The Application Of SiRNA In The Treatment Of Neonatal Rat Jaundice By Inhibiting The Expression Of HO-1 Gene

Neonatal jaundice caused by increased bilirubin load and decreased bilirubin excretion is a common disease in the newborn. About two thirds of newborn might suffer from neonatal jaundice. Bilirulin can do harm to the nerve system. Serious jaundice may develop to bilirubin toxic encephalopathy, which may lead to children dysnoesia and even death therefore resulted in high lethality and disability.HO is a rate-limiting enzyme in the metabolic pathway from hemin to bilirubin, among which HO-1 can be induced by many factors. In our study, RNAi (RNA interference) was used to reduce the expression of HO-1 in BRL cell to prove that bilirubin production could be disrupted by inhibiting HO-1 expression. Next, the most effective siRNA was screned and administered to rats with neonatal jaundice to examine its effect. Our ultimate aim is to find out a possible new strategy for early prevention and treatment of neonatal hyperbilirubinemia and bilirubin toxic encephalopathy.Four pairs of siRNAs were designed and synthesized according to the sequence of HO-1 mRNA. The efficiency of transfection in BRL cell line with INTERFERin could be up to 90%. siRNA-4 showed the most powerful inhibition effect on HO-1 mRNA about 77%, detected 24 hours after transfection with 10 nM siRNA-4. SiRNA-1 and siRNA-2 also exhibited some effect. The optimal transfection concentration was 10 nM, the optimal function time on mRNA level was 24 hours. HO-1 protein could be induced by hemin, the optimal function time and concentration were 18 hours and 3μg/mL. SiRNA-4 could also inhibit the production of HO-1 protein induced by hemin, the effect was up to 64% when detected 36 hours later.7~10-day-old SD rats were administered intraperitoneally with ALA(50μM/100 g). The HO-1 protein expression was 2 times more than that treated with saline, while the production level of TBIL was about 4 times, indicating that the model of neonatal rat jaundice was successfully established. The jaundice rats were administered intraperitoneally with siRNA-4 modified with 2'-O-methyl. The concentration of TBIL in serum could be reduced by 43% and 50% at dose of 5 OD/20 g body weight and 10 OD/20 g body weight respectively, while for the expression level of HO-1 protein in liver, the inhibition effects were 30% and 42%. At the dose of 5 OD/20 g body weight, Bilirubin was reduced by 36%,45%,27% and HO-1 protein was inhibited by 26%,33%,18% when detected 1 d,2 d,3 d after the last injection.Both in intro and in vivo experiments confirmed that siRNA-4 could effectively interfere in the expression of HO-1 to reduce the production of bilirubin, which provided a new possible strategy for early prevention and treatment of neonatal hyperbilirubinemia and bilirubin toxic encephalopathy.