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An Experiment about the Anti-Inflammatory Effects of Omega-3Fatty Acids on Human Hepatocytes and Macrophages

Author: HaoZuo
Tutor: WangYuLin; Kenneth Wong
School: Shandong University
Course: Pediatrics
Keywords: omega-3fatty acids (EPA) omega-6fatty acids (AA) PNALD TPN inflammation
CLC: R720.597
Type: PhD thesis
Year: 2012
Downloads: 479
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Abstract


Backgroud and objectivesTotal parenteral nutrition (TPN) is essential for survival of critical premature,congenital digestive tract,serious diseases of tract and pediatric patients with digestive tract operation, and it has been generally acknowledged as an effective and relatively safe method for supplying energy and nutrients to pediatric patients. TPN is one of the major advances of neonatal medicine and can be used successfully for prolonged periods in infants who cannot be fed enterally. Parenteral nutrition minimizes the harmful impact of multiple metabolic complications and helps to maintain an optimal nutrition level. However, long-term application of TPN can cause parental nutrition associated liver disease (PNALD), which was first described by Peden et al in1971. As the duration of total parenteral nutrition increases, so does the incidence of liver complications associated with parenteral nutrition. This incidence of PNALD is higher in neonates and infants, owing to physiological immaturity. In1998, Sondheimer reported that approximately40%-60%of children on long-term TPN will develop hepatic dysfunction. Also, in the same year, Sondheimer et al. performed a study of42infants on total parenteral nutrition and in the short time of six weeks, about13%of the patients progress to liver failure. The manifestation of liver complications differs in adults and in infants. In infants, intrahepatic cholestasis is the most common liver abnormality that manifests when the therapy is total parenteral nutrition. The development of parenteral nutrition-associated liver disease (PNALD) predisposes patients to an increased incidence of sepsis, higher mortality rates, and the potential to develop irreversible liver injury, and even to endanger the life, so the prevention and treatment of PNALD is critical.Thus far, the etiology of TPN-induced liver disease remains unknown, but TPN-associated cholcstasis (PNAC) is its main pathological basis, PNAC is thought to be the result under the action of various factors, such as liver immature of premature infants, early fasting, imbalance of nutrition composition of the parenteral nutrient solution, some components in lipid emulsion have hepatotoxicity, fetal age and early infection (such as sepsis, catheter-related infection, necrotizing enterocolitis), intestinal surgery and so on. PNAC is related to the immature bile excretion system of premature infants and newborn, the immature bile excretion system lead to the decrease of the uptake rate of liver and the disorder of bile acids recycling. The capacity of uptake and synthesis bile salts of liver is decreased and the disorder of enterohepatic circulation bilirubin because of the imperfect of the function of hepatobiliary transportation and metabolic disorder in premature infants.The decrease of the capacity of uptake and synthesis bile salts of liver and the disorder of bile acids recycling, the time of bile stay in the intestinal is prolonged, result in lithocholic acid formation increased under the effect of intestinal bacteria, and reabsorbed to liver, so that this produced toxicity to hepatocyte. Secondly, children patients receving TPN who can’t be tolerated feeding by oral, or feeding deficiency, feeding delay, lack of the effective gastrointestinal stimulation, which cause a decrease in some gastrointestinal hormones, such as motilin, gastrin, secretin, glucagon, porcine Vasoactive Intestinal Peptide (VIP), and then, the contractility of gall bladder is decreased which lead to PNAC. There was a research shows that the PNAC incidence decreased more significantly with caloric intake decreased. Deficiency in essential amino acids, the amount and components of amino acid which are related to the occurrence of PNAC. Hepatotoxicity of nutrient admixture may be related to the deficiency in essential amino acids such as tyrosine, cysteine, taurine and so on. Taurine is an