BACKGROUND
neonatal hyperbilirubinemia is a common medical condition in newborn mammalian.
about 60% term infants and 80% preterm infants develop hyperbilirubinemia within one week after birth.
neonatal hyperbilirubinemia is mainly due to the accumulation of bilirubin as a result of metabolic disturbance caused by various factors.
normally, serum bilirubin levels increase within  <dig> to  <dig> days after birth, and then begin to decline  <cit> .
however, under pathologic states, serum bilirubin, especially indirect bilirubin, can reach an exceedingly high level which leads to neurotoxicity, namely kernicterus, and eventually results in permanent neurological damage or even death  <cit> .
bilirubin is produced by the degradation of heme.
heme oxygenase  is the initial and rate-limiting enzyme in this catabolic process.
ho consists of three isozymes: ho- <dig>  ho- <dig> and ho- <dig>  heme released from degraded fetal red blood is a potent inducer of ho- <dig> in the neonatal period, which is responsible for the increase of serum bilirubin levels and the development of hyperbilirubinemia.
inhibition of ho- <dig> expression or its enzymatic activity concurs to the reduction of bilirubin levels  <cit> .
therefore, it is plausible to target ho- <dig> as a novel therapeutic rationale for the treatment of neonatal hyperbilirubinemia.
in this study, we aimed to assess the efficacy of direct inhibition of bilirubin production by suppressing ho- <dig> in a neonatal rat hyperbilirubinemia model.
small interference rna  technology utilizes short double-stranded rna to specifically inhibit the transcription of a given target gene  <cit> .
this technique has been proven to be a promising method in treating a number of diseases, such as hereditary diseases, viral hepatitis, and certain cancers  <cit> .
furthermore, rna interference as a means of treating age-related macular degeneration has entered into a clinical trial  <cit> .
these advances provide a theoretical and experimental basis to treat neonatal hyperbilirubinemia using sirna.
previously, we demonstrated that sirna specifically inhibits the expression of human ho- <dig>  in human liver cell line hl- <dig>  <cit> .
in this paper, methoxyl sirna targeting rat ho- <dig>  mrna was used to suppress ho- <dig> in neonatal hyperbilirubinemia rat model.
this study tested the feasibility of preventing and treating neonatal hyperbilirubinemia and bilirubin toxic encephalopathy by targeting ho- <dig> gene.
RESULTS
sirna transfection efficiency
to determine the transfection efficiency of sirna, a carboxy-fluorescein  labeled negative control -sirna was transfected into brl cells, and the nuclei were stained with 4',6-diamidino-2-phenylindole  after  <dig> hours.
confocal microscopy showed that  <dig> hours post-transfection, sirna entered the cells and primarily distributed in the cytoplasm .
furthermore, flow cytometry demonstrated the sirna transfection efficiency in brl cells was up to 90% .
down-regulation of rho- <dig> gene expression by sirna
quantitative gel gray scale scanning was used to compare the inhibitory effect of four pairs of sirna targeting the rho- <dig> gene.
sirna- <dig> showed the most potent inhibition on rho- <dig> mrna expression .
therefore, sirna- <dig> was selected for the rest of the experiments.
dose- and time-dependent down-regulation of rho- <dig> by sirna
brl cells were transfected with  <dig>   <dig> and  <dig> nmol/l sirna- <dig>  respectively, and collected at  <dig> hours post-transfection.
rt-pcr was performed to determine the ho- <dig> mrna level.
the expression of rho- <dig> in the brl cells transfected with  <dig>   <dig> and  <dig> nmol/l sirna were inhibited approximately 30%, 72% and 80%, respectively.
thus, the inhibitory effect of sirna- <dig> on rho- <dig> exhibited a dose-dependent relationship .
ten nmol/l sirna- <dig> was transfected into brl cells, and the rho- <dig> expression was examined at  <dig>   <dig> and  <dig> hours post-transfection as demonstrated in figure 3c and 3d.
the maximum inhibitory effect of sirna- <dig> was observed at  <dig> hours.
