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

OBJECTIVE:Constructing Lentivector encoding murine TREM-1, TREM-1vshRNA,to observe the effect of TREM-1vshRNA in the form of a hydrodynamic intravenous injection on the expression of TREM-1 in liver and spleen,TNF-α,IL-1β,IL-6 in liver and plasma,nuclear NF-kBp65,pDAP12 and IκBαin liver,the survival of septic mice,to explore the mechanism of TREM-1's influence on murine sepsis caused by Bacteroides fragilisMETHODS:(1) Constructing TREM-1 vshRNA Lentiviral Vector. Bacteroides fragilis was intraperitoneal injected in each mouse to build up murine septic model(2.5×109 CFU/mL,0.5 mL/mouse),twelve hours after instrumentation,sepsis was induced.(2) 115 mice were divided into normal control group(28 mice),sepsis group(28 mice),TREM-1 vshRNA group(28 mice),TREM-1 vshRNA hd group(28 mice),GFP group(28 mice) according to the table of random digit.Mice were firstly given saline,TREM-1 vshRNA 2×108TU,TREM-1 vshRNA 1×108TU, GFP siRNA by hydrodynamic tail vein injection and then subjected to sepsis 1h later.Control mice were intraperitoneal injected with an equal volume of saline.Twelve hours later three mice were chosen to be killed from every group,livers and blood plasma were obtained to determine the levels of TNF-α,IL-1βand IL-6,spleens and livers were harvested for TREM-1 mRNA and protein analysis,and nuclear NF-kBp65,pDAP12 and IκBαin livers were determined.The survival of rest of mice were monitored for 72h.(3) Sepsis was induced in 100 mice,every 25 mice were injected with TREM-1 vshRNA 2×108 TU at 1,2,4,6h after the intraperitoneal injection.The percentage of survival was followed and recorded for 72h after the intraperitoneal injection.RESULTS:(1) The administration of TREM-1vshRNA efficiently downregulated the expression of TNF-α,IL-1βand IL-6 in the plasma and livers of septic models(P＜0.05),the decrease in TREM-1 2.5×10~9 cfu/ ml group is more notably,whereas GFP siRNA had no effect on cytokines expression(P＞0.05).(2) Compared with GFP group,the administration of TREM-1vshRNA efficiently silenced TREM-1 expression in murine hepatic and splenic cells(P＜0.01),the depression in TREM-1 2.5×10~9 cfu/ml group is more notably(P＜0.01).Moreover, transfection of vshRNA into the murine model markedly inactivated nuclear NF-kBp65,reduced expression of pDAP12,and upregulated IκBα.(3) Seventy-six percent of the mice administered vshRNA at 2×10~8 TU 1h before injection survived sepsis compared to 44%of mice treated at 1×10~8 TU and 16%of control and GFP group(P＜0.05 and P＜0.01,respectively,compared to controls).(4) In the other 125 septic murine models,72h survival rate of animals in control and 1h,2h,4h,6h groups is 16%,72%,56%,40%,16%,respectively.The rate of 1h,2h,4h group is higher significantly(P＜0.05 or P＜0.01). CONCLUSION:The lentivirus RNAi vector of TREM-1 was constructed successfully.The interference of TREM-1/DAP12 signal pathway by TREM-1 vshRNA inhibits the genetic transcriptions of inflammatory factors,such as TNF-α,IL-1βand IL-6,etc,decreased the synthesis and secretion of them,therefore inhibits the inflammatory reaction induced by Bacteroides fragilis,turns out to improve the survival of septic mice caused by Bacteroides fragilis. CHAPTERⅠ:Expression of TREM-1 in murine septic model induced by Bacteroides fragilis.OBJECTIVE:To investigate the expression pattern of TREM-1 and its relationship with the secretion of proinflammatory factors in the hepatic cells of murine septic model induced by Bacteroides fragilis.METHODS:The clinical strain of Bacteroides fragilis was isolated and cultured under anaerobiotic condition.The mice received the intraperitoneally(i.p.) administration of 0.5ml of a suspension of Bacteroides fragilis and the livers and plasma were harvested for mRNA and protein