鼠李糖乳杆菌代谢物吲哚-3-乳酸拮抗SP3/TNF-α通路促进结直肠癌细胞凋亡的机制研究  

Mechanism of Lactobacillus rhamnosus metabolite indole-3-lactate promoting colorectal cancer cell apoptosis through antagonizing the SP3/TNF-α pathway

作　　者：史惠文 陶雪梅 朱延朋 SHI Huiwen;TAO Xuemei;ZHU Yanpeng(Department of General Surgery,the 971st Naval Hospital of PLA,Qingdao,Shandong 266000,China;Department of Military Medicine and Special Sciences,the 971st Naval Hospital of PLA,Qingdao,Shandong 266000,China)

机构地区：[1]中国人民解放军海军第九七一医院普通外科,山东青岛266000 [2]中国人民解放军海军第九七一医院军事医学与特种学科,山东青岛266000

出　　处：《中国普通外科杂志》2023年第4期529-537,共9页China Journal of General Surgery

摘　　要：背景与目的:众所周知,肠道系统的微生物群是调节肠道健康与否的重要微环境。拥有平衡的微生物群有助于预防疾病,特别是与胃肠系统有关的癌症。鼠李糖乳杆菌(L.rhamnosus)具有抗肿瘤作用,但是具体机制尚不明确,基于此,本研究探讨L.rhamnosus对人结直肠癌(CRC)细胞凋亡的影响和潜在机制。方法:将人CRC细胞HCT-116、HT-29以及正常结肠上皮细胞NCM-460用L.rhamnosus的培养上清液(LRCS)或大肠埃希菌(E.Coli)的培养上清液(ECCS)处理后,检测细胞活力、凋亡水平和细胞周期分布。使用3 kDa超滤管将细菌培养上清液分为低分子量组分(<3 kDa)和高分子量组分(>3 kDa),分析这两个组分对细胞凋亡的影响。非靶向LC-MS/MS以鉴定LRCS有效组分中的抗CRC细胞的代谢物,并使用5μmol/L浓度的上述代谢物筛选影响凋亡的关键代谢物。细胞凋亡通路siRNA筛选鉴定关键代谢物的作用靶标。分子对接分析关键代谢物与靶标分子的相互作用位点。结果:LRCS呈浓度依赖性降低CRC细胞的活力,且明显促进CRC细胞的凋亡(均P<0.05),而ECCS无上述作用(均P>0.05)。进一步分析发现,仅LRCS低分子量能产生上述作用。非靶向LC-MS/MS鉴定以及验证实验表明,吲哚-3-乳酸(I3L)影响CRC细胞凋亡的L.rhamnosus关键代谢物。通过siRNA筛选,将CRC细胞的特异性蛋白3(SP3)敲除后,I3L对CRC细胞的凋亡水平、TNF-α的表达与分泌水平均无明显影响(均P>0.05)。分子对接发现,I3L与SP3的K551、E551和E552存在相互作用。将SP3敲除CRC细胞分别转染SP3野生型过表达质粒和SP3突变型过表达质粒,I3L可以促进前者的细胞凋亡水平和TNF-α分泌水平(均P<0.05),但对后者无此作用(均P>0.05)。结论:L.rhamnosus能促进CRC细胞的凋亡,作用机制可能与其代谢物I3L通过结合SP3的K551、E551和E552位点,增加TNF-α的转录和分泌有关。Background and Aims:As is widely known,the microbiota in the gastrointestinal tract plays an important role in regulating intestinal health.Having a balanced microbiota can help prevent diseases,particularly cancer related to the gastrointestinal system.Lactobacillus rhamnosus(L.rhamnosus)has been found to have anti-tumor effects,but the specific mechanisms are still unclear.Based on this,this study was conducted to investigate the impact and potential mechanisms of L.rhamnosus on apoptosis of human colorectal cancer cells(CRC).Methods:Human CRC cells HCT-116,HT-29,and normal colon epithelial cells NCM-460 were treated with the culture supernatant of L.rhamnosus(LRCS)or Escherichia coli(E.coli)culture supernatant(ECCS),and cell viability,apoptosis level,and cell cycle distribution were measured.The bacterial culture supernatant was divided into low molecular weight(<3 kDa)and high molecular weight(>3 kDa)fractions using a 3 kDa ultrafiltration membrane,and the effects of these fractions on cell apoptosis were analyzed.Untargeted LC-MS/MS was used to identify anti-CRC metabolites in the effective fraction of LRCS,and these metabolites were screened at a concentration of 5μmol/L to identify key metabolites affecting apoptosis.SiRNA screening was performed to identify the target of the key metabolites in the apoptotic pathway.Molecular docking analysis was conducted to study the interaction sites between the key metabolites and the target molecules.Results:LRCS exhibited a concentration-dependent decreasing effect on cell viability of CRC cells and significantly promoted apoptosis in CRC cells(all P<0.05),while ECCS had no such effects(all P>0.05).Further analysis revealed that only the low molecular weight fraction of LRCS was capable of producing these effects.Untargeted LC-MS/MS identification and validation experiments indicated that indole-3-lactic acid(I3L)was a key metabolite of L.rhamnosus that influenced CRC apoptosis.Through siRNA screening,I3L showed no significant effect on apoptosis level or TNF-αexpr

关 键 词：结直肠肿瘤 鼠李糖乳杆菌 吲哚-3-乳酸 Sp3转录因子 肿瘤坏死因子α 细胞凋亡 

分 类 号：R735.3[医药卫生—肿瘤]