健脾养正消癥方对裸鼠胃癌皮下移植瘤细胞凋亡的影响及自噬机制的实验研究

Effect of Jianpi Yangzheng Xiaozheng Recipe on Apoptosis and Autophagy of Subcutaneous Transplanted Tumor in Nude Mice:an Experimental Study on Mechanism

目的观察健脾养正消瘢方对人胃癌MGC-803细胞BALB/c裸鼠皮下移植瘤抑瘤率的影响及可能的分子机制。方法取胃癌MGC-803细胞接种于裸鼠皮下,制备裸鼠移植性胃癌模型。32只BALB/c裸鼠按随机数字表法分为阴性对照组、阳性对照组和健脾养正消瘢方高、低剂量组,每组8只。阴性对照组给予生理盐水灌胃,阳性对照组给予5-氟尿嘧啶(5-Fu,2.5 mg/kg)灌胃,健脾养正消瘢方高、低剂量组分别以85、43 g/kg灌胃,每天1次,连续10天。观察健脾养正消瘢方对裸鼠皮下移植瘤抑瘤率的影响;采用RT-PCR法观察该复方对移植瘤中Bax、Bcl-2、Fas、Cyclin D1、Cyclin D2和Cyclin D3基因表达的影响;采用Western Blot法观察该复方对移植瘤中procaspase-3、procaspase-8和procaspase-9、cleavedPARP、Beclin-1和LC3B蛋白表达的影响。结果(1)与阴性对照组比较,阳性对照组和健脾养正消瘢方高、低剂量组瘤重明显减轻(P<0.05),健脾养正消瘢方高、低剂量组瘤重均高于阳性对照组(P<0.05)。(2)RT-PCR显示:与阴性对照组比较,阳性对照组和健脾养正消瘢方高、低剂量组Bax表达上调,Bcl-2、Cyclin D1、Cyclin D2和Cyclin D3表达下调(均P<0.05),阳性对照组及健脾养正消瘢方高剂量组Fas表达上调(P<0.05);与阳性对照组比较,健脾养正消瘢方高、低剂量组Fas、Bax表达均下调,Bcl-2、Cyclin D2和Cyclin D3表达上调(均P<0.05),健脾养正消瘢方高剂量组Cyclin D1下调,低剂量组Cyclin D1上调(均P<0.05)。(3)Western blot显示:与阴性对照组比较,健脾养正消瘢方高、低剂量组procaspase-3、procaspase-8、procaspase-9蛋白表达下调,cleaved-PARP、Beclin-1、LC3BⅡ蛋白表达上调(均P<0.05);阳性对照组procaspase-3、procaspase-8、procaspase-9和LC3BⅡ蛋白表达下调,cleaved-PARP、Beclin-1和LC3BⅠ蛋白表达上调(均P<0.05)。结论健脾养正消瘢方对胃癌MGC-803细胞裸鼠皮下移植瘤有显著的抑制作用,其作用可能与其激活凋亡、自噬相关因子有关。

Objective To observe the effect of Jianpi Yangzheng Xiaozheng Recipe（JYXR） on the tumor inhibition rate of subcutaneous transplanted tumor gastric cancer cell line MGC-803 in BALB/c nude mice,and to study its molecular mechanism of apoptosis and autophagy.Methods Gastric cancer cell line MGC-803 was subcutaneously inoculated to nude mice for preparing transplanted gastric cancer models.Totally 32 BALB/c nude mice were randomly divided into 4 groups according to random digit table,i.e.,the negative control group,the positive control group,the high dose JYXR group,the low dose JYXR group,8 in each group.Normal saline was administered to mice in the negative control group by gastrogavage.5-fluorouracil（5-Fu） at 2.5 mg/kg was administered to mice in the positive control group by gastrogavage.JYXR at 85 and 43 g/kg was administered to mice in the high dose JYXR group and the low dose JYXR group by gastrogavage,once per day for 10 successive days.The effect of JYXR on the tumor inhibition rate of subcutaneous transplanted tumor was observed.Effects of JYXR on gene expression levels of Bax,Bcl-2,Fas,Cyclin D1,Cyclin D2,and Cyclin D3 in transplanted tumor were observed by real-time PCR.Effects of JYXR on protein expression levels of Procaspase-3,Procaspase-8,Procaspase-9,cleaved-PARP,Beclin-1,and LC3 B were detected using Western blot.Results（1） Compared with the negative control group,the tumor weight was obviously reduced in the rest three groups（P0.05）.The tumor weight was higher in the high dose JYXR group and the low dose JYXR group than in the positive control group（P 0.05）.（2） Results of RT-PCR indicated that,compared with the negative control group,expression levels of Bax were up-regulated,but expression levels of Bcl-2,Cyclin D1,Cyclin D2,and Cyclin D3 were down-regulated in the positive control group and JYXR groups（P0.05）.The expression level of Fas was up-regulated in the positive control group and the high dose JYXR group（P0.05）.Compared with the positive control group,expression levels of Fas,and Bax were all down-regulated,but expression levels of Bcl-2,Cyclin D2,and Cyclin D3 were all up-regulated in the high dose JYXR group and the low dose JYXR group（all P 0.05）.The expression level of Cyclin D1 was down-regulated in the high dose JYXR group,but it was up-regulated in the low dose JYXR group（both P0.05）.（3） Results of Western blot showed,compared with the negative control group,expression levels of Procaspase-3,Procaspase-8,and Procaspase-9 were down-regulated,but expression levels of cleaved-PARP,Beclin-1,and LC3 B Ⅱ were up-regulated in the high dose JYXR group and the low dose JYXR group（all P 0.05）.Compared with the negative control group,expression levels of Procaspase-3,Procaspase-8,Procaspase-9,and LC3 B Ⅱ were down-regulated,but expression levels of cleaved-PARP,Beclin-1,and LC3 B Ⅰ were up-regulated in the positive control group（all P0.05）.Conclusions JYXR showed significant inhibition on subcutaneous transplanted tumor gastric cancer cell line MGC-803 in BALB/c nude mice.Its mechanism might be associated with activating apoptosis and autophagy correlated factors.