| Objective: To examine the regulatory effects of hazel leaf polyphenols on high-fat-fed obese mice and 3T3-L1 cells and to explore their mechanisms of action.Methods:1.Extraction and antioxidant and anti-inflammatory activity of polyphenols from hazel leaves: free phenols(FP),conjugated phenols(BCP,ACP)and bound phenols(BBP,ABP)were obtained by extraction of 70% methanol and 1% hydrochloric acid(V:V=1:1)in different acid-base states by extraction of ether-ethyl acetate(V:V=1:1),and the total polyphenols and total flavonoids of each component were detected,and the antioxidant capacity was preliminarily detected by DPPH method and FRAP method.The MTT method detected the effects of TBHP and five components on the survival rate of HUVEC cells,the effects of the five components on the survival rate of RAW264.7 cells,the levels of SOD,MDA and ROS of HUVEC cells,and the NO,IL6,IL-1β and TNF-α contents of RAW264.7cells to test their antioxidant and anti-inflammatory abilities.2.Analysis of the main components of hazel leaf polyphenols and pharmacological study of the targeting network: the UPLC-TOF-MS/MS method was used to analyse the main constituents of five extracts of hazel leaf polyphenols,using oxidative damage and inflammation as disease targets,combined with a targeted network pharmacological approach to predict their potential health benefits.3.Effects of hazel leaf polyphenols on obese mice: A high-fat diet was used to establish a model of obesity in mice,except for mice in the blank group(Control group),which were fed a high-fat diet(60% kcal)for 6 weeks.The mice were randomly divided into model group(Model group),orlistat group(30mg/kg),hazel leaf polyphenol high dose group(ZPH group,500mg/kg),hazel leaf polyphenol medium dose group(ZPM group,250mg/kg),hazel leaf polyphenol low dose group(ZPL group,125mg/kg)and administered continuously for 6 weeks.At the end of the experiment,biological X-ray scanning of the fat content of each group of mice;detection of serum TG,TCHO,LDL-C,HDL-C,ALT,AST levels;serum SOD,MDA levels and IL6,IL-1β levels;Hematoxylin-eosin staining(HE),oil red O staining to detect The pathological changes in liver tissue and perirenal white adipose tissue;GC/MS for faecal short-chain fatty acids;sequencing of the V3-V4 region of the16 sr RNA gene for faecal flora composition,species abundance,diversity and differences in intestinal flora composition between groups;Western blot for perirenal white adipose tissue lipid synthesis-related proteins(ACC,SREBP1 c,PPARγ,CEBPα)in perirenal white adipose tissue.4.Effects of hazel leaf polyphenols on 3T3-L1 cells: 3T3-L1 cells were induced to differentiate and divided into model group(Model group),hazel leaf polyphenol high dose group(ZP-H group,50μg/m L),hazel leaf polyphenol medium dose group(ZP-M group,30μg/m L),hazel leaf polyphenol low dose group(ZP-L group,10μg/m L),microscopic observation of cell morphology,oil red O staining The cellular lipid droplet distribution was detected;the cellular TG and TCHO contents were detected;the expression of lipogenic differentiation-related proteins(SREBP1c,PPARγ,CEBPα)and the expression levels of AMPK and ACC phosphorylated proteins were detected by Western blot.Results:1.Five hazel leaf polyphenol fractions(FP,BCP,ACP,BBP,ABP)were isolated,with the highest polyphenol content(14.8 mg GAE/g DW)and flavonoid content(4.76 mg CAE/g DW)of the free phenols,and the DPPH and FRAP assays showed that FP,BCP,ACP,BBP and ABP had antioxidant capacity,with FP having the strongest antioxidant capacity.The anti-inflammatory capacity of FP,BCP,ACP,BBP and ABP was increased by 10 μg/m L of different hazel leaf polyphenol fractions,which reduced the oxidative damage of HUVEC cells caused by TBHP by increasing the SOD content,decreasing the MDA level and reducing the ROS content(P < 0.01,P < 0.05),IL-1β,TNF-α,IL-6(P < 0.01,P < 0.05),thereby reducing inflammatory damage in RAW264.7 cells caused by LPS.2.UPLC-TOF-MS/MS analysis showed that the phenolics in hazel leaves were mainly present in free form and have been identified as being present in various forms of composition,with gallic acid being the most widely distributed phenolic.Pedalitin,chlorogenic acid and gallocatechin are only present in the free phenol,resveratrol and ellagic acid are only present in the conjugated phenol,and kaempferol 3-O-rhamnoside and hydroxybenzoic acid are only present in the bound phenol.Targeted network pharmacology predictions show that hazel leaf polyphenol targets are mainly enriched in cancer,abnormal lipid metabolism and atherosclerosis-related pathways.3.(1)After 6 weeks of high fat diet feeding,the body weight of Control group was24.409±1.141 g,that of Model group was 30.064±0.507 g,that of Orlistat group was29.627±0.752 g,that of ZPH group was 30.155±0.998 g,that of ZPM group was30.245±0.994 g and that of ZPL group was 30.300±1.066 g.Body weight was 30.300±1.066 g and all other groups weighed more than 20% of the weight of the blank group,and the obesity model was successfully constructed.Compared with the Model group,the hazel leaf polyphenol intervention significantly reduced the body weight of obese mice on a high-fat diet and significantly reduced the Lee’s index(P<0.01,P<0.05),but there was no significant difference in the daily food intake of the mice;at the same time,it significantly reduced the weight of liver tissue(P<0.01,P<0.05)and the percentage of fat content of the mice.