important amino acid for premature infants, which is related to many liver enzyme activities and promote bile flow. The deficiency of taurine will cause PNAC. Premature infants receiving TPN who have been infected, subsequently, PNAC occurred. After infected by bacteria or other pathogens, the liver enzyme became abnormal, and the bile secretion reduced. Lipopolysaccharide produced by pathogens inhibits Na+-K+-ATPase activity in liver plasma membrane. Intrahepatic bile duct transport system were damaged, so to inhibit the uptake and transport of bile acids. In liver biopsy, we can find the existence of the bile stasis, portal inflammation and ductular proliferation. PNALD also involves the downregulation of genes coding for transporters responsible for the bile acid-dependent system in endotoxin-mediated models, inducing cholestasis. Independent of how cholestasis is initiated, a cascade of events in the liver will occur in response to endotoxins. First, Kupffer cells generate hepatoxic cytokines, TNF-a, IL-1and IL-6. The intestines will upregulate the production of the same cytokines but in response to the parenteral nutrition. Fibrosis then occurs. It is caused by the overproduction of collagen by the Kupffer cells of the liver because of the stagnant bile acids caused by endotoxins slowing down the bile acid flow. This reduction in enterohepatic circulation of bile salts may further contribute to the liver injury as intestinal stasis and bacterial overgrowth of the small intestine promotes intraluminal bile salt deconjugation and the increase in the production of lithocholic acid, which impairs bile flow and produces cholestasis. This cholestasis promotes gastrointestinal sepsis, follow systemic inflammatory response includes the production of cytokines that directly inhibit the hepatocellular ion transporters essential to bile generation and also initiate a fibrogenic response by hepatic sinusoidal cells, sepsis aggravate original cholestasis,which creates a vicious cycle of severe cholestasis and sepsis. Furtherly, serious PNALD occurred.Recently, some study shows that PNALD is related to the lipid material in nutrient admixture. In clinic, parenteral lipid emulsion are used including fatty acid and triglyceride, triglycerides were classified into short-chain fatty acids (2C-4C), medium-chain fatty acids (6C-12C) and long-chain fatty acids (14C-24C). We couldn’t synthesize linoleic acid, linolenic acid and arachidonic acid which called necessary fatty acids. Other fatty acids called nonessential fatty acids. Fatty acids were classified into saturated fatty acid and unsaturated fatty acid. Multiple unsaturated double bond content is called polyunsaturated fatty acids (PUFAs). Long-chain PUFAs included omega-3PUFAs, omega-6PUFAs、omega-7PUFAs and omega-9PUFAs on the basis of the position of unsaturated double bond. The metabolic product of omega-3PUFAs and omega-6PUFAs are eicosanoids, but their products are different, the former are mainly triene-acid epoxide and pentaene-acid epoxide, the later are mainly diene-acid epoxide and arachidonic-acid lipid oxide. Omega-6PUFA is a pro-inflammatory mediator, which has potent biological effects upon the immune system, may cause constriction or dilation in vascular smooth muscle cells, they may cause aggregation of platelets, and they have leukocyte chemotaxis. Although, the metabolites of omega-3PUFAs are similar to the metabolites of omega-6PUFAs on the structure, and their physiological function are similar, the physiological function of omega-3PUFAs was one hundredth of that of omega-6PUFAs. We can add appropriate content omega-3PUFAs so that to reduce adverse effect of omega-6PUFAs on the systemic inflammatory response. Eicosapentaenoic acid (EPA) is an omega-3fatty acid, which is a breakdown product of a-linolenic acid, found in fish oils and breast milk. It suppresses the production of arachidonic acid-derived eicosanoids and is also a substrate for the synthesis of an alternative family of eicosanoids, which have many anti-inflammatory effects. Furthermore, a study showed that EPA can replace arachidonic acid (AA) on membrane phospholipid and compete cyclooxygenase and lipoxidase to relieve inflammation. EPA exists in cell membrane phospholipids and it affectes the membrane fluidity and the function of related signal molecules, receptor, enzyme on the membrane