Effects Of Intravenous Infusion Of Bilirubin On The Expression Of Phospho-tak1and Phospho-IKK In Neonatal Rats’ Splenocytes

Objective: By establishing the hyperbilirubinemia animalmodel, we aimed to investigate the effects of bilirubin on phospho-transforming growth factor-β activated kinase-1(TAK1), phospho-inhibitor of nuclear factor kB kinase (IKK) in splenocytes. Methods:1.Grouping:144seven-day-old Sprague Dawley rats (clean grade), male orfemale, weighing12.0～15.0gm, 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(Ⅳ a),30mg/kg group(Ⅳ b) and50mg/kg group(Ⅳ c), and thensubsequently divided into2h,5h and24h subgroups (n=8) in each groups.2. Process:⑴Rats were anesthesiaed and then the jugular vein wereexposed. Rats were injected at various doses of bilirubin (15mg/kg,30mg/kg and50mg/kg respectively) intravenously (i.v.) or0.1ml ofsaline(Ⅰ and Ⅱ groups) and then sutured the incision.⑵1h after theintravenous injection of bilirubin or saline, all groups were administeredLPS intraperitoneally (i.p.) at a dose of1mg/kg, except for blank controlgroup and15mg/kg bilirubin control (free-LPS) group (saline,0.05ml,i.p.).⑶Blood samples were obtained for measurement of plasma bilirubinconcentrations at2h,5h and24h following bilirubin administration.⑷phospho-TAK1and phospho-IKK were determined byimmunohistochemistry; Result:1. There were varying degrees ofyellowish discoloration of the skin in newborn rats from5to10min afterbilirubin injected;1h later, the yellowish discoloration were faded; At1h,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/L respectively. As there were positive correlation between theinjected doses of bilirubin and total plasma bilirubin concentrations (rs=0.9452, P＜0.01), the conclusion can be made that total plasma bilirubinconcentrations of the model met our desired requirements at1h.2.Expression of phospho-TAK1in each group at various times:⑴In thelow concentrations of range (106.31,123.49) μmol/L, bilirubin couldinhibit the expression of phospho-TAK1respectively (P＜0.01). It wasobserved at2h, reduced at5h, disappeared at24h.⑵Blirubin couldinhibit the expression of phospho-TAK1in response to stimulation ofLPS (P＜0.05). The inhibition strengthened with increasing concentrationof bilirubin. The inhibition was observed at2h, reduced at5h,disappeared in low concentrations of range, while the inhibitioncontinuously existed in mid-high concentrations of range (P＜0.01) at24h.3. The correlation between the expression of phospho-TAK1orphospho-IKK and the concentration of bilirubin.⑴There was negativecorrelation between the expression of phospho-TAK1and theconcentration of bilirubin (r=－0.9067and－0.9198, respectively at2h and5h, P＜0.01). As the concentration of bilirubin elevated, theinhibition was enhanced; There was no correlation between theexpression of phospho-TAK1and concentration of bilirubin (r=0.1373，P＞0.05) at24h.⑵There was negative correlation between theexpression of phospho-IKK and the concentration of bilirubin (r=－0.8247and－0.8479，P＜0.01, respectively at2h and5h, P＜0.01). Asthe concentration of bilirubin elevated, the inhibition was enhanced;There was no correlation between the expression of phospho-IKK andconcentration of bilirubin (r=0.1077，P＞0.05) at24h.4. The correlation between the expression of phospho-TAK1and phospho-IKK. There waspositive correlation between the expression of phospho-TAK1andphospho-IKK.(r=0.8739,0.9014and0.8487，P＜0.01, respectively at2h,5h and24h, P＜0.01). Conclusion:1. Intravenous infusion ofbilirubin to neonatal rats could make the hyperbilirubinemia model thatmet our desired requirements.2. In low concentrations of range, bilirubinalready inhibited the expression of phospho-TAK1and phospho-IKK inresponse to stimulation of LPS. The higher the concentration was，thestronger of the inhibition was, and the longer it would last; Our findingsimplied that the immune dysfunction in neonatal hyperbilirubinemia mayhave something to do with the regulation of expression ofphosphorylation of TAK1and phosphorylation of IKK.