Effect Of Liraglutide On Diet Induce Non-alcoholic Fatty Liver Disease And JNK Signaling In Rats

Background:NAFLD is a clinicopathological syndrome including a wide spectrum of liver damage, characterized by steatosis along with inflammation and hepatocellular damage in patients with no history of significant alcohol consumption. NAFLD,one of the most common liver diseases in the word,is highly associated with insulin resistance and is considered the hepatic manifestation of the metabolic syndrome. NAFLD represents a spectrum of disease ranging from simple steatosis to steatohepatitis (NASH), increasing fibrosis and eventually, cirrhosis.When lipid accumulation in liver exceed5%or1/3above of the liver cells have steatosisi in liver biopsy,the liver is called fatty liver.In recent years, with the improvement of living standards, a large amount of fat intake, increased incidence of obesity and diabetes, The incidence of NAFLD is apparently higher and a younger trend, which has become the second largest after chronic liver disease after a viral hepatitis. Furthermore, up to10%to25%patients with NAFLD can progress to NASH and10%to15%patients with NASH develop hepatocellular carcinoma. Although the pathogenesis of NAFLD remains elusive, the prevailing theory for development of NAFLD is the ’two-hit’ hypothesis. The first hit is the dysregulation of fatty acid metabolism, which leads to simple steatosis. The secondary hit is including oxidative stress, lipid peroxidation and inflammatory response, which finally lead from NAFL to NASH or even cirrhosis.JNK signal transduction pathway activation is the main factors affecting IR and inflammatory reaction,which has been shown to play an important role on occurrence and development of NAFLD. JNK1gene deletion can alleviate inflammation, IR and steatosis of the liver induced by high-fat diet, which can prevent inflammatory reaction and fibrosis of liver. So far it has been thought that TNF-a, FFAs, oxidative stress are all specificity agonists of JNK signaling pathway, and proved that it is they which can all interfere with the conduction of INS signal leading to IR. Adiponectin, a specific protein secreted by adipocytes, is considered to play an important role in the pathogenesis of NAFLD, because it has insulin-sensitizing, antifibrogenic and anti-inflammatory properties. A large number of animal and human experiments have found that it’s closely related to obesity, IR, coronary heart disease and NAFLD.The current treatment for NAFLD is limited to improving insulin sensitivity with lifestyle interventions and weight loss or surgery. Since the lifestyle modification, weight loss and bariatric surgery are exceedingly difficult to achieve by most people, an effective pharmacologic treatment for NAFLD is desperately needed.Liraglutide, a Glucagon-like peptide-1(GLP-1) analogue with97%sequence identity to human GLP-1, is known to have actions to decrease glucose, as potentiating glucose-dependent insulin secretion, enhancing beta cell growth, and reducing food intake and body weight. Several human and animal studies have demonstrated that GLP-1has an effect on NAFLD, which is conducted by improving insulin resistance and reducing hepatic steatosis; Whether Liraglutide can reverse oxidative stress, lipid peroxidation and inflammation in NAFLD still remains obscured. It is not known whether Liraglutide influenced these effects associated with ADP content and JNK signaling on NAFLD rats. We therefore conducted the present study to investigate effects and mechanisms of glucagon-like peptide-1(GLP-1) on diet induce NAFLD.Aim:This study created the NAFLD rat model with high fat diet,observing the changes of liver homogenates and serum levels of ADP、TG、TC、FFAs、MAD、 SOD、TNF-α、and ADP、TG、TC、TGF-β1、FBG and expression of JNK1and p-JNKl,to explore the effect of Liraglutide on the content of serum and liver tissue ADP、liver tissue TG and FFAs, and the insulin resistance, oxidative stress and inflammation.The aim of the investigation was to explor effects and mechanisms of Liraglutide on diet induce NAFLD and provide the basis for an effective pharmacologic