Nano-selenium Antagonizes Heat Stress-Induced Apoptosis of Rainbow Trout (Oncorhynchus mykiss) Hepatocytes by Activating the PI3K/AKT Pathway.

The cold-water fish rainbow trout (Oncorhynchus mykiss) shows poor resistance to heat, which is the main factor restricting their survival and yield. With the advancement of nanotechnology, nano-selenium (nano-Se) has emerged as a key nano-trace element, showing unique advantages, including high biological activity and low toxicity, for studying the response of animals to adverse environmental conditions. However, little is still known regarding the potential protective mechanisms of nano-Se against heat stress-induced cellular damage. Herein, we aimed to investigate the mechanism underlying the antagonistic effects of nano-Se on heat stress. Four groups were assessed: CG18 (0 µg/mL nano-Se, 18 °C), Se18 (5.0 µg/mL nano-Se, 18 °C), CG24 (0 µg/mL nano-Se, incubated at 18 °C for 24 h and then transferred to 24 °C culture), and Se24 (5.0 µg/mL nano-Se, incubated at 18 °C for 24 h and then transferred to 24 °C culture). We found that after heat treatment (CG24 group), T-AOC, GPx, and CAT activities in rainbow trout hepatocytes showed a decrease of 36%, 33%, and 19%, respectively, while ROS and MDA levels showed an increase of 67% and 93%, respectively (P < 0.05). Meanwhile, the mRNA levels of the apoptosis-related genes caspase3, caspase9, Cyt-c, Bax, and Bax/Bcl-2 in the CG24 group were 41%, 47%, 285%, 65%, and 151% higher than those in the CG18 group, respectively, while those of PI3K and AKT were 31% and 17% lower, respectively (P < 0.05). Besides, flow cytometry analysis showed an increase in the level of apoptotic cells after heat exposure. More importantly, we observed that nano-Se cotreatment (Se24 group) remarkably attenuated heat stress-induced effects (P < 0.05). We conclude that heat stress induces oxidative stress and apoptosis in rainbow trout hepatocytes. Nano-Se ameliorates heat stress-induced apoptosis by activating the PI3K/AKT pathway. Our results provide a new perspective to improve our understanding of the ability of nano-Se to confer heat stress resistance.

Dietary nano-selenium alleviates heat stress-induced intestinal damage through affecting intestinal antioxidant capacity and microbiota in rainbow trout (Oncorhynchus mykiss).

Heat stress-induced intestinal damage is a key event in fish pathology. Nano-selenium (nano-Se) shows remarkably high biological activity and low toxicity, making it an ideal and ecological Se formulation; however, to date, the protective effects of nano-Se against heat stress-induced intestinal injury and pertinent molecular mechanisms remain unknown. Herein, rainbow trout (Oncorhynchus mykiss) were fed either a basal diet or basal diet + 5 mg/kg nano-Se. Samples were collected before (18 °C for 9 days; CG18 and Se18 groups) and after (24 °C for 8 h; CG24 and Se24 groups) heat stress treatment. On heat stress exposure, intestinal villus height, muscularis thickness, and goblet cell number decreased, and expression of tight junction proteins (ZO-1, occludin, and claudin-8d) was downregulated; dietary supplementation with nano-Se alleviated these effects. Furthermore, in the presence of nano-Se, catalase activity was elevated, and expression of diverse heat shock proteins (Hsp70b, Hsp90α, and Hsp30), selenoproteins (Gpx1a, Gpx1b1, and Trx), and anti-inflammatory cytokine (TGF-ß) was upregulated. In contrast, nano-Se supplementation significantly alleviated the increase of the expression of pro-inflammatory cytokines (IL-1ß and TNF-α) and the malondialdehyde content. We also observed that heat stress markedly increased the relative abundance of Actinobacteria, Firmicutes, Methylobacterium, Akkermansia, and Deinococcus and decreased that of Proteobacteria; nano-Se supplementation restored these changes, making their distribution similar to that in the control group. Overall, our findings suggest that nano-Se plays a protective role against heat stress-induced intestinal damage in rainbow trout by promoting the recovery of antioxidant enzyme activity, enhancing protein repair, alleviating inflammatory responses, and restoring intestinal microbiota composition.

Transcriptome sequencing reveals the effect of selenium nanoparticles on primary hepatocytes of rainbow trout.

