Research On The Effects Of Curcumin On The Proliferation And Adipogenic Differentiation Of Porcine And Mouse Preadipocytes

In recent years,with the continuous improvement of people’s attention to food safety and ecological environment,the problems of antibiotic residues and antibiotic resistance in bacteria are gradually exposed,and the prohibition of antibiotics will be an inevitable trend in the development of animal husbandry and veterinary industry.Therefore,it is urgent to develop green and safe feed additives to replace antibiotic.Curcumin(Cur)is a phenolic substance extracted from the traditional Chinese medicine Curcumin.As a multi-effect natural active ingredient,Curcumin is expected to replace antibiotics and plays an important role in non-resistant breeding of livestock and poultry.Although curcumin has been widely used in food industry as a common natural pigment for a long time,there are relatively few studies on its application in animal husbandry and veterinary production,especially on the effect of reducing fat deposition.In the present study,porcine and mouse 3T3-L1 preadipocytes were used as in vitro models to investigate the effects of curcumin on the proliferation and adipogenic differentiation.In addition,Obese C57BL/6J mice induced by high-fat diet were used as in vivo models to further investigate the effects of curcumin on fat deposition reduction and the improvement effect on obese mice induced by high-fat diet.These results will provide scientific basis for curcumin as a safe and effective feed additive in animal husbandry and veterinary production.Part 1.Effect of Curcumin on Proliferation and Adipogenic Differentiation of 3T3-L1 Mouse PreadipocytesMouse 3T3-L1 preadipocytes were cultured until reached 80%-90%confluence,differentiation induction solution I containing 0.1 mmol/L 3-isobutyl-l-Xanthine(IBMX),2.5μmol/L dexamethasone(DEX),1 μg/mL Insulin and 2 μmol/L Rosigitazone was firstly used to induce adipogenic differentiation,meantime,different concentration of curcumin was added to the medium,according to the different dosages of curcumin used,the cells were divided into 6 groups:①Control group with no differentiation medium(None,normal culture medium);②Control group with differentiation medium(DM,differentiation medium+curcumin dissolving reagent DMSO);③Differentiation medium+2.5 μmol/L curcumin group(DM+2.5 μmol/L Cur);④Differentiation medium+5 μmol/L curcumin group(DM+5 μmol/L Cur);⑤Differentiation medium+10 μmol/L curcumin group(DM+10 μmol/L Cur);⑥Differentiation medium+15 μmol/L curcumin group(DM+15 μmol/L Cur);After the co-treatment with the differentiation medium Ⅰ and curcumin for 3 days,the cells were then induced differentiation continually by the differentiation medium Ⅱ containing only 1 μg/mL Insulin for another 6 days,the medium was changed once every 2 days,the amount of lipid droplets deposited is observed under a microscope after 9 days of total treatment,and CCK method was used to detect the cell proliferation activity,the TG content in cells is detected by Oil Red O staining and TG quantitative detection kit.The contents of glycerol and FFA in cell supernatant were determined by glycerol content determination kit and mouse FFA ELISA kit,and the effects of curcumin on proliferation and adipogenic differentiation of 3T3-L1 mouse preadipocytes were comprehensively evaluated by the above indexes.In addition,the effect of curcumin on the expression of adipogenic differentiation related genes of 3T3-L1 cells was further detected by q-PCR,to preliminarily determine the molecular mechanism of curcumin on adipogenic differentiation.The results showed that 2.5,5,10 and 15μmol/L curcumin treatment had no significant effect on cell viability when compared with DM cells,while 20 μmol/L curcumin treatment could significantly inhibit the cell proliferative activity.Microscopic observation showed that 2.5,5,10,and 15 μmol/L curcumin treatment could significantly reduce the lipid droplet deposition.Oil Red O staining results showed that TG deposition in curcumin treatment group was significantly reduced in a dose-dependent manner when compared with DM cells.In addition,curcumin treatment showed a significant dose-dependent inhibition of the contents of glycerol and FFA in the cell supernatant.These above results showed that curcumin treatment can significantly inhibit 3T3-L1 preadipocytes adipogenic differentiation to some extent.q-PCR results showed that curcumin treatment can significantly inhibit the expression of adipocyte differentiation-related marker genes C/EBP-α,PPAR-γ and C/EBP-β,as well as fat synthesis marker gene FAS,SCD and aP2,while significantly increased the mRNA expression of lipolysis marker genes ATGL.These results showed that curcumin reduced lipid accumulation of 3T3-L1 preadipocytes is not only related to the inhibited adipogenic differentiation and fat synthesis,but also related to the enhanced lipolysis.Part 2.Effect of Curcumin on Proliferation and Adipogenic Differentiation of Porcine PreadipocytesSubcutaneous adipose tissues in the neck and back of weaned piglets were seperated,and preadipocytes were obtained by type I collagenase digestion.When the cells reached 80%-90%fusion,the differentiation medium with the final concentration of 0.1 mmol/L IBMX,2.5 μmol/L DEX,1 μg/mL Insulin and 2 μmol/L Rosigitazone was used to induce adipogenic differentiation and curcumin was added at the same time.According to the concentration of curcumin,cells were divided into 5 groups:①Control group with only differentiation media and curcumin dissolving reagent DMSO(DM);②Differentiation media plus 2.5 μmol/L curcumin group(DM+2.5 pmol/L Cur);③Differentiation media plus 5 μmol/L curcumin group(DM+5 μmol/L Cur);④Differentiation media plus 10 μmol/L curcumin group(DM+10 μmol/L Cur);⑤Differentiation media plus 15 μmol/L curcumin group(DM+15 μmol/L