to change signal transduction. Furthermore, omega-3fatty acids inhibit the secretion of por-inflammatory factors, regulating the expression of adhesion molecule by affecting the expression of enzymes and cytokines. The protective benefits of omega-3fatty acids may be related to its ability to decrease the production of prostaglandins and subsequently, the release of other inflammatory cytokines. These reductions will inevitably lead to the decrease in the magnitude of inflammation and the severity of insult to the liver. Indeed, there have been a few case series published recently suggesting the advantages of omega-3fatty acids in the formulation of parenteral nutrition in the rescue of babies with PNALD. Despite all these significant and encouraging clinical findings, the exact mechanism of action of omega-3fatty acids in preventing PNALD is still not clear and has not been fully investigated.As the change from pro-inflammatory to anti-inflammatory state has implications for the status and progression of PNALD in response to the initial cholestatic and steatotic insult, it is likely that both pro-inflammatory and anti-inflammatory cytokines could play a role. The pro-inflammatory state is mediated by macrophages and Kupffer cells in liver through the release of cytokines such as tumor necrosis factor-a (TNF-a) and interleukin-6(IL-6), and the plasma levels of TNF-a and IL-6correlate positively with the degree of underlying liver damage. On the other hand, interleukin-10(IL-10), an anti-inflammatory cytokine, has pleiotropic effects in regulating exaggerated immune response and the eventual termination of inflammation.We therefore hypothesize that omega-3fatty acid (EPA) could exert its action on cells which produce these pro-inflammatory and anti-inflammatory cytokines. In this study, we observed TNF-a and IL-6secretion pattern in human marophages and Kupffer cells in different inflammatory models, in the same time, treating with omega-3fatty acid and omega-6fatty acid in the expriment, which demonstrated omega-3fatty acid play a role in inflammation. In addition, the treatment effect of omega-3fatty acid on the level of TNF-a and IL-6in a inflammatory model of co-culture human marophages and Kupffer cells was investigated. We try to elucidate that the anti-inflammatory effects of omega-3fatty acids on human marophages and Kupffer cells in order to demonstrate the protective action of the liver against hepatic steatosis and damage.Methods1. Cell sepration and culture(1)Peripheral blood mononuclear cells (PBMC) sepration and culturePeripheral blood mononuclear cells were obtained from donated blood (Hong Kong Red Cross Blood Transfusion Service) by Ficoll Paque gradient method. Briefly, ACK buffer and Ficoll were added to blood and left in room temperature for15minutes. The samples were then diluted with phosphate buffered solution (PBS) and centrifuged at2000rpm for25minutes.Cells at the interphase (lymphocytes, monocytes, and thrombocytes) were transferred to a new conical tube filled with PBS and centrifuged at1200rpm for7minutes at room temperature. The supernatant was carefully removed completely.For removal of platelets,the cell pellet was re-suspended in PBS and centrifuged at800rpm for7minutes.The supernatant was removed and repeated twice.35ml of RPMI1640medium was added and mixed, then centrifuged at1200rpm for7minutes.The pellet was re-suspended in50ml macrophage SFM medium, supplemented with L-glutamine2ml of the solution was added to6-well plates and cultured at37℃and5%CO2. After7days of incubation, mononuclear cells would change into macrophages,which could be used in further experiments.(2)Preparation of Liver THLE-3CellsThe human liver cell line THLE-3, was purchased from the American Type Culture Collection (ATCC, USA). The cells were maintained in precoated flasks with a mixture of fibronectin (0.01mg/ml), bovine collagen type1(0.03mg/ml), and bovine serum albumin (0.01mg/ml) dissolved in BEGM medium and incubated at37℃and5%CO2.Medium was changed every2to3days.2. Experimental DesignMacrophages and THLE-3cells were used when80%confluent.1×106of macrophages or THLE-3was seeded in each well of a6-well plate which could be used in further experiments.