effect of hemin and sirna on rho- <dig> protein
hemin, an ho- <dig> inducer, was selected to induce the expression of rho- <dig>  brl cells were incubated with  <dig> ,  <dig>  and  <dig> μmol/l hemin, and collected  <dig> hours post-induction.
the level of rho- <dig> protein was determined by western blot analysis.
hemin induced rho- <dig> in a dose-dependent manner.
however, cell toxicity was observed at the higher concentrations of hemin.
thus,  <dig>  μmol/l was selected as the working concentration for rho- <dig> induction .
cells treated with  <dig>  μmol/l hemin for  <dig>   <dig> and  <dig> hours were collected to determine the level of rho- <dig> protein by western blot analysis.
eighteen hours after induction, rho- <dig> level peaked and then declined over time.
therefore, the optimized induction time of rho- <dig> by hemin was determined to be <dig> hours .
the inhibitory effect of sirna- <dig> on rho- <dig> is shown in figure 5a and 5b.
compared with the negative control, sirna inhibited the hemin-induced expression of ho- <dig> in a time-dependent manner.
the inhibitory effect of sirna reached the maximum level of 64% at  <dig> hours post-transfection.
effect of sirna- <dig> on enzymatic activity of rho-1
in order to verify our assumption that the suppression of ho- <dig> will ultimately lead to a reduction of bilirubin in vitro, homogenates from brl cells transfected with and without sirna- <dig> were used to measure bilirubin production by the rho- <dig> enzymatic assay.
the activity of rho- <dig> treated with hemin and sirna- <dig> was  <dig>  ±  <dig>  and  <dig>  ±  <dig>  nmol bilirubin mg protein- <dig> h- <dig>  respectively .
* p <  <dig> , compared with control group
distribution of fam-sirna in neonatal rat
to confirm the targeting effect of sirna in vivo, fam labeled sirna- <dig> was injected intraperitoneally into 7-day-old neonatal sprague-dawley  rats.
confocal microscopy showed the distribution of fluorescence in the tissues at  <dig>   <dig> and  <dig> hours after injection, and the sirna distributed predominantly in the liver and spleen .
dose- and time-dependent reduction of the serum bilirubin and rho- <dig> levels by sirna in a neonatal hyperbilirubinemia rat model
in light of the in vitro results, we sought to determine if sirna inhibited serum bilirubin in vivo.
seven to 9-day-old sd rats were administered intraperitoneally with δ-aminolevulinic acid   <dig> μmol/ <dig> g body weight  to induce hyperbilirubinemia.
western blot analysis showed that the level of ho- <dig> significantly increased in the liver of these rats .
this is consistent with recent reports in the literature  <cit> .
in addition, the levels of serum total bilirubin, direct bilirubin and indirect bilirubin all increased .
this suggests that we have successfully established a rat model of neonatal hyperbilirubinemia.
western blot analysis revealed that in vivo sirna- <dig> treatment reduced rho- <dig> product in the liver in a concentration-dependent manner .
the levels of total, direct and indirect bilirubin decreased considerably after sirna- <dig> injection at doses of  <dig> od/ <dig> g and  <dig> od/ <dig> g bw .
this effect was concentration-dependent.
however, the decrease of total, direct and indirect bilirubin was not significant at  <dig> od/ <dig> g bw .
in rats treated intraperitoneally with  <dig> od/ <dig> g bw of sirna- <dig> for three times, no abnormal behavior was observed.
the changes of both serum bilirubin and rho- <dig> indicate that the effective treatment time of sirna- <dig> was  <dig> hours because the inhibitory effect decreased at  <dig> hours.
the control group showed no significant change in either bilirubin levels or rho- <dig> in the liver, suggesting the inhibitory effect of sirna- <dig> is ho- <dig> specific .
effect of sirna and snpp in a neonatal hyperbilirubinemia rat model
we next compared the suppressive effect of sirna and sn-protoporphyrin  on the rat neonatal model of hyperbilirubinema.
