LPS诱导的HepG2细胞NOTCH与LPS-TLR4-NF-κB炎症信号通路的“会话”研究

Cross-talk between Notch and LPS-TLR4-NF-κB inflammatory signaling pathways in LPS-activated HepG2 cells

目的探讨在脂多糖(LPS)刺激下HepG2细胞Notch与LPS-TLR4-NF-κB炎症信号通路的相互影响。方法给予LPS处理HepG2细胞,提取细胞RNA,采用qRT-PCR法检测HepG2细胞Notch信号通路受体及其配体mRNA水平,给予γ分泌酶抑制剂(DAPT)、LPS或LPS联合DAPT处理细胞,采用Western blot法检测Notch受体胞内区域(NICD)和LPS-Toll样受体4(TLR4)蛋白表达水平。结果经LPS处理HepG2细胞后,Notch 1和Jag 1 mRNA水平分别升高了2.25倍(P<0.001)和2.47倍(P<0.001),NOTCH 3仅增加的0.0700倍(P>0.05),Jag 2仅增加了0.420倍(P>0.05),Dll 4增加了0.947倍(P<0.01),而NOTCH 2却降低了0.857倍(P<0.01),NOTCH 4降低了0.283倍(P>0.05),Dll 1降低了0.750倍(P<0.01)、Dll 3降低了0.393倍(P>0.05);与对照组比,LPS处理组NICD和NF-κB蛋白表达水平显著增加,而DAPT处理组NICD和NF-κB蛋白表达显著减少。结论本研究结果揭示了在LPS刺激下,HepG2细胞Notch与TLR4-NF-κB信号通路之间的相互作用,抑制Notch信号通路可以显著改善LPS-TLR4引起的炎症反应。

Objective To investigate the interaction between Notch and LPS-LPS-Toll like receptor-4(TLR4)-NF-κB inflammatory signaling pathways in HepG2 cells stimulated with lipopolysaccharide(LPS).Methods HepG2 cells were cultured with LPS and cell RNAs were extracted,and Notch signaling pathway receptors and their ligands mRNA were detected by quantitative reverse transcription polymerase chain reaction.Western blot analysis was used to detect Notch intracellular domain(NICD)and NF-κB protein expression levels after theγ-secreting enzyme inhibitor(DAPT),LPS or combination of LPS and DAPT activation,respectively.Results After LPS activation of HepG2 cells,the mRNA level of Notch 1 was 2.25 times(P<0.001),Jag 1 was 2.47 times(P<0.001),NOTCH 3 was 0.0700 times(P>0.05),Jag 2 was 0.420 times(P>0.05),and Dll 4 was 0.947 times(P<0.01)increased,while NOTCH 2 was 0.857 times(P<0.01),NOTCH 4 was 0.283 times(P>0.05),Dll 1 was 0.750 times(P<0.01),and Dll 3 was 0.393 times(P>0.05)decreased;the expression of NICD and NF-κB proteins in LPS-intervened cells increased obviously,while those in DAPT-intervened cells decreased greatly as compared to those in the control.Conclusion Our findings reveals that the interaction might be going on between Notch and TLR4-NF-κB signaling pathways in HepG2 cells stimulated by LPS.The inhibition of Notch signaling pathway could significantly alleviate the inflammatory response caused by LPS-TLR4.