(1) LPS-induced and PGE2-induced IL-6and TNF-a expression in macrophages and THLE-3cell lineMacrophages and THLE-3cells were stimulated with LPS (0.lug/ml) and PGE2(0.1μg/ml),respectively, and continue to incubate for8h,16h,24h,32h. Then the supernatant was collected at different time points for the measurement of TNF-a and IL-6concentrations using enzyme-linked immunosorbent assay (ELISA).(2) LPS-induced and PGE2-induced IL-6and TNF-a expression in pretreated macrophages and THLE-3cells with EPA, AA and EPA+AA100μM EPA,100μM AA or100μM EPA+100μM AA (ratio,1:1) was added to the medium of macrophages and THLE-3cells, and were incubated for24hours. Then, the cells were washed twice with PBS. All groups were then stimulated with LPS (0.1μg/ml) or PGE2(0.1μg/ml) to induce an in-vitro inflammatory condition respectively.After24h,the supernatants of each group were collected and for the measurement of TNF-a and IL-6concentrations using enzyme-linked immunosorbent assay (ELISA).(3) Co-incubation of EPA, AA or EPA+AA with LPS or PGE2in macrophages and THLE-3cellsSimultaneously, macrophages and THLE-3cells were treated with100μM EPA,100μM AA or100μM EPA+100μM AA (ratio,1:1) and LPS (0.1μg/ml) or PGE2(0.1μg/ml) for24h. Then, the supernatants of each group were collected and for the measurement of TNF-a and IL-6concentrations using enzyme-linked immunosorbent assay (ELISA).(4) Post-incubation with EPA, AA or EPA+AA,24h after stimulation with LPS or PGE2in macrophages and THLE-3cellsMacrophages and THLE-3cells were stimulated with LPS (0.1μg/ml) or PGE2(0.1μg/ml) for24h, then, the cells were washed twice with PBS. All groups were then treated with100μM EPA,100μM AA or100μM EPA+100μM AA (ratio,1:1), respectively. After24h, the supernatants of each group was collected and for the measurement of TNF-a and IL-6concentrations using ELISA. (5) IL-10expression in EPA-treated macrophages with pre-stimulated with LPSMacrophages were stimulated with LPS (0.1μg/ml) for24h, then, the cells were washed twice with PBS.100μM EPA was added into each well, and continue to incubate for8h,16h,24h,32h. Then the supernatant was collected at different time points for the measurement of IL-10concentrations using ELISA.(6) Co-culture of EPA-treated macrophages with pre-stimulated THLE-3Firstly, macrophages were stimulated with LPS (0.1ug/ml) for24h, after washing twice with PBS,100μM EPA was added into the medium; and then, THLE-3cells were stimulated with LPS (0.1μg/ml) for24h; finally, we co-cultured EPA-treated, LPS pre-stimulated macrophages and LPS pre-stimulated THLE-3for24h.The supernatant was collected for the measurement of TNF-α and IL-6concentrations using ELISA.3. StatisticsAll values are measured as means±SD in experiments. ANOVA and the Student’s t-test were used for statistical analysis. Differences were considered significant at P<0.05(*) or P<0.01(**).Results1. The reaction was more sensitive under LPS stimulation than under PGE2stimulation in macrophages. In the presence of0.1μg/ml LPS and0.1μg/ml PGE2stimulation, there was an increase in TNF-α and IL-6production in macrophages when compared to the medium alone group, even to nearly ten fold above the baseline, peaking at24h time point. The same stimulation acting on THLE-3cells, a liver cell line. Here, although the variation trend of IL-6and TNF-α production were similar to those seen in macrophages, the overall response of THLE-3was much lower. We used LPS and PGE2to stimulate human peripheral blood mononuclear cells and human liver cell line (THLE-3) to induce an in-vitro inflammatory condition successfully. The level of response of different cell lines was different to different stimulation.We observed the reaction was most strongest at24-h time point, so in the following experiments, we made24-h time point as the investigative time point.2. Before stimulation, macrophages and THLE-3cells were pre-treated with 100μM EPA,100μM AA,100μM EPA+AA (ratio,1:1) for24h, then were stimulated with0.1μg/ml LPS or0.1μg/ml PGE2for24h, we obersved the level of TNF-a and IL-6decreased significantly in EPA treatment group(p<0.05)compared with control. In AA treatment group and EPA+AA (ratio,1:1) treatment group, we found there was varying decline of the concentration of TNF-a and IL-6.However, it’s descensive extent was lower than EPA treatment group(p<0.05).3. When macrophages and THLE-3cells were given stimulating factor LPS or PGE2and five different treatment measures simultaneously. We observed that the level of TNF-a and IL-6was