| Metabolic syndrome is a metabolic disorder syndrome with insulin resistance as the pathological basis, mainly including obesity, dyslipidemia, hypertension, hyperuricemia and so on, which is a clustering patterns of the pathological state with various metabolic disorders in the body. Epidemiologic investigations show that the significant increase in the incidence of metabolic syndrome is parallel with the long-term consumption of high fructose. High fructose intake can cause pathological features of metabolic syndrome including insulin resistance, hyperinsulinemia, hyperlipidemia, obesity, hyperuricemia, accompanied with liver injury (oxidative stress, decreased insulin sensitivity, inflammatory response and lipid deposition). But, the molecular pathological mechanism by which high fructose intake induces liver injury still remains to be further elucidated.High fructose induces reduction in insulin sensitivity in system as well as liver and other organs, and destruction of body lipid balance in humans and animals. Sterol regulatory element binding protein (SREBP-lc) can inhibit insulin receptor substrate 2 (IRS2) expression, down-regulate protein kinase B (Akt) phosphorylation, thereby decreasing hepatic insulin sensitivity. SREBP-lc also regulates fatty acid synthase (FAS) expression. Lipid transfer protein ATP-binding cassette transporter A1 (ABCA1) regulates hepatic lipid transport, associating with SREBP-1c. Insulin-induced gene 1 (insig-1) and Sirtuin (Sirtl) can regulate lipid metabolism via inhibiting SREBP-lc activation. miRNAs are small,19-25 nucleotide, non-coding, highly conserved regulatory RNAs. miR-29b and miR-34a are found to regulate insulin signaling and lipid metabolism. High miR-29b expression is detected in the liver of diabetes mice. Significant up-regulation of miR-34a expression is also observed in serum of patients with non-alcoholic fatty liver disease (NAFLD) as well as the liver of patients with non-alcoholic steatohepatitis (NASH), and mice with obesity and diabetes. miR-29b and miR-34a are predicted to bind the 3’UTR site of insig-1 and Sirtl mRNA by the algorithm targetscan (http://www.targetscan.org/), respectively. Whether high fructose up-regulates miR-29b and miR-34a expression to affect insig-1 and Sirtl expression and then cause SREBP-1c-related impairment of insulin signaling, disorder of lipid synthesis and transfer-related protein expression, resulting in hepatic lipid deposition? In addition, high fructose-induced reactive oxygen species (ROS) generation also causes hepatic inflammation and injury. Thioredoxin (TRX) and its endogenous inhibitor thioredoxin-interacting protein (TXNIP) regulats intracellular redox balance. Hydrogen peroxide (H2O2) as an important component of ROS, is reported to promote TXNIP dissociated from TRX and NOD-like receptor protein 3 (NLRP3) inflammasome activation to promote inflammatory cytokine interleukin-1β (IL-1β) maturation in human monocytes. Our research group has developed a rat model of metabolic syndrome induced by feeding 10% fructose solution. High ROS and H2O2 production, as well as TXNIP/NLRP3 inflammasome activation with IL-1β maturation have been observed in the kidney or/and liver of this animal model. On the other hand, down-regulation of miR-200a expression is detected in the liver of mice with NAFLD. MiR-200a expression may be regulated by H2O2-related oxidative stress in human macrophages. Furthermore, miR-200a is predicted to bind the 3’UTR site of TXNIP mRNA by the algorithm targetscan. Whether high fructose-induced high H2O2 is possible to induce low miR-200a expression and high TXNIP level, causing NLRP3 inflammasome activation and IL-1β production to promote hepatic inflammation?Clinical and basic researches have showed that stilbenes as pterostilbene and curcumin improve insulin resistance, hyperinsulinemia, hyperlipidemia and diabetic complications and so on, as well as have liver protection. Our research group demonstrated that pterostilbene alleviated metabolic syndrome in high fructose-fed rats, but its improvement of hepatic insulin sensitivity and lipid deposition as well as the molecular pathological mechanisms still need to be further elucidated. Curcumin has been confirmed to alleviate liver injury in fructose-induced metabolic syndrome in rats, but its improvement of high fructose-induced hepatic oxidative stress and inflammation as well as the possible molecular pathological mechanisms also need further investigation.To further explore the new ways of high fructose-induced hepatic lipid deposition, this thesis examined whether high fructose increased miR-29b and miR-34a expression to affect insig-1 and Sirtl expression, causing