serum bilirubin levels of rats treated by sirna- <dig> and snpp were significantly attenuated compared with the control group.
however, sirna- <dig> exhibited more inhibitory effect on indirect bilirubin than snpp .
real-time pcr also showed that ho- <dig> level in the liver significantly decreased in the rats treated with sirna- <dig> but not in those treated with snpp .
these results reveal that snpp possesses a dual role: it could up-regulate ho- <dig> expression but significantly inhibit ho- <dig> activity.
discussion
bilirubin exerts a mixture of helpful and harmful effects.
positive effects include its role as an anti-oxidant and anti-inflammatory agent  <cit> .
however, the abnormal increase of total serum bilirubin, particularly indirect bilirubin  in the neonatal period can cause devastating neurological complications.
indirect bilirubin, a lipid-soluble molecule, is taken up by hepatic cells and transformed into glucuronic acid bilirubin  in the presence of udp-glucuronyl transferase.
lipid-soluble indirect bilirubin can penetrate the cell membrane and the blood brain barrier, leading to neonatal kernicterus, whose central nervous system sequelae reflect both a predilection of bilirubin toxicity for neurons  and the regional topography of bilirubin-induced neuronal injury involving prominent basal ganglia, cochlear and oculomotor nuclei  <cit> .
currently, the treatments for neonatal hyperbilirubinnemia are traditional phototherapy, exchange transfusion and drug treatment  <cit> .
although these treatments are effective, they are limited to removing excessive bilirubin rather than preventing the production of bilirubin.
bilirubin is a product of complex heme metabolism that requires the participation of a series of key enzymes.
ho is an inducible, rate-limiting enzyme in the production of bilirubin.
ala is a heme precursor which can rapidly and consistently cause elevation of the serum bilirubin levels.
concurrent with increasing bilirubin, hepatic heme oxygenase activity increases considerably due to the increased availability of ala-derived heme  <cit> .
recently, ho- <dig> has become a novel therapeutic target for attempts to reduce bilirubin production.
sn-mesoporphyrin  and snpp, which are structural analogs of heme, competitively inhibit ho enzymatic activity and consequently decrease the synthesis of bilirubin  <cit> .
clinical trials have demonstrated that snmp effectively prevents hyperbilirubinemia caused by premature birth and hemolysis  <cit> .
however, the inhibitory mechanism of snmp on hyperbilirubinemia in vivo is uncertain  <cit> .
therefore, the safety issue concerning the application of this compound in clinics remains to be fully studied.
in this study, we investigated whether sirnas can effectively inhibit ho- <dig> expression that is up-regulated by hemin under hyperbilirubinemia conditions.
recent progress in sirna technology provides a novel means to treat many diseases including hyperbilirubinemia.
sirna shows a superior specificity and stability compared with antisense rna  <cit> .
since ho- <dig> is an inducible enzyme, targeted suppression by sirna is a promising therapeutic approach to suppress ho- <dig> both in vitro and in vivo.
we first compared four pairs of sirnas for their effects on inhibiting rho- <dig> in brl cells.
we found that sirna- <dig> was the most potent inhibitor of ho- <dig> expression and activity, and its inhibitory effect was both dose- and time-dependent.
to further test if sirna- <dig> could suppress ho- <dig> induced by endogenous heme in vivo, we established an animal model of neonatal hyperbilirubinemia according to the report of drummond et al  <cit> .
seven to 9-day-old neonatal sd rats were treated with ala intraperitoneally.
these animals subsequently developed hyperbilirubinemia that mimics human hyperbilirubinemia.
injection of sirna- <dig> significantly inhibited the rho- <dig> activity in the liver and reduced the levels of serum bilirubin in the hyperbilirubinemia rats.
snpp, as a chemical inhibitor, could also reduce the serum bilirubin levels.
however, the indirect bilirubin of sirna- <dig> treated rats decreased more significantly.
taken together, our results suggest that sirna may hold for the treatment of hyperbilirubinemia conditions in human neonates.
