Effect Of RNA Interference On TLR4 Signaling Pathway In Alveolar Macrophage Of Acute Lung Injury Rat Induced By Lipopolysaccharide

Background:Acute lung injury(ALI), mainly caused by infection, is one of the most common critical illnesses of respiratory system. ALI induced by Gram-negative bacteria infection is due to effects of cytokines from inflammatory cells activated by Lipopolysaccharides(LPS). TLR4, as a trans-membrane receptor in LPS target cells, its out-membrane leucine-rich repeats could recognize and bind LPS, and the domain homologuing to DL-1β in cytoplasma may transduce the signaling to EL-1β. So, TLR4 is the key molecular in LPS signaling pathway. TLR4 expression and its perfect function directly affect the effects of LPS signaling transduction. Otherwise, to depress the expression and function of TLR4 is helpful to decrease inflammatory cytokines excretion and relieve ALI induced by LPS.Alveolar macrophages(AM) are the important defense line of the respiratory tract and also the target cells of LPS. It is helpful and important to investigate LPS signaling transduction of AM and the blocking effects to understand the mechanism of ALI and find out a new therapy way.The dsRNAs synthesized in vitro or formed in vivo trigger the sequenced-specific mRNA destruction and the suppression of corresponding gene. This phenomenon is called RNA interference(RNAi). Because the effect of RNAi is on the level of mRNA. RNAi is a kind of PTGS(post-transcription gene silencing). In recent years, RNAi has been found in organisms from epiphytes to plants, and from invertebrates to mammalian animals. Compared with gene knockout, RNAi is simple, time saving and effective. RNAi has now been widely used in studies of gene function. With the breakthrough of RNAi technology in mammalian cells, more and more studies begin to apply the RNAi in the research of functional genomics and gene therapy.In order to investigate the role of TLR4 signaling pathway in ALI induced by LPS, the reporter gene, enhanced green fluorescence protein (EGFP) is helpful to establish a rapid screen system for siRNA targeting TLR4;then, chemically synthesized siRNAs were administered to the cultured AM cells before or after LPS stimulation. Expression of TLR4 mRNA and cytokine proteins were detected by real-time PCR and ELISA. Finally, a plasmid (pRNA-H1.rTLR4^shRNA) which contains a H1 promoter expressing shRNA targeting TLR4 in vivo was constructed. After transfected from respiratory tract, rats were treated with LPS. Expression of TLR4 mRNA of AM and cytokine proteins were detected both in vivo and in vitro.Methods:1. screening siRNA sequence which could suppression TLR4 expression most effectively in vitroFirst the full length gene of rat TLR4 was cloned and inserted to pEGFP-Cl to construct a new plasmid, pEGFP-rTLR4. Then, co-transfected with pEGFP-rTLR4, chemically synthesized siRNAs( 3 pairs of siRNA targeting rTLR4, one pair of siRNA targeting GFP and one pair of negative-control siRNA) were trnsfected into HEK-293 cell lines mediated by Lipofectamine2000. The cells were observed under inverted fluorescence microscope and flow cytometer to detect the suppression effect ofall siRNAs on EGFP expression.2.Experimental studies of siRNA targeting rat AM TLR4 in vitro before or after LPSstimulation.Alveolar macrophages were isolated and cultured, and transfected with siRNA before LPS stimulation (protocol A) or after LPS stimulation(protocol B). Expression of TLR4 mRNA in AM cells were detected by real-time PCR , cytokine proteins(TNF a , IL-10) were detected by ELISA.3. Construction of pRNA-Hl.rTLR4shRNA which could expression shRNA in vivoBased on the special sequence of siRNA screened before, the sense and antisense oligos were synthesized. After annealing, this short DNA was inserted into pRNA-Hl to construct a recombinant plasmid pRNA-Hl.rTLR4shRNA, which contain HI promoter of mammalian cells. Restriction analysis and sequence were performed to identify the plasmid.4.ExperimentaI studies of pRNA-Hl.rTLR4shRNA transfection in vivo before LPS stimulationAfter anesthetized, SD rats were intubated. pRNA-Hl .rTLR4shRNA mediated by jetPEI? in vivo was administered intratracally. The control group were treated with pRNA-Hl. Then, rats were extubated. Forty-eight hours after transfection, rats were anesthetized and treated with LPS intravenously. The lungs were excised and lavaged after^lh. Expression of TLR4 mRNA in AM cells were detected by real-time PCR, cytokine proteins in lavage fluid by ELISAResults:l.Restiction analysis and sequence results demonstrated that the recombinant pEGFP-rTLR4 containing full-length TLR4 was expected;Forty-hours after 5 pairs of siRNAs co-transfected with pEGFP-rTLR4 into HEK-293 cell lines, under inverted fluorescence microscope and flow cytometer, compared with control group, 3 pairs of siRNAs targeting TLR4 and one pair of siRNA targeting GFP suppress the EGFP expression significantly (PO.05), especially the effect of siRNA2, the interference efficiency was more than 75%. So the sequence of siRNA2(5'-GTC TCA GAT ATC TAG ATC T-3\19bp, 1352—1370) was the right sequence to suppress TLR4 expression.2.The cultured AM cells were in good condition. In protocol A, compared with control group, TLR4 mRNA in RNAi group shows no difference in Tl(12h), significant difference in T2(24h) and T3(48h)(P<0.05 and P<0.0\);TNF- a in RNAi group showed no difference in Tl(12h).significant difference in T2 and T3CPO.05);IL-10 in RNAi group showed no difference in Tl and T2, significant difference in T3(P<0.05). In protocol B, compared with control group, TLR4 mRNA in RNAi group showed no difference in Tl (12h), significant difference in T2(24h) and T3(48h)(P<0.05 1);TNF- a in RNAi group showed no difference in Tl(12h). significant difference in T2 and T3(P<0.05);IL-10 in RNAi group showed no difference in Tl and T2, significant difference in T3(P<0.01)3.Restriction analysis and sequence results showed the segment length of the DNA sequence which could express shRNA in mammalian cells and the right direction was identical.4.Forty-eight hours after transfected the pRNA-Hl.rTLR4shRNA intratracally mediated by jetPEI? in vivo, the rats were normal in vital signs. In BALF, compared with control group, expression of TLR4 mRNA of RNAi group showed significant down-regulation(/><0.05). the interference efficiency is 70.3%. TNF- a and IL-10 expression in BALF were significantly lower than those in control group(.P<0.05).Conclusion:1 .Using EGFP reporter genes, the special siRNA sequence which could efficiently suppress the expression of TLR4 mRNA was screened. This method proved to be rapid and high efficient.2.1n cultured AM cells, the chemically synthesized siRNA could effectively suppress the expression of TLR4 mRNA after transfected for 48 hours, and also suppress the expression of the down-stream cytokines like TNF- a and IL-10.3.Construct the pRNA-Hl.rTLR4shRNA, which contains HI promoters and could express shRNA targeting rat TLR4 in mammalian cells.4.With the new transfection agent, jetPEI? in vivo, pRNA-Hl.rTLR4shRNA could be transfected intratracheally safe and effectively. pRNA-Hl.rTLR4shRNA could down-regulate the expression of TLR4 mRNA in AM cells and TNF-a and IL-10 in BALF significantly.