.22±0.04,0.10±0.09,0.45±0.06 and 1.00±0.00;Ki67 mRNA were 0.48±0.08,0.10±0.03,0.68±0.11 and 1.00±0.00;PCNA mRNA were 0.46±0.08,0.20±0.02,0.61±0.16 and 1.00±0.00.Comparing with the model group,all the above indexes of control group and experimental-M,-H groups had statistically significant differences(all P<0.05).Conclusion Shenqi Yiliu decoction has a significant anti-tumor effect on H22 hepatoma mice,and significantly reduces the expression of Ki67 and PCNA.The mechanism may be related to the inhibition of GSK-3β/NF-kB signal path response.