SREBP-1c and the related insulin signaling impairment, disorder of lipid synthesis and transfer-related protein expression in rats of high fructose feeding-induced liver injury in metabolic syndrome and high fructose-exposed hepatocytes models. Moreover, to finding the new molecular pathological mechanisms, this thesis analyzed whether high H2O2 production caused by high fructose decreased miR-200a expression to activate TXNIP/NLRP3 inflammasome and promoted IL-1β maturation in these animal and cell models. Subsequently, this thesis importantly investigated the improvement of pterostilbene on hepatic insulin sensitivity reduction and lipid deposition and its molecular pathological mechanisms, and examined restoration of hepatic oxidative stress and inflammation by curcumin and its molecular pathological mechanisms in these animal and cell models.This thesis developed the rat model of metabolic syndrome in this thesis. Rats were feeding with 10% fructose solution for continuous 12 weeks. At week 7, high fructose-fed rats were received 10,20 and 40 mg/kg pterostilbene,15,30 and 60 mg/kg curcumin, 4 mg/kg pioglitazone (positive control), and 5 mg/kg allopurinol (positive control) by intragastric gavage for next 6 weeks, respectively. Compared to normal rats, decreased glucose tolerance and insulin sensitivity was confirmed by oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) in this animal model, in parallel with increased body weight, serum insulin, triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), uric acid and IL-1β levels. Compared with fructose-vehicle group, pterostilbene, curcumin, pioglitazone and allopurinol markedly reduced body weight, serum levels of insulin, TG, TC, LDL-C, uric acid and mature IL-1β.Based on the above, this thesis examined whether high fructose increased miR-29b and miR-34a to affect insig-1 and Sirtl expression, causing SREBP-lc and the related insulin signaling impairment, disorder of lipid synthesis and transfer-related protein expression, resulting in hepatic lipid deposition, and the molecular pathological mechanism by which pterostilbene ameliorates hepatic lipid deposition.Compared with control group, hepatic TG, TC levels and lipid deposition were increased in high fructose-fed rats, accompany with increased hepatic SREBP-lc and FAS protein levels and decreased ABCA1 protein level in high fructose rat model. Increased SREBP-1c (rat liver cell line BRL-3A:12 h; human hepatocarcinoma cell line HepG2:8 h), FAS protein levels, as well as decreased ABCA1 protein level and lipid deposition (BRL-3A:24 h; HepG2:12 h) were observed in high fructose (5 mM) cell models in vitro. In addition, this animal model appeared hepatic insulin signaling impairment, as hepatic protein levels of IRS2 and phosphorylated Akt were significantly reduced, and confirmed in cell models in vitro (BRL-3A:12 h; HepG2:8 h). These data indicated that high fructose caused insulin signaling impairment and lipid dysregulation, resulting in lipid deposition in the liver of rats and hepatocytes. Compared with fructose-vehicle group, pterostilbene and pioglitazone significantly up-regulated IRS2 and p-Akt protein levels, down-regulated SREBP-lc and FAS protein levels, increased ABC A1 protein level and reduced lipid deposition in these animal and cell models.Compared with control group, insig-1 and Sirtl protein levels were significantly reduced in the liver of high fructose rat model and high fructose-exposed hepatocytes of BRL-3A (12 h) and HepG2 (8 h), which were reversed by pterostilbene and pioglitazone. Compared with control group, high miR-29b and miR-34a expression was observed in microarray-based analysis of miRNAs in the liver of this animal model, which was further confirmed by qPCR. Compared with normal cell group, high fructose increased miR-29b and miR-34a expression levels in BRL-3A (12 h) and HepG2 (4 h) cells. Compared with fructose-vehicle group, pterostilbene and pioglitazone significantly down regulated miR-29b and 34a expression levels in the liver of fructose animal model and hepatocytes models.To verify whether miR-29b and miR-34a is possible to target insig-1 and Sirtl respectively, this thesis further used miR-29b or miR-34a inhibitor or mimic transfection. Compared with negative control-transfected cells under high fructose condition, miR-29b and miR-34a inhibitor significantly increased insig-1 or Sirtl protein levels, respectively, decreased SREBP-lc protein level, increased IRS2 and p-Akt protein levels (BRL-3A:12 h; HepG2:8 h), down-regulated FAS protein level, up-regulated ABCA1 protein level and reduced lipid deposition in high fructose-exposed hepatocytes (BRL-3A:24 h; HepG2:12 h). Compared