| Our previous studies have shown that excessive fluoride leads to abnormal growth and development,oxidative stress,histopathological damage and dysfunction of multiple organs in fish.Selenomethionine(Se-Met)is the principal source of selenium(Se)absorption in fish and is usually supplemented to the diet to improve immunity and antioxidant properties.Moreover,Se has long been known for its ability to alleviate the toxic effects of some metals(such as Cd,Li,Hg).Therefore,in the present study,zebrafish were selected as a model organism to explore the alleviating effects of Se-Met on fluorosis in fish and its underlying mechanisms.1 Effects of Se-Met on the developmental toxicity of fluoride-exposed zebrafish embryosHealthy zebrafish embryos at 6 h post-fertilization(hpf)were randomly divided into 8 groups,namely control(C)group,0.25,0.5,and 1.0 μg Se/L Se-Met(0.25,0.5 and 1Se)groups,80 mg/L of fluoride(F)group,and 0.25,0.5,1Se+F(0.25,0.5 and 1SF)groups.The alleviating effects of Se-Met on developmental toxicity,oxidative stress and inflammation in fluoride-induced zebrafish embryos were investigated after treatment of zebrafish embryos for different periods(24,48,72,96 and 120 hpf).The results showed that F exposure significantly increased F contents in larvae(P<0.05),and induced developmental toxicity,oxidative damage and apoptosis,increased significantly the MPO and LZM activities and the mRNA levels of the inflammation-related genes IL-1β,IL-6,TNF-α,IL-10 and TGF-β(P<0.05).Moreover,F exposure significantly elevated the mRNA levels of ERK2,JNK,p38 and p65 in MAPKs and NF-κB pathways(P<0.05).Co-treatment with Se-Met and F significantly reduced the F contents in larvae,mortality and deformity rate(P<0.05),inhibited oxidative stress and apoptosis,reversed the transcriptional levels of inflammatory cytokines and related genes in the MAPKs and NF-κB pathways.However,treatment with Se-Met alone also markedly increased the levels of IL-6,TNF-α,IL-10,TGF-β,ERK2 and JNK mRNA(P<0.05).In short,these data demonstrate that Se-Met could alleviate the developmental toxicity of F-exposed zebrafish embryos,although low levels of Se-Met alone had certain toxic effects on zebrafish embryos.2 Effects of Se-Met on growth performance and liver damage in F-exposed zebrafishAdult zebrafish were randomly divided into 8 groups(half of male and female in each group):the blank control group(C),fed the control diet;Se-Met 1 group(Sel),fed 0.25 μg Se/g diet;Se-Met 2 group(Se2),fed the 0.5 μg Se/g diet;Se-Met 3 group(Se3),fed 1.0 μg Se/g diet;fluoride group(F),80 mg/L F+control diet;F+Sel group(SF1),80 mg/L F+0.25 μg Se/g diet;F+Se2 group(SF2),80 mg/L F+0.5 μg Se/g diet;F+Se3 group(SF3),80 mg/L F+1.0 μg Se/g diet.Zebrafish liver was collected after 30,60 and 90 d of treatment for further study.The results showed that:(1)growth performance:F exposure inhibited the body weight,body length,weight gain rate and length gain rate in zebrafish(P<0.05).After co-treatment with Se-Met and F,the body weight,body length,weight gain rate and length gain rate showed a variable degree of increases.Treatment with Se-Met alone,the body weight,body length,weight gain rate and length gain rate in the Se2 and Se3 groups were significantly increased at 60 d(P<0.05).(2)histopathological changes:F exposure resulted in severe liver histopathological damage,including cell swelling,nuclear pyknosis or lysis,diffuse vacuolation,and mitochondrial swelling,endoplasmic reticulum dilates or ruptures.Treatments with Se-Met and F had slightly reduced diffuse vacuolar degeneration and nuclear pyrosis.Furthermore,the blurred mitochondrial ridges,regular round nuclei with uniformly dispersed chromatin,and clear nuclear membranes were found in the three SF groups.The liver tissue structure of the three Se groups fish was normal.(2)changes in oxidative stress indicators and immune-related enzyme levels:F exposure significantly increased the contents of ROS,MDA and the activities of SOD,AKP,MPO,decreased the activities of CAT,GSH,GPx,ACP and the content of LZM(P<0.05).Treatments with Se-Met and F reversed the levels of these indicators.Se-Met treatment alone significantly increased the activities of SOD and CAT in the liver of female zebrafish,and decreased LZM content in the liver of male zebrafish(P<0.05).(4)liver inflammation:F exposure significantly up-regulated the transcription levels of IL-6,IL-1β,TNF-α,IL-10 and TGF-β(P<0.05).Treatments with Se-Met and F regulated the transcription levels of these inflammatory cytokines.After Se-Met treatment alone,the transcripts of IL-6 and IL-10 were significantly up-regulated in male zebrafish livers after 30 and 60 d(P<0.05).These results suggest that dietary Se-Met treatment alleviates F-induced liver histopathological damage and attenuated oxidative stress and inflammation in zebrafish.3 Effects of Se-Met on intestinal barrier function in F-exposed zebrafishAdult zebrafish were used in this study to investigate the effects of Se-Met on the intestinal barrier function in F-exposed zebrafish.The results showed that:(1)histopathological changes:F exposure led to atrophy of intestinal villi,disorder of intestinal epithelial cells,and decreased V/C ratio and number of goblet cells.After co-treatment with Se-Met and F,the villi were intact,and the epithelial cells were neatly arranged,the V/C ratio and the number of goblet cells were slightly increased,but there was still a minor separation between lamina propria and epithelial layer.No obvious histopathological changes were observed after Se-Met treatment alone.