(2)Compared with the Control group,mice in the Model group had significantly higher serum levels of TG(1.7±0.179 mmol/L,P<0.01),T-CHO(5.295±0.78 mmol/L,P<0.01)and LDL-C(2.371±0.387 mmol/L,P<0.01),ALT(26.058±4.566 U/L,P<0.01),AST(30.759±4.557 U/L,P<0.01)levels were significantly increased and HDL-C levels were significantly decreased(2.267±0.511 mmol/L,P<0.01).Compared to the Model group,the ZPH group showed significantly higher levels of TG(0.949±0.168 mmol/L,P<0.01),TCHO(3.557±0.62 mmol/L,P<0.01)and LDL-C(1.527±0.148 mmol/L,P<0.01),ALT(12.892±1.107 U/L,P<0.01),AST(14.718±3.016U/L,P<0.01)levels were significantly reduced and HDL-C levels were significantly increased(3.208±0.473mmol/L,P<0.01),and the other hazel leaf polyphenol dose groups also showed improvement in lipid profile compared to the Model group,but there was no statistical difference.(3)Compared with the Control group,the Model group showed a significant decrease in SOD level(P<0.01),a significant increase in MDA level(P<0.01)and a significant increase in IL-6 and IL-1β levels(P<0.01).The administration of hazel leaf polyphenols significantly increased the serum SOD level and decreased the MDA level and IL-6 and IL-1β levels in obese mice.(4)The results of HE staining showed that the liver tissue structure of mice in Control group was intact,the morphology of hepatocytes was normal in size,the cells were evenly arranged and tight,no fatty degeneration was seen,and no lipid droplets appeared in the hepatocytes;compared with Control group,the liver cells in Model group were scattered in arrangement,vacuoles of different sizes and numbers appeared,and lipid accumulation occurred in the liver to form lipid droplets;compared with Model group The number of cell vacuoles and lipid droplets was significantly reduced in each of the hazel leaf polyphenol administration groups.the perirenal adipocytes in the Control group were smaller in size,with clear morphology and intact structure;compared to the Control group,the Model group had significantly larger adipocytes,with uneven cell size and irregular arrangement,and within the same field of view,the Model group had significantly fewer adipocytes and significantly larger adipocyte volume;Compared with the Model group,the adipocyte volume decreased to different degrees and the number of cells increased to different degrees in the same field of view in each administration group of hazel leaf polyphenols.The results of oil red O staining showed that the Model group had significantly more red staining of hepatocyte lipid droplets than the Control group,and the hazel leaf polyphenols significantly reduced the red staining of hepatocyte lipid droplets in each administration group.Compared with the Control group,the perirenal adipocytes in the Model group had significantly larger lipid droplet diameters and larger lipid droplet volumes,and the lipid droplet volumes of the cells in each dose group of hazel leaf polyphenols were reduced to different degrees compared with the Model group.(5)Compared to the Control group,the high-fat diet induced a significant decrease in the fecal content of short-chain fatty acids acetic acid,propionic acid and butyric acid in obese mice(P<0.01),which was significantly increased by the administration of hazel leaf polyphenols(P<0.05).The high-fat diet also reduced the abundance,diversity and homogeneity of the intestinal flora in obese mice,and the administration of hazel leaf polyphenols adjusted these changes in the intestinal flora to normal.(6)The expression levels of intracellular lipid synthesis-related proteins SREBP1 c,PPARγ and CEBPα were significantly increased in the Model group compared to the Control group(P<0.01),and the expression levels of the above adipose tissue-related proteins were significantly decreased after the administration of different concentrations of hazel leaf polyphenols compared to the Model group(P<0.01)4.(1)Hazel leaf polyphenols could inhibit 3T3-L1 cell differentiation and reduce lipid droplet formation.Under the microscope,cell morphology was observed.Undifferentiated3T3-L1 cells were fibroblast-like,with flat and narrow cells and a normal pink colour in the microscopic view;after differentiation and maturation,the cells were rounded and rounded lipid droplets appeared,and yellow highlights were observed under the microscope.After induction of differentiation,the cellular TG and TCHO content levels were significantly elevated in the Model group compared to the Control group(P<0.01),and after administration of hazel leaf polyphenols,the cellular TG and TCHO content levels were significantly decreased compared to the Model group(P<0.01).(2)Compared with the Control group,the expression levels of intracellular lipid synthesis-related proteins SREBP1 c,PPARγ and CEBPα were significantly increased in the Model group(P<0.01),and their adipose tissue-related protein expression content was significantly decreased after the administration of different concentrations of hazel leaf polyphenols compared with the differentiated mature 3T3-L1 cell group(P<0.01).(3)Hazel leaf polyphenols significantly increased the p-AMPK and p-ACC protein expression levels in 3T3-L1 cells.p-AMPK levels were significantly lower in the Model group compared to the Control group(P < 0.01),thereby inhibiting ACC phosphorylation and promoting ACC expression,and administration of hazel leaf polyphenols significantly enhanced the expression of AMPK phosphorylation levels(P < 0.01),increased the level of p-ACC expression and reduced cellular lipid formation.Conclusion: Hazel leaf polyphenols have some antioxidant and anti-inflammatory ability,can reduce obese body weight,improve blood lipid levels and inhibit lipid accumulation in 3T3-L1 cells in high-fat diet-induced obese mice,and their mechanism of action may be related to the regulation of SREBP1 c,PPARγ,CEBPα protein expression,and the regulation of AMPK/ACC. |
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