雷公藤甲素调控nSMase2-Cer鞘脂代谢信号通路发挥抗胰腺癌的作用及其相关机制研究  

Triptolide regulates nSMase2-Cer sphingolipid metabolism signal pathway against-pancreatic cancer and its related mechanism

作　　者：唐科 罗余琴 谭亲友[1,2,3] TANG Ke;LUO Yu-qin;TAN Qin-you(Department of Clinical Pharmacy and Clinical Pharmacology,Affiliated Hospital of Guilin Medical College,Guilin Guangxi 541001;School of Pharmacy,Guilin Medical College,Guilin Guangxi 541199;China-US Lipid Research Center for Health and Diseases,Guilin Guangxi 541001)

机构地区：[1]桂林医学院附属医院临床药学与临床药理学教研室,广西桂林541001 [2]桂林医学院药学院,广西桂林541199 [3]中美健康与疾病脂质研究中心,广西桂林541001

出　　处：《中南药学》2024年第3期598-605,共8页Central South Pharmacy

基　　金：国家自然科学基金项目(No.82160765);广西自然科学基金面上项目(No.2018GXNSFAA050147);广西研究生教育创新计划项目(No.YCSW2023415)。

摘　　要：目的探究雷公藤甲素抑制胰腺癌细胞系PANC-1细胞增殖的作用机制,揭示其抗胰腺癌的作用与神经鞘脂之间的相互联系。方法用不同浓度梯度的雷公藤甲素处理胰腺癌PANC-1细胞后,检测细胞增殖、迁移能力及凋亡情况。通过PCR array实验筛选给药后PANC-1细胞上有变化的神经鞘脂基因,并通过q-PCR和Western blot实验验证所筛选基因的转录翻译情况。构建PANC-1细胞相关基因的过表达模型并进行细胞功能性实验验证。Western blot实验检测瞬时转染的PANC-1细胞给药前后及不同药物浓度下nSMase2蛋白水平的变化及Caspase-3蛋白水平的变化。免疫荧光实验检测不同药物梯度和过表达模型下神经酰胺(Cer)含量的变化。建立BALB/C裸鼠PANC-1皮下成瘤模型,在体内验证雷公藤甲素对裸鼠肿瘤大小的影响。提取肿瘤组织进行Western blot实验,肝组织进行HE染色实验。结果雷公藤甲素呈浓度梯度抑制PANC-1细胞的增殖、迁移能力并促进细胞凋亡。神经鞘脂相关基因SMPD3与雷公藤甲素抑制PANC-1细胞活力相关性最强,且雷公藤甲素可下调其在PANC-1细胞中的表达。过表达SMPD3后PANC-1细胞增殖、迁移能力明显增强,细胞凋亡减少。雷公藤甲素可促使PANC-1细胞内Cer含量的升高,而过表达SMPD3后是可以降低PANC-1细胞内Cer的含量,此外,雷公藤甲素还能够促进PANC-1细胞Caspase家族蛋白Caspase-3表达的上调。体内实验表明雷公藤甲素能够下调SMPD3蛋白的表达,促进Caspase-3活化进而促进肿瘤发生细胞凋亡,发挥抑制裸鼠体内肿瘤生长的作用。结论体内外实验均表明雷公藤甲素通过下调SMPD3的表达,影响nSMase2-Cer信号通路抑制PANC-1细胞的生长。Objective To determine the mechanism of triptolide inhibiting the growth and proliferation of pancreatic cancer(PAC)cell line PANC-1,and to reveal the interaction between triptolide anti-PAC effect and neurosphingolipids.Methods Different concentrations of gradients of triptolide were used to treat PANC-1 cells,and the proliferation,migration and apoptosis of cells were detected after drug treatment.The changes of neurosphingolipid genes in PANC-1 cells after the treatment were screened by PCR array,and the transcription and translation of the genes screened by PCR array were verified by q-PCR and Western blot.The overexpression model of related genes in PANC-1 cells was constructed and verified by cell function experiment.The changes of nSMase2 protein level and Caspase-3 protein level in transiently transfected PANC-1 cells before and after the treatment and at different drug concentrations were detected by Western blot.The changes of ceramide(Cer)with different drug gradients and in the overexpression model were detected by immunofluorescence test.The subcutaneous tumorigenic model of PANC-1 in BALB/C nude mice was established,and the effect of triptolide on the tumor size in nude mice was verified in vivo.The tumor tissue was extracted for Western blot,and the liver tissue was stained with HE.Results Triptolide inhibited the proliferation,and migration and promoted the apoptosis of PANC-1 cells in a concentration gradient manner.The inhibition of triptolide on PANC-1 cell viability was most related to SMPD3 gene,and triptolide down-regulated its expression in PANC-1 cells.After overexpression of SMPD3,the proliferation and migration of PANC-1 cells were significantly enhanced,and the apoptosis was decreased.Triptolide promoted the increase of Cer ceramide content in PANC-1 cells,while overexpression of SMPD3 reduced the content of Cer in PANC-1 cells.In addition,triptolide up-regulated Caspase-3 expression in PANC-1 cells.In vivo experiments showed that triptolide down-regulated the expression of SMPD3 protei

关 键 词：雷公藤甲素 PANC-1细胞 中性鞘磷脂酶2 细胞凋亡 

分 类 号：R286[医药卫生—中药学]