although sirna has been widely applied to therapeutic research on various diseases, there are still many difficulties that prevent its clinical application at the present time.
one of the major challenges is the stability of sirna in vivo.
after entering the body, unmodified sirna is quickly degraded by endogenous nuclease  <cit> .
various methods have been employed to stabilize the sirna.
for example, certain vectors are used to synthesize cationic polymer.
additionally, chemical modification of sirna can improve sirna stability and enhance its delivery efficacy as well as therapeutic specificity  <cit> .
as demonstrated in our study, the therapeutic effect of methoxy-sirna can last more than  <dig> hours after a single administration.
previous studies have shown that similar modification of sirna can enhance the affinity of sirna to target sequences, improve resistance to degradation by nuclease, and increase the interference of a target gene  <cit> .
another obstacle hindering sirna treatment progress is that sirna may not reach its therapeutic target or organ.
presently, this obstacle is overcome by local administration of sirna.
for instance, sirna targeting vascular endothelial growth factor for age-related macular degeneration is delivered through retinal injection.
in addition, sirna can treat chronic neuropathic pain through intrathecal administration  <cit> .
systemic delivery of sirna is used to target multiple organs  <cit> .
for example, sirna administrated intraperitoneally is mainly distributed in the liver, spleen and bone marrow  <cit> .
it is well documented that ho- <dig> is primarily expressed in liver and spleen.
thus, we selected the intraperitoneal injection as the administration method for this study.
we demonstrated that sirna- <dig> was mainly distributed in the liver and spleen, and both serum bilirubin levels and ho- <dig> expression were significantly reduced in a dose- and time-dependent manner after intraperitoneal injection of sirna.
the maximal inhibitory effect was observed at  <dig> hours after single injection of sirna.
these findings are consistent with other published studies  <cit> .
CONCLUSIONS
compared to the traditional phototherapy and exchange transfusion, the mechanism of sirna treatment is different from the traditional methods in view of directly inhibiting the production of bilirubin.
our study provides a novel rationale to treat neonatal hyperbilirubinemia via further development of rna interference techniques.

 neonatal hyperbilirubinemia is a common medical condition in newborn mammalian. about 60% term infants and 80% preterm infants develop hyperbilirubinemia within one week after birth. neonatal hyperbilirubinemia is mainly due to the accumulation of bilirubin as a result of metabolic disturbance caused by various factors. normally, serum bilirubin levels increase within to days after birth, and then begin to decline. however, under pathologic states, serum bilirubin, especially indirect bilirubin, can reach an exceedingly high level which leads to neurotoxicity, namely kernicterus, and eventually results in permanent neurological damage or even death. bilirubin is produced by the degradation of heme. heme oxygenase is the initial and rate-limiting enzyme in this catabolic process. ho consists of three isozymes: ho- ho- and ho- heme released from degraded fetal red blood is a potent inducer of ho- in the neonatal period, which is responsible for the increase of serum bilirubin levels and the development of hyperbilirubinemia. inhibition of ho- expression or its enzymatic activity concurs to the reduction of bilirubin levels. therefore, it is plausible to target ho- as a novel therapeutic rationale for the treatment of neonatal hyperbilirubinemia. in this study, we aimed to assess the efficacy of direct inhibition of bilirubin production by suppressing ho- in a neonatal rat hyperbilirubinemia model. small interference rna technology utilizes short double-stranded rna to specifically inhibit the transcription of a given target gene. this technique has been proven to be a promising method in treating a number of diseases, such as hereditary diseases, viral hepatitis, and certain cancers. furthermore, rna interference as a means of treating age-related macular degeneration has entered into a clinical trial. these advances provide a theoretical and experimental basis to treat neonatal hyperbilirubinemia using sirna. previously, we demonstrated that sirna specifically inhibits the expression of human ho- in human liver cell line hl-. in this paper, methoxyl sirna targeting rat ho- mrna was used to suppress ho- in neonatal