Eurycomanone Stimulates Lipolysis In 3T3-L1 Adipocytes

As people's living standards gradually increase,the number of obese people is also increasing year by year.The incidence of obesity in the world has grown at an alarming rate and has become a major threat to human health.Obesity promotes the development of hypertension,diabetes,stroke,osteoarthritis and cancer.Therefore,an effective method for prevention and treatment is sought.Obesity is urgent and necessary.At present,it is generally believed that the cause of obesity is due to the imbalance of energy absorption and energy expenditure in the body,which leads to the storage of excess energy in the form of lipids in white adipose tissue(WAT).The more lipids are stored,the more lipids are stored.The larger the drop,the more adipocytes are formed,and the more fat the body becomes.Therefore,the key to inhibiting obesity is to inhibit the differentiation of pre-adipocytes and promote the decomposition of mature adipocytes.Eurycoma lon gifolia Jack is abundant in Malaysia.It is also known as the three national treasures in the same place as yanwo and tin.It has good lipolysis effect.In addition,it has anti-cancer,anti-malarial and male sexual function.Obstacles and other effects.The lignin diterpenoid is one of the main chemical constituents of Tongkat Ali,and Eurycomanone(EN)is one of its main active ingredients.At present,the mechanism of lipid lowering of EN is not clear,which also limits the further development and utilization of this component.Objective: To establish an in vitro 3T3-L1 adipocyte model to study the effects of EN on lipolysis and its regulation mechanism to understand its effects on fat differentiation and metabolism.It provides a scientific basis for the utilization of EN resources and lays a theoretical foundation for the development of EN into new drugs.Methods:(1)To promote the decomposition of triglyceride in adipocytes and the inhibition of differentiation of 3T3-L1 preadipocytes into mature adipocytes by detecting EN,and observe mouse 3T3-by microscopic pHotograpHing and oil red O staining.Changes in cell morpHology and lipid droplet formation during differentiation of L1 preadipocytes.(2)The mechanism of fat decomposition of mature fat cells by EN was fully explained by immunofluorescence,Western blottin,real-time PCR,siRNA cell transfection,and(Q-PCR).Results:(1)When EN concentration was ?200 ?M,the proliferation of mouse 3T3-L1 preadipocytes was normal within 72 h after EN treatment,and the cell survival rate was about 90%,and the blank control group(NC)There was no significant difference(p>0.05).The effect of EN on mature adipocytes was detected by trypan blue.The results showed that when EN?200 ?M,the survival rate of mature adipocytes was about 100% within 24 h after EN treatment.(2)Oil red O staining,it was observed that the area of the NC group stained with oil red O was larger than the EN group,and then the oil red O in the dyed cells was re-dissolved with isopropyl alcohol,and the oil red O in the EN and NC groups was quantitatively detected.As a result,the detected value of the NC group is larger than the value of the EN group.The differentiation of adipocytes was observed by laser confocal scanning microscopy.The pHotograpHed results were consistent with the results of oil red O detection.(3)EN significantly promoted the lipolysis of adipocytes.The expression levels of PKA,HSL,AKT and AMPK phosphorylated proteins were significantly increased after drug treatment.After H89 treatment,the expression levels of PKA and HSL phosphorylated proteins were significantly decreased,and H89 was inhibited.It can significantly inhibit the decomposition of fat promoted by the drug EN to a certain extent.Real-time quantitative PCR was used to detect the mRNA expression of fat metabolism-related genes in each group.The results showed that the key genes C-AMP(Cyclic Adenosine monop hosp hate)and PKA(Protein kinase A)that promote the decomposition of triglycerides during the whole process of adipocytes after EN treatment The mRNA and protein expression levels of HSL(Hormone-sensitive lipase)were significantly upregulated,and the expression of Adipose triglyceride ATGL(Adipose tri glyceride lipase)mRNA was not significantly affected.siRNA was transfected into adipocytes to reduce the expression of PKA-CA gene.After EN treatment,PKA-CA and HSL were up-regulated,but PKA-CB gene was not affected.Conclusion: In summary,EN significantly stimulates the lipolysis of mouse adipocytes and effectively inhibits the differentiation of preadipocytes.The cAMP/PKA pathway and the AKT/PDEG-3B pathway are involved in EN-stimulated lipolysis.At the same time,it can inhibit the differentiation of 3T3-L1 preadipocytes into mature adipocytes.This study established a scientific research foundation for the development and application of obesity.