Effect Of ω-3 Polyunsaturated Fatty Acids On Parenteral Nutrition Associated Liver Disease-clinical Observation And Experimental Study

Intestinal function damage occurs when there is reduced intestinal digestion,absorption and/or injury of mucosal barrier function,so that macronutrient and/or water and electrolyte supplements are needed to maintain health and/or growth.Parenteral nutrition(PN)can be a life-saving therapy for patients who are unable to absorb enteral nutrients.In patients with short-bowel syndrome(SBS),PN provides the main source of calories for survival and growth.Although PN is life saving,its long-term use is associated with severe adverse effects,including septic infection,metabolic imbalance and hepatobiliary dysfunction.Steatosis is a common feature in adult recipients of parenteral nutrition while cholestasis is a common feature in infants and young children.Biliary cirrhosis is a late finding in both adults and infants and worsen with increasing time on PN.Parenteral nutrition associated liver disease(PNALD)is defined as a decrease in bile flow,independent of mechanical obstruction in patients receiving prolonged PN and with no other underlying cause of liver disease.PNALD is a common complication in patients with intestinal failure and develops in 40%-60%of infants compared with 15%-40%of adults on long-term PN.The mechanisms underlying PNALD remain to be elucidated.Currently,lack of enteral feeding,extended duration of PN,recurrent sepsis,and nutrient deficiency or excess may play important roles in its pathogenesis.Recent studies found that intravenous lipid emulsion(ILE)might play key role in the development of PNALD.The role of ILE in the development of PNALD is not clear,but at least some factors may contribute to the problem.First,ILE is an important source of energy and if inadvertently delivered in excess,it may increase the burden of the liver.Secondly,standard soybean oil-based ILE,which predominantly contains omega-6 polyunsaturated fatty acids(ω-6 PUFAs),has been shown to impair biliary secretion,generate a proinflammatory response,and impair immune function.Furthermore,phytosterols,present as contaminants in soy-based lipid emulsions,may be an important factor in the pathogenesis of PNALD.The switch from a soybean oil-based ILE to a fish oil based ILE in PN-dependent children with cholestasis was associated with a dramatic improvement in serum liver function tests.Bile acid(BA)-activated farnesoid X receptor(FXR)mediates hepatoprotection from excess BAs by coordinating a transcriptional reprogramming of hepatocytes:(1)reducing sinusoidal BA import via suppression of Na+taurocholate cotransporter polypeptide(NTCP);(2)reducing BA synthesis via repression of cholesterol 7 alpha-hydroxylase(CYP7A1);and(3)increasing intrahepatic BA efflux across canalicular and sinusoidal membranes via up-regulation of bile salt export pump(BSEP)and organic solute transporter(OST)α/β genes,respectively.Recent studies rarely mention effect of ω-3 polyunsaturated fatty acids on adult patients with PNALD and effect of ILE on metabolism of bile acids.Besides,studies on mechanisms and treatment of PNALD with FO and the relationship between high dose of lipids and bile acid transport protein were less discussed.The main purpose of this paper is to assess the effect of ω-3 polyunsaturated fatty acids on adult patients with PNALD,to observe the effect of high dose of lipids on mice liver and establish a mice model of PNALD.Besides,we observed the critical protein and receptors in BA metabolism,and we also observed the effects of fish oil on these molecules in PNALD model to study the mechanisms by which parenteral fish oil ameliorated liver function.Section Ⅰ:Effect of omega-3 polyunsaturated fatty acids on parenteral nutrition associated liver disease in adultsStudy 1.1 Effect of omega-3 polyunsaturated fatty acids to reverse biopsy-proven parenteral nutrition associated liver disease in adultsObjective To investigate the effects of n-3PUFAs supplemented intravenous lipid emulsion(ILE)on treatment of PNALD in adults.Methods We performed an open-labeled study of a n-3PUFAs-based ILE in 30 adults with SBS who developed cholestasis(serum direct bilirubin≧2mg/dL)while receiving soybean oil-based ILE.All patients received a formula with NPC/N 125:1 andlipid/sugar 1:1.Lipid consisted of soybean oil(Lipofundin 20%,B.Braun,Germany)plus a novel fish oil supplement(Omegaven 10%;Fresenius,Germany).The u-6 PUFAs