Effects Of Bilirubin On The Expression Of TLR4、NF-κB、IL-12 In Monocytes In Blood Of Neonates With ABO-HDN

Objective:To explore effect of bilirubin on the expression of TLR4,NF-κB,IL-12 in monocytes in blood of neonates with ABO-HDN.Methods : The newborn infants admitted to the pediatric ward and the east ward in Provincial People’s Hospital,Longquan District Maternity and Child Hospital from September 1st,2015 to February 29 th,2016 were selected as the research objects including ABOHDN,transient tachypnea of newborn,neonatal hypoglycemia,neonatal hypocalcemia and swallowing syndrome of newborn.The newborns also should meet these criterions:37 to 42 weeks gestational age,birth weight 2500 ~ 4000 g,Apgar score is normal,clear amniotic fluid.The subjects were divided into two groups according to hemolyzed conditions : hemolytic group and non-hemolytic control group.Hemolytic group was also divided into three subgroups based on the concentrations of bilirubin : lowhemolytic subgroup(IBIL < 220.6μmol/L),mid-hemolytic subgroup(IBIL between 220.6 ~ 307.8μmol/L)and highhemolytic subgroup(IBIL≥307.8μmol/L).The newborns were been taken 2ml venous blood through the femoral vein to extract mononuclear cells when they were admitted to hospital.Lymphocytes and monocytes were differentiated and the expression of TLR4,NF-κB andIL-12 in monocytes were detected by flow cytometry.The newborns in hemolytic group were also been taken 2ml venous blood after the treatment of IVIG to differentiate lymphocytes and monocytes,and to detect the expression of TLR4,NF-κB and IL-12 in monocytes by flow cytometry.Results:1.According to the standard of include,exclude and suspend,74 qualified cases were included.Because there were only four cases in low-hemolytic subgroup,so the four cases were not been analyzed.The three subgroups have no statistically significant in gender,gestational age,birth way,birth weight and age in days.The newborns in hemolytic subgroups were comparable.2.The expression of TLR4 of the midhemolytic subgroup and the high-hemolytic subgroup were lower than that of non-hemolytic control group(P<0.01),and the expression of TLR4 of the high-hemolytic subgroup was lower than that of the mid-hemolytic subgroup(P<0.01),the expression of TLR4 of the low-hemolytic subgroup were close to that of non-hemolytic control group.3.The expression of NF-κB protein of the mid-hemolytic subgroup and the high-hemolytic subgroup were lower than that of non-hemolytic control group(P<0.01),and the expression of NF-κB protein of the high-hemolytic subgroup was lower than that of the mid-hemolytic subgroup(P < 0.01),the expression of NF-κB of the low-hemolytic subgroup were close to that of non-hemolytic control group.4.The expression of cytokine IL-12 of the mid-hemolytic subgroup and the high-hemolytic subgroup were lower than that of non-hemolytic control group(P<0.01),and the expression of IL-12 of the high-hemolytic subgroup was lower than that of the mid-hemolytic subgroup(P<0.01),the expression of IL-12 of the low-hemolytic subgroup were close to that of non-hemolytic control group.5.Correlation between the expression levels of TLR4,NF-κB,IL-12 and the concentrations of bilirubin :(1)The expression of TLR4 and the concentration of plasma bilirubin were negatively correlated(r =-0.6426,P<0.01).(2)The expression of NF-κB and the concentration of plasma bilirubin were negatively correlated(r =-0.5366,P < 0.01).(3)The expression of cytokine IL-12 and the concentration of plasma bilirubin were negatively correlated(r =-0.3859,P<0.01).6.Correlation between the expression of TLR4,NF-κB and IL-12 :(1)There was positively correlation between the expression of TLR4 and the expression of NF-κB protein(r = 0.3994,P < 0.01).(2)There was no correlation between the expression of TLR4 and the expression of cytokine IL-12(r = 0.1916,P> 0.05).(3)There was no correlation between the expression of NF-κB protein and the expression of cytokine IL-12(r = 0.2116,P> 0.05).7.The expression of TLR4,NF-κB and IL-12 in hemolytic group before and after the treatment of IVIG:(1)The expression of TLR4 in monocytes after the treatment of IVIG were higher than those before the treatment of IVIG(P<0.01).(2)The expression of NF-κB protein in monocytes after the treatment of IVIG were higher than those before the treatment of IVIG(P < 0.01).(3)The expression of cytokine IL-12 in monocytes after the treatment of IVIG were higher than those before the treatment of IVIG(P<0.01).Conclusion:1.The mid-high concentration of plasma bilirubin could inhibit the expression of TLR4 in monocytes of newborns with ABO-HDN in a concentration-dependent manner.Plasma bilirubin concentration is higher,the stronger its inhibition effect,the lower the expression of TLR4 in monocyte.2.The mid-high concentration of plasma bilirubin could inhibit the expression of NF-κB in monocytes of newborns with ABO-HDN in a concentrationdependent manner.Plasma bilirubin concentration is higher,the stronger its inhibition effect,the lower the expression of NF-κB in monocyte.3.The mid-high concentration of plasma bilirubin could inhibit the expression of IL-12 in monocytes of newborns with ABO-HDN in a concentration-dependent manner.Plasma bilirubin concentration is higher,the stronger its inhibition effect,the lower the expression of IL-12 in monocyte.4.IVIG could recover the immune function in monocyte of newborns with ABO-HDN.Our findings implied that the influence of bilirubin on neonatal immune cells might be related to the regulation of the expression of TLR4,NF-κB and IL-12 in TLRs signaling pathway,and IVIG could recover the immune function in monocyte of newborns with ABO-HDN.