| Linoleic acid is a polyunsaturated fatty acid containing with 18 carbon atoms and 2 unsaturated double bonds.For most animals,it is an essential fatty acids due to lacking ofΔ12 and Δ15 desaturase for further desaturation of oleic acid(C18:1 n-9)to synthesize linoleic acid.Linoleic acid has a variety of biological functions.Linoleic acid is one of the raw materials for phospholipid,which can regulate the fluidity of cell membranes.Further,its transfer,metabolism and transformation in vivo affect lipid metabolism.Linoleic acid can also be used as synthesis of prostatic hormones,involved in the proliferation and differentiation of immune cells.Insufficient dietary linoleic acid causes growth retardation and weaked immunity in aquatic animals.Linoleic acid is mainly sourced from vegetable oils such as soybean oil,corn oil and safflower oil.Some scholars found that the replacement of fish oil with vegetable oil changed the flavor characteristics of aquatic animals.With the extensive use of vegetable oils,the content of linoleic acid in the diet increases year by year.Excessive intake of linoleic acid was first found to have negative health effects in mammals.Subsequently,it has also been reported in aquatic animals that dietary linoleic acid increases the content of oxygen free radicals,induces oxidative stress and increases the sustained release of various pro-inflammatory factors.These studies suggest that linoleic acid promotes the growth and development of aquatic animals,and at the same time may pose a threaten on health.With increasing dietary linoleic acid content,it is meaningful to explore the optimal dietary linoleic acid for chinese mitten crabs(Eriocheir sinensis).Bearing these in mind,the present study was conducted to evaluate the effect of dietary linoleic acid on growth,lipid metabolism and muscle flavor quality of Eriocheir Sinensis.This study is divided into five parts as follows:Experiment 1 Effects of dietary linoleic acid on growth performance,oxidative stress and inflammatory factorsThe purpose of this experiment was to investigate the effect of dietary linoleic acid on growth performance,oxidative stress and inflammatory factors of Eriocheir Sinensis.Five isonitrogen isoenergetic diets supplementing with different five 5 different levels linoleic acid(7,14,21,28 and 35 g/kg)were made.A total of 200 Eriocheir sinensis with an initial weight of 28.69 ± 0.43 g were selected and randomly divided into 5 groups.Each group was set up with 4 replicates and fed the corresponding diets respectively for 12 weeks.The results showed that when the dietary linoleic acid increased from 7 g/kg to 21 g/kg,the final weight,weight gain,weight gain rate and specific growth rate of Eriocheir sinensis increased significantly(P<0.05),and when its content was further increased,these four growth performance indicators showed no significant change(P>0.05).The level of linoleic acid in the diet showed no significant effect on the survival rate of Eriocheir sinensis,but the survival rate was highest in the 28 g/kg linoleic acid groups(P>0.05).Based on the evaluation of weight gain rate,the optimal demand for linoleic acid was predicted to be 19.77 g/kg.With the increase of dietary linoleic acid,the hepatopancreatic index and number of G2/M cells showed a trend of a significant increase followed by a significant decrease(P<0.05),reached the maximum value when the dietary linoleic acid was 28 g/kg.The level of dietary linoleic acid showed no significant effect(P<0.05)on the enzyme activities of T-AOC,SOD and CAT in lymph.When diets were supplemented with 35 g/kg linoleic acid,the GSH content in the lymphatic was significantly decreased(P<0.05),while the MDA content was significantly increased(P<0.05).The level of dietary linoleic acid showed no significant(P>0.05)effect on SOD and CAT enzyme activities in hepatopancreas.The GSH content and T-AOC activity in the hepatopancreas were significantly decreased(P<0.05),while the MDA content was significantly(P<0.05)increased in the high-level(28 and 35 g/kg)linoleic acid groups.The level of dietary linoleic acid showed no significant(P>0.05)effect on p38-MAPK,LITAF,ADAM 17,and ILF2 genes expression.The gene expression ofMyD88 was significantly increase in the 35g/kg linoleic acid group.In conclusion,based on growth performance,the optimal dietary linoleic acid requirement was 19.77 g/kg;21 and 28 g/kg dietary linoleic acid could improve the development of hepatopancreas;High levels of linoleic acid do not induce inflammation but cause oxidative stress.Experiment 2 Effects of dietary linoleic acid on fatty acid composition and lipid metabolismThe purpose of this experiment was to investigate the effect of dietary linoleic acid on fatty acid composition and lipid metabolism.The results showed that the level of linoleic acid in the diet showed no significant effect on GOT and GPT enzyme