analysis.The expression levels of TREM-1 mRNA and protein were assayed by semi-quantitative RT-PCR and Western blot, respectively.Meanwhile,the proinflammatory factors TNF-α,IL-1βand IL-6 in the plasma were detected by ELISA.RESULTS:TREM-1 was expressed on murine livers.Expression of TREM-1 mRNA and protein on livers were markedly up-regulated after priming with Bacteroides fragilis and peaked at 12h.Moreover, expression of TREM-1 were up-regulated by different concentration of Bacteroides fragilis with dose dependence and peaked at 2.5×10~9 CFU/ mL Bacteroides fragilis.Bacteroides fragilis also induced massive secretion of TNF-α,IL-1βand IL-6 in plasma,which were positively correlated with TREM-1,respectively(P＜0.01).CONCLUSION:There is a positive correlation between the time and concentration of Bacteroides fragilis and the level of TREM-1 expression,which may contribute to the secretion of proinflammatory factors. CHAPTERⅡ:Construction and identification of RNAi Lentiviral Vector targeting for TREM-1 OBJECTIVE:Constructing a lentiviral vector of RNA interference (RNAi) of murine TREM-1 gene to explore the effect of TREM-1 on the inflammatory response caused by Bacteroides fragilis.METHODS:Four target sequences were selected according to TREM-1 mRNA sequence firstly,then four pairs of double-strand DNA oligo according to these target sequences and one pair of negative control double-strand DNA oligo were designed and synthesized.These fragments were subcloned into pGCSIL-GFP/Lenti plasmid.After being identified by PCR and sequencing,these plasmids were cotransfected into 293T cells along with pHelper 1.0(gag/pol) and Helper 2.0(VSVG) to package lentiviral particles.Then the lentiviral vector particles were transfected into Raw264.7 cells and TREM-1 expression in the transfected cells was assayed by Real-Time PCR and Western blotting. Different concentrations of Bacteroides fragilis LPS was administrated in the Raw264.7 cells in a blind fashion,then the cells were stimulated with LPS for 12h.TREM-1 expression was determined at the time points indicated.RESULTS:It was confirmed by PCR and sequencing that lentiviral vectors had the correct structure and could express high titer of virus.After transfected into Raw264.7 cells,TREM-1 expression was knocked down significantly by all of these lentiviral vectors at both the protein and mRNA level and the first vector has the best interefence efficiency.TREM-1 is upregulated in the presence of Bacteroides fragilis LPS,and this increase was partly abrogated in the TREM-1 siRNA-treated cell models of endotoxemia,depending on the sequence chosen.CONCLUSION:The lentivirus RNAi vector of TREM-1 was constructed successfully.It appealed that TREM-1 is involved in the endotoxemia caused by Bacteroides fragilis LPS CHAPTERⅢ:The effect of Lentiviral Vector encoding murine TREM-1 on the inflammatory reaction during sepsis in mice caused by Bacteroides fragilisOBJECTIVE:Constructing Lentivector encoding murine TREM-1,TREM-1vshRNA,to observe the effect of TREM-1vshRNA on the survival of septic mice caused by Bacteroides fragilis and the expression of TNF-α,IL-1βand IL-6,to explore the mechanism of TREM-1's influence on murine survival.METHODS:(1) Constructing TREM-1 vshRNA Lentiviral Vector. Bacteroides fragilis was intraperitoneal injected in each mouse to build up murine septic model(2.5×10~9 CFU/mL,0.5 mL/mouse) as we described before,twelve hours after instrumentation,sepsis was induced.