摘要
目的:探究柚皮苷在卵巢癌中的抗肿瘤活性并评估其潜在信号传导机制.方法:⑴将雌性Balb/c裸鼠随机分为6组:阴性对照组(PBS溶液)、0.5 mg·kg-1柚皮苷组(柚皮苷0.5 mg·kg-1)、1.0 mg·kg-1柚皮苷组(柚皮苷1.0 mg·kg-1)、2.0 mg·kg-1 柚皮苷组(柚皮苷2.0 mg·kg-1)、阳性对照组(顺铂25.0 mg·kg-1)、联合用药组(顺铂25.0 mg·kg-1、柚皮苷2.0 mg·kg-1),取对数生长期的SKOV3细胞皮下注射Balb/c裸鼠,当肿瘤大小达到50 mm3时,按照分组情况在腹膜内注射相应试剂.给药10 d后处死并切除小鼠整个肿瘤,测量肿瘤大小及体积并通过RT-PCR检测各组肿瘤凋亡相关基因mRNA的表达.⑵将SKOV3细胞随机分为4组:阴性对照组(PBS溶液)、10 µmol·L-1柚皮苷组、20 µmol·L-1柚皮苷组、40 µmol·L-1柚皮苷组.ELISA检测各组相关蛋白表达.⑶取对数生长期SKOV3细胞,以每孔2×105个细胞接种于6孔板中.将SKOV3细胞随机分8组:空白对照组(不导入质粒及siRNA)、空质粒对照组、siRNA对照组、NF-κB过表达组、NF-κB siRNA组、P38MAPK过表达组、P38MAPK siRNA组、PKC抑制剂组.RT-PCR检测各组细胞P-gp和ERK mRNA表达.⑷对NF-κB过表达组和P38MAPK过表达组SKOV3细胞进行柚皮苷干预,测量各组细胞P-gp和ERK mRNA表达.结果:⑴给药10 d后,肿瘤体积及重量由大到小依次为阴性对照组、0.5 mg·kg-1柚皮苷组、1.0 mg·kg-1柚皮苷组、2.0 mg·kg-1柚皮苷组、阳性对照组、联合用药组,且与阴性对照组相比,差异均有统计学意义(P＜0.05).⑵与阴性对照组相比,2.0 mg·kg-1柚皮苷组、阳性对照组、联合用药组Caspase-3和Caspase-7 mRNA表达增加,差异均有统计学意义(P＜0.05);与阴性对照组相比,0.5 mg·kg-1柚皮苷组、1.0 mg·kg-1柚皮苷组、2.0 mg·kg-1柚皮苷组、阳性对照组、联合用药组Bcl-2、Bcl-xl mRNA表达降低,差异均有统计学意义(P＜0.05);与阴性对照组相比,2.0 mg·kg-1柚皮苷组、阳性对照组、联合用药组Cyclin D1、C-Myc和Survivin mRNA表达降低,差异均有统计学意义(P＜0.05).⑶与阴性对照组相比,40 µmol·L-1柚皮苷组PKC、P38MAPK、ERK、NF-κB、P-gp、COX-2蛋白表达降低,Caspase-3和Caspase-7蛋白表达增加,差异均有统计学意义(P＜0.05).⑷与空白对照组相比,空质粒及siRNA对照组的ERK和P-gp mRNA表达差异无统计学意义(P＞0.05),P38MAPK过表达组ERK mRNA表达增加,P38MAPK siRNA组及PKC抑制剂组ERK mRNA表达降低,NF-κB过表达组P-gp mRNA表达增加,NF-κB siRNA组及P-gp mRNA表达降低,差异均有统计学意义(P＜0.05).⑸与空质粒组比较,柚皮苷+P38MAPK过表达组PKC、ERK、COX-2 mRNA表达降低,差异均有统计学意义(P＜0.05),P38MAPK mRNA无明显差异;柚皮苷+NF-κB过表达组PKC、NF-κB、P-gp表达降低,Caspase-3、Caspase-7表达增加,差异均有统计学意义(P＜0.05).结论:柚皮苷能够通过抑制PKC所介导的不同信号路径调控卵巢癌细胞凋亡与生长,并具有剂量依赖性.