(2)intestinal permeability:F exposure significantly declined the transcription levels of Muc2,ZO-1,Claudin-1 and Occludin in the intestine of male zebrafish,while only the transcription level of Muc2 was significantly reduced in the intestine of female zebrafish(P<0.05).However,the transcripts of Muc2,ZO-1,Claudin-1 and Occludin were significantly elevated after the co-treatment with Se-Met and F(P<0.05).Treatment with Se-Met alone,the transcripts of Claudin-1 and Occludin in the intestine of female zebrafish were elevated in the Se2 and Se3 groups at 90 d(P<0.05).(3)intestinal inflammation:F exposure significantly increased the transcriptional levels of IL-6,IL-1β,TNF-αand IL-10 and significantly decreased the transcriptional levels of TGF-β in the intestine of male zebrafish,whereas the transcripts of these inflammatory factors were significantly increased in the intestine of female zebrafish(P<0.05).Treatments with Se-Met and F reversed the transcriptional levels of these inflammatory factors.After treatment with Se-Met alone,the transcripts of IL-6,IL-10,TNF-α and TGF-βof the Se2 and Se3 groups were significantly increased in the intestine of male zebrafish(P<0.05).These results indicate that dietary Se-Met treatment could ameliorate F-induced intestinal histopathological damage,reduce intestinal permeability and inflammation,and restore intestinal barrier function in zebrafish.4 Effects of Se-Met on intestinal microbiota in F-exposed zebrafishAfter exposure for 90 d,the intestinal contents of male zebrafish(C,Se2,F,SF2 groups)were collected,and high-throughput sequencing of 16S rRNA gene sequences was performed.Bioinformatics analysis and qPCR were used to investigate the effects of Se-Met on intestinal microbiota,and LPS level,TLR4a and NF-κB p65 genes expression in intestine and liver in F-exposed zebrafish.The results showed that:(1)intestinal microbial communities:the richness and diversity of microbiota were decreased in the F group,and the intestinal microbial communities were altered at different taxonomic levels.Treatments with Se-Met and F increased the richness and diversity of microbiota,and the microbial community structure recovered to control levels.After Se-Met treatment alone,the richness and diversity of microbiota were decreased,and the microbial communities were also altered at different taxonomic levels.(2)Changes in the gut-derived LPS:LPS levels in the intestine and liver were significantly increased in the F group(P<0.05).Treatments with Se-Met and F significantly decreased the LPS level in the liver(P<0.05);after Se-Met treatment alone,the LPS level in the intestine and liver were similar to those in the C group.(3)expression of TLR4a and NF-κB p65 genes in the intestine and liver:the transcripts of TLR4a and p65 in the intestine were increased significantly in the F group,but the transcripts of IκBaA were significantly decreased.In addition,the transcripts of p65 and IκBaA in the liver were significantly decreased(P<0.05).After the co-treatment with Se-Met and F,the transcriptional levels of TLR4a and p65 in the intestine were significantly lower than those in the F group(P<0.05).The transcription levels of TLR4a and P65 in the liver were similar to those in the C group,but the difference was not significant compared with the F group.However,the transcription level of IκBaA in the liver was significantly increased(P<0.05).After Se-Met treatment alone,the transcription level of TLR4a in the intestine and the transcription level of p65 in the liver were significantly higher than those in the C group(P<0.05).These results suggest that Se-Met modulates F-induced intestinal microbiota imbalance in zebrafish,prevents translocation of gut-derived LPS and subsequent inflammatory responses.This study confirms that:(1)Se-Met alleviates F-induced toxicity in zebrafish embryos,although low levels of Se-Met alone has certain toxic effects on zebrafish embryos.(2)Se-Met can prevent the vicious cycle of intestinal and liver injury by modulating the intestinal microbiota,improving the intestinal barrier function,and inhibiting the translocation of gut-derived LPS to the liver,thereby ameliorating the intestinal and liver damage of zebrafish induced by F.The indirect gut-liver axis pathway may serve as the base for the mechanism underlying its alleviative effects on F-induced liver and intestinal damage.This study provides new insight into the protective effects of Se-Met against fluorosis and helps understand the essentiality of Se-Met for future applications. |
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