| Ovotransferrin（OVT）is the second most ample protein in chicken egg white,which can able to bind and release iron,and has abundant nutritional value and various physiological activities,such as antisepsis,antioxidation and immunoregulation.However,the mediating mechanisms of physiological and biochemical parameters from OVT in vivo are underdeveloped,and it is needed to be further researched.The Caenorhabditis elegans（C.elegans）,as a model organism,has many advantages,like a clear genetic landscape,some intestinal cells being similar to humans’,several homologous signal pathways associated with nutrient metabolism,and especially no ethical reviews.In the present study,the organism C.elegans was the testing animal to investigate the effects of OVT on its physiological function by constructing testing models under its different physiological states.The transcriptomic and metabolomic techniques were applied to explore the possible mechanisms of action by OVT.The research aimed to provide a theoretical basis for the application of OVT as an active factor in nutraceuticals,and had guiding significance for the in-depth development and utilization of poultry eggs.The main methods,content,results and conclusions of the research were as follows:（1）The effects of OVT on the growth and development,motility and lifespan of C.elegans under health states were tested by stereomicroscope observation.Its lipofuscin accumulation level was determined by using upright fluorescence microscope observation,specific fluorescent probes and enzyme activity detection kits were used to detect changes in its oxidative stress system.Two acute oxidative impairments induced by paraquat（PQ）and tertiary butyl hydroperoxide（t BHP）were used to investigate the influences of OVT on its stress resistance.The results showed that the body length and motility of C.elegans were both improved,the body length was increased by 6.38%,and the frequencies in 30 s of its head swings and body bends were increased by 9.99%and 39.09%,respectively.Its mean lifespan was prolonged by8.2%.Its antioxidant enzyme activities/level were elevated by OVT,the activities of SOD,POD and CAT were separately increased by 50%,5%,25%,and the GSH level was increased by 10%.The level of its oxidation metabolites was also decreased,including ROS,MDA and lipofuscin,which were decreased by 10.19%,14.5%and5.61%,respectively.And after C.elegans was exposed to 113 m M PQ and 2 m M t BHP its survival rate was separately increased by 48.56%and 15.96%because of OVT intake.Therefore,OVT could increase the body length and motility of C.elegans under healthy states and also enhance its oxidative stress resistance and prolong its lifespan.（2）The C.elegans-Pseudomonas aeruginosa O1（PAO1）infection model was used to study the effects of OVT on the C.elegans under pathogenic bacterium infection.The results showed that the C.elegans being infected for 24 h by PAO1 were all survival with growth and development stunting.Compared to that being fed with OP50 strain,the C.elegans infected by PAO1,its body length was significantly decreased by 3.39%and the motility was impaired,in which the frequencies in 30 s of its head swings and body bends were decreased by 1.75%and 27.67%,respectively.The GSH level in C.elegans was increased by 2.41%,but not obviously.The level of the initial oxidation products O2·-and ROS,and lipofuscin,were increased by 29.71%,6.23%and 22.31%,respectively,which derived from oxidative stress caused by the accumulation of PAO1and bacterial metabolites in the gut.Its intestinal barrier function was damaged too,and the intestinal barrier permeability was increased by 20.45%.In the infectious states,physiological and biochemical damages in C.elegans were mitigated after being fed with OVT for 48 h,and its growth and development as well as motility were partially recovered.Especially,its body length was increased by 9.29%,and the frequencies of its head swings in 30 s and body bends were increased by 31.52%and 4.40%,respectively.Its oxidative stress was also inhibited,the GSH level was significantly increased by 5.91%and the level of O2·-,ROS and lipofuscin were decreased by 16.46%,4.55%and 14.1%,respectively.And the permeability of its intestinal barrier was decreased by 11.41%,but failed to recover to its normal levels.Therefore,OVT could enhance the antibacterial and antioxidative stress capacities of C.elegans infected by PAO1,and facilitate it repairing the damaged intestinal barrier,eventually its resistance to PAO1 infection was improved.