The Regulation Of Eucommia Ulmoides And Its Active Components On Growth,Lipid Metabolism And Flesh Quality Of Grass Carp (Ctenopharyngodon Idellus) In Vitro And In Vivo

As a traditional herb widely used in East Asia,Eucommia ulmoides(EU)has many functions in human,including enhancing bones strength,antioxidant capacity and reducing body fattiness.In the study,grass carp(Ctenopharyngodon idellus)was used as the experimental fish.Firstly,EU and its active components were selected through the growth,lipid metabolism and collagen metabolism of hepatocyte and intramuscular fibroblast,and then they were supplemented into diets to feed grass carp.Biochemical measurement,flesh quality evaluation,histology and transcriptomics analysis were combined to investigate the modulation and mechanism of EU and its active components on the growth,lipid metabolism and flesh quality of grass carp.Expt.1 Effects of Eucommia ulmoides extracts and its active components on growth and lipid metabolism of grass carp hepatocyteThe study investigated the effects of Eucommia ulmoides extracts and its four active components on growth and lipid metabolism of grass carp hepatocytes(L8824)in vitro.Hepatocytes were treated with 2.5,5,10,20,40 and 80 μg/ml Eucommia bark extract(EBE),Eucommia leaf extract(ELE),pinoresinol diglucoside(PDG),chlorogenic acid(CGA),quercetin(QC)and aucubin(AU)for 24 h to observe the cell proliferation rate by MTT method,then cells were collect to detect triglyceride content,growth and lipid metabolism-related genes expression.The results showed that the cell proliferation rate was significantly increased by the five supplements except PDG(P<0.05),which was increased firstly,and then decreased with the increasing concentration.The highest cell proliferation rate was observed at the concentration of40 μg/ml for EBE and ELE,10 μg/ml for CGA and QC,and 20 μg/ml for AU(P<0.05), respectively.In addition,the triglyceride contents in EBE,ELE,CGA and QC groups rather than in PDG and AU groups were significantly lower than that in the control group(P<0.05).In genes expression,EBE,ELE,CGA,QC and AU significantly promoted the relative transcript levels of insulin-like growth factor-1(IGF-1),protein kinase B(AKT),target of rapamycin(TOR)and eukaryotic initiation factor 4E binding protein 1(4E-BP1)genes,while the m RNA level of ribosomal protein S6 kinase 1(S6K1)was significantly increased only by ELE,CGA and QC(P<0.05).The expressions of peroxisome proliferator activated receptor α(PPARα),carnitine O-palmitoyltransferase 1(CPT1),adipose triglyceride lipase(ATGL),hormonesensitive lipase(HSL)and peroxisome proliferator activated receptor γ(PPARγ)genes m RNA were significantly up-regulated by EBE,ELE,CGA and QC(P<0.05).Moreover,EBE and ELE significantly increased the lipoprotein lipase(LPL)m RNA expression(P<0.05).In conclusion,EU and its active components could improve the growth and lipid metabolism of grass carp hepatocytes,which may be realized through m TOR and PPAR signaling pathways.CGA and QC may be the main active components in EU to improve the growth and lipid metabolism of grass carp.Expt.2 Effects of Eucommia ulmoides extracts and its active components on growth and collagen metabolism of grass carp intramuscular fibroblastThe study investigated the effects of Eucommia ulmoides extracts and its four active components on growth and collagen metabolism of grass carp intramuscular fibroblasts in vitro.Intramuscular fibroblasts were treated with 2.5,5,10,20,40 and 80μg/ml Eucommia bark extract(EBE),Eucommia leaf extract(ELE),pinoresinol diglucoside(PDG),chlorogenic acid(CGA),quercetin(QC)and aucubin(AU)for 24 h to detect the cell proliferation rate,hydroxyproline content in culture medium and collagen metabolism-related genes expression.The results showed that the cell proliferation rate were significantly increased by the five supplements except PDG(P<0.05),which was increased firstly and then decreased with the increasing concentration,and