摘要
目的:研究冬凌草甲素(Ori)体内外抗卵巢癌(OC)活性及相关机制。方法:采用网络药理学方法分析Ori抗OC的核心靶点,并通过GO和KEGG富集分析筛选其相关生物学过程和通路。通过分子对接验证Ori与核心靶点的结合活性。用不同浓度(0、5、10、15、20、25、30μmol/L)Ori干预SKOV3细胞24和48 h,采用CCK-8法检测细胞存活率。将SKOV3细胞分为3组:对照组、8μmol/L Ori组、16μmol/L Ori组,给予相应药物干预24 h后,采用EdU染色和克隆形成实验检测细胞增殖能力,Annexin V-PE/7-AAD染色后流式细胞术检测细胞凋亡率,Western blot检测凋亡相关蛋白(Cleaved-Caspase-3、Bax、Bcl-2)和PI3K/AKT通路相关蛋白(p-PI3K、PI3K、p-AKT、AKT)的表达。加入PI3K激活剂740Y-P后,将细胞分为3组:对照组、16μmol/L Ori组、Ori+740Y-P组,给予相应药物干预24 h后,采用Western blot检测上述凋亡相关蛋白和PI3K/AKT通路相关蛋白的表达,CCK-8法检测细胞存活率。构建OC裸鼠荷瘤模型,将裸鼠分为3组:对照组、15 mg/kg Ori组、30 mg/kg Ori组,每组5只,给予相应药物治疗3周。给药结束后,测量肿瘤体积,采用免疫组化检测瘤组织中Ki-67的表达,HE染色观察主要器官的病理变化。结果:网络药理学研究显示,Ori抗OC的核心靶点是TP53、AKT1、ALB、EGFR、ESR1等,KEGG富集分析显示共有145条通路显著富集,包括癌症通路、PI3K/AKT通路等。分子对接显示大部分核心靶点与Ori有较强的结合活性。细胞实验结果表明,与对照组相比,8和16μmol/L Ori组的EdU阳性细胞率和克隆形成率均降低,细胞凋亡率增加,Cleaved-Caspase-3和Bax表达增加,Bcl-2表达、p-PI3K/PI3K和p-AKT/AKT均降低(P<0.05)。与16μmol/L Ori组相比,Ori+740Y-P组的p-PI3K/PI3K、p-AKT/AKT和Bcl-2表达增加,细胞存活率升高,Cleaved-Caspase-3和Bax表达降低(P<0.05)。动物实验结果表明,与对照组相比,15和30 mg/kg Ori组肿瘤体积缩小,Ki-67表达降低(P<0.05),且裸鼠的肝、肾、心、肺未见明显病理改变。结论:Ori在体内外均表现出抗OC活性,其机制可能与抑制PI3K/AKT通路进而抑制细胞增殖并诱导凋亡有关,且体内用药安全,可能是一种潜在的抗OC药物。
Aim:To investigate the anti-ovarian cancer(OC)activity of oridonin(Ori)in vitro and in vivo and its mechanism.Methods:The network pharmacology method was used to analyze the core targets of Ori anti-OC.GO and KEGG enrichment analysis were used to screen related biological processes and pathways.The binding activity of Ori to the core targets was verified by molecular docking.SKOV3 cells were treated with different concentrations(0,5,10,15,20,25,30μmol/L)of Ori for 24 and 48 hours,and the cell viability was detected by CCK-8.SKOV3 cells were divided into control group,8μmol/L Ori group and 16μmol/L Ori group,treated with corresponding drugs for 24 hours,EdU staining and colony formation assay were used to detect cell proliferation ability,and Annexin V-PE/7-AAD staining and flow cytometry were used to detect cell apoptosis rate.Western blot was used to detect the expressions of apoptosis-related proteins(Cleaved-Caspase-3,Bax,Bcl-2)and PI3K/AKT pathway-related proteins(p-PI3K,PI3K,p-AKT,AKT).After adding PI3K activator 740Y-P,the cells were divided into control group,16μmol/L Ori group and Ori+740Y-P group.After 24 hours of corresponding drug intervention,the expressions of apoptosis-related proteins and PI3K/AKT pathway-related proteins were detected by Western blot,and cell viability was detected by CCK-8.The tumor-bearing nude mice were divided into control group,15 mg/kg Ori group and 30 mg/kg Ori group,with 5 mice in each group,and treated with corresponding drugs for 3 weeks.The tumor volume was measured,the expression of Ki-67 in tumor tissue was detected by immunohistochemistry,and the pathological changes of major organs were observed by HE staining.Results:Network pharmacology study showed that the core targets of Ori anti-OC were TP53,AKT1,ALB,EGFR,ESR1,etc.KEGG enrichment analysis showed that 145 pathways were significantly enriched,including cancer pathway,PI3K/AKT pathway,etc.Molecular docking showed that most of the core targets had strong binding activity with Ori.Compared with control group,EdU positive cell rate and colony formation rate of 8 and 16μmol/L Ori groups decreased,the apoptosis rate increased,the expressions of Cleaved-Caspase-3 and Bax increased,while the expression of Bcl-2,p-PI3K/PI3K and p-AKT/AKT decreased(P<0.05).Compared with 16μmol/L Ori group,p-PI3K/PI3K,p-AKT/AKT and the expression of Bcl-2 in Ori+740Y-P group increased,cell viability increased,and the expressions of Cleaved-Caspase-3 and Bax decreased(P<0.05).Compared with control group,the tumor volume of 15 and 30 mg/kg Ori groups decreased,the expression of Ki-67 decreased(P<0.05),and no obvious pathological changes were observed in the liver,kidney,heart or lung of nude mice.Conclusion:Ori exhibits anti-OC activity both in vitro and in vivo,and its mechanism may be related to inhibiting cell proliferation and inducing apoptosis by inhibiting PI3K/AKT pathway.It is safe to use in vivo,and may be a potential anti-OC drug.
作者
何静
严如根
金国钰
苏娜
李长忠
HE Jing;YAN Rugen;JIN Guoyu;SU Na;LI Changzhong(The First College for Clinical Medicine,Shandong University of Traditional Chinese Medicine,Jinan 250014;School of Traditional Chinese Medicine,Nanjing University of Chinese Medicine,Nanjing 210023;Department of Gynecology,the Affiliated Shandong Provincial Hospital,Shandong First Medical University,Jinan 250021;Department of Obstetrics and Gynecology,Shenzhen Hospital,Peking University,Shenzhen,Guangdong 518036)
出处
《郑州大学学报（医学版）》
CAS
北大核心
2023年第4期480-489,共10页
Journal of Zhengzhou University(Medical Sciences)
基金
山东大学荣祥再生医学基金项目(2019SDRX-21)
济南市科学计划项目(202019161)。
