The Effect Of DHA On Adipocytes Apoptosis And The Molecular Mechanism In Grass Carrp (Ctenopharyngodon Idellus)

Adipose tissue is one of the main energy storage organs in animals.Its main function is to store excess energy accumulated in the body in the form of triglycerides.Adipocytes,as the main constituent unit of adipose tissue,are the basic units for storing triglycerides.Docosahexaenoic acid(DHA),one of the main n-3 long-chain polyunsaturated fatty acids(LC-PUFAs),plays an inmportant role in growth and development,immune regulation,lipid metabolism.Studies have shown that DHA can regulate fish lipid accumulation,but the underlying mechanism is not clear.This rsearch aimed to study the effect and potential mechanism of DHA on lipid accumulation and adipocytes apoptosis in grass carp,and provide some references for the study of lipid accumulation in freshwater fish.The main results of this study are as follows:1)Dietary 0.52% DHA decreased lipid accumulation in grass carp.Diets containing 0%(control)and 0.52%(test)of DHA,which were fed to fish for 3 and 6 weeks,fatty acid composition analysis was used to determine by gas chromatography,hematox-ylin and eosin(H&E)staining was used to observe the morphology of adipocytes.Triglyceride kits were used to detect adipose tissue triglyceride level,mRNA levels of lipid metabolism related genes were used to detected by RT-PCR,lipid metabolism related protein expression levels were used to detect by western blot.The results showed that DHA significantly decreased the IPFI and triglyceride(TG)content of grass carp,but larger the adipocytes.Further,DHA significantly up-regulated the expression of adipogenesis-related genes and apoptosis-related genes and protein expression levels.2)DHA induced adipocyte apoptosis in a PPARγ dependent manner in grass carp.The adipose tissue of grass carp was isolated,preadipocytes were obtained andinduced to differention.Grass carp adipocytes were treated with DHA at different concentrations(0,25,50,100,200,400 μM)for 24 h or treated with 200 μM DHA for different times(0,12,24).Besides,adipocytes were exposed with 200 μM DHA and the presence of PPARγ antagonist or the inhibitor of certain key enzymes of apoptosis,CCK-8 was used to detect the adipocyte viability,flow cytometry was used to detect the cell apoptosis rate,RT-PCR was used to detect the mRNA level of apoptosis-related genes,western blotting was used to detect the protein expression levels of apoptotic gene,caspase 3/8/9 enzyme activity kit was used to detect the enzyme activity of caspase3/8/9.These results showed that DHA induced the adipocytes apoptosis in a time-and dose-dependent manner in grass carp.DHA significantly up-regulated the gene and protein expression levels of PPARγ arrcording the results of quantitative PCR and WB.After co-incubation with PPARγ inhibitor and DHA,PPARγ inhibitor significantly attenuated DHA-induced apoptosis in grass carp adipocytes.Further study found that caspase 9 inhibitor significantly inhibited DHA-induced apoptosis.In conclusion,DHA can reduce the lipid accumulation of grass carp by inducing apoptosis of grass carp adipocytes,and further,we found that that PPARγ may fully or at least partially mediate the DHA pro-apoptotic effects via the mitochondrial apoptotic pathway in grass carp preadipocytes.