| Nonalcoholic fatty liver disease(NAFLD) is one of the most common clinical chronic liver disease, and there is no specific treatment for its complex pathogenic mechanism at present. Researches showed ω-3PUFA has an active effect in reducing blood fat and systemic inflammation, improving cardiovascular function, and increasing insulin sensitivity. ω-3PUFA also has an obvious effect in alleviating NAFLD, but the specific mechanism has not been investigated thoroughly.We investigate the effect and mechanism ofω-3PUFA PUFA in improving the NAFLD by establishing the NAFLD rat model and ERS liver cells, to provide new ideas for the development and utilization ofω-3PUFA and prevention of NAFLD.Part 1: The effect of fish oil and Perilla oil in improving NAFLDObject: Establish the NAFLD animal model by feeding high fat and cholesterol to observe the effect of fish oil and Perilla oil in improving NAFLD.Methods: 80 male SD rats were randomly divided into four groups: normal control group(CON), high fat and cholesterol group(HFD), fish oil intervened group(FOH) and Perilla oil intervened group(POH). The rats were beheaded at the 4th, 8th, 12 th,16th week to collect blood, achieve body and liver weight, and detect the changes of rat serum(ALT, AST), blood lipid(TC, TG, HDL- C, LDL- C), SOD, GST and FFA.Rat liver were embedded in paraffin for hematoxylin-eosin and Masson staining. The expression of marker genes(BIP, ATF6, XBP-1) and marker protein(CHOP, ATF6, XBP- 1) in endoplasmic reticulum stress were detected by RT-PCR and Westen- Blot respectively.Results: After 8 weeks’ feeding, compared with the CON group, the liver index and the level of serum ALT, TCH, FFA increased significantly in HFD group, but HDL-C decreased. And the liver morphology changes, lipid accumulation and inflammatory infiltration could be easily observed on HFD liver cells, which means NASH had happened. Compared with the HFD group, liver index in FOH and POH groups decreased significantly, and serum lipid indexes and liver enzyme activity were significantly decreased. The syndrome of hepatocyte ballooning, steatosis, lobular and collect abbacy inflammatory cells infiltration and fibrosis had been improved. After 16 weeks’ feeding, hepatocyte fatty degeneration, lipid accumulation, liver tissue inflammatory infiltration and fibrosis in HFD group became serious, but the FOH and POH groups improved this syndrome obviously.RT-PCR and Westen Blot test results showed the BIP, ATF6, XBP-1 and CHOP expression in the HFD group were higher than the normal group obviously, and Perilla oil and fish oil can reduce the gene and protein expression significantly. Conclusion: The NAFLD rat model were established by high fat and cholesterol, and fish oi and Perilla oil had an active effect in preventing and treating NAFLD, such as reducing blood lipid, improving liver function, relieving the endoplasmic reticulum stress.Part2: The mechanism of ω-3PUFA in relieving rat BRL liver cells’ endoplasmic reticulum stress, induced by overloaded saturated fatty acidObjective: To explore the mechanism of ω-3PUFA in relieving BRL liver cells’ endoplasmic reticulum stress by establishing the damaged BRL liver cellsMethods: Incubate the BRL cells with different PA concentrations and test its proliferative activity by MTT assay to get an optimal concentration. Set the normal, PA, PA + DHA, PA + EPA, PA + ALA, PA + AA groups. The RNA expression of ATF6, BIP, SERCA, PEMT were detected by RT- PCR.HPLC/MS assay was used to test the components of cell membrane phosphatidylcholine(PC) and phosphatidyl ethanolamine(PE), GC/MS for cell membrane fatty acid composition detection and Fura- 2 am fluorescent probe detection for intracellular free Ca2+ concentration.Results: The best PA incubating concentration was 75 μM, if it surpassed 75μM the cells’ proliferative activity decreased significantly and appeared large-scale apoptosis. Compared with normal group, the mRNA expression of ATF6, XBP- 1, BIP, PEMT were up-regulated in PA group, but SERCA down-regulated. PC/PE in cell membrane was up-regulated, and intracellular free Ca2+ increased significantly. Compared with PA group, the SERCA mRNA expression in ω-3PUFA groups increased but every other indicator decreased accordingly. Compared with normal group, the mRNA expression of ATF6, BIP, PEMT in PA+AA group increased, SERCA decreased, and PC/PE in cell membrane and intracellular free Ca2+ increased, but there was no significant difference compared with PA group.Conclusion: Overloaded PA transformed PE on the cell membrane to PC by up-regulating PEMT expression, and then lead a large number of Ca2+ in endoplasmic reticulum Ca2+ releasing into the cytoplasm, resulting in higher intracellular free Ca2+ concentration, Ca2+ homeostasis imbalance, and endoplasmic reticulum stress further more。Continued endoplasmic reticulum stress led to liver cell damage. The use of ω-3PUFA down-regulated PEMT gene expression and inhibited the transformation of PE to PC, and raised SERCA gene expression at the same time. All of these pumped the free Ca2+ back to endoplasmic reticulum and alleviated the endoplasmic reticulum stress, which had an obvious improvement on hepatocyte damage, and AA had no such a function. |
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