with negative control-transfected cells under high fructose condition, pterostilbene (10 μM) and pioglitazone (10 μM) up-regulated insig-1 or Sirtl protein levels, down-regulated SREBP-lc protein level, increased IRS2 and p-Akt protein levels (BRL-3A:12 h; HepG2:8 h), down-regulated FAS protein level, up-regulated ABCA1 protein levels and reduced lipid deposition (BRL-3A:24 h; HepG2:12 h) in miR-29b or 34a inhibitor transfected and high fructose-exposed hepatocytes. Compared with negative control-transfected cells under high fructose condition, miR-29b and miR-34a mimic further significantly down-regulated insig-1 or Sirtl protein levels, respectively, up-regulated SREBP-lc protein level, down-regulated IRS2 and p-Akt protein levels (BRL-3A:12 h; HepG2:8 h), increased FAS protein level, decreased ABCA1 protein level, and increased lipid deposition (BRL-3A:24 h; HepG2:12 h) in high fructose-exposed hepatocytes. Compared with negative control-transfected cells under high fructose condition, miR-29b or miR-34a mimic in partially prevented pterostilbene and pioglitazone-mediated up-regulation of insig-1 or Sirtl protein levels, respectively, partially prevented their improvement of high SREBP-lc protein level, low IRS2 and p-Akt protein levels, high FAS protein level, low ABCA1 protein level, and amelioration of lipid deposition in miR-29b or 34a mimic transfected and high fructose-exposed hepatocytes. These data indicated that high fructose may up-regulate miR-29b and miR-34a expression to down-regulate insig-1 and Sirtl expression, respectively, leading to SREBP-lc and the related insulin signaling impairment, disorder of lipid synthesis and transfer-related protein expression, resulting in hepatic lipid deposition. Pterostilbene may down-regulate miR-29b and miR-34a to up-regulate insig-1 and Sirtl expression, improve SREBP-1c and the related insulin signaling impairment, disorder of lipid synthesis and transfer-related protein expression, thereby ameliorating lipid deposition in the liver of animal model and hepatocytes models.Another key investigation point of this thesis is examining whether high H2O2 production caused by high fructose decreased miR-200a expression to affect TXNIP expression, activating NLRP3 inflammasome and IL-1β maturation, thereby promoting hepatic inflammation, and the reverse improvement and the molecular pathological mechanism by curcumin.Compared with control group, the liver of high fructose rat model appeared inflammatory cell infiltration, high protein expression levels of TXNIP, NLRP3, ASC, caspase-1 and IL-1β. Increased protein levels of TXNIP, NLRP3, ASC, caspase-1 and IL-1β were observed in high fructose BRL-3A and HepG2 cell models in vitro (BRL-3A:12 h; HepG2:48 h). These data indicated that high fructose may cause high TXNIP expression, activate NLRP3 inflammasome and IL-1β production, promote inflammatory response in the liver of rat and hepatocytes. Compared with fructose-vehicle group, curcumin and allopurinol significantly down-regulated TXNIP, NLRP3, ASC, caspase-1 and IL-1β protein levels and alleviated inflammatory response in these animal and cell models.Decreased miR-200a expression compared with control group was observed with microarray-based analysis of miRNAs in the liver of high fructose rat model, which was further confirmed by qPCR. Compared with normal cell group, high fructose decreased miR-200a expression level in hepatocytes (BRL-3A:4 h; HepG2:8 h). Compared with fructose-vehicle animal and cell models, curcumin and allopurinol significantly up-regulated miR-200a expression in these animal and cell models.To verify whether miR-200a is possible to target TXNIP, this thesis further used miR-200a mimic or inhibitor transfection. Compared with negative control-transfected cells under high fructose condition, miR-200a mimic down-regulated TXNIP, NLRP3, ASC, caspase-1 and IL-1β protein levels (BRL-3A:12 h; HepG2:48 h). Compared with negative control-transfected cells under high fructose condition, miR-200a inhibitor further promoted high fructose-induced high TXNIP protein level, in parallel further up-regulated the protein levels of NLRP3, ASC, caspase-1 and IL-1β (BRL-3A:12 h; HepG2:48 h), thereby aggravating inflammatory response in hepatocytes. These findings indicated low miR-200a expression caused by high fructose may mediate TXNIP/NLRP3 inflammasome activation and IL-1β maturation to induce inflammatory response in hepatocytes. Compared with negative control-transfected cells under high fructose condition, curcumin and allopurinol down-regulated TXNIP, NLRP3, ASC, caspase-1 and IL-1β protein levels (BRL-3A:12 h; HepG2:48 h) in miR-200a mimic transfected hepatocytes. Compared