The Role Of LRP16in Regulating LPS Induced TLR4Signaling Pathway And Its Mechanism

Objective: To investigate the role of LRP16in regulating LPS induced TLR4signaling pathway and its mechanism.Methods:(1). Construction the3T3-L1adipocytes with over/down-expression of LRP16a. The3T3-L1adipocytes were seeded in6cm culture dish with density of80%,24hours later, pcDNA-3.1-16and pcDNA-3.1were transfected intothese cells using lipofectamine2000according to the introduction. Another24hours later, the DMEM containing G418(800μg/ml) was used to Screeningfor resistance to3weeks. Then the cells was cultured with the DMEMcontaining G418(400μg/ml) to construct cell lines with LRP16over-expression and the control cells.b. Synthesis the oligo DNA according to the siRNA target sequence whichhave been identified for screening with hairpin structure of the LRP16gene.The Oligo DNA was annealed to form double-stranded DNA, then connectedto pENTR/U6vector to produce lentiviral vector LV-siLRP16. The connectedvector was transformed into competent cells DH5α, and Recombinant colonieswith positive identification were picked for PCR and sequencing. VectorLV-siLRP16and the Plasmid packaging system were cotransfected into293Tcell and produce virus particle. Cells which was infected by virus particlewere cultured with DMEM containing Blasticidin (4μg/ml) for three weeks.Then the cells was cultured with the DMEM containing Blasticidin (2μg/ml)to conruct cell lines with LRP16over-expression and the control cells.(2). The effect of LRP16on LPS/TLR4signal pathway of3T3-L1adipocytea. Collection of different concentrations of LPS stimulation after4,8,12,24h,cell protein, and the expression of LRP16protein was detected by Western Blot.b. The3T3-L1-374adipocytes with down-expression of LRP16and theconrtol cell3T3-L1-GFPi were incubated with1μg/ml LPS medium for1min,the mRNA expressions of TLR4, MyD88, IRAK-1and TRAF6weredetermined by RT-PCR; expression levels of the receptors protein weredetected by Western Blot.c. Expression levels of the above receptors protein without LPS stimulatedwere detected by Western Blot.d. The nuclear protein expression of NF-κB was detected by Western blot.e. The concentration of TNF-α and IL-6were measured by ELISA kits.(3). Effect of LRP16gene on MAPK signal pathway by LPS-inducedCollect the protein by stimulated of LPS (1μg/ml) for60min, the proteinexpression of ERK1/2, p38and JNK in over/down-expression of LRP16cellswere detected by Western blot.(4). Effect of LRP16gene on proliferation induced by LPS in3T3-L1preadipocytea. MTT.b. Western blot was used to detect the expression of Akt protein in over/down-expression of LRP16cells.Results:(1). Construction the3T3-L1cell lines with over/down-expression of LRP16a. Lipidosome transfection was used to construct the cell lines withover-expression of LRP16. The stable transfected cells were selected withG418. Western blot indicated that LRP16protein in3T3-L1-16adipocytes was2.37times of that of3T3-L1-3.1adipocytes.b. Lentivirus mediated siRNA technology was used to construct the cell lineswith reduced-expression of LRP16. The stable transfected cells were selectedwith blasticidin for three weeks. Western blot indicated that LRP16protein in3T3-L1-374adipocytes was reduced to40.6%of that of3T3-L1-GFPi adipocytes.(2).The effect of LRP16on LPS/TLR4signal pathway of3T3-L1adipocytea. LPS can affect the LRP16the protein level: the LRP16protein expressionafter LPS stimulation4h did not change significantly,8h and12h increased in adose-dependent,24h decreased in a dose-dependent.b. RT-PCR indicated that TLR4,MyD88,IRAK-1and TRAF6mRNA in3T3-L1-374adipocytes was reduced to58%,62.4%,60%and56%of that of3T3-L1-GFPi adipocytes respectively; Western Blot indicated that TLR4,MyD88protein in3T3-L1-374adipocytes was reduced to58.1%and60%ofthat in3T3-L1-GFPi adipocyte respectively; IRAK-1and TRAF6proteinexpression was decreased to50%and42%respectively, significantly lowerthan control cells.c. Western Blot indicated that there is no change on above receptors between3T3-L1-374and3T3-L1-GFPi cells without LPS stimulated.d. The nuclear protein expression of NF-κB in3T3-L1-374adipocytes werereduced to61%of that in3T3-L1-GFPi adipocytes (P<0.05).e. The concentrations of TNF-α and IL-6in the supernatants of cultured3T3-L1-374adipocytes was reduced to65%and68%of that in3T3-L1-GFPiadipocytes.(3). Effect of LRP16gene on MAPK signal pathway by LPS-inducedWestern Blot indicated that the phosphorylation of ERK1/2, p38and JNK in3T3-L1-16preadipocytes were significantly increased; meanwhile,phosphorylation of ERK1/2, p38and JNK in3T3-L1-374adipocytes weresignificantly reduced.(4).Effect of LRP16gene on proliferation induced by LPS in3T3-L1preadipocytea. The MTT results demonstrated that there was a remarkable decrease of cellviability in the3T3-L1-16adipocytes;b. Western Blot indicated that serine-473phosphorylation of Akt and in3T3-L1-16preadipocytes were significantly reduced; meanwhile, serine-473 phosphorylation of Akt in3T3-L1-374preadipocytes were significantlyincrease.Conclusions:(1). We successfully constructed the cell lines with over-expression of LRP16,down-expression of LRP16and control cell lines, which provided cell modelfor the follow up experiment.(2). Inhibition of LRP16gene can block the activation of the LPS/TLR4signalingpathways, down-regulating TLR4, MyD88, IRAK-1and TRAF6expression,which may be one of the negative regulatory mechanisms of the TLR4signaling pathway; and LRP16may by down-regulating TLR4signalingpathway, inhibition of NF-κB pathway activity, and reduce the pathway downstream inflammatory factors TNF-alpha and IL-6release.(3). The LRP16can up-regulate the activity of LPS-induced mitogen-activatedprotein kinase (MAPK) activation.(4). The LRP16inhibition of cell proliferation of3T3-L1preadipocytes may berelated to the inhibition of PI3-K/Akt signaling pathway.