| The aims of present study was to investigate the effect of persimmon tannin on nonalcoholic fatty liver disease (NAFLD) and peradipocyte differentiation and to confirm whether persimmon tannin accounts for the hypolipidemic effect of persimmon and its extract. The underlying mechanisms were also explored. The hypolipidemic effect of persimmon tannin was evaluated both in a high-cholesterol diet fed rats and gastrointestinal environment. Persimmon tannin was confirmed to be the main component associated with the anti-hyperlipidemic effect of consuming persimmon in high-fat diet (lard and cholesterol) induced rats’NAFLD. The mechanisms of persimmon tannin against NAFLD based on AMP-activated protein kinase (AMPK) pathway by real time RT-PCR and western blot. Third, we studied the effect of persimmon tannin and its characteristic units on oleic acid induced hepatic steatosis in normal human liver cells L02and its underlying mechanism. The relationship between persimmon tannin structure and its hypolipidemic effect was also confirmed. Finally, the effect and mechanism of persimmon tannin and its characteristic units on preadipocyte differentiation were also studied. The results were showed as follows:1. The hypolipidemic effect of persimmon in vivo and in vitroTo investigate the hypolipidemic effect of persimmon tannin, rats were fed a high-cholesterol diet and with or without persimmon tannin treatment and serum and hepatic lipids were checked. The results indicated that persimmon tannin effectively reduced serum and hepatic triglyceride (TG), total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C) while increased high density lipoprotein cholesterol (HDL-C)(p<0.05). Morphology showed that accumulation of hepatic lipid was inhibited markedly by persimmon tannin. In addition, persimmon tannin treatment could enhance the serum lecithin cholesterol acyl transferase (LCAT) activity and fecal bile acids excretion. Besides, the results also suggested persimmon tannin could reduced the lipid peroxidation which induced by high-cholesterol diet while the serum antioxidant enzyme activities were increased by administration of persimmon tannin. The effect of persimmon tannin on inhibition of pancreatic lipase and pancreatic cholesterol esterase activities and cholesterol micelle as well as bile acid binding, cholesterol binding were evaluated in vitro model systems, The results indicated that persimmon tannin could inhibit pancreatic lipase (IC50,0.445mg/mL) activity by an un-competitive way as well as cholesterol esterase (IC50,0.442mg/mL). Persimmon tannin could also decrease the cholesterol micellar solubility and showed potent bile acid-binding activity, which resulted in poor absorption of lipids in intestinal tract. These results indicated that persimmon tannin may exert the anti-hyperlipidemic activity through inhibition of pancreatic lipase and pancreatic cholesterol esterase, inhibition of cholesterol micellization and binding bile acids.2. Persimmon tannin is the main componentof persimmon fruit accounting for improvement of NAFLD through AMPK pathwayTo investigate the effect and mechanism of persimmon tannin and persimmon fruit on NAFLD, rats were fed a high-fat diet (HF,38%calorie come from fat) in the presence or absence of persimmon tannin and4.2%of lyophilized fresh persimmon fruit (with the same diet persimmon tannin content in the two groups) for9weeks. Administration of persimmon tannin or persimmon fruit significantly (p<0.05) lowered serum TG and free fatty acid, enhanced HDL-C level and the excretion of TG, TC and bile acids, and improved hepatic steatosis and inflammatory cell infiltration in rats fed a high-fat diet. The results of real time RT-PCR indicated that dietary PERSIMMON TANNIN or persimmon fruit could significantly reduce (p<0.05) genes involved in lipogenesis, including sterol regulatory element binding protein1(SREBP1), Acetyl Co A carboxylase1(ACC1) and stearoyl CoA desaturase1(SCD1). While the genes involved in fatty acid oxidation, such as carnitine palmitoyltransferase-1(CPT-1), were notably up-regulated (p<0.05). Western blot results showed that treatment of persimmon tannin or persimmon fruit significantly increased (p<0.05) AMPK phosphorylation levels. Moreover, the concentration of inflammatory factor and protein levels of nuclear factorkB (NFkB) were significantly suppressed (p<0.05) by persimmon tannin and persimmon fruit. Taken together, persimmon tannin is the main component of persimmon fruit for improvement of high-fat diet induced hepatic steatosis and chronic inflammatory response through AMPK pathway.3. Persimmon tannin regulated simple fat accumulation in normal human liver cells L02through miR-122and miR-33bThe effect of persimmon tannin and its characteristic units on hepatic steatosis in normal human liver cells L02induced by0.4mmol/L oleic acid and the mechanisms were studied. The results indicated that persimmon tannin and its characteristic units decreased intracellular TG accumulation effectively, the inhibition of fat accumulation by A type ECG dimer and A type EGCG dimer were similar to persimmon tannin, but stronger than A type EC dimer and B type EC dimer. Persimmon tannin and its characteristic units reduced the expression of SREBP1C and fatty acid synthetase (FAS) in L02cells, but didn’t increase the expression of CPT and AMPK phosphorylation levels. These results indicated that persimmon tannin repressed lipid accumulation in L02cells not through AMPK. In addition, persimmon tannin and its characteristic units could repress the expression of miR-122and miR-33b and their downstream genes involved in SREBP1C and FAS in human hepatocytes L02. The inhibition begin at3h by treatment of persimmon tannin while dimers begin at0.5h. In conclusion, persimmon tannin repressed hepatic steatosis in L02cells through mediated miR-122and miR-33b, these effects were due to persimmon tannin units ECG, EGCG, and the A type interflavan linkages.4. Persimmon tannin inhibited3T3-L1preadipocyte differentiation via miR-27a/bThe inhibition rato of persimmon tannin (60μg/mL) on3T3-L1preadipocyte differentiation was68.6%, and the inhibition effect was largely limited to the early stage of adipocyte differentiation, which resulted in delay cell clone expansion. The expression of transcription factor peroxisome proliferatoractivated receptory (PPARy), CCAAT/enhancer binding proteina (C/EBPa) and SREBP1C and their downstream genes, such as FAS and ACC1, were reduced by persimmon tannin during preadipocyte differentiation. Persimmon tannin could not activate AMPK, and the inhibitory effect of persimmon tannin on preadipocyte differentiation was not abolished by compound C. furthermore, the expression of miR-27a and miR-27b was up-regulated by persimmon tannin, while PPARy, the target gene of miR-27, was down-regulated. Collectively, Persimmon tannin inhibited3T3-L1preadipocyte differentiation and reduced fat accumulation via miR-27a/b-PPARy pathway.5. Persimmon tannin characteristic units inhibited3T3-L1preadipocyte differentiation via miR-27a/bA type ECG dimer and A type EGCG dimer, the main units of persimmon tannin, showed potent inhibition on3T3-L1preadipocyte differentiation. And the inhibitory action of the two dimers were largely limited to the early stage of adipocyte differentiation, which resulted in delay mitotic clonal expansion (MCE). A type EC dimer could also inhibit preadipocyte differentiation, but the inhibitory effect was not strong as A type ECG dimer and A type EGCG dimer. B type EC dimerhad no effect on preadipocyte differentiation. Supplementation with A type ECG dimer and A type EGCG dimer increased the expression of miR-27a and miR-27b, this, in turn, results in inhibition of expression of PPARy, and then down-regulated lipogenesis, including FAS and SCD1, blocked preadipocyte differentiation. These results indicated that the inhibitory effect of persimmon tannin on preadipocyte differentiation was closely associated with the structure that rich of A type ECG dimer and EGCG dimer. |
PDF Full Text Request