the highest cell proliferation rate was found at concentration of 40μg/ml for EBE and ELE,20 μg/ml for QC and AU,and 10 μg/ml for CGA(P<0.05),respectively.The EBE,ELE,CGA,QC and AU groups rather than PDG group had higher hydroxyproline content in culture medium than control group(P<0.05).In genes expression,EBE,ELE,CGA,QC and AU significantly promoted the relative transcript levels of the transforming growth factor-β1(TGF-β1),connective tissue growth factor(CTGF),collagen type I alpha 1(COL1A1),collagen type I alpha 2(COL1A2),lysine oxidase(LOX)and tissue inhibitor of matrix metalloproteinase-2(TIMP-2)genes,and decreased matrix metalloproteinase-2(MMP-2)gene m RNA level(P<0.05).Moreover,the expressions of drosophila mothers against decapentaplegic protein 2(Smad2),drosophila mothers against decapentaplegic protein 4(Smad4)and proline hydroxylase(PHD)were also significantly up-regulated by ELE,CGA,QC and AU(P<0.05).In conclusion,EU and its active components could promote the synthesis of collagen through TGF-β/Smads/CTGF signaling pathways,and up-regulate collagen synthesisrelated genes and down-regulate collagen decomposition-related genes to improve collagen metabolism.CGA,QC and AU may be the key active components in EU to improve collagen metabolism of grass carp.Expt.3 Dietary effects of Eucommia bark,leaf and their extracts on the growth,flesh quality and collagen-related genes expression in grass carpThe study investigated the effects of dietary Eucommia bark,leaf and their extracts on growth performance,flesh quality and collagen-related genes expression of grass carp.Eucommia bark(EB),Eucommia leaf(EL),Eucommia bark extract(EBE)and Eucommia leaf extract(ELE)were individually supplemented into basal diet(control)with inclusions of 20 g/kg,40 g/kg,4 g/kg and 4 g/kg,respectively,and then the five diets were fed to grass carp(59.7 ± 0.3 g)for 60 days.The results indicated that dietary EB,EL and ELE significantly increased weight gain(+3.38%,+5.71%,+4.22%),and decreased feed conversion ratio(-0.06,-0.10,-0.07)(P<0.05),while EBE did not significantly affect the growth performance of grass carp(P>0.05).Compared to the control,dietary EB,EL and ELE increased calcium,total collagen,heat-insoluble collagen contents in muscle as well as flesh hardness,chewiness and gumminess(P<0.05),and decreased crude lipid in flesh(P<0.05).In all the treatment groups,the flesh steaming loss,centrifugal loss,drip loss24 h and lactic acid,glucose,total cholesterol and triglycerides contents in serum were significantly lower than those in the control(P<0.05).Moreover,the dietary four supplementations also increased flesh DHA,n-3 polyunsaturated fatty acids levels as well as flesh superoxide dismutase,flesh glutathione peroxidase(GSH-Px)and serum GSH-Px activities,and decreased the protein carbonyl(PC)and malondialdehyde(MDA)contents in flesh(P<0.05).In collagen-related genes expression,the EB,EL and ELE supplementations significantly promoted the expression of collagen type Ⅰ(COL1A1,COL1A2)and lysine oxidase(LOX)in muscle,and decreased the expression of matrix metalloproteinase-2(MMP-2)and matrix metalloproteinase-9(MMP-9)(P<0.05).In addition,proline hydroxylase(PHD)gene in muscle was up-regulated by dietary EL and ELE(P<0.05).In conclusion,dietary EB,EL and ELE improved the growth performance,promoted the antioxidant capacity,flesh water-holding capacity,flesh texture parameters,lipid metabolism and the expression of collagen synthesis-related genes,and decreased the expression of collagen degradation-related genes to improve the flesh quality of grass carp.Expt.4 Effects of Eucommia bark,leaf supplementation on muscle and liver transcriptomics of grass carpBased on the results of Expt.3,the liver and muscle of grass carp were conducted by transcriptomics to evaluate the underlying mechanism of dietary EB and EL on growth,lipid metabolism and flesh quality of grass carp.A total of 1814 and 979 differentially expressed genes(DEGs)were