decreased in all groups, however, EPA treatment group was most significant.Compare the two different time point (pre-incubation or co-incubation), it was obvious that pre-incubation was more better than co-incubation.4. After stimulated with LPS or PGE2for24h, then macrophages and THLE-3cells were treated with100μM EPA,100μM AA,100μM EPA+AA (ratio,1:1) for24h. We were surprised to find that there was a most significant decline of TNF-a and IL-6in post-incubation groups. In addition, EPA treatment group was the best than the other four treatment measures (p<0.01).5. Since EPA could effectively suppress the production of pro-inflammatory cytokines, we next asked if it could also alter the production of an anti-inflammatory cytokine, IL-10. Here, a time chase experiment was carried out after EPA had been added to LPS-stimulated macrophages(8h,16h,24h,32h). IL-10secretion was found to be significantly increased after treatment with EPA. At24-h time point, its level reached the peak at280.32±11.86pg/ml. Interestingly, at the same time point, the levels of IL-6(110.72±12.23pg/ml) and TNF-a (170.75±15.76pg/ml) were at the lowest correspondingly.6. The above data showed that EPA could significantly decrease the production of IL-6and TNF-a when macrophages were stimulated by LPS,with a corresponding increase in IL-10secretion. So we next asked whether this effect could be mediated indirectly through macrophages on liver cells.We co-cultured EPA-treated, LPS pre-stimulated macrophages with LPS pre-stimulated THLE-3cells for24h.Results showed that when compared with untreated group, the production of IL-6and TNF-a decreased by75.9%and by85.2%in the EPA-treated group.Conclusions1. We used LPS and PGE2for stimulation to mimic an in-vitro inflammatory condition in liver. Macrophages and THLE-3cells were stimulated to secrete massive pro-inflammatory cytokines TNF-a and IL-6. From the experiment data, we can draw a conclusion that the level of response of different cell lines to different stimulation was various.2. EPA, AA and the mixture of EPA and AA (ratio,1:1) could control inflammation of macrophages and THLE-3cells stimulated by LPS and PGE2. But, the group of EPA was added alone was best among all groups.3. Our data indicate that different treatment time had different therapentic efficacy. Among three treatment time, the effect was found to be most dramatic in the post-incubation group.4. EPA could effectively suppress the production of pro-inflammatory cytokines, and could significantly increase anti-inflammatory cytokine IL-10.In addition, at24-h time point, the two effectes were most effective.5. We co-cultured EPA-treated,LPS pre-stimulated macrophages and LPS pre-stimulated THLE-3,and detected the level of TNF-a and IL-6. We concluded that EPA not only had anti-inflammatory effect on macrophages and hepatocytes directly, but could indirectly reduce inflammations in hepatocytes through activated macrophages.Innovation and significance1. The study firstly used stimulated-human peripheral blood mononuclear cells to make a comparative study on the anti-inflammatory effect of omega-3fatty acids in different treatment time successfully. In addition, in the experiment we imitate real circumstances in liver, a mass of Kupffer cells and liver cells gathered in liver. So, the experiment results have relatively strong theory persuasiveness.2. The study descrided that EPA (omega-3fatty acids) at100μM concentrations effectively reduced LPS-induced and PGE2-induced TNF-a and IL-6expression in macrophages and THLE-3cells.In addition,AA (omega-6fatty acids) and the mixture of EPA and AA did not seem to have the same effect. These findings may help explain the clinical benefits of EPA in pediatric patients receiving long term TPN.3. In this study we firstly co-cultured EPA-treated,LPS pre-stimulated macrophages and LPS pre-stimulated THLE-3to carry out experiment. We demonstrated that EPA not only had anti-inflammatory effect on macrophages and hepatocytes directly, but could indirectly reduce inflammations in hepatocytes through activated macrophages.The benefits of omega-3fatty acids in TPN preserves immune function and suppress the inflammatory response.In the future, we hope that omega-3fatty acids will become a standard for both the rescue as well as prevention of PNALD.

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