| Objective: With the significant change in lifestyle and diet structure,hyperlipidemia(HLP)has become one of the biggest security hidden danger in china. It is well-known that liver is a vital organ of the lipid metabolism,involves the transport,synthesis and decomposition process of fat. Lipid metabolism disorder can lead to ectopic fat deposition in the liver, causing nonalcoholic fatty liver disease(NAFLD), which is closely associated with diabetes and obesity. Nearly 20 years, the prevalence of NAFLD is doubled,and become the highest liver disease in western countries. NAFLD included nonalcoholic simple fatty liver(NAFL), nonalcoholicsteatohepatitis(NASH)even liver cirrhosis and hepatocellular carcinoma(HCC). Liver lipid metabolic process is complex. The beta oxidation of fatty acid is an important form of adipose decompose. Camitine palmitoyltransferase(CTP-1A)is the rate-limiting enzyme of beta oxidation of fatty acid. The change of the level of CTP-1A is associated with the occurrence of hyperlipidemia closely.Peroxisome proliferativeactivated receptor-alpha(PPAR-alpha) is the upstream transcription factors of the fatty acid oxcidation process. CPT-1A is the key target genes in downstream region. The expression of CTP-1A is regulated by the PPAR-alpha. PPAR-alpha/CTP-1A is the important pathway of liver lipid metabolism. Curcumin is extracted from curcuma rhizome of a kind of natural polyphenols, with a number of roles such as lipid-decreasing,antioxidation, anti-inflammatory, analgesia and antitumor. Jianzhi Tongluo capsule, called “patent” herbal medicine, was made of curcumin compounds as the main raw material, becoming one of lipid-lowering drugs in clinical application. But it is not clear that the lipid-lowering mechanism is involved in tne pathway of PPAR-alpha/CPT-1A. The experiment establishes an acute hyperlipidemia model of Sprague Dawley(SD) rats using the egg yolkemulsion method, to observe the liver lipid deposition, the changes of the liver lipid metabolism and related enzymes in acute hyperlipiddemia state, and clear whether curcumin compounds improving liver lipid metabolism by PPAR-alpha/CPT-1A signaling pathway, and discuss the lipid-lowering mechanism and possible liver protection, provide theoretical basis and guide clinical medication?Methods:1 Sixty healthy male SD rats(5-6 weeks, quality of 180±20g) were randomly divided into 6 groups: normal control group(NC), hyperlipidemia model group(model group), low dosage of curcumin compounds group(LC,125 mg/kg of curcumin solution), middle dosage of curcumin compounds group(MC,250 mg/kg of curcumin solution), high dosage of curcumin compounds group(HC,500 mg/kg of curcumin solution) and the fenofibrate group(FF,30 mg/kg fenofibrate). NC group and model group rats were given by gavage once a day for 10 days with corn oil(2.5 ml/kg), the experimental groups were given corresponding drug at the sametime?2 On the 10 th day, the model and experimental groups rats were given disposable intraperitoneal injection of 75% yolk emulsion(25 ml/kg),while the NC group of saline(25 ml/kg) in the same way?3 24 hours later,we obtained tail blood(fasting water 10 days) to determine the levels of serum TG,TC,LDL-C,HDL-C,ALT,AST,free fatty acid(FFA). Besides, the levels of TG and FFA in liver tissue were examined also. Optical microscope to observe the liver structure. The PPAR-alpha/CPT-1A mRNA and protein content were detected by Real-time PCR and Western blotting in rats' liver?4 All data were analyzed by SPSS 13.0 software. Measurement data expressed as mean±standard deviation( x|-±s). Comparison between the two groups were analyzed using Oneway ANOVA, while multiple comparisons LSD and SNK- q test.When P < 0.05, we think the difference is statistically significant?Results:1 The effect of the acute hyperlipidemia model on the level of serum TG,TC and LDL-C:The level of serum lipid(TG 9.66±10.98mmol/L, TC8.72±2.17mmol/L, LDL-C 0.78±0.57mmol/L) began to rise in the 6th hour after the intraperitoneal injection of yolk emulsion while the peak maximum of Lipid level(TG 20.97±15.85mmol/L, TC 10.34±4.24mmol/L, LDL-C0.58±0.25 mmol/L) waiting for 24 th hour. And HDL-C(2.49±0.88mmol/L)had decreased. Compaired with the levels of 0h, the diffirence was statisically significant(P<0.01), promoting the acute hyperlipidemia model successfully established;2 Comparison in blood lipid and FFA levels between groups:The serum lipid and FFA levels of acute hyperlipidemia model group were obviously higher than that of normal control group(P < 0.01). Compared with model group,curcumin compounds in each dose group and fenofibrate group