The Molecular Mechanism Of Gene Up-regulated Expression Of Inflammation-related Factors Induced By Tumor Necrosis Factor Alpha In Human Mesenchymal Stem Cells

Objective:To study the molecular mechanism of gene up-regulated expression of inflammation-related factors induced by tumor necrosis factor-alpha(TNF-a)in mesenchymal stem cells(MSC)which derived from three human different tissues.Methods:The MSCs were isolated from human umbilical cord,bone marrow,or adipose tissue which provided by volunteers,and were treated with 5,10,or 20 ng/ml TNF-?for different hours.The mRNA level of interleukin-8(IL-8),monocyte chemoattractant protein-1(MCP-1),glucose-regulated protein 78(GRP78),or C/EBP homologous protein(CHOP)gene was detected by real-time reverse transcription-polymerase chain reaction(RT-PCR),and the protein level of IL-8,MCP-1,GRP78,glucose-regulated protein 94(GRP94),eukaryotic translation initiation factor 2a(eIF2a),CHOP,Caspase-3,nuclear factor-kappa B(NF-?B)gene was analyzed by western blot in MSC.The level of soluble cytokine of IL-8 or MCP-1 gene was measured by ELISA in MSC supernatant.The level of endoplasmic reticulum stress-related proteins,NF-?B signal molecules,and IL-8 or MCP-1 protein were analyzed in MSC which treated with TNF-? combined to salubrinal or pyrrolidine dithiocarbamate(PDTC).Results:The mRNA level of IL-8 and MCP-1 gene was elevated in three different tissue derived MSC treated with different concentration TNF-?(The level of cytokines was increased about 59,328,or 506 multiples and 3.7,132,or 521 multiples in UCMSC,BMMSC,or ADMSC with 10 ng/ml drug for 48 hours respectively compared with that of controls.).The protein level of above cytokines was increased significantly,and the secretion level of soluble IL-8 or MCP-1 also elevated obviously treated with 10 ng/ml TNF-a(The level of cytokines was increased about 1.7,11.4,or 7.7 multiples and 5.7,4.8,or 5.5 multiples in three type MSC respectively compared with that of controls.P values<0.0.01).The mRNA level of endoplasmic reticulum stress protein GRP78 or CHOP gene was increased,however,that of XBP-1s gene was not changed in cells stimulated by TNF-?.The protein level of GRP78,GRP94,eIF2a,CHOP,or cleaved Caspase-3 was also elevated in MSC treated with same drug.Furthermore,the increase of eIF2a or CHOP protein level could be inhibited by salubrinal,and the protein and secretion level of IL-8 and MCP-1 could also be suppressed by the same drug in MSC.And NF-?B protein was activated by TNF-? in cells.However,the phosphorylation of NF-?B,the expression of IL-8,MCP-1 protein,and the secretion of two cytokines was inhibited in UCMSC pre-treated with PDTC.Simultaneously,NF-?B pathway could be blocked by salubrinal through the suppression of the NF-?B protein phosphorylation.Conclusions:The secretion of inflammation-related factors was elevated in three different human tissues derived MSCs treated with TNF-?.The eIF2a/CHOP pathway and NF-?B pathway were activated by TNF-?,which resulted in the increase of the protein expression and secretion of IL-8 or MCP-1.The expression of IL-8 or MCP-1 gene was partly suppressed by TNF-? in UCMSC through inhibition of the NF-?B phosphorylation,which induced by increased phosphorylation of eIF2a.