广藿香醇下调PD-L1的表达对人胃癌细胞抗肿瘤作用的研究  

Effect of Patchouli alcohol on anti-tumor effect of gastric cancer cells by down-regulation of PD-L1 expression

作　　者：侯坤 王振飞 HOU Kun;WANG Zhenfei(The People’s Hospital Affiliated to Inner Mongolia Medical University/Inner Mongolia Cancer Hospital,Inner Mongolia,Hohhot 010020,China)

机构地区：[1]内蒙古医科大学附属人民医院/内蒙古自治区肿瘤医院,内蒙古自治区呼和浩特市010020

出　　处：《河北医药》2023年第5期645-650,共6页Hebei Medical Journal

基　　金：国家自然科学基金(编号:82074144);内蒙古自治区重点研发和成果转化项目(编号:2022YFHH0095);内蒙古自治区科技计划项目(编号:201802152);内蒙古自然科学基金杰出青年培育基金(编号:2021JQ09);内蒙古医科大学致远人才计划(编号:ZY0130014);内蒙古医科大学科技创新团队(编号:YKD2022TD003);内蒙古医科大学重点项目(编号:YKD2022ZD004);内蒙古医科大学联合项目(编号:YKD2022LH004)。

摘　　要：目的研究广藿香醇能否通过下调人胃癌细胞中关键促癌因子PD-L1的表达进而抑制人胃癌细胞的恶性行为以及下调PD-L1表达的机制。方法使用不同剂量的广藿香醇作用于常规培养的人胃癌细胞SGC-7901和BGC-823,用CCK-8实验、细胞划痕实验和流式细胞技术分别检测不同浓度的广藿香醇对SGC-7901和BGC-823细胞的增殖能力、迁移能力和凋亡能力的影响,用荧光定量PCR法检测SGC-7901和BGC-823细胞中PD-L1、NF-κB的mRNA表达水平的变化,用Western-blot法检测SGC-7901和BGC-823细胞中PD-L1、NF-κB的蛋白表达水平的变化。结果经实时荧光定量PCR法检测人胃正常上皮细胞GSY和胃癌细胞系细胞BGC-823、MGC-803、SGC-7901、MKN-74中mRNAPD-L1、NF-κB mRNA表达水平,最终筛选出BGC823、SGC7901进行后续实验。与对照组比较,0.08、0.1、0.3、0.5 mmol/L药物组中BGC-823、SGC-7901细胞的增殖能力显著降低,且降低幅度随加药浓度升高而增大。经计算SGC-7901的IC_(50)浓度为0.37 mmol/L,BGC-823的IC_(50)浓度为0.27 mmol/L,因此选择浓度为0.3 mmol/L、0.5 mmol/L的广藿香醇进行后续实验。与人胃癌细胞SGC-7901、BGC-823的对照组比较,广藿香醇各加药组细胞的划痕愈合率均有明显下降,差异有统计学意义(P<0.05),并且0.3、0.5 mmol/L的广藿香醇对BGC823细胞的划痕愈合率的影响更为显著。经AnnexinV-FITC/PI双染后,0.3、0.5 mmol/L广藿香醇作用后能明显诱导SGC-7901、BGC-823细胞发生凋亡。加药组(广藿香醇浓度分别为0.3,0.5 mmol/L)作用于SGC-7901、BGC-823细胞24 h后,细胞凋亡率显著高于对照组(P<0.05),并且广藿香醇对SGC-7901、BGC-823细胞的凋亡作用呈剂量依赖,与对照组比较,0.3、0.5 mmol/L加药组的两种胃癌细胞的凋亡率均显著提高(P<0.05)。在SGC-7901中,与对照组比较,广藿香醇各加药组细胞中PD-L1、NF-κB的mRNA均明显下降(P<0.05);在BGC-823中,与对照组比较,广藿香醇各加药组细胞中PDObjective To investigate the inhibitory effect of Patchouli alcohol on the malignant behaviors of gastric cancer cells by down-regulation of programmed cell death-ligand 1(PD-L1)expression,and its mechanism.Methods In vitro cultured BGC-823 and SGC-7901 gastric cancer cells were treated with different concentrations of patchouli alcohol.The effects of different concentrations of patchouli alcohol on the proliferation,migration and apoptosis of SGC-7901 and BGC-823 cells were detected by the cell counting kit-8(CCK-8)assays,cell scratch test and flow cytometry.The quantitative real time polymerase chain reaction(qRT-PCR)was conducted to detect the level of mRNA expression level of PD-L1 and nuclear factor-kappaB(NF-κB)in SGC-7901 and BGC-823 cells;Western blot was performed to detect the protein expressions of PD-L1 and NF-κB.Results The expression levels of mRNA of PD L1 and NF-κB in normal human gastric epithelial cells GSY and gastric cancer cell lines involving BGC-823,MGC-803,SGC-7901 and MKN-74 were detected by qRT-PCR,BGC-823 and SGC-7901 were finally screened for subsequent experiments.Compared with the blank control group,the proliferative capacity of BGC-823 and SGC-7901 cells in the 0.08mmol/L,0.1mmol/L,0.3mmol/L and 0.5 mmol/L drug groups were significantly decreased,and the degree of decrease rose with increase in the drug concentration.The inhibitory concentration 50%(IC_(50))of SGC-7901 upon calculation was 0.37mmol/L,and that of BGC-823 was 0.27mmol/L.Consequently,Patchouli alcohol with the concentration of 0.3mmol/L or 0.5mmol/L was selected for subsequent experiments.Patchouli alcohol in 0.3mmol/L and 0.5mmol/L after double staining of the Annexin V-Fluorescein Isothiocyanate(FITC)/Propidium Iodide(PI)could significantly induce SGC-7901 and BGC-823 cells to have apoptosis.After 24h,the apoptosis rate of SGC-7901 and BGC-823 cells in the treatment group(the concentration of Patchouli alcohol was 0.3mmol/L and 0.5mmol/L,respectively)were significantly higher relative to the corresponding control

关 键 词：广藿香醇 人胃癌细胞 恶性行为 PD-L1 NF-κB 

分 类 号：R735.2[医药卫生—肿瘤]