Heat stress is one of the important threats in rainbow trout culture, and selenium nanoparticles (SeNPs) have an important role in combating heat stress and enhancing immunity. In this study, to enable rainbow trout to survive at higher temperatures, we added 5 µg/mL SeNPs to hepatocytes to study the resistance effect and immune effect of SeNPs against heat stress, thus enabling rainbow trout to adapt to summer temperatures (Average 26 °C) in Lanzhou, Gansu Province, China. Therefore, we investigated the transcriptome expression profile of hepatocytes spiked with SeNPs when exposed to heat stress. A total of 234 differentially expressed genes (DEGs) were firmly established in SeNPs-added group when exposed to heat stress compared to non-SeNPs-added group. Of these DEGs, 156 were up-regulated and 78 were down-regulated. These DEGs were grouped into different Gene Ontology (GO) functional terms and enriched in 75 significantly different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, of which approximately-one-third (19) were associated with immunity. STRING was used to identify 39 key immune DEGs belonging to 5 immune pathways (C-type lectin receptor signaling pathway, FoxO signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, and Rachidonic acid metabolism). These pathways interact extensively and formed a complex network to regulate heat stress. These results provided an important basis for future elucidation of the role of SeNPs in heat stress resistance and immune enhancement in rainbow trout.

Effect of selenium nanoparticles on alternative splicing in heat-stressed rainbow trout primary hepatocytes.

Alternative splicing (AS) is a ubiquitous post-transcriptional regulatory mechanism in eukaryotes that generates multiple mRNA isoforms from a single gene, increasing diversity of mRNAs and proteins that are essential for eukaryotic developmental processes and responses to environmental stress. Results showed that a total of 37,463 AS events were identified in rainbow trout hepatocytes. In addition, a total of 364 differential alternative splicing (DAS) events were identified in hepatocytes under selenium nanoparticles (SeNPs) and 3632 DAS events were identified under a combination of SeNPs and heat stress (24 °C). Gene Ontology (GO) enrichment showed that some subcategories "immune effector processes", "response to stimuli" and "antioxidant activity" were associated with immunity, abiotic stimuli and antioxidants. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment showed that differentially expressed genes (DEGs) were significantly enriched in spliceosomes by adding SeNPs in heat-stressed hepatocytes. Splicing factor family (SRSF3, SRSF7, SRSF9, U2AF1 and U2AF2) and pre-RNA splicing factors (ACIN1 and PPRF18) were significantly upregulated and promoted AS. Furthermore, addition of SeNPs activated the phosphatidylinositol signaling system and upregulated the related genes PI4KA, DGKH, ITPK1 and Ocrl, and thus attenuated the inflammatory response to heat stress and enhanced resistance to heat stress by activating the adherent plaque kinase-PI3K-Akt signaling pathway and calcium channels. Those findings suggested that AS could be an essential regulatory mechanism in adaptation of rainbow trout to heat-stressed environments.

Comprehensive proteomic analysis to elucidate the anti-heat stress effects of nano-selenium in rainbow trout (Oncorhynchus mykiss).

Because of the continuous intensification of global warming, extreme climate fluctuations, and high-density farming, cold-water rainbow trout (Oncorhynchus mykiss) are exposed to conditions of heat stress, which has severely impacted their survival and yield. Nano-selenium (nano-Se) shows higher biological activity and lower toxicity and has emerged as an ideal and ecological Se formulation. Herein rainbow trout were fed either a basal diet (control group) or basal diet plus 5 mg/kg nano-Se (treatment group). Samples were collected before (18 °C for 9 days; CG18, Se18) and after (24 °C for 8 h; CG24, Se24) heat stress. The DIA/SWATH approach was then applied to compare changes at the proteome level. We found 223 and 269 differentially abundant proteins in the CG18-CG24 and Se18-Se24 groups, respectively, which mainly included apoptosis-, heat stress-, and lipid-related proteins. In comparison with the CG18-CG24 group, the Se18-Se24 group showed higher abundance of molecular chaperone, such as Hsp70, Hsp90a.1, Hspa8, Hsp30, DNAJA4, Dnajb1, Bag2 and Ahsa1; on nano-Se supplementation, the heat stress-induced decline in the abundance of the selenoprotein MsrB2 was partially restored. Furthermore, nano-Se supplementation downregulated the abundance of lipid-related (CYP51, EBP, DHCR7, DHCR24, and APOB) and pro-apoptotic (caspase-8 and Bad) proteins. Protein-protein interaction analyses suggested that nano-Se inhibits apoptosis by upregulating the expression of Hsp70, Hsp90a.1, Hspa8, and Dnajb1; further, Hsp70/Hspa8 and MsrB2 appear to play a synergistic role in antioxidant defense under heat stress. Overall, our findings provide novel insights into nano-Se-mediated tolerance of heat stress, demonstrating that 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.

Dietary nanoselenium supplementation for heat-stressed rainbow trout: effects on organizational structure, lipid changes, and biochemical parameters as well as heat-shock-protein- and selenoprotein-related gene expression.

