Effects Of Intravenous Infusion Of Bilirubin On The Expression Of IRAK-4and P-JNK In Neonatal Rats’ Splenocytes

Abstract:Objective:By establishing the hyperbilirubinemia animal model, we aimed to investigate the effects of bilirubin on IRAK-4and p-JNK in splenocytes. Methods:1. Grouping:144seven-day-old Sprague Dawley rats were randomly assigned to6groups (n=24). There were blank control group (I), lipopolysaccharide control group (Ⅱ),15mg/kg bilirubin control (free-LPS) group (Ⅲ),15mg/kg group (Ⅳa),30mg/kg group (IVb) and50mg/kg group (Ⅳc), and then subsequently divided into2h,5h and24h subgroups (n=8) in each groups.2. Process:(1) Rats were anesthesiaed and injected at various doses of bilirubin (15mg/kg,30mg/kg and50mg/kg respectively) intravenously (i.v.) or0.1ml of saline (Ⅰand Ⅱ groups) and then sutured the incision.(2)1h after the intravenous injection of bilirubin or saline, all groups were administered LPS intraperitoneally (i.p.) at a dose of lmg/kg, except for blank control group and15mg/kg bilirubin control (free-LPS) group (saline,0.05ml, i.p.).(3) Blood samples were obtained for measurement of plasma bilirubin concentrations at2h,5h and24h following bilirubin administration.(4) IRAK-4and p-JNK were determined by immunohistochemistry. Result:1. There were varying degrees of yellowish discoloration of the skin in newborn rats from different time after bilirubin injected;30to40minutes after bilirubin injected, yellow discoloration reached the peak;1h later, the yellowish discoloration were faded. At1h,95%confidence intervals for total levels of plasma bilirubin concentration 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 the injected doses of bilirubin and total plasma bilirubin concentrations (rs=0.9452, P<0.01), the conclusion can be made that total plasma bilirubin concentrations of the model met our desired requirements at1h.2. LPS could stimulate the expression of IRAK-4and p-JNK (P<0.01), The stimulation was observed at2h, strengthened at5h, weakened and continuously existed on IRAK-4and disappeared on p-JNK at24h.3. In the low concentrations of range bilirubin could inhibit the expression of IRAK-4and p-JNK (P<0.01).On IRAK-4,the inhibition was observed at2h, strengthened at5h, disappeared at24h; on p-JNK, the inhibition was more obvious at2h, and then gradually weakened, and continuously existed at24h.4.In low-mid-high concentrations of range bilirubin could inhibit the stimulation of LPS on IRAK-4(P<0.01).It was observed at2h, strengthened at5h, disappeared in low-mid concentrations of range, while the inhibition continuously existed in high concentrations of range at24h (P<0.01).5. In mid-high concentrations of range bilirubin could promote the stimulation of LPS on p-JNK (P<0.01).It was observed at2h, strengthened at5h, weakened and continuously existed at24h (P <0.01).In the low concentrations of range bilirubin could inhibit the stimulation the of LPS on p-JNK (P<0.01), the inhibition was observed at2h, then gradually increase.6. Correlation analysis between expression of IRAK-4and p-JNK and the concentration of bilirubin:(1) There was negative correlation between the expression of ERAK-4and the concentration of bilirubin (r=-0.838、-0.845,-0.580, respectively at2h,5h and24h, P<0.01).(2) There was positive correlation between the expression of p-JNK and the concentration of bilirubin (r=0.897,0.935, respectively at2h and5h, P<0.01; r=0.466, at24h, P<0.05,).7. Correlation analysis between expression of IRAK-4and p-JNK:There was no correlation between expression of IRAK-4and p-JNK (r=-0.384, P=0.064) at24h, while negative correlation at2h and5h (r=-0.846,-0.946, P<0.01). Conclusion:l.LPS can promote expression of IRAK-4and p-JNK.2. Bilirubin in the range of low concentration can be suppressed expression of IRAK-4and p-JNK.3.In low-mid-high concentrations of range, bilirubin could inhibit the expression of IRAK-4in response to stimulation of LPS; the inhibition in a concentration-dependent manner. The inhibition of bilirubin strengthened and prolonged with increasing concentration of bilirubin.4. Med-high concentrations of bilirubin can promote the phosphorylation of JNK, with increasing concentration of bilirubin, the role in promoting strengthened and prolonged. In low concentrations of range, bilirubin could inhibit phosphorylation of JNK. Our findings implied that the immune dysfunction in neonatal hyperbilirubinemia may have something to do with the regulation of expression of IRAK-4and p-JNK in TLR4signaling pathway.