identified in liver and muscle of the EB group,including 683,534 up-regulated and 1131,445 down-regulated DEGs,and a total of 1915 and 1980 DEGs were identified in liver and muscle of the EL group,including 846,1319 up-regulated and 1069,661 down-regulated DEGs when compared with the control.The results of KEGG showed that 47,28 and 36,65 KEGG pathways significantly were enriched in liver,muscle of the EB and EL groups,respectively.Based on the above-mentioned DEGs and signaling pathways,some key pathways involved in the promotion of growth and lipid metabolism and key genes involved in the improvement of flesh quality were selected,including m TOR,PPAR signaling pathways and muscle cytoskeleton-related genes(myosin and collagen).Expt.5 Dietary effects of chlorogenic acid and quercetin on growth and flesh quality of grass carpThe study investigated the effects of dietary chlorogenic acid and quercetin on growth performance,flesh quality of grass carp.Chlorogenic acid(CGA,400 mg/kg),quercetin(QC,400 mg/kg)and their combinations(CGA:QC=1:1,the mixture)with400 mg/kg and 800 mg/kg were individually supplemented into basal diet(control),and then the five diets were fed to grass carp(59.7 ± 0.3 g)for 60 days.The results indicated that dietary CGA and QC significantly increased weight gain(+4.00%,+4.41%),and decreased feed conversion ratio(-0.07,-0.08)(P<0.05),while the two CGA:QC mixtures did not significantly affect the growth performance(P>0.05).Compared to the control,dietary CGA and QC increased apparent digestibility coefficient of dry matter,crude protein and intestinal digestive enzyme activity(P<0.05),and decreased glucose,total cholesterol(TCHO),triglycerides(TG)and lactic acid(LA)contents in serum(P<0.05).The total collagen,heat-insoluble collagen,docosahexaenoic acid(DHA),n-3 polyunsaturated fatty acids contents and n-3/n-6 ratio in muscle as well as flesh hardness,chewiness and gumminess were significantly increased(P<0.05),and the steaming loss,centrifugal loss and drip loss24 h in flesh were significantly decreased by dietary CGA,QC and 800 mg/kg CGA:QC mixture(P<0.05).Moreover,dietary 800 mg/kg CGA:QC mixture decreased TCHO,TG and LA in serum as well as crude lipid content in flesh(P<0.05).CGA,QC and 800 mg/kg CGA:QC mixture groups had significantly higher flesh superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)activities and serum GSH-Px activity,and lower flesh protein carbonyl(PC),malondialdehyde content(MDA)contents and serum MDA level than the control group(P<0.05).In conclusion,dietary CGA and QC promoted the growth performance and flesh quality of grass carp by increasing intestinal digestive enzyme activities and nutrient digestibility as well as flesh water-holding capacity,collagen content,texture parameters and antioxidant capacity.Expt.6 Effects of chlorogenic acid and quercetin supplementation on muscle and liver transcriptomics of grass carpBased on the results of Expt.5,the liver and muscle of grass carp were conducted by transcriptomics to evaluate the underlying mechanism of dietary chlorogenic acid(CGA)and quercetin(QC)on growth,carbohydrate metabolism,lipid metabolism and flesh quality of grass carp.A total of 3125 and 706 differentially expressed genes(DEGs)were identified in liver and muscle of the CGA group,including 1272,391up-regulated and 1853,315 down-regulated DEGs,and a total of 2340 and 658 DEGs were identified in liver and muscle of the QC group,including 1049,411 up-regulated and 1291,247 down-regulated DEGs when compared with the control.The results of KEGG showed that 63,15 and 48,35 KEGG pathways significantly were enriched in liver,muscle of the CGA and QC groups,respectively.Based on the above-mentioned DEGs and signaling pathways,some key pathways involved in the improvement of CGA and QC on growth,carbohydrate metabolism,lipid metabolism and flesh quality of grass carp were selected,including m TOR,PPAR,AMPK,insulin,Fox O and Glycolysis/Gluconeogenesis signaling pathways.