| Objective:Pomegranate Flower is an advanced medicine in Xinjiang. Our experiment is toinvestigate the effects of blood-lipid regulation and possible mechanism of Pomegranateextract--Pomegranate polyphenol. To detect the contents of ellagic acid and gallic acid inthe Pomegranate polyphenol, as well as to investigate the possible effects of these twoactivity materials in the prevention of nonalcoholic fatty liver disease (NAFLD).Methods:1．Model mice with apolipoprotein E (ApoE-/-) deficiency were fed with high fat dietfor8weeks, and established NAFLD animal model.2．The oleic acid content was judged by MTT, which was incubation chang liver cellby0.2mM for24h, then detected intracellular TG level and observed intracellular lipidacceleration by oil red O stain, established NAFLD cellular model.3． Use Pomegranate polyphenol at the dose of140mg·kg-1by intragastricaladministration to the mice after8weeks. Blood glucose, lipid, AST and ALT in plasmawere detected by automatic biochemical analyzer. Plasma insulin level was determinedby ELISA method, then calculated insulin resistance index. Hepatic lipid level wasdetected in hepatic homogenate by automatic biochemical analyzer, the content of ROS,MDA, SOD, GSH-PX were detected by kit. Hepatic pathological changes were observedfrom HE stain. The AMPK, SirT3, ACC2, CPT-1A protein expression level wereobserved by immunohistochemical method. 4．Cellular model was interfered with different content of Pomegranate polyphenolfor24h. Intracellular lipid acceleration was observed by oil red O stain. Cells werecollected for protein quantification. Cellular TG level, activity of SOD, MDA andGSH-PX were detected by kit. Using ELISA method to detect TNF-α and IL-8level.ROS activity was detected by flow cytometry. SirT3, AMPK, ACC2and CPT-1A mRNAexpression were detected by RT-PCR. SirT3, AMPK, P-AMPK, P-ACC2, ACC2andCPT-1A protein expression were detected by Western-blot.5．Content of ellagic acid and gallic acid in the Pomegranate polyphenol weredetected by HPLC.6．Cell model was interfered with different doses of ellagic acid and gallic acid for24h. Intracellular lipid acceleration was observed by oil red O stain. Cells were collectedfor protein quantification. Cellular TG level, activity of SOD, MDA and GSH-PX weredetected by kit. Using ELISA method to detect TNF-α and IL-8level. ROS activity wasdetected by flow cytometry. SirT3, AMPK, ACC2and CPT-1A mRNA expression weredetected by RT-PCR. SirT3, AMPK, P-AMPK, P-ACC2, ACC2and CPT-1A proteinexpression were detected by Western-blot.Results:Level of plasma lipid and hepatic lipid were significantly increased inapolipoprotein E (ApoE-/-) deficiency mice fed with high fat diet, which occurred insulinresistance. Inflammatory cytokines such as TNF-α and IL-8level became higher clearly.All of above fit for the human pathogenetic characteristics of NAFLD, which confirmedsuccessful replication of animal model.Compared with sham group, MTT results demonstrated cellular TG level incubationby oleic acid during the content of0.2～1mM for24h was significant increased (P＜0.01), which was no effect on the normal cell growth. Furthermore, oil red O staindisplayed that cellular lipid deposition also increased compared with sham group,suggesting a successful establish of cellular model.Compared with model group, plasma TC and TG level significantly decreased (P＜0.01), plasma AST and ALT level decreased (P＜0.01), and also occurred in the plasmainsulin content (P＜0.05). Whereas, hepatic TC and TG level decreased significantly (P＜0.05, P＜0.01). SOD and GSH-PX level became higher clearly (P＜0.01) in thehepatic homogenate, meanwhile MDA level decreased (P＜0.01). HE stain resultsdisplayed hepatic cell edema eased in the Pomegranate polyphenol group, lipid droplets in the cytoplasm decreased compared with model group, as well as the inflammatory cellinfiltration. Immunohistochemical results suggested SirT3, AMPK, and CPT-1A proteinexpression increased in Pomegranate polyphenol group.According to the MTT method, we screened a dose of Pomegranate polyphenolwhich had no effect on cell growth interfered with model cell