treatment for NAFLD.Method:Grouping and establishment of animal model Thirty5-week-old male SD rats with the weight of140±10g were fed with normal diet for one week, then randomly assigned as the normal group (ND group) and continued to feed normal diet, the rest20were regarded as the model-making group which were fed with high-fat and high cholesterol diet (88%ordinary forage+10%lard+2%cholesterol) until the end of the12th week. Then one rat of each group was executed randomly and after detection, liver index, serum lipid, hepatic transaminase, etc. of model-making group increased significantly, liver cells of it had significant steatosis after giving HE stain of liver tissue section, to confirm that the model was set up. The remaining19rats of the model-making group were randomly divided into2groups:9in HFHC group continued to be fed with high fat diet,10in GLP-1group with high fat diet and intraperitoneal injection of liraglutide. Then the rats were received daily injections of either saline or liraglutide (0.6mg/(kg-d) for4weeks. At the end of the16th week, all the rats were given chloral hydrate solution intraperitoneal anesthesia and killed after12hours of fasting, and then we collected their blood from postcava and separate the serum. At last, we removed part of the liver tissue cryopreservation after liquid nitrogen flash freezing and took another part to conduct histopathological examination.Liver index After the experiment,we ’weight the body weight and liver wet weight, calculating liver index (liver weight/body weightx100%),Blood chemistry Blood from postcava was collected to measure fasting blood glucose (FBG), aminotransferase (ALT), triglyceride (TG), total-cholesterol (TC), fasting insulin (FINS) and Adiponectin (ADP). FBG, ALT and lipids were detected by biochemistry automatic analyzer. FINS, ADP and TGF-β1were measured by ELISA. According to manufacturer’s instructions, homeostasis assessment-insulin resistance (HOMA-IR) was expressed as [FBG×FINS]/22.5.Liver homogenates indicators Approximately0.2g frozen liver tissue was rinsed of blood and placed in1.8ml of ice-cold homogenization buffer and then sliced into sections on ice. The tissue was homogenized on ice for3-5minutes.The homogenate was then centrifuged at2000×g for15minutes. The clarified supernatant was then apportioned into lml aliquots and stored at-70℃until ready for use. Intrahepatic lipids were quantified spectrophotometrically using an enzymatic assay and expressed as lipid (mmol)/g wet liver weight. The levels of superoxide dismutase (SOD) and malondial-dehyde (MAD), free fatty acid (FFAs), ADP and tumor necrosis factor-a (TNF-a) in liver homogenates were detected by RIA and ELISA respectively.Pathology10%neutral-buffered formalin-fixed section and frozen samples. Formalin-fixed liver tissue was then paraffin-embedded, and mounted and stained with hematoxylin and eosin. Frozen samples were used for Oil Red O staining.Western blot analyses Approximately0.1g liver tissue was crushed in liquid nitrogen, and then homogenized in1ml of ice-cold lysis buffer with protease inhibitors and phosphatase inhibitors (Roche). The homogenate was then centrifuged at12000×g for30minutes and the supernatant harvested. Protein concentration of the supernatant was measured with a BCA protein quantification kit.60ug was separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore). Membranes were then blocked in TBS containing0.1%Tween-20and5%skimmed milk for2h at room temperature and incubated with primary antibody including anti-JNKl (Abeam)(1:2000dilution with BSA), anti-phospho-JNKl (Abeam)(1:1000dilution with BSA) and β-action (1:1000dilution with BSA) overnight at4℃. Following three consecutive5min washes in TBST and incubation with HRP-conjugated secondary antibody (Invitrogen)(1:5000dilution with BSA) for2h at4℃, protein expression was detected with enhanced chemiluminescence, and densitometry performed using FluorChem8900System.Statistical analyses Data were analyzed and curves fitted with SPSS16.0. Dates were presented as mean±SE. Multiple comparisons were analyzed by one way ANOVA followed by post hoc testing