with negative control-transfected cells under high fructose condition, miR-200a inhibitor interferenced the suppression of TXNIP/NLRP3 inflammasome activation and IL-1β production (BRL-3A:12 h; HepG2: 48 h) by curcumin and allopurinol in miR-200a inhibitor transfected high fructose-exposed hepatocytes. These findings indicated the suppression of TXNIP/NLRP3 inflammasome activation by curcumin and allopurinol may partly depend on the up-regulation of miR-200a. These dates above indicated that low miR-200a expression caused by high fructose may elevate TXNIP expression, activate NLRP3 inflammasome to produce IL-1β maturation, and induce inflammation in the liver of rats and hepatocytes. Curcumin and allopurinol may up-regulate miR-200a expression, inhibit TXNIP/NLRP3 inflammasome activation and IL-1β production to alleviate inflammation in the liver of these animal model and hepatocytes.Compared with control group, the levels of superoxide anion (O2·-) in situ, H2O2 and ROS were increased, and the levels of superoxide dismutase (SOD), catalase (CAT) activity and glutathione (GSH) were decreased in the liver of high fructose rat model, and confirmed in hepatocytes models in vitro, as 5 mM fructose induced an increase of cellular ROS, H2O2 and O2- levels, decreased CAT activity (BRL-3A:4 h; HepG2:8 h). These findings further confirmed high fructose-induced oxidative stress in the liver of rats and hepatocytes. Compared with fructose-vehicle group, curcumin and allopurinol markedly reduced O2-, H2O2 and ROS levels, increased SOD, CAT activity and GSH levels in the liver of this animal model. Curcumin and allopurinol also down-regulated cellular ROS, H2O2 and O2·- levels, increased CAT activity (BRL-3A:4 h; HepG2:8 h) in hepatocytes models. These data indicated the inhibition of oxidative stress by curcumin and allopurinol in these animal and cell models.Compared with high fructose-exposed hepatocytes, CAT inhibitor 3-Amino-1,2,4-triazole (ATA) further aggravated CAT hypoactivity, high H2O2 levels (BRL-3A:4 h; HepG2:8 h), further up-regulated TXNIP protein level, promoted high fructose induced NLRP3 inflammasome activation and IL-1β maturation (BRL-3A:12 h; HepG2:48 h). Compared with high fructose-exposed hepatocytes, ATA partly prevented the improvement of high fructose-induced CAT hypoactivity, high H2O2 level (BRL-3A:4 h; HepG2:8 h) and TXNIP protein level, NLRP3 inflammasome activation and IL-1β maturation (BRL-3A:12 h; HepG2:48 h) by curcumin and allopurinol in hepatocytes. These results indicated that high H2O2 level caused by high fructose intake may increase TXNIP protein level, activate NLRP3 inflammasome and produce IL-1β, thereby causing inflammation in hepatocytes. Curcumin and allopurinol inhibited high fructose-induced TXNIP/NLRP3 infiammasome activation and IL-1β production to ameliorate inflammation, probably associating with their antioxidant activity and H2O2 elimination. In addition, miR-200a suppression was confirmed in 50 μM H2O2-treated hepatocytes (BRL-3A:4 h; HepG2:4 h), and reversed by curcumin and allopurinol.In conclusion, high fructose may up-regulate hepatic miR-29b and miR-34a expression to down-regulate insig-1 and Sirtl protein levels, leading to SREBP-lc and its related insulin signaling impairment and disorder of lipid synthesis and transfer-related protein expression, thereby causing hepatic lipid deposition. Moreover, high fructose-induced H2O2 over-production may reduce miR-200a expression to increase TXNIP protein level and activate NLRP3 inflammasome with IL-1β production, inducing hepatic inflammation. Pterostilbene alleviated lipid deposition in the liver of high fructose-fed rats and high fructose-exposed hepatocytes possibly through decreasing miR-29b and miR-34a expression to increase insig-1 and Sirtl protein levels and then improve SREBP-lc and its related insulin signaling and regulate lipid synthesis and transfer-related proteins. Curcumin restored high fructose-induced hepatic inflammation possibly via by up-regulating miR-200a expression to inhibit TXNIP/NLRP3 inflammasome activation and IL-1β production in these animal and cell models, which may be associated with its antioxidant activity. These results illustrated the new ways of high fructose intake-induced hepatic insulin sensitivity reduction, oxidative stress, inflammation and lipid deposition, as well as revealed the new molecular pathological mechanisms of the hepatoprotection by pterostilbene and curcumin. These findings provided experiment basis for pterostilbene and curcumin used in the treatment and prevention of metabolic syndrome and associated liver injury in the clinic. |
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