Abstract
Objective:To investigate the antitumor activity of Naringin in ovarian cancer and to evaluate its potential signaling mechanism.Methods:⑴ Female Balb/c nude mice were divided into six groups:a negative control group(PBS solution),three naringin groups with doses of 0.5 mg·kg-1,1.0 mg·kg-1,and 2.0 mg·kg-1 respectively;a positive control group(treated with 25.0 mg·kg-1 cisplatin),and a combination group(given both cisplatin 25.0 mg·kg-1 and naringin 2.0 mg·kg-1).SKOV3 cells were then injected subcutaneously into these mice during the logarithmic growth stage.Once the tumor size reached 50 mm3,the corresponding treatment was administered intraperitoneally.After 10 days,the tumors were removed,and their size,volume,and mRNA expression of tumor apoptosis-related genes were evaluated using RT-PCR.⑵ In a separate experiment,SKOV3 cells were divided into four groups:a negative control group(treated with PBS solution),and three groups treated with different concentrations of naringin(10 µmol·L-1,20 µmol·L-1,and 40 µmol·L-1).The expression of related proteins in each group was measured using ELISA.⑶ Moreover,SKOV3 cells in the logarithmic growth phase were plated into 6-well plates at a density of 2×105 cells per well.These cells were randomly divided into eight groups:a blank control group(without plasmid or siRNA introduction),an empty plasmid control group,a siRNA control group,NF-κB overexpression and siRNA groups,P38MAPK overexpression and siRNA groups,and a PKC inhibitor group.The mRNA expressions of P-gp and ERK were detected using RT-PCR.⑷ Finally,naringin intervention was applied to SKOV3 cells overexpressing NF-κB and P38MAPK,and the mRNA expression levels of P-gp and ERK were measured in each group.Results:⑴ After 10 days of treatment,tumor volume and weight were significantly different across groups,including the negative control,0.5 mg·kg-1 naringenin,1.0 mg·kg-1 naringenin,2.0 mg·kg-1 naringenin,positive control,and combined treatment groups,and compared with negative control group,the differences were statistically significant(P＜0.05).⑵ Compared with the negative control group,mRNA expressions of Caspase-3 and Caspase-7 increased in the 2.0 mg·kg-1 naringin,positive control,and combined treatment groups,with statistical significance(P＜0.05).Conversely,mRNA expressions of Bcl-2 and Bcl-xl were notably reduced in the 0.5 mg·kg-1 naringenin,1.0 mg·kg-1 naringenin,2.0 mg·kg-1 naringenin,positive control,and combined treatment groups,with statistical significance(P＜0.05).Additionally,mRNA expressions of Cyclin D1,C-Myc,and Survivin were decreased in the 2.0 mg·kg-1 naringin,positive control,and combined treatment groups compared to the negative control group,with statistical significance(P＜0.05).⑶ In contrast to the negative control group,the protein expressions of PKC,P38MAPK,ERK,NF-κB,P-GP,and COX-2 were reduced in the 40 µmo·l L-1 naringin group,while the protein expressions of Caspase-3 and Caspase-7 were increased,with statistical significance(P＜0.05).⑷ Compared with blank control group,there was no statistically significant difference in the expression of ERK and P-gp mRNA between the empty plasmid and siRNA control groups(P＞0.05).The expression of ERK mRNA increased in the P38MAPK overexpression group and decreased in the P38MAPK siRNA group and the PKC inhibitor group.NF-κB overexpression led to increased P-gp mRNA expression,whereas NF-κB siRNA resulted in decreased P-gp mRNA expression,with statistical significance(P＜0.05).⑸ Compared with the empty plasmid group,mRNA expressions of PKC,ERK,and COX-2 were decreased in the naringin+P38MAPK overexpression group,with statistical significance(P＜0.05).However,there was no significant difference in P38MAPK mRNA expression.In the naringin+NF-κB overexpression group,expressions of PKC,NF-κB,and P-GP were decreased,while expressions of Caspase-3 and Caspase-7 were increased,with statistical significance(P＜0.05).Conclusion:Naringin can regulate the apoptosis and growth of ovarian cancer cells in a dose-dependent manner by inhibiting different signaling pathways mediated by PKC.
基金项目
江西省自然科学基金项目(20192ACBL20038)
江西省中医药科技计划基金项目(2020Z001)
维普
万方数据