Study On The Mechanism Of Lps-induced Apoptosis In Normal Human Colonic Epithelial Cells(NCM460)

Mucosal barriers are the first line of defense against external pathogens for life,a dynamic structure that separates intestinal contents from the host tissues,regulates nutrient absorption and the balance of mucosal immune system.Abnormal apoptosis of the epithelial cells and the defective of tight junction proteins can cause damage of intestinal mucosa.lipopolysaccharide(LPS)is a major cell wall constituent of Gram-negative bacteria,the outcome is the production of various pro-inflammatory mediators and ultimately cellular injury,leading to the various vascular sequelae of sepsis.Mucosa damage induced by LPS associated with epithelial cell apoptosis and defective of tightly protein,but the specific mechanism remains to be further studied.In present study,using NCM460 cells as an research object,the effect of LPS on NCM460(proliferation,apoptosis and tight junction protein expression)and the molecular mechanism of LPS induced its apoptosis were evaluated in order to provide additional evidences about mucosal barriers injury that induced by LPS.First,the effect of LPS on NCM460 cell proliferation or apoptosis and the expression of ZO-1 and Occludin were measured by MTT assay,flow-cytometry,fluorometer,western-blot et al.The results showed,(1)LPS could reduced the viability of NCM460 in concentration-dependent manners.(2)LPS could induce concentration-and time-dependent apoptosis in NCM460.(3)LPS inhibited the expression of ZO-1 and Occludin in concentration-dependent manners.These results showed that LPS induced NCM460 cells apoptosis and inhibited the expression of tight junction proteins(ZO-1 and Occludin)and these may related with mucosal barriers injury that induced by LPS.Then a deep understanding of apoptotic mechanisms induced by LPS was elucidated.Results as showed below,(1)the amount of TNF-a and the abundance of mRNA increased significantly in NCM460 after treated by LPS(p<0.05).(2)the expression of TNFR1 and FADD increased in NCM460 after treated by LPS.(3)comparing with the control,the activity of Caspase 8 increased significantly in NCM460 after treated by LPS(p<0.05),while there was no significant difference about Caspase 9(p>0.05.(4)the expression of MyD88 increased in NCM460 after treated by LPS while there was no significant difference about TIRAP compared to control.(5)the expression of MyD88/FADD/TNFR1 decreased significantly induced by LPS after TLR4 was blocked with TLR4 antibody compared to treated by LPS only,as well as Caspase 3/8 activity(p<0.05).(6)the expression of FADD increased induced by TNF-a recognized with TNFR1.Based on the above,we can obtain three conclusions that,(1)LPS induced NCM460 cell apoptosis through death receptors pathway.(2)LPS was recognized by TLR4 to initiate a MyD88-dependent pathway in NCM460.(3)LPS induced the secretion of TNF-? in NCM460 cells and then promoted apoptosis through death receptors pathway.In order to study the role of pathway and transcription factors in LPS inducing cell opoptosis,we investigated the changes of mTORC1 and NF-?B and STAT1 in LPS signaling in NCM460.The datas showed that,(1)LPS activated mTORC1 pathway,NF-?B and STAT1,(2)Rapamycin impaired LPS induced-STAT and NF-?B activation and the higher expression of MyD88/FADD/TNFR1 and the mRNA expression of FLIP,(3)Rapamycin decreased the secretion of TNF-? and the activity of Caspase 3/8 induced by LPS in NCM460.The above results suggested that mTORC1 pathway play an important role in regulating LPS-induce apoptosis of NCM460,while the mechanisms were still not well understood and needed further study.In conclusion,LPS could induce NCM460 apoptosis though MyD88 dependent pathway.LPS promoted the secretion of TNF-a by TLR4/MyD88 pathway.And then TNF-a induced cell apoptosis by TNFR1/FADD/Caspase 8 pathway.mTORC1 regulated apoptosis of NCM460 cells induced by LPS by mediating the level of phosphorylation of transcription factors NF-?B and STAT1,the protein synthesis and gene transcription related with apoptosis pathway.All these results provided some new ideas for understanding cellular and molecular mechanism of LPS induced apoptosis and mucosal barriers.