(2) 115 mice were divided into normal control group(3 mice),sepsis group(28 mice),TREM-1 vshRNA group(28 mice),TREM-1 vshRNA hd group(28 mice),GFP group(28 mice) according to the table of random digit.Mice were firstly given saline,TREM-1 vshRNA 2×10~8TU,TREM-1 vshRNA 1×10~8TU,GFP siRNA by hydrodynamic tail vein injection and then subjected to sepsis 1h later.Control mice were intraperitoneal injected with an equal volume of saline.Twelve hours later three mice were chosen to be killed from every group,blood plasma was obtained to determine the levels of TNF-α,IL-1βand IL-6,and livers were harvested for TREM-1mRNA and protein analysis.The survival of rest of mice were monitored for 72h.(3) Sepsis was induced in 100 mice,every 25 mice were injected with TREM-1 vshRNA 2×10~8 TU at 1,2,4,6h after the intraperitoneal injection.The percentage of survival was followed and recorded for 72h after the intraperitoneal injection.RESULTS:(1) The administration of TREM-1vshRNA efficiently downregulated the expression of plasma TNF-α,IL-1βand IL-6 in septic models(P<0.05),the decrease in TREM-1 2.5×10~9 cfu/ml group is more notably,whereas GFP siRNA had no effect on cytokines expression (P>0.05).(2) Compared with GFP group,the administration of TREM-1 vshRNA efficiently silenced TREM-1 expression in murine hepatic cells(P<0.01),the depression in TREM-1 2.5×10~9 cfu/ml group is more notably(P<0.01).A similar pattern was found when total TREM-1 protein concentration was determined.(3) Seventy-six percent of the mice administered vshRNA at 2×10~8 TU 1 h before injection survived sepsis compared to 44%of mice treated at 1×108 TU and 16% of control and GFP group(P<0.05 and P<0.01,respectively,compared to controls).(4) In the other 125 septic murine models,72h survival rate of animals in control and 1h,2h,4h,6h groups is 16%,72%,56%,40%, 16%,respectively.The rate of 1h,2h,4h group is higher significantly(P <0.05 or P<0.01).CONCLUSION:The interference of TREM-1 signal pathway by TREM-1 vshRNA inhibits the genetic transcriptions of TREM-1, therefore inhibits the secretion of inflammatory factors(TNF-α,IL-1βand IL-6),turns out to improve the survival of septic mice caused by Bacteroides fragilis. CHAPTERⅣ:The role of TREM-1 in sepsis in mice caused by Bacteroides fragilisOBJECTIVE:Constructing Lentivector encoding murine TREM-1, TREM-1vshRNA,to observe the effect of TREM-1vshRNA in the form of a hydrodynamic intravenous injection on the expression of TREM-1 in spleen,TNF-α,IL-1β,IL-6,nuclear NF-kBp65,pDAP12 and IκBαin liver,to explore the mechanism of TREM-l's influence on murine sepsis caused by Bacteroides fragilis,so as to provide latest theorical evidence.METHODS:Male mice(BALB/c) were randomly divided into several groups:positive control,TREM-1 vshRNA,GFPvshRNA control vshRNA and half dose TREM-1 vshRNA.Bacteroides fragilis(25mg/klg) was injected via caudal veins in each group to build up murine septic model.The expression of TREM-1 in the spleens,TNF-α,IL-1β,IL-6, nuclear NF-kBp65,pDAP12 and IκBαin the livers was observed in each group.RESULTS:The administration of TREM-1vshRNA efficiently silenced TREM-1 expression in murine splenic cells in a concentration-dependent manner.Meanwhile,the transfection can downregulate the expression of TNF-α,IL-1βand IL-6 in livers efficiently.Moreover,transfection of vshRNA into the murine model markedly inactivated nuclear NF-kBp65,reduced expression of pDAP12, and upregulated IκBα.CONCLUSION:TREM-1 is involved in the sepsis caused by Bacteroides fragilis.We can confirm that TREM-1 synergizes the effects of sepsis induced by Bacteroides fragilis,amplifies the synthesis of the proinflammatory cytokines TNF-α,IL-1βand IL-6.The lentivirus RNAi vector of TREM-1 was constructed successfully.The interference of TREM-1/DAP12 signal pathway by TREM-1 vshRNA inhibits the genetic transcriptions of inflammatory factors,such as TNF-α,IL-1βand IL-6,etc,decreased the synthesis and secretion of them,therefore inhibits the inflammatory reaction induced by Bacteroides fragilis,turns out to improve the survival of septic mice caused by Bacteroides fragilis.