for24h. Compared withmodel group, cellular TG level significantly decreased (P＜0.05, P＜0.01), cellular lipiddroplets also decreased by oil red O stain. Moreover, cellular MDA decreased clearly (P＜0.05, P＜0.01), SOD and GSH-PX level increased significantly (P＜0.05, P＜0.01),ROS, TNF-α and IL-8level was also decreased (P＜0.01, P＜0.01). SirT3and CPT-1AmRNA expression level were much higher than model group (P＜0.05, P＜0.01), ACC2mRNA expression level were much lower than model group (P＜0.01). Furthermore,SirT3and CPT-1A protein expression level significantly increased than model group (P＜0.01), the degree of phosphorylation in AMPK and ACC2increased clearly (P＜0.01).HPLC results demonstrated gallic acid content in the Pomegranate polyphenol was1.91mg·g-1, ellagic acid content was12.09mg·g-1. We screened each of gallic acid andellagic acid content had no effect on cell growth interfered with model cell for24h. Theresults demonstrated that compared with model group, cellular TG level significantlydecreased (P＜0.05, P＜0.01) in the gallic acid and ellagic acid group, cellular lipiddeposition also decreased by oil red O stain. Moreover, cellular MDA decreased clearly(P＜0.05, P＜0.01), SOD and GSH-PX activity increased significantly (P＜0.05, P＜0.01). ROS level decreased only in the ellagic acid group (P＜0.01). TNF-α and IL-8level was also decreased in both groups (P＜0.01). SirT3, CPT-1A and AMPK mRNAexpression level were much higher than model group (P＜0.05, P＜0.01), ACC2mRNAexpression level were much lower than model group (P＜0.01). Furthermore, SirT3andCPT-1A protein expression level significantly increased than model group (P＜0.01), thedegree of phosphorylation in AMPK and ACC2increased clearly (P＜0.01).Conclusion:Replication and establishment of NAFLD animal and cellular model.We established NAFLD animal model and cellular model in our research, in whichmice with apolipoprotein E (ApoE-/-) deficiency were fed with high fat diet, and changliver cells were incubation with oleic acid. Results demonstrated that the pathogeneticcharacteristic in model animal and cell were both similar with human NAFLD, such asincreased lipid level, hepatic pathological changes, insulin resistance production, imbalance of antioxidant and oxidative stress system and so on. Thus, we concludedmodel was established.Pomegranate polyphenol had a good effect of lipid regulation and hepaticprotection.Pomegranate polyphenol decreased plasma lipid and hepatic lipid level effectivelyof model animal and significantly decreased the intracellular acceleration of TG in thecellular model, which proved Pomegranate polyphenol had a good effect of lipidregulation. As well, in the model animal AST and ALT level became lower suggesting ahepatic protection effect in the NAFLD animal.The possible mechanism of lipid regulation effects of Pomegranate polyphenol:Relieve insulin resistance of Pomegranate polyphenolAntioxidant effect of Pomegranate polyphenolDecreased inflammatory cytokines production significantly such as TNF-α and IL-8,relieved the development of NAFLDPomegranate polyphenol relieved TG acceleration in the hepatic cell may throughimproving SirT3level and degree of phosphorylation in AMPK resulted in ACC2phosphorylation increased after AMPK activation, which leaded to ACC inactivation,relieved CPT-1A inhibition from rate limiting enzyme ACC2in the fatty acid oxidationprocess, then strengthen fatty acid oxidation and fatty acid synthesis decreased.Effect of lipid regulation of Pomegranate polyphenol may related to containing ofellagic acid and gallic acidHPLC results demonstrated gallic acid content in the Pomegranate polyphenol was1.91mg·g-1, ellagic acid content was12.09mg·g-1. Through cellular experiment we foundthat lipid regulation effect and influence of inflammatory cytokines and lipid metabolismrelated enzyme gene expression of gallic acid and ellagic acid were very similar withPomegranate polyphenol. Thus, we concluded that lipid regulation effect of Pomegranatepolyphenol may be associated with these two active substance gallic acid and ellagicacid. |
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