hyperbilirubinemia rat model. this study tested the feasibility of preventing and treating neonatal hyperbilirubinemia and bilirubin toxic encephalopathy by targeting ho- gene.  sirna transfection efficiency to determine the transfection efficiency of sirna, a carboxy-fluorescein labeled negative control -sirna was transfected into brl cells, and the nuclei were stained with 4',6-diamidino-2-phenylindole after hours. confocal microscopy showed that hours post-transfection, sirna entered the cells and primarily distributed in the cytoplasm. furthermore, flow cytometry demonstrated the sirna transfection efficiency in brl cells was up to 90%. down-regulation of rho- gene expression by sirna quantitative gel gray scale scanning was used to compare the inhibitory effect of four pairs of sirna targeting the rho- gene. sirna- showed the most potent inhibition on rho- mrna expression. therefore, sirna- was selected for the rest of the experiments. dose- and time-dependent down-regulation of rho- by sirna brl cells were transfected with and nmol/l sirna- respectively, and collected at hours post-transfection. rt-pcr was performed to determine the ho- mrna level. the expression of rho- in the brl cells transfected with and nmol/l sirna were inhibited approximately 30%, 72% and 80%, respectively. thus, the inhibitory effect of sirna- on rho- exhibited a dose-dependent relationship. ten nmol/l sirna- was transfected into brl cells, and the rho- expression was examined at and hours post-transfection as demonstrated in figure 3c and 3d. the maximum inhibitory effect of sirna- was observed at hours. effect of hemin and sirna on rho- protein hemin, an ho- inducer, was selected to induce the expression of rho- brl cells were incubated with, and μmol/l hemin, and collected hours post-induction. the level of rho- protein was determined by western blot analysis. hemin induced rho- in a dose-dependent manner. however, cell toxicity was observed at the higher concentrations of hemin. thus, μmol/l was selected as the working concentration for rho- induction. cells treated with μmol/l hemin for and hours were collected to determine the level of rho- protein by western blot analysis. eighteen hours after induction, rho- level peaked and then declined over time. therefore, the optimized induction time of rho- by hemin was determined to be hours. the inhibitory effect of sirna- on rho- is shown in figure 5a and 5b. compared with the negative control, sirna inhibited the hemin-induced expression of ho- in a time-dependent manner. the inhibitory effect of sirna reached the maximum level of 64% at hours post-transfection. effect of sirna- on enzymatic activity of rho-1 in order to verify our assumption that the suppression of ho- will ultimately lead to a reduction of bilirubin in vitro, homogenates from brl cells transfected with and without sirna- were used to measure bilirubin production by the rho- enzymatic assay. the activity of rho- treated with hemin and sirna- was ± and ± nmol bilirubin mg protein- h- respectively. * p <, compared with control group distribution of fam-sirna in neonatal rat to confirm the targeting effect of sirna in vivo, fam labeled sirna- was injected intraperitoneally into 7-day-old neonatal sprague-dawley rats. confocal microscopy showed the distribution of fluorescence in the tissues at and hours after injection, and the sirna distributed predominantly in the liver and spleen. dose- and time-dependent reduction of the serum bilirubin and rho- levels by sirna in a neonatal hyperbilirubinemia rat model in light of the in vitro results, we sought to determine if sirna inhibited serum bilirubin in vivo. seven to 9-day-old sd rats were administered intraperitoneally with δ-aminolevulinic acid μmol/ g body weight to induce hyperbilirubinemia. western blot analysis showed that the level of ho- significantly increased in the liver of these rats. this is consistent with recent reports in the literature. in addition, the levels of serum total bilirubin, direct bilirubin and indirect bilirubin all increased. this suggests that we have successfully established a rat model of neonatal hyperbilirubinemia. western blot analysis revealed that in vivo sirna- treatment reduced rho- product in the liver in a concentration-dependent manner. the levels of total, direct and indirect bilirubin decreased considerably after sirna- injection at doses of od/ g and od/ g bw. this effect was concentration-dependent. however, the decrease of total, direct and indirect bilirubin was not significant at od/ g bw. in rats treated intraperitoneally with od/ g bw of sirna- for three times, no