| Adipose tissue is formed by a combination of proliferation,differentiation and apoptosis of pre-adipocytes.Obesity and its related diseases are caused by excessive growth of adipose tissue.Therefore,regulation of differentiation of adipocytes and regulation of degradation of mature adipocytes are key points in the study of obesity and related diseases.Lipid droplets(LD)act as subcellular structures in cells and are present in somatic cells of all organisms.In adipocytes,lipid droplets have long been considered only as a storage structure for lipids and are a reservoir for providing energy to the body.It also provides lipid raw materials for the synthesis of biomacromolecules,such as neonatal cell membranes and lipoproteins,but studies in the past decade have shown that the diversity of lipid droplets far exceeds people's imagination.In the regulation of fat metabolism,lipid droplets are not passive storage sites,but highly active and important organelles.Scientists have revealed the diversity of lipid droplets by studying fat-related diseases.Related diseases caused by abnormal lipid droplets include obesity,chlamydial infection,diabetes,atherosclerosis and liver diseases,and the starting point of these diseases is the disorder of lipid metabolism.Cordycepin(3'-deoxyadenosine)was originally isolated from cordyceps militaris.It has a good development prospect as a natural compound for weight loss and lipid-lowering.It has the advantages of multiple targets,multiple pathways,and no toxic side effects.And it has a variety of biological and pharmacological activities,including hypoglycemia,anti-diabetes,anti-cancer,immune stimulation,anti-viral,and anti-infection.Due to its broad range of pharmacology and treatment,it has received more and more attention.The previous study of our group found that cordycepin also significantly reduced the fat droplet content in fat cells while losing weight.Can Cordycepin regulate fat cells by inhibiting fat cell fat droplet formation,growth and inducing degradation of fat droplets? Lipid metabolism is a scientific issue worth exploring.This study firstly evaluated the weight loss effect of cordycepin and the effects of different adipose tissue(white fat,yellow fat and brown fat)and blood lipid and lipid droplets in adipocytes by constructing a rat obesity model;then 3T3-L1 fat precursor cells were used,as a model cell of fat metabolism,whether cordycepin can inhibit lipid droplets formation,growth and induce lipid droplet degradation.Finally,the 3T3-L1 cells were used to study the possible mechanism of the effect of cordycepin on the lipid metabolism of adipocytes in vitro.First,we established an animal model of obesity in a representative disease of adipocytes lipid metabolism disorder by administering high fat diet to SD rats.The rats were given low,medium and high doses(12.5,25,50 mg/kg)of cordycepin.After 8 weeks of continuous gavage,the body weight,fat coefficient and related biochemical parameters were measured,and liver tissue and adipose tissue were observed.Pathological changes.Rat adipocytes were extracted,white fat,yellow fat and brown fat were labeled with ASC-1,PAT2 and P2RX5,respectively,and the change of fat droplet content was detected by BODIPY staining.The results showed that rats given high dose(50 mg/kg)of cordycepin could significantly reduce body weight and fat coefficient(P<0.01),and rats with medium dose(50 mg/kg)cordycepin could be significantly reduced.Body weight and fat coefficient(P<0.05),while low dose of cordycepin(12.5 mg/kg/d)did not reduce the body weight and fat coefficient of rats.Cordycepin significantly reduces serum triglyceride(TG),cholesterol(TC),low density lipoprotein(LDL),aspartate aminotransferase(AST),alanine aminotransferase(ALT),malondialdehyde(MDA)and peripheral fat The content of drip protein(Perilipin1,Perilipin2,Perilipin3)(P<0.01),and significantly increased the activity of superoxide dismutase(SOD)(P<0.01).The high dose of cordycepin(50 mg/kg)significantly increased high-density lipoprotein(HDL)(P<0.01),and the middle-dose group(25 mg/kg)significantly increased high-density lipoprotein(HDL)(P<0.05).The HE staining results of liver pathological sections showed that low,medium and high doses(12.5,25,50 mg/kg)of cordycepin could protect the liver damage induced by high