（3）The exploration of the response mechanism at the transcription level of C.elegans after OVT intake was completed by the DNBSEQ gene sequencing platform.There were 201 significant differential expressed genes（DEGs,Fold change>1.5,Qvalue<0.05）being identified based on the reference genome of C.elegans（GCF＿000002985.6＿WBcel235）,including 58 upregulated genes and 143downregulated genes.According to the analysis results of Gene Ontology（GO）database in the biological processes,the enriched DEGs were mainly involved in the metabolic process,the regulation of growth and development,all of which were affected by energy metabolism,for example,the cuticle development involved in collagen and cuticulin-based cuticle molting cycle,collagen and cuticulin-based cuticle development,proteolysis,organic acid metabolic process,xenobiotic metabolic process and oxidation-reduction process,etc..In brief,the mechanism of OVT affecting the growth and development of C.elegans was mainly through regulating the expression of molting cycle function genes of it.The mechanism of OVT enhancing the enzyme activities/level of SOD,CAT,POD and GSH,and reducing the formation of ROS,MDA and lipofuscin was mainly through improving the catalytic activity of enzymes in C.elegans.The mechanism of OVT enhancing the survival rate of C.elegans under PQ and t BHP induced oxidative stress states was mainly through increasing the activities of related enzymes in its phase I and phase II biotransformation reactions,thereby increasing the ability to metabolize exogenous toxic compounds.Moreover,SJ4143strain was used to evaluate the intestinal energy transfer of C.elegans,the result showed that the activity of intestinal mitochondria was significantly enhanced by OVT.Kyoto Encyclopedia of Genes and Genomes（KEGG）database was used to analyze the DEGs,and there were 5 pathways finally being enriched,which related to lipids metabolism,including fatty acid metabolism,biosynthesis of unsaturated fatty acids,peroxisome,PPAR signaling pathway and fatty acid degradation.The mechanism of OVT on lipid metabolism in C.elegans was mainly through regulating its various genes related to lipases,lipid synthesis and decomposition,which led to the above all signaling pathways being activated.The regulation of energy metabolism was also one of the main mechanisms.（4）The analysis of metabolomics in C.elegans being fed with OVT was completed by UHPLC-Q-TOF MS technology.Results showed that 246 metabolites were totally screened and 127 differential metabolites were effectively annotated,in which the number of lipids and lipid-like molecules was the most,but there were also a small amount of protein and carbohydrate metabolites.The functional annotation and enrichment analysis for differential metabolites were compared based on the KEGG database and the Human Metabolome Database（HMDB）.Those results were mainly involved in lipid metabolism,amino acid metabolism and energy metabolism processes.In brief,the mechanism of OVT affecting the growth and development,lifespan of C.elegans was partly through regulating the content of energy-supplying substances,such as glucose and trehalose.The mechanism of OVT enhancing the catalytic activity of antioxidant enzymes of C.elegans was partly through activating lipid metabolism pathways to increase the content of polyunsaturated fatty acids.The mechanism of OVT enhancing the stress resistance of C.elegans was partly through increasing the content of intermediate metabolites of the electronic respiratory chain,and also through regulating the content of glutamine,cysteine and glycine of C.elegans.The mechanism of OVT affecting the energy metabolism of C.elegans was partly through increasing the expression of glycerophosphocholine and eicosane compounds.The mechanism of OVT affecting the lipid metabolism of C.elegans was partly through the activation of PPAR signaling pathway.（5）Combined with the analysis results of transcriptome data and metabolomics data,the upregulation of cyp-35A5 and sod-2,and the content increase of the choline glycerophosphate,EPA and salidroside should be closely related to the increased activity of antioxidant enzymes and enhanced resistance to oxidative stress of C.elegans by OVT,indicating that the mechanism of OVT on C.elegans perhaps partly through the inducement of upstream genes encoding cytochrome oxidase and downstream lipid metabolism pathway,polyunsaturated fatty acids might involve in the process.Besides,the upregulation of acox-1.2,acox-1.3 and maoc-1,and the content increase of the histidine and pyroglutamate might be closely related to promoting the growth and development of C.elegans by OVT,indicating that OVT could promote the lipids degradation,increase the content of key amino acids,in turn,increased the activity of mitochondria to affect the synthesis of ATP and cell metabolism,and finally promoted the energy metabolism of C.elegans,thereby promoting its growth.（6）Salidroside was selected to verify the mechanism of OVT on C.elegans,which was completed by the Alpha Fold2 database and molecular docking simulations.The binding energy data showed that salidroside could directly bind to the key upstream regulators and their downstream targets in the IIS and MAPK pathways through non-covalent actions,such as hydrogen bonds and van der Waals forces,and produce stable conformation.Therefore,OVT-mediated changes in the physiological levels of C.elegans might part through the metabolite-salidroside,and might further activate downstream stress-inducible genes together with other metabolites.However,further studies should be done to elucidate the more precise regulatory mechanisms of OVT in vivo. |
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