abnormal behavior was observed. the changes of both serum bilirubin and rho- indicate that the effective treatment time of sirna- was hours because the inhibitory effect decreased at hours. the control group showed no significant change in either bilirubin levels or rho- in the liver, suggesting the inhibitory effect of sirna- is ho- specific. effect of sirna and snpp in a neonatal hyperbilirubinemia rat model we next compared the suppressive effect of sirna and sn-protoporphyrin on the rat neonatal model of hyperbilirubinema. serum bilirubin levels of rats treated by sirna- and snpp were significantly attenuated compared with the control group. however, sirna- exhibited more inhibitory effect on indirect bilirubin than snpp. real-time pcr also showed that ho- level in the liver significantly decreased in the rats treated with sirna- but not in those treated with snpp. these results reveal that snpp possesses a dual role: it could up-regulate ho- expression but significantly inhibit ho- activity. discussion bilirubin exerts a mixture of helpful and harmful effects. positive effects include its role as an anti-oxidant and anti-inflammatory agent. however, the abnormal increase of total serum bilirubin, particularly indirect bilirubin in the neonatal period can cause devastating neurological complications. indirect bilirubin, a lipid-soluble molecule, is taken up by hepatic cells and transformed into glucuronic acid bilirubin in the presence of udp-glucuronyl transferase. lipid-soluble indirect bilirubin can penetrate the cell membrane and the blood brain barrier, leading to neonatal kernicterus, whose central nervous system sequelae reflect both a predilection of bilirubin toxicity for neurons and the regional topography of bilirubin-induced neuronal injury involving prominent basal ganglia, cochlear and oculomotor nuclei. currently, the treatments for neonatal hyperbilirubinnemia are traditional phototherapy, exchange transfusion and drug treatment. although these treatments are effective, they are limited to removing excessive bilirubin rather than preventing the production of bilirubin. bilirubin is a product of complex heme metabolism that requires the participation of a series of key enzymes. ho is an inducible, rate-limiting enzyme in the production of bilirubin. ala is a heme precursor which can rapidly and consistently cause elevation of the serum bilirubin levels. concurrent with increasing bilirubin, hepatic heme oxygenase activity increases considerably due to the increased availability of ala-derived heme. recently, ho- has become a novel therapeutic target for attempts to reduce bilirubin production. sn-mesoporphyrin and snpp, which are structural analogs of heme, competitively inhibit ho enzymatic activity and consequently decrease the synthesis of bilirubin. clinical trials have demonstrated that snmp effectively prevents hyperbilirubinemia caused by premature birth and hemolysis. however, the inhibitory mechanism of snmp on hyperbilirubinemia in vivo is uncertain. therefore, the safety issue concerning the application of this compound in clinics remains to be fully studied. in this study, we investigated whether sirnas can effectively inhibit ho- expression that is up-regulated by hemin under hyperbilirubinemia conditions. recent progress in sirna technology provides a novel means to treat many diseases including hyperbilirubinemia. sirna shows a superior specificity and stability compared with antisense rna. since ho- is an inducible enzyme, targeted suppression by sirna is a promising therapeutic approach to suppress ho- both in vitro and in vivo. we first compared four pairs of sirnas for their effects on inhibiting rho- in brl cells. we found that sirna- was the most potent inhibitor of ho- expression and activity, and its inhibitory effect was both dose- and time-dependent. to further test if sirna- could suppress ho- induced by endogenous heme in vivo, we established an animal model of neonatal hyperbilirubinemia according to the report of drummond et al. seven to 9-day-old neonatal sd rats were treated with ala intraperitoneally. these animals subsequently developed hyperbilirubinemia that mimics human hyperbilirubinemia. injection of sirna- significantly inhibited the rho- activity in the liver and reduced the levels of serum bilirubin in the hyperbilirubinemia rats. snpp, as a chemical inhibitor, could also reduce the serum bilirubin levels. however, the indirect bilirubin of sirna- treated rats decreased more significantly. taken together, our results suggest that sirna may hold for the treatment of hyperbilirubinemia conditions in human