| Teprenone(Geranylgeranylacetone)is one kind of safe and effective agent in gastrointestinal mucosa,which have been widely used in human and veterinary,but rarely used in aquaculture animals.Lateolabrax maculatus,one of the commercial aquaculture species in south China,was used in this study.At first,this paper studied the protective effects of teprenone against hypoxia stress in intestine.On this basis,the research explored the mechanism of the protective effects by combining intestinal morphology,microbiome composition,the related factors activity or gene expression level of oxidative stress,nonspecific immunity,and cell apoptosis.The results are as following:1.The effects of teprenone on the intestine morphology in hypoxia stress of L.maculatus were studied.Results showed that long-term periodic hypoxia stress resulted in the decrease of intestinal villi length and the shedding of intestinal epithelial cells,while there was no significant difference between the intestinal villi length,width and muscle layer thickness of the HT group(HT200,HT400 and HT800 group)(P>0.05),and no significant difference between the HT group and the normoxic control group(NC group)(P>0.05).Compared with the hypoxic control group(HC group),the intestinal tissue structure of the HT group was relatively complete without obvious shedding of epithelial cell.Results indicated that the pretreatment with different concentrations of teprenone could maintain the homeostasis of intestinal structural homeostasis in periodic hypoxia stress.2.Based on high throughput sequencing technology Illumina MiSeq,we analyzed the effects of teprenone on the intestinal microflora in periodic hypoxia stress of L.maculatus.Results showed that periodic hypoxia resulted in a significant increase in the diversity and richness of the intestinal flora(P<0.05).The operational taxonomic units(OTUs)of the hypoxic control group(HC group)were significantly higher than other groups(P<0.05),with a unique number of 334 OTUs.At the level of class classification,the dominant bacteria of each groups were Proteobacteria,Firmicutes and Bacteroidetes.Compared with NC group,the relative abundance of Proteobacteria was significantly decreased in the HC group(P<0.05),while that of Bacteroidetes was significantly increased(P<0.05).The composition of intestinal microflora between HT and NC groups was no significant difference(P>0.05).Meanwhile,at the level of genus classification,the relative abundance of Alphaproteobacteria and Bacilli decreased significantly(P<0.05),and that of Clostridia,Gammaproteobacteria and Bacteroidia increased significantly(P<0.05).The relative abundance of Alphaproteobacteria in HT400 group was between NC and HC groups,and the relative abundance of Clostridia,Gammaproteobacteria and Bacilli was no significant difference between HT400 and NC groups(P>0.05).In addition,periodic hypoxia stress also led to an increase of the relative abundance of anaerobes or facultative anaerobes(Anaerolineaceae,Lachnospiraceae,and Ruminococcaceae)and oxygen-producing bacteria(Chlorobiaceae)in the intestines of L.maculatus.Results indicated that periodic hypoxia leads to a complicated community structure of the intestinal microflora.The relative abundance of beneficial bacteria(such as Alphaproteobacteria)decreased in the HC group,and the relative abundance of conditional pathogens(such as Bacteroides)increased in the HC group,while 400 mg/kg of has a good effect on maintaining intestinal microflora homeostasis.3.Based on the analysis of immune-related enzyme activity,apoptosis-related protein and gene expression,we analyzed the effects of teprenone on the intestinal immunity in periodic hypoxia stress of L.maculatus.(a)Results of oxidative stress showed that superoxide dismutase(SOD),catalase(CAT)activity and MDA content were significantly increased in HC group(P<0.05),and total antioxidant capacity(T-AOC)was significantly lower than NC group(P<0.05).The antioxidant enzyme activity and MDA content of HT200 and HT400 groups were between HC and NC groups.(b)Results of nonspecific immunity showed that the activity of lysozyme(LZM)in HC group was the lowest,and the difference between HC group and NC group was significant(P<0.05).LZM activity in HT200 group was significantly higher than that in HC group(P<0.05).HSP70 content and relative gene expression level were higher in NC,HT200 and HT400 group,but lower in HC and HT800 group.The relative expression level of HSP90 mRNA in HC group was significantly lower than that in other groups(P<0.05),and the relative expression level of HSP90 gene in HT200 and HT400 groups was slightly lower than that in NC group(P > 0.05).(c)Results of cell apoptosis showed that the levels of cytochrome C(Cyt-C),caspase-3 activity and caspase-9 mRNA expression in HC group were significantly higher than those in NC group(P<0.05),and the levels of Bcl-2 mRNA were significantly lower than that in NC group(P<0.05).Caspase9 activity and gene expression level in HT group were close to those in NC group.The activity of caspase3 in HT400 group was the lowest,and the difference between HT400 and NC group was not significant(P>0.05).The expression level of Bcl-2 mRNA in HT group was not significantly different from that in NC group(P>0.05).Results indicated that periodic hypoxia causes oxidative stress in intestinal tissues,reduces the level of non-specific immunity,and induces apoptosis.4.In the present study,the effects of teprenone on the intestinal immunity in acute hypoxia stress of L.maculatus were studied.(a)For oxidative stress parameters,there was no significant difference in SOD activity between the hypoxic control group(Control group)and the teprenone treatment group(Teprenone group)at each time point(P>0.05).CAT activity at 0h was significantly higher than that in Teprenone group(P<0.05).T-AOC was significantly higher than Teprenone group at 6,12,24 and 48h(P<0.05).MDA content in B and 12 h was significantly higher than that in Teprenone group(P<0.05).(b)For immune associated proteins,LZM activity in the Control group was lower than that in the Teprenone group except at 12 h,and the difference between the two groups at 6 and 48 h was significant(P<0.05).Compared with time point B,the content of HSP70 in the Control group increased significantly at 0h(P<0.05).At 0-48 h,the HSP70 content in the Control group showed a gradually decreasing trend,which was higher than that in the Teprenone group.(c)For apoptosis-related factors,the activity of Cyt-C,caspase9 and caspase3 increased first and then decreased in both groups.The content of Cyt-C in the Control group at 3h and 48 h was significantly higher than that in the Teprenone group(P<0.05),and the activity of caspase9 and caspase3 was higher than that in the Teprenone group at B-48 h.Results indicated that acute hypoxia causes the expression levels of oxidative stress and apoptosis-related factors in the intestine increased gradually within 0-12 h and decreased within 12-48 h.Acute hypoxia also causes the level of nonspecific immunity decreased first and then increased at 0-48 h.400mg/kg treatment of teprenone can protect intestinal tissues to a certain extent.It can effectively protect intestinal oxidative stress and apoptosis within 0-48 h after acute hypoxia,and enhance non-specific immunity.In conclusion,the results indicated that hypoxia caused stress damage to the intestinal tissue of the L.maculatus,and affected the morphology of intestinal tissue and the structure of intestinal flora.Besides,teprenone had protective effects on hypoxia stress injury.Among them,400 mg/kg teprenone had good effects on the maintenance of intestinal tissue and the stabilization of intestinal microflora,which could reduce the oxidative damage caused by periodic and acute hypoxic stress,enhance the activity of non-specific immune enzymes and the expression of related protein,inhibit the activity of apoptosis factors and gene expression. |
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