Cur);The CCK method was used to detect the cell proliferation activity,the TG content in cells was detected by Oil Red O staining,and the mRNA expressions of adipogenic marker genes PPAR-y and C/EBP-β,fatty synthesis key enzymes FAS and ACC and lipolysis rate limiting enzymes ATGL and LPL were detected by quantitative PCR.The enzyme activity of lipolytic enzymes in cells was determined by enzyme chemistry.CCK results showed that when compared with DM cells,2.5,5,10 μmol/L curcumin treatment for 48 h and 72 h had no significant effect on the proliferation activity of porcine preadipocytes,but 20μmol/L curcumin treatment for 48 h and 72 h significantly inhibited the proliferation activity of cells.Oil Red O staining results showed that when compared with DM cells,10 μmol/L curcumin treatment for 48 h and 72 h significantly reduced the TG content in adipocytes.In addition,microscopic observation also found that when compared with DM cells,the amount of lipid droplets deposited in 10 μmol/L curcumin treated cells was significantly reduced.These above results showed that curcumin also had inhibitory roles on the proliferation and adipogenic differentiation of porcine preadipocytes in a dose-and time-dependent manner.q-PCR results showed that when compared with DM cells,10 μmol/L curcumin treatment significantly reduced the mRNA expression of PPAR-y and C/EBP-β,two important transcription factors during adipogenic differentiation.In addition,the mRNA expression of ACC and FAS,two important synthetases during fat synthesis,were also significantly decreased.Furthermore,curcumin treatment can significantly increase the mRNA expression and enzyme activity of lipolytic rate-limiting enzymes HSL and ATGL.In addition,the 10 μmol/L curcumin treatment can significantly increase the mRNA and protein expression of the autophagy marker gene LC3,and the ratio of LC3 II and LC3I,as well as the mRNA expression of lysosome acid lipase LIPA.These above results indicate that curcumin inhibits adipogenic differentiation of porcine preadipocytes,may be related to the enhanced lipolysis,the inhibited fat synthesis and autophagy activation mechanism.Part 3.Effect of Addition of Curcumin to Daily Food on Fat Deposition of High Fat Diet Induced C57BL/6J Obese Mouse ModelSixty C57BL/6J male mice(4 weeks old,15-17g)with similar breeding environment,genetic background,etc.were select and randomly divide into 5 groups.The mice were feeded 12 weeks with normal diet(ND),high fat diet(HFD),high fat diet plus 0.1%curcumin(HFD+0.1%Cur),high fat diet plus 0.5%curcumin(HFD+0.5%Cur)and high fat diet plus 1%curcumin(HFD+1%Cur)respectively.Body weight(BW)and food intake were recorded weekly,and the oral glucose tolerance tests(OGTT)were conducted in 4,8,and 12 weeks,and the area under the curve(AUC)were calculated.At the end of the experiment,the BW of the mice was recorded and the weight gain was calculated,the weight of epididymis fat and the ratio of epididymis fat weight/BW,the weight of the liver,heart,spleen,kidneys,testicles,gastroenteric muscle and soleus muscles were recorded and the ratios between organs and body weight were calculated.The content of biochemical indicators related to lipid metabolism were detected.HE staining was used to determine the amount of lipid droplets deposited in the epididymal adipose and liver tissues,and pathological analysis was performed on the tissue samples of the heart,spleen,and kidneys.The results showed that when compared with HFD mice,HFD plus Cur treatment could significantly reduce the body weight and body weight gain,and significantly inhibit the epididymal adipose tissue weight and the ratio of epididymal adipose to body weight,and the inhibition effect presented a dose-dependent manner.Furthermore,food intake and feed utilization rate showed no difference among HFD mice and HFD plus Cur groups.The statistics results of relative organ weight showed that when compared with HFD mice,HFD plus Cur treatment had no significant effect on the liver,heart,spleen,kidney,testis,gastriculus muscle and testis weight,but significantly increased the weight of soleus muscle,liver/body weight and soleus muscle/body weight.In addition,when compared with HFD mice,HFD+0.1%Cur,HFD+0.5%Cur treatment has no obvious effect on FBG,but HFD+1%Cur treatment significantly decreased the FBG level.OGTT results showed that when compared with HFD mice,HFD+0.1%Cur,HFD+0.5%Cur and HFD+1%Cur treatment can inhibited the blood glucose concentration to varying degrees,and significantly inhibited the AUC value,and the inhibition effect presented a dose-dependent manner.Blood biochemical indexes showed that low concentration of Cur significantly inhibited LDL-c content,and high concentration of Cur significantly inhibited plasma TG,Glucose,TCH and NEFA content,but had no significant effect on AST,ALT and AST/ALT.HE results showed that treatment with HFD+0.1%Cur.HFD+0.5%Cur and HFD+1%Cur could significantly reduce the number of lipid drops in epididymal adipose tissue and liver tissue,as well as the volume of adipose cells in epididymal adipose tissue,but had no significant pathological changes in heart,spleen,kidney,gastriculointestinal muscle and soleus muscle.These above results showed that dietary curcumin could significantly inhibit epididymal fat deposition and reduce blood lipid content,and had a good reversing effect on obesity induced by high-fat diet.In addition,curcumin can significantly reduce blood glucose levels and improve glucose tolerance.It is demonstrated that curcumin can be used as a physiological regulator to effectively reduce body fat deposition,blood lipid content and blood glucose level,and increase the glucose tolerance,thus providing theoretical support for the application of curcumin as a safe and effective lipid and glucose-lowering feed additive in animal husbandry and veterinary production.