Effects Of Intravenous Infusion Of Bilirubin On The Expression Of P-ERK And IκBα In Neonatal Rats’ Splenocytes

Objective：By establishing the hyperbilirubinemia animalmodel,we aimed to investigate the effects of bilirubin on p44/42mitogenactivated protein kinases （p-ERK）,Inhibitor of nuclear factor kappa-Bα（IκBα）in splenocytes. Methods：1. Grouping:144Seven-day-old SpragueDawley rats (clean grade), male or female, were randomly assigned to6groups(n=24). There were blank control group (Ⅰ), lipopolysaccharide controlgroup (LPS, Ⅱ),15mg/kg bilirubin control (free-LPS) group (Ⅲ),15mg/kggroup (Ⅳa),30mg/kg group (Ⅳb) and50mg/kg group (Ⅳc), and thensubsequently divided into2h,5h and24h subgroups (n=8) in each groups.2.Process:⑴Rats were anesthesia and then the jugular vein were exposed. Ratswere injected at various doses of bilirubin (15mg/kg,30mg/kg and50mg/kgrespectively) intravenously (i.v.) or0.1ml of saline (Ⅰand Ⅱ groups) and thensutured the incision.⑵1h after the intravenous injection of bilirubin or saline,all groups were administered LPS intraperitoneally (i.p.) at a dose of1mg/kg,except for blank control group and15mg/kg bilirubin control (free-LPS) group(saline,0.05ml,i.p.).⑶Blood samples were obtained for measurement ofplasma bilirubin concentrations at1h,4h and23h following bilirubinadministration.⑷The expression of p-ERK and IκBα in neonatal rats’splenocytes was determined by immunohistochemistry. Result:1.1hour afterbilirubin injection,95％confidence intervals for total levels of plasma bilirubinconcentration in three doses (15mg/kg,30mg/kg and50mg/kg) were (106.31,123.49) μmol/L,(196.58,238.90) μmol/L and (325.15,349.07) μmol/Lrespectively. As there were positive correlation between the injected doses ofbilirubin and total plasma bilirubin concentrations (rs=0.9452).2. LPS couldstimulate the expression of p-ERK and degradation of IκBα（P＜0.01）, the stimulation of p-ERK was more obvious at2h,and then gradually weakened,and vanished at24h,the inhibition of IκBα was more obvious at2h,and thengradually weakened， and continuously existed at24h.3.In the lowconcentrations of range bilirubin could stimulate the expression of p-ERK andinhibit the expression of IκBα. the stimulation of p-ERK was more obvious at2h,As the metabolism of bilirubin in vivo, simulative effect was abating, untilthe effects vanished at24h,the inhibition of IκBα was more obvious at2h，andthen gradually weakened, and abating at24h. but the effect of bilirubin werelower than that of LPS(P＜0.01).4.Different doses of bilirubin group effects onthe expression of p-ERK in response to stimulation of LPS. In theconcentrations of range （106.31，349.07）μmol/L,bilirubin could promote thestimulation of LPS on p-ERK，As the concentration of bilirubin elevated, thesynergistic effect was enhanced. With the time prolonged,weakened andvanished at24h.5.Different doses of bilirubin group effects on the expression ofIκBα in response to stimulation of LPS.In the concentrations of range（106.31,349.07）μmol/L,bilirubin could inhibit the expression of IκBα,theinhibition of IκBα was more obvious at2h,and then gradually weakened, andabating at24h. As the concentration of bilirubin elevated, the synergistic effectwas enhanced. With the time prolonged， the effect was weakened andcontinuously existed at24h.6. Correlation analysis between expression ofp-ERK and IκBα and the concentration of bilirubin：⑴There was positivecorrelation between the expression of p-ERK and the concentration of bilirubinat2h,5h.（rs=0.9428、0.9945respectively, P＜0.01）. There was nocorrelation between the expression of p-ERK and the concentration of bilirubinat24h(-0.1809,P＞0.05）,⑵There was negative correlation between theexpression of IκBα and the concentration of bilirubin at2h,5h.（rs=-0.9391、-0.969respectively,P＜0.01）. There was no correlation between the expression of IκBα and the concentration of bilirubin at24h（r=-0.098,P＞0.05）.7. Correlation analysis between expression of p-ERK and IκBα Therewas negative correlation between the expression of IκBα and the concentrationof bilirubin at2h,5h （r=-0.9763-0.9687respectively,P＜0.01）. There wasno correlation between the expression of IκBα and the concentration ofbilirubin at24h(r=-0.185,P＞0.05）.Conclusion:1.LPS can promote expression of p-ERK and degradation ofIκBα.2. Bilirubin in the range of low concentration can promote expression ofp-ERK and inhibit expression of IκBα.3. In the concentrations of range（106.31,349.07）μmol/L, bilirubin could promote the phosphorylation of ERKin response to stimulation of LPS,with increasing concentration of bilirubin,the role in promoting strengthened4.In the concentrations of range（106.31,349.07）μmol/L, bilirubin could inhibit expression of IκBα inresponse to stimulation of LPS,with increasing concentration of bilirubin, therole in a concentration-dependent manner. The role in promoting of bilirubinstrengthened with increasing concentration of bilirubin. Our findings impliedthat the immune dysfunction in neonatal hyperbilirubinemia may havesomething to do with the regulation of expression of p-ERK and IκBα in TLR4signaling pathway.