fat diet.HE staining results of fatty pathological sections showed that low,medium and high doses(12.5,25,50 mg/kg)of cordycepin can significantly inhibit the growth of fat cells;cordycepin can also significantly reduce white fat cells in a dose-dependent manner The content(P<0.05)significantly promoted the burning of beige fat cells(P<0.01)and significantly increased the content of brown fat cells(P<0.01).This indicates that cordycepin can accelerate the conversion of adipocytes,thereby promoting the consumption of fat.In addition,the results of lipid droplets from adipocytes in the body showed that low,medium and high doses(12.5,25,50 mg/kg)of cordycepin decreased the content of fat droplets in fat cells in a dose-dependent manner.Secondly,we cultured 3T3-L1 cells in vitro with inducer 1(DMEM medium containing IBMX,insulin,dexamethasone,penicillin and 10% fetal bovine serum)and inducer 2(DMEM medium containing insulin,penicillin and 10% fetal bovine serum)inducing differentiation of 3T3-L1 cells into mature adipocytes.Cordycepin was added at different time points of the inducing agent to detect the effects of cordycepin on the three stages of fat droplet formation,growth and degradation.Cordycepin was given different concentrations(1.563,3.125,6.25,12.5,25 ?g/mL)for 168 h,stained with BODIPY,detected by fluorescence microscopy,and the content of fat droplets was quantified by Image J software.The experimental results show that cordycepin significantly inhibits the formation and growth of lipid droplets in a concentration-dependent manner,and induces lipid droplet degradation in mature adipocytes to promote dedifferentiation of fat cells.Finally,for inducer 1(DMEM medium containing IBMX,insulin,dexamethasone,penicillin,and 10% fetal bovine serum)and inducer 2(DMEM medium containing insulin,penicillin,and 10% fetal bovine serum)induces the differentiation of 3T3-L1 cells into mature adipocytes,the expressions of Perilipin2,Perilipin3,Fsp27 and PPAR-? related to lipid droplets formation were detected by western blot;the expressions of Rab5,Rab11 and Fsp27 related to lipid droplets growth were detected by western blot;the expression of ATGL,CGI-58 and Perilipin1 related to fat droplet degradation was detected by western blot.The results showed that cordycepin significantly inhibited the formation of lipid droplets by down-regulating the expression of Fsp27,Perilipin2 and Perilipin3,whereas PPAR-? was not involved in this process.The protein expression of Rab5 was slightly decreased after treatment with 6.25 ?g/mL,12.5 ?g/mL and 25 ?g/mL cordycepin;Rab11 expression level was significantly decreased after treatment with 6.25 ?g/mL and 25 ?g/mL cordycepin;after treatment with 25 ?g/mL cordycepin,the protein expression of Fsp27 was significantly reduced.This indicates that cordycepin inhibits the growth of lipid droplets in differentiated 3T3-L1 cells by modulating Rab5,Rab11 and Fsp27.The treatment with cordycepin resulted in a significant decrease in the expression levels of Perilipin1 and CGI-58.Conversely,the expression of ATGL was significantly increased,indicating that cordycepin can exert a degradative effect by increasing ATGL expression and decreasing Perilipin1 expression rather than by affecting CGI-58 expression.These results indicate that cordycepin inhibits the formation of lipid droplets,the expression of growth-related genes in adipocytes,inhibits and induces the expression of genes related to lipid droplets degradation,thereby affecting the content of lipid droplets and regulating the lipid metabolism of adipocytes.In conclusion,the results of this study indicate that cordycepin has a weight-loss effect on obese model rats;the mechanism by which cordycepin regulates lipid metabolism in adipocytes in vitro is achieved by affecting the content of fat droplets,including down-regulating the expression of Fsp27,Perilipin2 and Perilipin3 to reduce the formation of lipid droplets;down-regulation of the expression of Rab5,Rab11 and Fsp27 inhibited lipid droplets differentiation;increased ATGL expression and decreased Perilipin1 expression to degrade lipid droplets.These results provide an experimental basis and theoretical basis for the further development of cordycepin as a new weight-loss drug. |
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