content of PN was partiallyreplaced by u-3 PUFAs up to 10 g daily,about 0.15e0.2 g/kg body weight.Thus,the u-3/u-6 PUFAs ratio was about 1:4 in our research group.Liver biopsy were performed before and one month after the initiation of n-3PUFAs to confirm the presence and improvement of cholestasis.Blood samples were collected every week for analysis of fatty acid composition and liver function.Results Direct bilirubin normalized within 4 weeks and total bilirubin markedly decreased over time in our study.There were ameliorations of functional hepatic indexes and fatty acids pattern in all adults patients and serial liver biopsy specimens showed progressive histologic improvement.Conclusions Parenteral n-3PUFAs based fat emulsions are safe and effective in the treatment of PNALD in adults.Study 1.2 Effect of omega-3 polyunsaturated fatty acids to prevent parenteral nutrition associated liver disease in adultsObjective To investigate the effects of n-3PUFAs supplemented intravenous lipid emulsion(ILE)on prevention of PNALD in adultsMethods A total of 30 patients who need TPN for more than 30 days between September 2012 and March 2013,were randomly assigned to control group or experimental group.All patients received a formula with non-protein calories about 30kcal/(kgxday)and lipid/sugar 1:1.In the experimental group,the lipids of PN was partially replaced by fish oil up to 10 g daily,about 0.15-0.2 g/kg body weight.Blood samples were collected every week for analysis of bilirubin,liver function,and cytokine(IL-4,IL-6 and TNF-a).Results Serum bilirubin,liver enzyme and Il-6 were highly increased in control group than experimental group(p<0.05),while IL-4 and TNF-a did not have significant difference between control group and experimental group.Conclusions Parenteral fish oil supplemental fat emulsions are effective in the prvention of PNALD in adults.Section II The role of parenteral lipids in the development of hepatic dysfunction and hepatic steatosis in an adult mouse model of total parenteral nutritionObjective We examined whether different doses of parenteral lipids would affect the development of PNALD in an adult mouse model,and explore a mouse model to further investigate pathogenesis and treatment of PNALD.Methods Adults female mice with a central catheter placed in the right jugular vein were divided into three groups.Control Group received normal mouse chow with intravenous NaCl;Lipids Group received normal TPN solution;D-lipids Group received TPN solution with twice the amount of lipids emulsion in Lipids Group.Control mice received normal mouse chow,which supplied 260 to 280 kcal/kg body weight per day.To allow for a matched amount of caloric delivery,the mice in group Lipids were infused with TPN admixture with 1.3 g of nitrogen per kilogram body weight per day and 255 kcal of nonprotein calorie(NPC)per kilogram body weight daily(NPC:N ratio of 200:1).The mice in group D-lipid were infused with twice the amount of lipids in TPN Group,and TPN solution provide 390 kcal of NPC per kilogram body weight daily with the similar NPC:N ratio.Body weight was measured immediately before the start of catheterization and at the time of sacrifice body weight and liver weight were both measured.Blood samples were drawn for analysis of serum bilirubin,y-glutamyl transferase(GGT),alanine aminotransferase(ALT)and cholesterol(CHOL).After blood collection,random biopsy of liver were stained with hematoxylin/eosin(H&E)and periodic acid/Schiff(PAS)for light microscopy.Results Body weight decreased significantly in TPN groups at day 5,the differences of body weight became smaller among groups over time.The values of serum direct bilirubin,total bilirubin and cholesterol were markedly increased in D-lipids group at day 7.Lipids group had mild to moderate steatosis at day 5 or day 7(steatosis score 1.410.5 and 2.1±0.7,respectively),while mice in D-lipis group had more severe steatosis(steatosis score 2.6±0.9 and 3.1±0.5,respectively).Conclusions High doses of parenteral lipids are more likely to develop PNALD in an adult mouse model of TPN,and this model is useful to further investigate pathogenesis and treatment of PNALD for adults with PNALD.Section Ⅲ Changes of critical receptors in BA metabolism and mechanism ofω-3PUFAs on BA metabolism in mice model with parenteral nutrition associated liver diseaseStudy 3.1 Changes of critical protein and receptors in BA metabolism on mice with parenteral nutrition associated liver