Study Of Effect And Mechanism Of TREM-1 Gene Silencing By RNA Interference On Inflammatory Responses Induced By Lipoplysaccharide From Bacteroides Fragilis

CHAPTERⅠ: Expression of TREM-1 in inflammatory responsesinduced by lipoplysaccharide from Bacteroides fragilis.OBJECTIVE: To investigate the expression pattem of TREM-1 andits relationship with the secretion of proinflammatory factors duringTHP-1 and HL-60 cell inflammatory response induced by LPS fromBacteroides fragilis.METHODS: The clinical strain of Bacteroides fragilis was isolatedand cultured under anaerobiotic condition, then the LPS was extractedfrom the strain by optimized phenol-water method. The differentiatedTHP-1 and HL-60 cell lines were stimulated with the LPS in vitro.Theexpression levels of TREM-1 mRNA and protein were assayed bysemi-quantitative RT-PCR and Westem blot, respectively. Meanwhile, theproinflammatory factors TNF-α,IL-1βin the supematants of THP-1 andIL-6,IL-8 in the supematants of HL-60 were detected by ELISA.RESULTS: TREM-1 was expressed on both the differentiated THP-1and HL-60 cell lines. Expression of TREM-1 mRNA and protein onTHP-1 and HL-60 were markedly up-regulated after priming with theLPS and peaked at 8h and 12h, respectively. Moreover, expression ofTREM-1 were up-regulated by different concentration of the LPS withdose dependence and peaked by 1μg/ml LPS. The LPS also induced massive secretion of TNF-α,IL-1βand IL-6,IL-8,which were positivelycorrelated with TREM-1, respectively (r_s=0.82,0.85 and 0.83,0.88,P＜0.01).CONCLUSION: Expression of TREM-1 can be up-regulated by LPSfrom Bacteroides fragilis time-dose dependently, which may contribute tothe secretion of proinflammatory factors. CHAPTERⅡ: Inhibition of shRNA targeting TREM-1 gene oninflammatory responses induced by lipoplysaccharide fromBacteroides fragilis.OBJECTIVE: To study the effect of shRNA targeting TREM-1 geneon inflammatory responses induced by LPS from Bacteroides fragilis.METHODS: Specific TREM-1 shRNAs were synthesized bytranscription method in vitro, and transiently transfected into thedifferentiated THP-1 and HL-60 cell lines by electroporation method,then the THP-1 and HL-60 were stimulated with the LPS fromBacteroides fragilis for 8h or 12h, respectively. At 48h post-transfection,the subsequent changes in TREM-1 mRNA and protein level weredetected by semi-quantitative RT-PCR and Western blot, respectively.ELISA was applied to examine the secretion of proinflammatory factorsTNF-α, IL-1βin the supernatants of THP-1 and IL-6, IL-8 in thesupernatants of HL-60.RESULTS: All three TREM-1 shRNAs obviously down-regulated theexpression of TREM-1 in both mRNA and protein levels, the mosteffective one was shRNA-2. Simultaneously, shRNA transfectionobviously inhibited the secretion of TNF-α,IL-1βin THP-1 and IL-6,IL-8in HL-60. However there were no similar effects in control groups.CONCLUSION: TREM-1 shRNA transfection can selectivelysuppress TREM-1 expression, and reduce the secretion of proinflammatory factors induced by LPS from Bacteroides fragilis, theninhibit the inflammatory response. CHAPTERⅢ: Possible mechanism of inhibition of TREM-1 genesilencing by RNA interference on inflammatory responses induced bylipoplysaccharide from Bacteroides fragilisOBJECTIVE: To explore the mechanism of inhibition of TREM-1gene silencing by RNA interference on inflammatory responses inducedby lipoplysaccharide from Bacteroides fragilis.METHODS: TREM-1 shRNA-2 or shRNA-NC were transfectedinto the differentiated THP-1 and HL-60 cell lines by electroporationmethod and stimulated with the LPS from Bacteroides fragilis aspreviously. DAP12,NF-κB p65,IκBαprotein levels were detected byWestern blot, TNF-α,IL-1βmRNA in THP-1 and IL-6,IL-8 mRNA inHL-60 were measured by semi-quantitative RT-PCR.RESULTS: TREM-1 shRNA-2 transfection down-regulated DAP12,NF-κBp65 expression, but elevated IκBαexpression conversely.Meanwhile, it suppressed TNFα, IL-1β, IL-6 and IL-8 mRNA expressiondramatically. However, shRNA-NC transfection had not the same effects.CONCLUSION: TREM-1 shRNA maybe down-regulate DAP12expression,stabilize the Iκ3α/NF-κB complex, inhibit the transcripitionof TNFα,IL-1β,IL-6,IL-8 gene, thus inhibit inflammatory responsesinduced by lipoplysaccharide from Bacteroides fragilis.