| Because of the continuous intensification of global warming,extreme climate fluctuations,and high-density farming,cold-water rainbow trout(Oncorhynchus mykiss)are exposed to heat stress conditions,which has severely impacted their survival and yield.In our previous studies,we found that the strategy for the response to heat stress in rainbow trout was to upregulate the expression of heat shock proteins(HSPs)and then activate various repair and defense mechanisms and antioxidant capacity.Selenium has been shown to upregulate the expression of HSPs and activate antioxidant reactions in the organism.With recent advancements in nanotechnology,Nano-selenium(nano-Se)has attracted widespread attention as it shows high biological activity,and low toxicity,making it the ideal selenium supplement and free radical scavenger.However,the overall molecular mechanism via which nano-Se participates in heat stress tolerance remains to be comprehensively understood.This study used full-sibling rainbow trout as experimental materials,by analyzing different times point of different levels of nano-Se(0,5,and 10 mg/kg)before and after heat stress and the dynamic changes of typical stress biomarkers in rainbow trout liver to determine the appropriate nano-Se level and heat treatment time;then liver samples at typical time points were selected for quantitative proteomics and metabolomics analysis,and differentially expressed proteins and differential metabolites were compared between groups added and without added selenium(parallel comparison)and before and after heat stress(longitudinal comparison),and the metabolic pathways involved in the differential molecules were analyzed using bioinformatics methods,and preliminary association analysis was performed on proteomics and metabolomics data;finally,based on the key pathways discovered by multi-omics data,in rainbow trout hepatocytes further evaluated the role of the PI3K/AKT pathway in the inhibition of heat stress-induced apoptosis by nano-Se.The main results are summarized as follows:1.Study on the suitable treatment level and treatment duration of nano-Se: Under heat stress,the fish supplementation 5 mg/kg nano-Se significantly increased liver glutathione peroxidase(GPx)activity and upregulated expression levels of HSP70 b,HSP90a1,GPx1 a,and Trx m RNAs,while liver alanine aminotransferase(ALT),aspartate aminotransferase(AST),superoxide dismutase(SOD),and malondialdehyde(MDA)levels as well as tissue structure damage and lipid accumulation were decreased.Combining the trends for the above indicators indicated that stress began to increase significantly at 8 h.It can be concluded that supplementation with 5 mg/kg nano-Se effectively alleviates stress damage in rainbow trout.Furthermore,stress at 24°C for 8 h can be thought of as a critical time point for the study of heat stress in rainbow trout.2.Proteomic study of heat stress in rainbow trout liver in response to nano-Se:Choose 18°C without added nano-Se(CG18),18°C added nano-Se(Se18)and 24°C without added nano-Se(CG24),24°C added nano-Se(Se24)liver samples from four groups were analyzed by proteome sequencing.In comparison with the CG18-CG24 group,the Se18-Se24 group showed higher abundance of molecular chaperones,such as Hsp70,Hsp90 a.1,Hspa8,Hsp30,DNAJA4,Dnajb1,Bag2 and Ahsa1;on nano-Se supplementation,the heat stress-induced decline in the abundance of the selenoprotein Msr B2 was partially restored.Furthermore,nano-Se supplementation downregulated the abundance of lipid-related(CYP51,EBP,DHCR7,DHCR24,and APOB)and proapoptotic(caspase-8 and Bad)proteins.Protein–protein interaction analyses suggested that nano-Se inhibits apoptosis by upregulating the expression of Hsp70,Hsp90 a.1,Hspa8,and Dnajb1;further,Hsp70/Hspa8 and Msr B2 appear to play a synergistic role in antioxidant defense under heat stress.3.Metabolomics study of heat stress in rainbow trout liver in response to nano-Se:Untargeted metabolomics detection was performed using the same four sets of samples as protein sequencing.The results showed that most glycerophospholipids and Co A levels were decreased in the CG18-CG24 group,and pathway enrichment analysis showed that it mainly interfered with glycerophospholipids and fatty acid metabolism.Se18-Se24 group inhibits galactose metabolism and activates the glutamate-glutamine metabolic pathway.Moreover,an integrated analysis of the two kinds of-omics data was performed by calculating the Pearson correlation coefficient of the abundance of differential expression protein and different metabolite and searching the Co-pathway that shared between the two-omics data.Correlation analysis suggests that the interaction of HSPs with phospholipids may have functional significance in the stress response.Common pathway studies showed that PI3K/AKT,and MAPK signaling pathways were significantly enriched in both types of omics studies.4.Role of PI3K/AKT pathway in the inhibition of heat stress-induced apoptosis by nano-Se: Nano-Se supplementation mitigated the decrease of heat stress-induced total antioxidant capacity,glutathione peroxidase and catalase activities,whereas it inhibited elevated levels of reactive oxygen species(ROS)levels and malondialdehyde content.Apoptosis results indicated that nano-Se supplementation decreased elevated levels of heat stress-induced apoptotic factors(Bax,Cyt-c,Bax/Bcl-2,Caspase3,and Caspase9)gene expression while increasing the decrease of anti-apoptotic factors(Bcl-2)gene expression.Moreover,nano-Se pretreatment alleviated the decrease in PI3 K and AKT m RNA levels.Our data indicate that nano-Se ameliorates heat stress-induced apoptosis of rainbow trout hepatocytes by activating the PI3K/AKT pathway.In conclusion,the anti-heat stress effect of nano-Se and its regulatory mechanisms were revealed at the multi-level levels of cells,proteins(enzymes),genes and metabolites.Heat stress-induced excess ROS leads to lipid accumulation by attacking phospholipid membranes,ultimately causing oxidative damage and apoptosis in the organism.Nano-Se exerts its anti-heat stress effects in rainbow trout by promoting protein repair,enhancing recovery of antioxidant enzyme activity,and alleviating lipid metabolism and apoptosis.Furthermore,the mechanism of heat stress-induced cell injury inhibition by nano-Se mainly involves glutamate-glutamine metabolism,PI3K/AKT and MAPK signaling pathways.The research results can provide scientific basis and new ideas for promoting the development of high temperature tolerance rainbow trout seed industry and solving the problem of rainbow trout breeding in summer. |
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