基于PTEN/PI3K/Akt信号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

Effect of Shenqi Yiliu Prescription Combined with Cisplatin on Tumor in Hepatoma H22-bearing Mice Based on PTEN/PI3K/Akt Signaling Pathway

目的:探讨参芪抑瘤方联合顺铂在磷酸酶及张力蛋白同源物(PTEN)/磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路中对H22肝癌荷瘤小鼠的抑瘤作用。方法:建立H22肝癌荷瘤小鼠模型,随机分为模型组、顺铂组、参芪抑瘤方高、中、低剂量联合顺铂组,10只/组,另随机筛选10只健康小鼠作为正常组;干预组小鼠灌胃给予参芪抑瘤方高、中、低剂量(54.06、27.03、13.515 g·kg^(-1)·d^(-1)),腹腔注射用顺铂(2.5 mg·kg^(-1)),1周2次,正常组、模型组给予生理盐水,连续治疗13 d后处死小鼠计算抑瘤率;苏木素-伊红(HE)染色观察小鼠瘤体病理形态学变化;酶联免疫吸附测定法(ELISA)和免疫荧光法检测小鼠瘤组织中细胞周期蛋白依赖性激酶抑制剂1A(p21),细胞周期蛋白依赖性激酶抑制剂1B(p27)含量;蛋白免疫印迹法(Western blot)检测瘤组织中PTEN、PI3K、磷酸化蛋白激酶B(p-Akt)蛋白含量。结果:与模型组比较,顺铂组和参芪抑瘤方高、中、低剂量联合顺铂组移植瘤瘤质量明显降低(P<0.05),参芪抑瘤方高剂量联合顺铂组抑瘤作用更明显;各组肿瘤组织中出现不同程度的坏死,参芪抑瘤方高剂量联合顺铂组最为明显。与模型组比较,顺铂联合参芪抑瘤方高、中、低剂量及顺铂组p21、p27、PTEN表达均明显升高(P<0.05);PI3K、p-Akt蛋白相对表达量降低(P<0.05),其中参芪抑瘤方高剂量联合顺铂组最为明显。结论:参芪抑瘤方可能通过调控PTEN/PI3K/Akt信号通路中关键分子表达,从而上调下游抑增殖蛋白p21、p27蛋白表达,进而发挥对H22肝癌小鼠的显著抑瘤作用,并且增强化疗效果。

Objective:To investigate the tumor-suppressing effect of Shenqi Yiliu prescription combined with cisplatin in hepatoma H22-bearing mice based on the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)pathway.Method:H22-bearing mice were prepared and randomized into model group,cisplatin group,and cisplatin combined with high-,medium-,and low-dose Shenqi Yiliu prescription groups,with 10 mice in each group.Another 10healthy mice were randomly selected as normal group.Shenqi Yiliu prescription was given by gavage with the high,medium,low dose of 54.06,27.03,13.515 g·kg^(-1)·d^(-1),respectively,and cisplatin(2.5 mg·kg^(-1))was administered by intraperitoneal injection,twice a week.Normal group and model group received normal saline.After 13 days of treatment,mice were killed and the tumor inhibition rate was calculated.The pathomorphological changes of tumor were observed based on hematoxylin-eosin(HE)staining,and enzymelinked immunosorbent assay(ELISA)and immunofluorescence method were used to detect the content of cyclindependent kinase inhibitor 1A(p21)and cyclin-dependent kinase inhibitor 1B(p27)in tumor tissue of mice.The levels of PTEN,PI3K and phosphorylated protein kinase B(p-Akt)in tumor tissue were measured by Western blot.Result:Compared with the model group,cisplatin alone and cisplatin in combination with the high-,medium-,and low-dose Shenqi Yiliu prescription decreased tumor mass(P<0.05),particularly the cisplatin in combination with the high-dose Shenqi Yiliu prescription.Necrosis of the tumor tissue was observed in each group,especially the cisplatin combined with high-dose Shenqi Yiliu prescription group.As compared with the model group,cisplatin alone and cisplatin in combination with the high-,medium-,and low-dose Shenqi Yiliu prescription raised the expression of p21,p27,and PTEN(P<0.05)and lowered the expression of PI3K and p-Akt(P<0.05),particularly the cisplatin in combination with high-dose Shenqi Yiliu prescription.Conclusion:Shenqi Yiliu prescription may regulate the expression of key molecules in PTEN/PI3K/Akt signaling pathway,thereby upregulating the expression of downstream proliferation inhibitors p21 and p27,further suppressing the tumor in H22-bearing mice,and enhancing the effect of chemotherapy.