Effects Of Intravenous Infusion Of Bilirubin On The Expression Of MYD88, Phospho-p38MAP Kinase And The Apoptosis In Neonatal Rats’ Splenocytes

Abstract：Objective：By establishing the hyperbilirubinemia animalmodel, we aimed to investigate the effects of bilirubin on Myeloiddifferentiation factor88(MyD88), phospho-p38MAP Kinase(p-p38MAPK) and apoptosis in splenocytes. Methods：1. Grouping:144seven-day-old Sprague Dawley rats (clean grade), male or female,weighing12.0～15.0gm, were randomly assigned to6groups(n=24).There were blank control group (Ⅰ), lipopolysaccharide control group(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 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 of saline(Ⅰand 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 plasmabilirubin concentrations at2h,5h and24h following bilirubinadministration.⑷MyD88and p-p38MAPK were determined byimmunohistochemistry; Apoptosis in splenocytes was determined by TUNEL methods. Result:1. There were varying degrees of yellowishdiscoloration of the skin in newborn rats from5to10min after bilirubininjected;1h later, the yellowish discoloration were faded; At1h,95％confidence intervals for total levels of plasma bilirubin concentration inthree 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 injecteddoses 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 MyD88in each groups:⑴In the low concentrations ofrange (106.31,123.49μmol/L), bilirubin and LPS could stimulate theexpression of MyD88respectively (P＜0.01), but the effect of bilirubinwere lower than that of LPS. The stimulation was observed at5h,disappeared at24h.⑵The low concentrations of range of bilirubin hadno significant effect on the expression of MyD88in response tostimulation of LPS (P＞0.05).⑶In mid-high concentrations of range[(196.58,238.90) and (325.15,349.07) μmol/L], bilirubin could inhibitthe stimulation of LPS (P＜0.01). The inhibition strengthened withincreasing concentration of bilirubin. It was observed at2h, strengthenedat5h, disappeared in mid concentrations of range, while the inhibitioncontinuously existed in high concentrations of range (P＜0.01) at24h.3.Expression of p-p38MAPK in each group:⑴In the low concentrationsof range, bilirubin and LPS could induced the phosphorylation ofp38MAPK respectively (P＜0.01), but the effect of bilirubin were lowerthan that of LPS (P＜0.01). It was observed at2h, strengthened at5h,continuously existed at24h.⑵In low-mid concentrations of range, bilirubin could inhibit LPS-induced p38MAPK activation (P＜0.01). Theinhibition strengthened with increasing concentration of bilirubin. It wasobserved at2h, strengthened at5h, disappeared at24h.⑶In the highconcentrations of range (325.15,349.07μmol/L), bilirubin could stimulatethe expression of p-p38MAPK (P＜0.01), observed at5h, reduced at24h.4. There was negative correlation between the expression of MyD88andthe concentration of bilirubin (r=－0.856,－0.791,－0.875, respectivelyat2h,5h and24h, P＜0.01). As the concentration of bilirubin elevated,the inhibition was enhanced; There was no correlation between theexpression of p-p38MAPK and concentration of bilirubin (r=－0.123, P＞0.05) at2h, while positively correlation at5h and24h (r=0.897,0.827,P＜0.01). It was suggested that there was a trend of increasing as theconcentration of bilirubin elevated.5. Effects of bilirubin on apoptosis insplenocytes:⑴The apoptosis index (AI) of splenocytes increased in lowconcentrations of range of bilirubin (P＜0.01).⑵Low-mid concentrationof bilirubin with LPS reduced the AI of splenocytes (P＜0.01), whilehigh concentration of bilirubin with LPS increased the AI of splenocytes(P＜0.01). Conclusion:1. In low concentrations of range, bilirubin hadno significant effects on the expression of MyD88in response tostimulation of LPS;⑶In mid-high concentrations of range, bilirubincould inhibit the stimulation of LPS in a concentration-dependent manner.The inhibition of bilirubin strengthened and prolonged with increasingconcentration of bilirubin.2. In low-mid concentrations of range,bilirubin could inhibit LPS-induced p38MAPK activation, whilestimulated activation in high concentrations of range. As theconcentration of bilirubin elevated, its inhibition was prolonged.3. Inhigh concentrations of range, bilirubin could induce apoptosis in splenocytes. Our findings implied that the immune dysfunction inneonatal hyperbilirubinemia may have something to do with theregulation of expression of MyD88and phosphorylation of p38MAPKand activation of apoptotic pathways.