can extremly reduce the blood TG,TC,LDL – C,FFA(P < 0.05), increase HDL- C(P <0.05). The change of high dose curcumin group was the most obvious, and Fenofibrate group and MC group were secondly. But there was no significant difference of the levels between Fenofibrate group and MC group(P > 0.05);3 Comparison in liver TG, FFA content between groups:The liver TG, FFA levels of acute hyperlipidemia model group were significantly higher than the normal control group(P < 0.01); Compared with model group, curcumin compounds in each dose group can obviously reduce the levels(P < 0.05), and this effect had a dose dependent increase; Fenofibrate group lower the levels than Model group(P < 0.05), but had no obvious difference with MC group(P > 0.05);4 Comparison in liver function between groups: The serum of ALT, AST levels had no statistical difference between each groups(P > 0.05);5 Pathomorphism changes in the liver tissue of rats:5.1 HE staining: We could see the structure of the liver cell integrally,regularly and clearly through microscope in the NC group. Its cytoplasm was dyed red uniformly. No lipid droplets cavitation in the cytoplasm. The liver cell in Model group showed swell significantly and hyaline degeneration, in which some cells dissolved; There were different number and sizes of lipid droplets in cytoplasm. With the increase of curcumin compounds doses, liver cell swelling and lipid drops gradually reduce. The HC group had the most significant change. Its cells were only slightly edema, no lipid drops;Fenofibrate group showed mildly hydropic degeneration, did not see fat vacuoles;5.2 Oil red O staining: The liver cells of NC group were arranged regularly. Its structure was integrity. Its cytoplasm was dyed light blue, no red lipid drops; A small amount of tiny roundlike red lipid drops can be seen in Model group, but lipid deposition not seen; Red lipid drops of all drug intervention groups were reduced in different degrees. The HC group had the most extreme change. We couldn't see red lipid droplet;6 Influence of curcumin extracts on CPT-1A,the fatty acid oxidation key enzyme in lipid metabolic pathways:6.1 Comparision in the expression level of CPT-1A mRNA between groups:Compared with the NC group, CPT–1A mRNA expression level of Model group rats was significantly reduced(P < 0.01); Compared with the Model group, CPT–1A mRNA expression levels of LC, MC, HC groups were extremely elevated(P < 0.01), and had a dose dependent increase; This level of Fenofibrate group was also higher than Model group(P < 0.01). But there was no obvious difference between FF group and MC group(P > 0.05);6.2 Comparision in the expression level of CPT-1A protein between groups:Compared with the NC group, CPT–1A protein expression level of Model group rats was significantly reduced(P < 0.01); Compared with the Model group, CPT–1A protein expression levels of LC, MC, HC groups were extremely elevated(P < 0.01), and had a dose dependent increase; This level of Fenofibrate group was also higher than Model group(P < 0.01). But there was no obvious difference between FF group and MC group(P > 0.05);7 Influence of curcumin extracts on the PPAR-alpha: Compared with the NC group, PPAR-? mRNA and protein expression levels of Model group rats were significantly reduced(P < 0.01); Compared with the Model group, PPAR-?mRNA and protein expression levels of LC, MC, HC groups were extremely elevated(P < 0.01), and had a dose dependent increase; This levels of Fenofibrate group were also higher than Model group(P < 0.01). But there was no obvious difference between FF group and MC group(P > 0.05)?Conclusion:1 Intraperitoneal injection of 75% yolk emulsion established an acute hyperlipidemia rat model successfully : 24 h after injection, the serum TG,TC,LDL-C reached peak, the level of FFA increased, fat deposited in liver mildly, and liver cell mitochondria structure damaged. No obvious difference was found in transaminase?2 Different curcumin compounds dose groups and fenofibrate group improved the dyslipidemia and decreased FFA in different degrees, alleviated liver fat deposition, and protect liver mitochondrial functionsthe effectively.And the high doses of curcumin group has the most significant role?3 Acute hyperlipdemia inhibited the fatty acid oxidation pathway PPAR-alpha/CPT-1A expression in rat liver. Different curcumin compounds dose groups and fenofibrate group activated PPAR-alpha/CPT-1A expression to different degrees, promoted the oxidation of fatty acid. And the high doses of curcumin group has the most significant role?... |
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