Effects of intravenous infusion of bilirubin on the expression of myeloid differentiation factor 88 and interleukin-1 receptor associated kinase-4 in splenocytes with neonatal rats

Abstract

Objective To explore the effects of bilirubin on myeloid differentiation factor 88(MyD88)and interleukin-1 receptor associated kinase-4(IRAK-4). Methods Seven-day-old Sprague Dawley rats (clean grade), male or female, weighing 12.0 to 15.0 g, were randomly assigned to 6 groups.There were normal saline group(Ⅰ), lipopolysaccharide(LPS) control group (LPS, Ⅱ), 15 mg/kg bilirubin control (free-LPS) group (Ⅲ), 15 mg/kg group (Ⅳa), 30 mg/kg group (Ⅳb) and 50 mg/kg group (Ⅳc), and then subsequently divided into 2 h, 5 h and 24 h subgroups in each groups.Some of the 200 newborn rats died amid the experiment.Finally a total of 144 were involved in the analysis of results, and 8 rats in each subgroups.Newborn Sprague Dawley rats were administered at various doses of bilirubin(15 mg/kg, 30 mg/kg and 50 mg/kg respectively)intravenously; 1 h after injection, the rats were administered LPS intraperitoneally at a dose of 1 mg/kg; MyD88 and IRAK-4 were detected by immunohistochemistry at 2 h, 5 h and 24 h after the injection of bilirubin. Results (1) LPS could stimulate the expression of MyD88 and IRAK-4 in spleen cells (qMyD88 2 h=49.89, qMyD88 5 h=139.54, qIRAK-4 2 h=7.93, qIRAK-4 5 h=24.30, qIRAK-4 24 h=6.97, P 0.05). Effects of medium and high concentration of bilirubin on LPS stimulation MyD88 were inhibitory(qⅣb 2 h=42.87, qⅣc 2 h=51.38, qⅣb 5 h=103.61, qⅣc 5 h=115.44, qⅣb 24 h=1.18, qⅣc 24 h=11.66, P<0.01). (4) Effects of low, medium and high concentration of bilirubin on LPS stimulation IRAK-4 were inhibitory(qⅣa 2 h=9.52, qⅣb 2 h=14.39, qⅣc 2 h=25.55, qⅣa 5 h=38.83, qⅣb 5 h=54.62, qⅣc 5 h=60.51, qⅣa 24 h=2.41, qⅣb 24 h=1.47, qⅣc 24 h=7.61, P<0.01). (5) The inhibition of bilirubin to MyD88 and IRAK-4 was observed at 2 h, strengthened at 5 h, disappeared at 24 h in low-mid concentrations of bilirubin(P<0.01)while still visible at 24 h in high concentration of bilirubin.(6) There was negatively correlation between the expression level of MyD88, IRAK-4 and bilirubin concentration(rsMyD88 2 h =-0.86, rsMyD88 5 h=-0.92, rsMyD88 24 h=-0.53, rsIRAK-4 2 h=-0.82, rsIRAK-4 5 h=-0.86, rsIRAK-4 24 h=-0.57, P<0.01). (7) Under the effect of bilirubin and LPS, there were positively correlation between the expression levels of MyD88 and IRAK-4 of spleen cells(r2 h=0.77, r5 h=0.9, r24 h=0.67, P<0.01). Conclusion Bilirubin could inhibit the expression of MyD88 and IRAK-4.As the concentration of bilirubin increasing, its inhibition is more obvious and prolonged.The mechanism that bilirubin affects immune function of newborn rat may be related to regulation of expression of MyD88 and IRAK-4 at toll-like receptor 4 signal pathway. Key words: Bilirubin; Hyperbilirubinemia; Myeloid differentiation factor 88; Interleukin-1 receptor associated kinase-4; Rat, newborn,