using Bonferroni correction for more groups. If variance is not homogenous,data would be analyzed by T3method after data tested via welch for vatiance.Rank data were analyzed by Kruskal-Wallis non-Parametric test. A value of P<0.05was considered statistically significant.Result:The changes of body weight and liver index in rats Body weight and liver index of rats in the HFHC group were significantly higher than those in the ND group (P<0.05), which was apparently reduced with in the GLP-1group. Body weight of rats was reduced by10.88%after treatment with liraglutide for4weeks, which suggested that liraglutide had a beneficial effect on weight loss.The changes of serum chemistry including TC, TLT ALT and TGF-β1in rats Compared with the ND group, serum levels of TC, TQALT and TGF-β1in the HFD-fed rats were apparently higher (1.98±0.40vs.1.26±0.24mmol/L,0.80±0.14vs.0.41±0.13mmol/L,59.44±12.43vs.46.56±6.39U/L,0.89±0.86vs.0.74±0.90ug/mL P<0.05). Liraglutide treatment prevented the increase in TC, TG,ALT and TGF-β1levels compared with the HFHC group (1.36±0.20vs.1.98±0.40mmol/L,0.47±0.10vs.0.80±0.14mmol/L,48.90±10.57vs.59.44±12.43U/L,0.75±0.91vs.0.89±0.86ug/mL P<0.05).The changes of oxidative stress, lipid peroxidation and inflammation in rats It is well known that excess FFA can proceed with lipid peroxidation,and then produce a large number of ROS, which causes oxidative stress.Compared with the ND group, The liver homogenates levels of MAD, FFAs, TNF-a of rats in the HFD group were apparently higher while the level of SOD decreased obviously (9.12±2.60vs.6.50±1.79nmol/mg,153.1±32.9vs.105.2±26.9umol/g,3.01±0.54vs.1.83±0.64ug/ml,172.3±6.3vs.188.7±13.0U/mg, P<0.05).We observed that the liver homogenates levels of MAD, FFAs, TNF-a of rats were significantly reduced while the level of SOD increased after liraglutide treatment (6.95±2.99vs.9.12±2.60nmol/mg,118.7±36.1vs.153.1±32.9umol/g,2.23±0.30vs.3.01±0.54ug/ml,193.4±7.1vs.172.3±6.3U/mg, P<0.05).The changes of lipid accumulation and pathological indexes in the liver in ratsRats in the ND and GLP-1group revealed no evidence of steatosis or nonalcoholic steatohepatitis.In contrast the HFHC-fed rats showed obvious pathology including marked hepatic steatosis and ballooning degeneration of hepatocytes and Oli Red O staining revealed obvious lipid accumulation.Liraglutide treatment protected HFHC-fed rats from hepatic fat accumulation as seen by Oil Red O staining and hepatic lipid content.The changes of ADP in serum and liver homogenates in rats As is known adiponectin can improve glucose and lipid metabolism, which plays an important role in the occurrence and development of NAFLD.Therefore,we investigated the effect of liraglutide on Adiponectin ADP in serum and liver homogenates. Compared with the ND group, the level of ADP in serum and liver homogenates decreased obviously(68.1±2.5vs.74.9±6.4ug/L,124.6±12.9vs.150.1±18.9ug/L, P<0.05). We observed that the level of ADP in serum and liver homogenates were apparently higher after liraglutide treatment (74.8±7.1vs.68.1±2.5ug/L,148.4±19.2vs.124.6±12.9ug/L,P<0.05).The changes of expression of JNK1and p-JNK1in rats Compared with the ND group, the expression of JNK1, p-JNK1and p-JNK1/JNK1increased obviously (P<0.05).However,such changes were significantly reversed by4-week treatment with liraglutide treatment.Conclusion:We successfully created the NAFLD rat model with high fat diet for12weeks, and the model was characterized as IR, hyperlipidemia, hepatic steatosis, oxidative stress, lipid peroxidation, and chronic inflammation, according with the characteristic and evolution of human fatty live.Liraglutide treatment can improve liver function,insulin resistance and reduce hepatic steatosis.Liraglutide treatment can alleviate oxidative stress, lipid peroxidation and liver inflammatory reaction.Liraglutide treatment can increase the level of ADP in serum and liver homogenates while down-regulated the expression of JNK1, p-JNK1,which is one of the most important mechanisms to decrease liver tissue inflammation index level and improve insulin resistance and liver pathological indexes significantly.