Paeoniflorin Protects Against Nonalcoholic Fatty Liver Disease Induced By A High-Fat Diet In Mice

Chapter 1 The effects of Paeoniflorin on glycolipid, inflammation and insulin resistance in a mouse model of Nonalcoholic Fatty Liver DiseaseObjective:To explore the effect of Paeoniflorin on glycolipid, inflammation and insulin resistance against Nonalcoholic Fatty Liver Disease by a high-fat diet.Methods:Experiments were obtained from the Animal Care Use Committee of the Renmin hospital of Wuhan University, seven weeks old male C57BL/6J mice were kept in an standard cages (the temperature:23±2?, humidity:60±5%, and 12 h dark/light cycle). The animals were given free food and water. After 1 week of acclimatization, mice were divided into four groups:normal control mice (Control) (n=10), normal control mice treated with paeoniflorin (Con+Pa) (n=10), high-fat diet-fed mice (HF) (n=10), and high-fat diet-fed mice treated with paeoniflorin (HF+Pa) (n=10). The control and the HF groups were fed with AIN-76A diet [containing (energy%) 11.5% fat,67.7% carbohydrate, and 20.8% protein] and high-fat diet [containing (energy%) 37.4% fat, 42.6% carbohydrate, and 20.0% protein], respectively. While the other groups were fed with corresponding diet supplemented with 0.05% paeoniflorin (>98% pure). The mouses were given free food for 24weeks. The food consumption and weight gain were measured one and two weeks, respectively.At the end of the study, animals were fasted overnight and were sacrificed under anesthesia (ether inhalation anesthesia). Before being sacrificed, body weight and length were measured. Blood samples were collected and centrifuged at 4000 rpm,30 min at 4? for serum. Liver were removed for weight and liver index calculation [Index (%)=liver weight (g)xbody weight (g)-1×100]. One part was cut and fixed in 10%(v/v) neutral formalin solution for histological analysis. Visceral fat, including perirenal, retroperitoneal,epididymal fat pad,was also removed and weighed for index calculation..Results:1. Basic data in mice:During the 24-week feeding period, the mice in HF group developed central obesity, hyperglycemia,insulin resistance,dyslipidemia, inflammatory state and hepatic steatosis. At the end of study, compared to control group, the the average body weight in model group and high-fat diet-fed mice treated with paeoniflorin (HF+Pa) acheieved 41.53±0.799,37.24±0.964, (p<0.01). Con+Pa group and Control group have no evident changes. It menifested that Paeoniflorin produce a marked effect only in a obese state. Other indicators, such as:liver weight, fat weight, abdominal circumference, Lee's index, fat index and liver index, has also undergone a corresponding change. Paeoniflorin prevent these changes effectively. It reduced fat weight, liver weight and fat index and liver index.2. Glycolipid metabolism and liver function in mice:In our study, hyperlipemia, insulin resistance, hyperlipidemia and Liver Function Damage formed by the high-fat diet. The levels of serum cholesterol were significantly increased by 145.4% in the HF group (p<0.01) and decreased to 68.7% in paeoniflorin-treated group (p<0.01). The levels of serum LDL and HDL showed the same changes as cholesterol. Triglyceride concentrations were not altered. Long-term high-fat diet resulted in serious liver function damage—elevated serum ALT and AST levels. After paeoniflorin treatment, impaired liver function returned to normal.3. Fasting glucose and insulin resistance in mice:In the HF group, the HOMA-IR index was 8 times higher than that of Control group (p<0.01). While in the HF+Pa group, insulin sensitivity was increased because the HOMA-IR index dropped to 27.3%. The levels of fasting glucose and insulin exhibited similar changes as the HOMA-IR index. Long-term paeoniflorin treatment alleviated these effects, which improve the insulin resistance.4. The pathological changes in mice's liver:Long-term high-fat diet resulted in steatosis, inflammation,ballooning degeneration and necrosis in HF group. But the lipid inclusions were strikingly reduced in both size and number in the liver of Pa-treated animals.Conclusions1. Paeoniflorin could lower weight gain, organ index and Lee's index of Nonalcoholic Fatty Liver Disease;2. Paeoniflorin could improve the Glycolipid metabolism disorder, and played a hepatoprotective effect;3. Paeoniflorin could alleviate high-fat diet-induced hyperglycemia and insulin resistance, which improve insulin sensitivity and glucose tolerance in mice.4. Paeoniflorin could inhibit the lipid ectopic deposition, which relieved the lipotoxicity in liver.Chapter 2 The protective mechanism of paeoniflorin on Nonalcoholic Fatty Liver Disease in a mouse modelObjective:To clarify the mechanisms of paeoniflorin on metabolism syndrome from lipid-lowering, anti-inflammatory and insulin resistance improvement.Methods:Animal grouping and model setting were the same as those in part one. The lipid metabolism pathways, regulated by paeoniflorin, were measured by Real-time PCR, including:the beginning synthesis way, PPARa-mediated fatty acid oxidation metabolism, PPARy-mediated lipid synthesis pathway, triglyceride synthesis. Choles-terol output related genes, Glucose metabolism related genes, and the Inflammation related genes.Results:1. Comparison of the De novo synthesis way in each group:High-fat diet induced the SREBP-1c, FAS, and ACCa mRNA increased apparently. Paeoniflorin down-regulated the expressions of the three genes, suppressing the De novo synthesis way.2. Comparison of the PPAR?-mediated fatty acid oxidation metabolism:The high-fat diet reduced the expressions of PPAR?, CPT-1a, and ACOX mRNA. the expressions of UCP2 up-regulated after high-fat diet.The lipid oxidative metabolism was blocked. However, paeoniflorin changes the target genes, which increased lipid oxidation.3.Comparison of the PPAR?-mediated Cholesterol synthesis pathway:High-fat diet activated PPAR?-mediated Cholesterol synthesis pathway. After s Paeoniflorin made the expressions of PPARy mRNA return to normal levels, which suggested that paeoniflorin could inhibit PPARy-mediated Cholesterol synthesis pathway. HMGCR and LDLR showed the same change.4. Comparison of the Cholesterol output related genesThe hepatic LXR-a and ABCA-1 mRNA levels increased by high-fat diet in HF group. Treated with paeoniflorin, the changes were reversed. Our results suggest that upregulation of LXR-a and ABCA-1 expressions in high-fat diet mice might be a compensatory and self-protection mechanism.;5. Comparison of the triglyceride synthesis:Great changes occurred in the levels of SCD-1 mRNA, which were increased 1.1-fold in model mice, but no significant difference was observed in high-fat fed mice supplemented with paeoniflorin.6. Comparison of glucose metabolism related gene:PEPCK and G6Pase mRNA levels were increased by 301% and 178%, respectively in obese mice, and were attenuated in high-fat fed mice supplemented with paeoniflorin.7. Comparison of inflammation related genes pathway:long-term high-fat diet resulted in high expressions of liver inflammatory factors (like tumor necrosis factor-a (TNF-a), interleukin-1 (IL)-1, IL-6,MCP-1). Paeoniflorin supplementation had a significant effect on the levels of inflammation related genes.Conclusions:1. Peaoniflorin could down-regulate the expressions of the three genes for suppressing the beginning synthesis way; down-regulated the expressions of PPARy, HMGCR and LDLR mRNA for decreasing the cholesterol synthesis; up-regulated the PPARa, CPT-1a, ACOX and UCP2 mRNA expressions for increasing lipid oxidation; had little effect on SCD-1 mRNA, which cannot lower the hypertriglyceridemia.2. Peaoniflorin could inhibit the expressions of inflammation related genes, which showed a strong anti-inflammatory effect.