activities.When the dietary linoleic acid increased from 7 g/kg to 28 g/kg,the contents of triglycerides and free fatty acids in the lymph were significantly increased(P<0.05),when its content further increased,these two indexes decreased significantly(P<0.05).The proportion of linoleic acid and total n-6 unsaturated fatty acids in hepatopancreas and muscle were significantly increased with increasing dietary linoleic acid levels(P<0.05),while C16:0,C18:1,saturated fatty acids and monounsaturated fatty acids showed the opposite trend.Fluorescence staining for lipid showed that 21 and 28 g/kg dietary linoleic acid increased lipid deposition in the hepatopancreas.Quantitative analysis of lipids showed that dietary supplementation with 21 g/kg and 28 g/kg linoleic acid significantly increased the content of total fat and triglyceride(P<0.05).The expression of genes related to β-oxidation showed that when dietary linoleic acid increased from 7 g/kg to 28 g/kg,the expression of CPT1 and ACO increased significantly(P<0.05),and when its content further increased,the expression of CPT1 decreased significantly,and while ACO expression showed no significant change.The expression of fatty acid transport-related genes showed that the FATP4 gene expression was significantly increased when dietary linoleic acid was higher than 21 g/kg(P<0.05).The gene expression of FABP3 and FABP9 showed a trend of increase and then followed by decrease,and reached the maximum when the dietary linoleic acid was 28 g/kg and 21 g/kg respectively(P<0.05).Dietary linoleic acid showed no significant effect on FAS gene expression.The gene expression of DTGA1 increased significantly with the increase of dietary linoleic acid between 7 g/kg and 28g/kg,and the its expression decreased significantly when its content increased at 35 g/kg.In conclusion,diets supplemented 21 or 28 g/kg linoleic acid increased lipid accumulation in the hepatopancreas,and enhanced the expression of genes related to β-oxidation,fatty acid transport and triglyceride synthesis.Experiment 3 Effects of linoleic acid on odor compound and lipoxygenase metabolic pathwaysThe purpose of this experiment was to study the effect of dietary linoleic acid level on odor compound and lipoxygenase metabolic pathways.The nutritional intervention experiment is the same as experiment 1.We also constructed a model of short-term gavage of linoleic acid to promote aldehyde synthesis,and in vitro linoleic acid was incubated to stimulate primary muscle cells.The chemical inhibitor(Zileuton)and siRNA were used to inhibit lipoxygenase metabolic pathway in vitro and in vivo,respectively.The experimental results were showed as follows:The principal component analysis of the electronic nose showed that dietary linoleic acid brought an distinguish on the flavor profile of muscle.The flavors of the high-grade linoleic acid group were completely differentiated from the lowgrade ones.Dietary linoleic acid showed a significant effect on 9 compounds:4 aldehydes(pentanal,hexanal,2-hexenal and nonanal),2 alcohols(1-pentanal en-3-ol and 1-hexanol),2 ketones(2-nonanone and 2-decanone)and 1 hydrocarbon(dodecane).Except for two aldehydes,the contents of the other seven substances increased significantly with the increase of dietary linoleic acid level(P<0.05).The content of aldehydes was further verified by fluorescence method and liquid chromatography.The results showed that dietary linoleic acid significantly increased the content of total aldehyde,hexanal,2-hexenal and nonanal(P<0.05).The lipoxygenase activity,protein and gene expression in muscle showed the same trend where they all increased significantly with the increase of dietary linoleic acid level(P<0.05).We next examined whether the promotion of aldehyde synthesis by linoleic acid is dependent on the activation of lipoxygenase.Gvage linoleic acid continuously for 15 days significantly increased the content of aldehydes in muscle.After inhibiting lipoxygenase activity with Zileuton,the content of aldehydes was significantly decreased(P<0.05),and returned to the normal value with no significant difference with the control group(P<0.05).The content of total aldehydes was significantly increased after incubation with linoleic acid in muscle primary cell culture medium and then significantly decreased after inhibition of transcription and translation of lipoxygenase with siRNA.The results of this experiment show that linoleic acid increases the production of aldehydes by activating the lipoxygenase metabolic pathway,which further influence the flavor characteristics of muscle.Experiment 4 Cloning and functional identification of lipoxygenase from Eriocheir sinensisIn the previous chapter,we found that linoleic acid can activate the lipoxygenase metabolic pathway,but there is no relevant report on lipoxygenase in Eriocheir sinensis,and little is known about its structure and