Nanoselenium (nano-Se) shows unique protective effects against environmental heat stress in rainbow trout as a selenium source additive and free radical scavenger. Accordingly, we investigated the effects of supplementation with different levels of nano-Se (0, 5, and 10 mg/kg) and before and after heat stress (24°C) for different treatment times on the dynamic changes of rainbow trout liver tissue structure, lipid changes, biochemical properties, and gene expression. The results showed that, under heat stress, the fish supplementation of 5 mg/kg nano-Se significantly increased liver glutathione peroxidase (GPx) activity and upregulated expression levels of HSP70b, HSP90a1, GPx1a, and Trx mRNAs, 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, with significant changes in response but no serious damage. Thus, these results provide a reference for the addition of nano-Se to rainbow trout feed and provide theoretical and practical guidance for enhancing the resistance of rainbow trout to heat stress.

Metabonomics analysis reveals the protective effect of nano­selenium against heat stress of rainbow trout (Oncorhynchus mykiss).

Nano­selenium (nano-Se) shows high biological activity and low toxicity, and has emerged as an ideal antioxidant. Our goal was to determine the underlying mechanism of nano-Se-mediated heat stress tolerance in rainbow trout (Oncorhynchus mykiss). Using liquid chromatography-mass spectrometry (LC-MS) metabolomics, histomorphology, and conventional biochemical assays, we investigated the physiological responses of heat-stressed rainbow trout to nano-Se. Fish were fed to two levels nano-Se at 18 °C for 9 days: CG18 (0 mg/kg) and Se18 (5 mg/kg). The water temperature of all groups was increased to 24 °C and maintained for 8 h (CG24, Se24). The results showed that most glycerophospholipids and CoA levels were decreased in CG18-CG24, and pathway enrichment analysis showed that it mainly interfered with glycerophospholipids and fatty acid metabolism. Meanwhile, hematoxylin and eosin and Oil Red O staining showed significant damage to CG18-CG24, which was ameliorated by Se18-Se24. The results combining analysis of antioxidant enzymes and heat shock proteins further support the notion that nano-Se supplementation inhibited galactose metabolism and activated the glutamate-glutamine metabolic pathway as the key metabolic strategy against heat stress. These results could establish heat stress defense strategies and increase our understanding of the mechanism of nutrient participation in fish's response to adverse environments. SIGNIFICANCE: Global warming is affecting the distribution and survival of cold-water fish worldwide, through seasonal water temperature increases and an increase in the frequency of extreme heat wave events. Surprisingly, Nano­selenium (Nano-Se) with its outstanding advantages of high biological activity and low toxicity, making it a good Se nutrient supplement and free radical scavenger, and also an ideal and ecological way to supplement Se. How to utilize the metabolome to better address the complexity of the interactions that may occur with Nano-Se during the process of heat stress resistance is an important challenge. In the present study, this is the first publicly available metabonomics study of the anti-heat stress effect of Nano-Se as a nutrient on rainbow trout liver. These data indicated that Nano-Se effectively alleviated stress damage in rainbow trout, in which heat stress interfered with the metabolism of glycerophospholipids and fatty acids significantly, causing liver cell membrane damage and lipid metabolism disorders in rainbow trout. Meanwhile, supplementation of Nano-Se downregulated galactose metabolism and activated glutamate and glutamine metabolic pathways, which seems to be a key metabolic strategy to combat heat stress. The results provide a scientific basis for the development of an anti-heat-stress feed for rainbow trout that help maintain their health, productivity and welfare under unfavorable heat conditions.

Hypothermia Effectively Treats Tumors with Temperature-Sensitive p53 Mutations.

The p53 tumor suppressor is frequently inactivated by mutations in cancer. Most p53 mutations are located in the DNA-binding domain, causing local disruption of DNA-binding surface or global misfolding. Rescuing the structural defect of mutant p53 is an attractive therapeutic strategy, but its potential remains unproven due to a lack of drugs capable of efficiently rescuing misfolded p53. Although mutant p53 in tumors is inactive at 37°C, approximately 15% are temperature sensitive (ts) and regain DNA-binding activity at 32°C to 34°C (ts mutants). This temperature is achievable using a therapeutic hypothermia procedure established for resuscitated cardiac arrest patients. To test whether hypothermia can be used to target tumors with ts p53 mutations, the core temperature of tumor-bearing mice was lowered to 32°C using the adenosine A1 receptor agonist N6-cyclohexyladenoxine that suppresses brain-regulated thermogenesis. Hypothermia treatment (32 hours at 32°C × 5 cycles) activated endogenous ts mutant p53 in xenograft tumors and inhibited tumor growth in a p53-dependent fashion. Tumor regression and durable remission in a ts p53 lymphoma model was achieved by combining hypothermia with chemotherapy. The results raise the possibility of treating tumors expressing ts p53 mutations with hypothermia. SIGNIFICANCE: Pharmacologic inhibition of brain-regulated thermogenesis and induction of 32°C whole-body hypothermia specifically targets tumors with temperature-sensitive p53 mutations, rescuing p53 transcriptional activity and inducing tumor regression.See related commentary by Hu and Feng, p. 3762.