neonates. although sirna has been widely applied to therapeutic research on various diseases, there are still many difficulties that prevent its clinical application at the present time. one of the major challenges is the stability of sirna in vivo. after entering the body, unmodified sirna is quickly degraded by endogenous nuclease. various methods have been employed to stabilize the sirna. for example, certain vectors are used to synthesize cationic polymer. additionally, chemical modification of sirna can improve sirna stability and enhance its delivery efficacy as well as therapeutic specificity. as demonstrated in our study, the therapeutic effect of methoxy-sirna can last more than hours after a single administration. previous studies have shown that similar modification of sirna can enhance the affinity of sirna to target sequences, improve resistance to degradation by nuclease, and increase the interference of a target gene. another obstacle hindering sirna treatment progress is that sirna may not reach its therapeutic target or organ. presently, this obstacle is overcome by local administration of sirna. for instance, sirna targeting vascular endothelial growth factor for age-related macular degeneration is delivered through retinal injection. in addition, sirna can treat chronic neuropathic pain through intrathecal administration. systemic delivery of sirna is used to target multiple organs. for example, sirna administrated intraperitoneally is mainly distributed in the liver, spleen and bone marrow. it is well documented that ho- is primarily expressed in liver and spleen. thus, we selected the intraperitoneal injection as the administration method for this study. we demonstrated that sirna- was mainly distributed in the liver and spleen, and both serum bilirubin levels and ho- expression were significantly reduced in a dose- and time-dependent manner after intraperitoneal injection of sirna. the maximal inhibitory effect was observed at hours after single injection of sirna. these findings are consistent with other published studies.  compared to the traditional phototherapy and exchange transfusion, the mechanism of sirna treatment is different from the traditional methods in view of directly inhibiting the production of bilirubin. our study provides a novel rationale to treat neonatal hyperbilirubinemia via further development of rna interference techniques. 
systemic treatment of sirna targeting rho- reduced hepatic ho- expression and decreased the serum bilirubin levels in a time- and dose-dependent manner, and sirna decreased the indirect bilirubin levels more effectively than sn-protoporphyrin, an ho- inhibitor. the sirna distributed mostly in the liver and spleen of neonatal rat. four pairs of sirna targeting rho- mrna were introduced into brl cells and compared for their inhibitory effect on the expression of rho- gene and production of rho- protein. thus this study provides a novel therapeutic rationale for the prevention and treatment of neonatal hyperbilirubinemia. heme oxygenase is one of the rate-limiting enzymes in catabolizing heme to bilirubin. excessive accumulation of bilirubin contributes to neonatal hyperbilirubinemia in rats. serum bilirubin levels and hepatic ho- expression were further evaluated. sirna targeting rho-l attenuates hepatic ho- expression and serum bilirubin levels.
 excessive accumulation of bilirubin contributes to neonatal hyperbilirubinemia in rats. heme oxygenase is one of the rate-limiting enzymes in catabolizing heme to bilirubin. in the present study, we investigated whether suppression of rat ho- expression by small interference rnas reduces bilirubin levels in hyperbilirubinemic rats.  four pairs of sirna targeting rho- mrna were introduced into brl cells and compared for their inhibitory effect on the expression of rho- gene and production of rho- protein. the sirna exhibiting the most potent effect on ho- expression and activity was then administered intraperitoneally to to 9-day-old rats with hyperbilirubinemia. the sirna distributed mostly in the liver and spleen of neonatal rat. serum bilirubin levels and hepatic ho- expression were further evaluated. systemic treatment of sirna targeting rho- reduced hepatic ho- expression and decreased the serum bilirubin levels in a time- and dose-dependent manner, and sirna decreased the indirect bilirubin levels more effectively than sn-protoporphyrin, an ho- inhibitor.  sirna targeting rho-l attenuates hepatic ho- expression and serum bilirubin levels. thus this study provides a novel therapeutic rationale for the prevention and treatment of neonatal hyperbilirubinemia. 