function.In this experiment,the full-length cDNA of lipoxygenase from Eriocheir sinensis was cloned by the terminal rapid amplification method,and the recombinant protein of lipoxygenase was expressed and purified by yeast in vitro.The open reading frame of LOX is 2157 bp,encoding 718 amino acids.It is predicted that its molecular weight is 82kd,and its isoelectric point is 8.62.Based on its amino acid sequence,the functionally predicted structure showed that PLAT and Lipoxygenase domains were included.The analysis of phylogenetic tree showed that LOX in Eriocheir sinensis shared a high homology with LOX5 in mouse.The three-dimensional structure alignment found that the spatial structure of the two proteins was highly similar.We examined the product of arachidonic acid catalyzed by recombinant LOX to further clarify whether it shared the function of LOX5.The natural oxidation of AA did not produce 5HETE.After adding recombinant LOX,AA was catalyzed to generates 5HETE.In the presence of calcium ions,the catalytic activity of LOX for AA was enhanced and the produce of 5HETE was significantly increased(P<0.001).The interaction between recombinant LOX and fatty acids with different degrees of saturation was examined by microscale thermophoresis.The results showed that there was no interaction between recombinant LOX and stearic acid and oleic acid.The interaction was detected between recombinant LOX and linoleic acid and linolenic acid.Quantitative analysis result showed that the interaction between LOX and linoleic acid was significantly higher than its and linolenic acid(P<0.001).Finally,we observed the distribution of LOX in muscle cells by immunofluorescence,and the results showed that LOX was mainly distributed in the nucleus in the resting state.The results of this experiment showed that LOX in Eriocheir sinens was highly similar in structure and function to LOX5 in mouse.Experiment 5 Effects of linoleic acid on the distribution of intracellular lipoxygenaseThe purpose of this experiment was to explore the mechanism by which linoleic acid activates the LOX metabolic pathway.The experimental results showed that neither dietary linoleic acid nor continuous gavage linoleic acid showed no significant effect on the content of leukotriene B4 in muscle(P>0.05).The intracellular distribution of LOX was analyzed by immunofluorescence,and observable LOX protein was found in the muscle cytoplasm in Eriocheir sinens fed with diets supplementing with 28 and 35 g/kg linoleic acid.Quantitative analysis of its protein found that dietary linoleic acid showed no significant effect on the protein expression of LOX in the nucleus(P>0.05),but significantly increased(P<0.05)the protein expression of LOX in the cytoplasm.Likewise,incubation of linoleic acid in L6 muscle cell medium induced LOX distribution in the cytoplasm.Compared with the control group(0 h time point),after 12 hours of linoleic acid incubation,the protein expression of LOX5 in the cytoplasm increased significantly(P<0.05),while the abundance of LOX protein in the nucleus did not change significantly throughout the incubation process(P>0.05).High-grade dietary linoleic acid significantly increased cAMP content and PKA protein expression.Next,we inhibited the expression of both types of PKA with siRNA to verify its role in linoleic acid-induced LOX cytoplasmic distribution.The experimental results showed that incubation of linoleic acid in L6 cell culture medium significantly increased the expression of LOX in the cytoplasm,but had no effect on the expression in the nucleus.After siRNA inhibited the PKA signaling pathway,the expression of LOX in the cytoplasm was significantly inhibited(P<0.05),while the abundance of LOX in the nucleus increased significantly(P<0.05),at the same time the content of total aldehyde also decreased significantly(P<0.05).Finally,we validated the conservation of this pathway in muscle primary cells,Drosophila cell line and C2C12 muscle cell line using H-89 as an inhibitor of PKA.It was found that PKA signaling pathway mediates the process of linoleic acid-induced LOX cytoplasmic expression in various cells.The experimental results show that linoleic acid affects the intracellular distribution of LOX through the PKA signaling pathway to activate its biological function involved in aldehyde synthesis.In conclusion,dietary linoleic acid is able to improve the growth performance and the development of hepatopancreas in Eriocheir sinensis.On the one hand,it increases the rate of cell proliferation,and on the other hand,it up-regulates the gene expression of lipid synthesis and transport-related proteins to increase lipid accumulation in cells;in addition,dietary linoleic acid activates PKA signaling.The pathway regulates the distribution of cytoplasmic LOX to increase the synthesis of flavor aldehydes,which in turn affects the flavor quality of muscles. |
