网络药理学预测西洋参抗胃癌作用及体内实验验证

Prediction of anti-gastric cancer effect of Panacis Quinquefoli Radix by network pharmacology and in vivo validation

考察西洋参抗胃癌的活性及机制,并初步对进口(美国)西洋参水煎液(JK-AG)与国产(山东)西洋参水煎液(SD-AG)体内抗胃癌的药效进行比较。在网络药理学预测基础上,建立LUC-MGC803细胞异位接种胃癌裸小鼠模型,分别灌胃给予JK-AG和SD-AG 1 g·kg^(-1),连续21 d,阳性组隔日腹腔注射5-氟尿嘧啶(5-FU)20 mg·kg^(-1),期间测量瘤体体积;实验结束时,采用超高分辨超声成像系统进行瘤体大小以及活体成像仪观察瘤体荧光表达量;剖取瘤体组织称重和进行病理组织学检查;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)及TUNEL+含半胱氨酸的天冬氨酸蛋白水解酶1(caspase-1)荧光双染检测组织凋亡和caspase-1表达;采用免疫组织化学、蛋白免疫印迹和实时荧光定量PCR(RT-qPCR)等技术检测瘤体组织中预测的凋亡和焦亡关键蛋白和基因的表达。网络药理学预测显示,西洋参具有抗胃癌活性,主要有效物质为人参皂苷类;肿瘤坏死因子-α(TNF-α)激活诱导的细胞凋亡和caspase-1可能是西洋参抗胃癌的重要分子机制之一。在体内抗肿瘤研究中,与模型组比较,JK-AG和SD-AG均不影响小鼠的体质量及脏器指数,而5-FU使小鼠体质量明显降低(P<0.05),肺、肝脏指数明显升高(P<0.05),JK-AG和SD-AG的抑瘤率分别为29.76%、27.97%,低于5-FU;但两者均可明显降低瘤组织荧光强度和三维瘤体体积(P<0.05或P<0.01);HE染色结果显示给药组的瘤体组织坏死病变和范围更大;TUNEL及TUNEL+caspase-1荧光染色显示JK-AG和SD-AG均诱导了瘤体组织细胞凋亡和增加了caspase-1表达;JK-AG和SD-AG均明显增加cleaved-caspase-8、cleaved-caspase-3蛋白以及TNF-α、caspase-8、caspase-9 mRNA表达,降低B淋巴细胞瘤-2基因(Bcl-2)蛋白表达;与JK-AG组相比,SD-AG可明显增加TNF-α、caspase-9、cleaved-caspase-9、肿瘤坏死因子受体1(TNFR1)蛋白表达,还可增加Bcl-2关联X蛋白(Bax)/Bcl-2比值;与SD-AG组相比,JK-AG可明显增加Bax mRNA表达。另外,JK-AG和SD-AG可明显增加NLRP3、caspase-1、cleaved-caspase-1、焦孔素D(GSDMD)、cleaved-GSDMD、IL-18、焦孔素E(GSDME)、GSDME-NT蛋白表达和caspase-1、GSDME mRNA的表达,其中蛋白免疫印迹检测中JK-AG增加cleaved-caspase-1蛋白表达较SD-AG更显著,同时JK-AG增加GSDMD mRNA表达比SD-AG明显;对于GSDME-NT蛋白表达,SD-AG增加比JK-AG明显。以上研究表明,西洋参具有抗胃癌作用,且美国与山东西洋参的药效作用相当,其机制不仅与TNF-α介导的外源性凋亡途径有关,还与诱导细胞焦亡有关,美国与山东西洋参的作用灵敏靶点存在不同。

This study investigated the anti-gastric cancer activity and mechanism of Panacis Quinquefolii Radix(Panax quinquefolium L.),and preliminarily compared the in vivo anti-gastric cancer efficacy of American-imported(JK-AG)and domestically produced(Shandong)Panacis Quinquefolii Radix decoctions(SD-AG).Based on network pharmacology predictions,a LUC-MGC803 cell ectopic gastric cancer nude mouse model was established.Mice were administered JK-AG and SD-AG at 1 g·kg^(-1) via gavage for 21 consecutive days.The positive control group received intraperitoneal injections of 5-fluorouracil(5-FU)at 20 mg·kg^(-1) every other day.Tumor volume was measured during the experiment.At the end of the experiment,high-resolution ultrasound imaging was used to measure tumor size,and an in vivo imaging system was used to observe tumor fluorescence expression.Tumor tissues were excised,weighed,and subjected to pathological histological examination.The terminal deoxynucleotidyl transferase-mediated nick end labeling(TUNEL)assay and TUNEL+caspase-1 fluorescence double staining were used to detect tissue apoptosis and caspase-1 expression.Immunohistochemistry(IHC),Western blot(WB),and quantitative real-time PCR(RT-qPCR)techniques were used to detect the expression of predicted key proteins and genes involved in apoptosis and pyroptosis in tumor tissues.Network pharmacology predictions indicated that Panacis Quinquefolii Radix had anti-gastric cancer activity,primarily due to its ginsenoside components.Apoptosis induced by tumor necrosis factor(TNF)-αactivation and caspase-1 may be key molecular mechanisms of its anti-cancer effects.In in vivo anti-tumor studies,compared to the model group,JK-AG and SD-AG did not affect the body weight or organ indices of the mice,whereas 5-FU significantly reduced body weight(P<0.05)and increased lung and liver indices(P<0.05).The tumor inhibition rates of JK-AG and SD-AG were 29.76%and 27.97%,respectively,which were lower than that of 5-FU.However,both JK-AG and SD-AG significantly reduced tumor tissue fluorescence intensity and three-dimensional tumor volume(P<0.05 or P<0.01).HE staining results showed that the drug groups had larger areas of tumor tissue necrosis.TUNEL and TUNEL+caspase-1 fluorescence staining indicated that both groups induced tumor cell apoptosis and increased caspase-1 expression.Both JK-AG and SD-AG significantly increased the expression of cleaved-caspase-8 and cleaved-caspase-3 proteins in tumor tissues detected by IHC and WB,as well as the mRNA expression of TNF-α,caspase-8,and caspase-9 detected by PCR,while reducing B-cell lymphoma 2(Bcl-2)protein expression.IHC showed that SD-AG increased the expression of TNF-α,caspase-9,cleaved-caspase-9,and tumor necrosis factor receptor 1(TNFR1)proteins more significantly than JK-AG,and also increased the Bax/Bcl-2 ratio,whereas JK-AG increased Bax mRNA expression more significantly than SD-AG.Additionally,JK-AG and SD-AG significantly increased the expression of NLRP3,caspase-1,cleaved-caspase-1,Gasdermin D(GSDMD),cleaved-GSDMD,IL-18,Gasdermin E(GSDME),and GSDME-NT proteins and the mRNA expression of caspase-1 and GSDME.JK-AG increased cleaved-caspase-1 protein expression more significantly than SD-AG in WB analysis.At the same time,JK-AG increased the expression of GSDMD mRNA,which was significantly higher than that of SD-AG group.For GSDME-NT protein expression,SD-AG increased it more significantly than JK-AG.The above studies indicate that Panacis Quinquefolii Radix has anti-gastric cancer effects,and the efficacy of American and Shandong ginsengs is comparable.Their mechanisms are related to TNF-α-mediated extrinsic apoptosis pathways and the induction of pyroptosis.The sensitivity of their action targets varies between American and Shandong ginsengs.