diseaseObjective We observed the critical protein and receptors in BA metabolism in mice model with parenteral nutrition associated liver diseaseMethods Adults female mice with a central catheter placed in the right jugular vein were divided into control group and experimental group.Control Group received normal mouse chow with intravenous normal saline;experimental group were fasted and infused with TPN admixture with 390 kcal of nonprotein calorie(NPC)per kilogram body weight daily(NPC:N ratio of 200:1).At day 5,7 and 10 after infusion with TPN,the expression of bile salt export pump(BSEP)and organic solute transporter(OSTα/β)in liver were analyzed by Western-blot.Immunohistochemistry was performed to observe the expression of BSEP and OSTα/β in liver.Results Compared with control group,WB results showed the expression of BSEP and OSTα/β in experimental group were significantly lower than control group(p<0.05),and the decrease was more obvious with increasing time.Immunohistochemical results also showed the similar results that the expression of BSEP and OSTα/β in control group were significantly higher than experimental group.Conclusions The expression of BSEP and OSTα/β were significantly lower in mice with intravenous lipid emulsion(ILE),and the decrease was more obvious with increasing time and increasing dose of lipids.The suppression of BSEP and OSTα/β in liver might paly a role in the pathogenesis of parenteral nutrition-associated liver disease induced by high dose of ILE.Study 3.2 Therapeutic effect and mechanism of ω-3PUFAs on BA metabolism in mice model with parenteral nutrition associated liver diseaseObjective We observed the therapeutic effect and mechanism of ω-3PUFAs on BA metabolism in mice model with parenteral nutrition associated liver diseaseMethods Adults female mice with a central catheter placed in the right jugular vein were divided into control group and experimental group.The experiment lasted 2 weeks.ControlGroup received normal mouse chow with intravenous normal saline for 2 weeks.Experimental group were fasted and infused with TPN admixture with 390 kcal of nonprotein calorie(NPC)per kilogram body weight daily(NPC:N ratio of 200:1)during the first week of the experiment.All mice in experiment group were then randomly divided into three groups for a second week infusion of TPN according to different dose or source of lipids:Group D-lipids,Group Lipids and Group FO.Lipids in Group D-lipids were the same as the TPN during the first week.Lipids were infused with TPN admixture with a lipids dose of 30g/kg/d from co-6PUFAs,the lipids dose in Group Lipids was 15g/kg/d from co-6PUFAs and the lipids dose in Group FO was 15g/kg/d from co-3PUFAs.Two weeks later blood samples were drawn for analysis of serum bilirubin and liver functions.After blood collection,random biopsy of liver were stained with hematoxylin/eosin(H&E)for light microscopy.The expression of bile salt export pump(BSEP)and organic solute transporter(OSTa/β)in liver were analyzed by Western-blot.Immunohistochemistry was performed to observe the expression of BSEP and OSTa/β in liver.Results The values of serum direct bilirubin,total bilirubin and cholesterol in Group D-lipids and Group Lipids were markedly increased than values in control group(P<0.05),while values of serum direct bilirubin,total bilirubin and cholesterol in Group FO were markedly decrease than values in D-lipids group(P<0.05).Mice in D-lipis group had diffuse involvement of hepatocytes by macrovesicular and microvesicular steatosis,mice in Lipids group had mild steatosis which were most prominent within hepatocytes around the portal and central vein regions,while mice in control and FO group appeared grossly normal and had normal liver morphology with no steatosis.The steatosis score in D-lipids and Lipis group were much higher than other two groups(P<0.05).Compared with control and FO groups,WB results showed the expression of BSEP and OSTa/β in D-lipids group were significantly lower(p<0.05),while there was no significant difference between control and FO groups.Immunohistochemistry confirmed RT-PCR and Western-blot findings.Conclusions Intravenous supplementation with fish oil release the suppression of BSEP and OSTa/β in liver induced by high dose of intravenous lipid emulsions.Therefore,it decreased levels of serum bilirubin and liver enzymes significantly and reduce hepatic steatosis.This may be one of the mechanisms of its therapeutic effect on PNALD...