Study On The Role And Mechanism Of Toll-like Receptor 2 In Sepsis

Objective:By establishing the sepsis model of the wild-type infant mice,the wild-type adult mice,TLR2 gene deficient(TLR2-/-)infant mice and TLR2 gene deficient(TLR2-/-)adult mice,to test the levels of IL-6 and TNF-?in every group mice,the expression of TLR2 of the wild-type infant mice and the wild-type adult mice,and phosphorylation of TLR2 signaling pathway P38MAPK?ERK1/2 and P65 in macrophages of wild-type infants and wild-type adult mice,and then detecting the maturation of phagosome in peritoneal macrophages of the wild-type infant mice,TLR2 gene deficient(TLR2-/-)infant mice and the wild-type adult mice which are normal,to analyze the role and possible mechanism of TLR2 in mice with sepsis which may provide new ideas and experimental basis for clinical treatment of neonatal sepsis.Methods:1.The model of sepsis was established by intraperitoneal injection of Gram-positive bacterial Staphylococcus aureus(SA).The mice were divided into four groups:wild-type infant mice,TLR2 gene deficient(TLR2-/-)infant mice,wild-type adult mice and TLR2gene deficient(TLR2-/-)adult mice.Each mouse was injected intraperitoneally with 1.25×10~6 CFU/g of Gram-positive bacteria,and the concentration of the Gram-positive Staphylococcus aureus(SA)strain was set to 5×10~6 CFU/ml,and the amount of liquid injected with Gram-positive bacteria Staphylococcus aureus(SA)per mouse was0.025ml/g;2.After the establishment of the mouse sepsis model,mice were sacrificed by cervical dislocation,and the macrophages were isolated and purified by peritoneal lavage;3.The levels of IL-6 and TNF-?in macrophages of four groups mice were measured by enzyme-linked immunosorbent assay(ELISA kit).The absorbance(OD)was measured by microplate reader and IL-6 and TNF-?concentration;4.The expression of TLR2 on the surface of macrophages in wild-type infants and wild-type adult mice was detected by flow cytometry;5.The activation of TLR2 signal transduction pathway P38MAPK,ERK1/2 and P65in the peritoneal macrophages of wild-type infant and wild-type adult mice was detected by Western Blot at 0,15,30,60,120 min;6.The maturation of peritoneal macrophages phagosome(which means the PH value of the phagosome)in the normal wild-type infants mice,TLR2-/-infant mice and wild-type adult mice was detected.Results:1.Compared with the wild-type adult mice group,the levels of IL-6 and TNF-?in the peritoneal macrophages of the wild-type infant mice group were significantly decreased,and the difference was statistically significant(P<0.05);Compared with the wild-type infant mice,the production of cytokines IL-6 and TNF-?of TLR2-/-infants mice was not significantly different(P>0.05);Compared with the wild-type adult mice group,the production of cytokines IL-6 and TNF-?in TLR2-/-adult mice was significantly lower,and the difference was statistically significant(P<0.05).2.TLR2 was expressed highly on the surface of macrophages both in wild-type infants mice and wild-type adult mice by flow cytometry,and the expression of TLR2 on the macrophages in wild-type adult mice was higher than that in wild-type infants,but there was no significant difference between the two groups(P>0.05).3.The TLR2 signal transduction molecules P38MAPK?ERK1/2 and P65 were expressed both in wild-type infants and wild-type adult mice.Compared with the wild-type adult mice,the phosphorylation of P38MAPK?ERK1/2 and P65 in the wild-type infant mice was significantly lower at 0,15,30,60,120 min(P<0.05).4.Compared with the wild-type adult mice group,the decline of the PH value of the macrophage phagosome in the wild-type infant mice was delayed and the acidification was insufficient;Compared with the wild-type infant mice group,the acidification of macrophage phagosome in the TLR2-/-infants mice group was further impaired.Conclusion:1.TLR2 plays an important role in regulating macrophage secretion of inflammatory factors;2.The TLR2-mediated signal transduction pathway in infant mice is not mature,causing the production of proinflammatory response factors decreased;3.The maturation of peritoneal macrophages phagosome in infant mice is insufficient in the resistance to Gram-positive bacterial infection;4.TLR2 plays an important role in regulating the maturation of macrophage phagosome in infant mice against Gram-positive bacterial infection.