| Fish is often exposed to anoxia in the water environment due to natural factors(such as temperature,season and precipitation)or human factors(such as eutrophication and high culture densities).Current studies have shown that aquatic organisms have evolved complex physiological and biochemical systems to adapt to hypoxia stress in water.However,the change of oxygen concentration in water will have adverse effects on the cells and tissues of aquatic organisms,such as the obstruction of the metabolic processes,tissue lesions,reduced transport efficiency of nutrients and insufficient energy supply,which will lead to diseases or death of fish.Pelteobagrus vachelli is the largest species in Pelteobagrus genus,with unique meat quality and less intermuscular spines,which are of great economic value.Scholars at home and abroad,have carried out a large number of studies on disease resistance,temperature tolerance and nutritional value of this fish,and discussed the molecular mechanism of its response to hypoxic stress.However,no reports have been found on the intestinal tissue as the research object under hypoxic stress.Therefore,this paper takes the intestinal tissue of P.vachelli as the entry point.The changes of intestinal tissue lesions,oxidative stress,cell apoptosis and intestinal microbial composition were studied to explore the effects of hypoxia stress on the intestine of P.vachelli,so as to provide reference for elucidating the dynamic regulation mechanism of intestinal environment in intestine.The main research results are as follows:1)Effects of hypoxia stress on intestinal lesions and oxidative stress of P.vachelliThe floating head point and suffocation point of P.vachelli were 0.78±0.06mg/L and 0.52±0.05 mg/L,respectively,according to which the hypoxia concentration was set as(1.0±0.05 mg/L).At the beginning of the experiment,the intestinal tissues of P.vachelli under the normal condition as well as under the treatments of hypoxia stress for 24 h and 72 h,and recorvering to normal oxygen concentration for 24 h were observed using H&E staining.The results showed that vacuolation,villi erosion and mucosal layer hyperplasia appeared in goblet cell swelling under hypoxia stress.After restoring normal oxygen concentration for 24 h,the physiological changes of tissue caused by hypoxia did not recover,and a large number of cavitation areas appeared.At the same time,the activity of oxidative kinase in intestinal tissue was measured,and the results showed that the activity of superoxide dismutase(SOD)continuously increased under hypoxia stress,while decreased slightly with no significant changes after restoring the normal oxygen concentration for 24 h.The activities of catalase(CAT)and glutathione peroxidase(GSH-Px)both increased,and reached to the peat values after 24 h of hypoxia stress,then decreased after restoring the normal oxygen concentration for 24 h.The activity of malondialdehyde(MDA)in P.vachelli after hypoxic stress was significantly higher than that of the control group,reached to the highest value at 72 h after hypoxic stress,showing a significant change.The activity of MDA decreased after restoring the normal oxygen concentration for 24 h,which showed no significant diference compared to that under hypoxic stress for 72 h.2)Effects of hypoxia stress on intestinal tissue cell apoptosis of P.vachelliTunel staining was performed on the intestinal tissue of P.vachelli to evaluate the proportion of apoptotic cells to normal cells.It was found that with the prolongation of hypoxia stress time,the degree of apoptosis of cells was increased.The experimental results showed that the apoptotic index was about 60%.At the same time,the expression patterns of five apoptosis-related genes(p53,Caspase-3,Caspase-9,Bax and Bcl-2)in the gut tissues of P.vachelli were detected by fluorescence quantitative PCR.The results showed that the expression levels of p53,Caspase-3,Caspase-9 and Bax increased with the duration of hypoxia stress,and all reached to the peak value after 72 h of hypoxia stress(P < 0.05).In contrast,the expression of Bcl-2 gene was negatively correlated with the duration of hypoxic stress(P < 0.05).After returning to normal oxygen concentration for 24 h,the expression levels of p53 gene and Bax gene decreased.However,the expression levels of Caspase-3,Caspase-9 and Bcl-2 were slightly changed compared with those at 72 h,and there was no significant difference.3)Effects of hypoxia stress on intestinal microorganisms of P.vachelliHigh-throughput 16 S r RNA sequencing analysis was performed on the intestinal microorganisms of P.vachelli.According to the Alpha diversity index,the abundance of microorganisms in the intestinal tissues of this fish was relatively high,but with the prolongation of hypoxia stress time,the abundance gradually decreased,but the evenness did not change.Through Beta diversity analysis,individual confidence ellipse regions were formed in each time period,and there was little difference in the distance between the three samples in each region.According to Venn plot,there were 156 microbial species in total,accounting for 4.32%.With the change of time,the common microbial species of the two adjacent periods remained unchanged.Eight phyla were detected in the intestinal microflora of P.vachelli.They were Firmicutes,Bacteroidetes,Cyanobacteria,Fusobacteria,Proteobacteria,Actinobacteria,Planctomycetes,Deinococcus-Thermus and Verrucomicrobia.Preliminary statistical analysis showed that 4 kinds of microorganisms were related to the changes of antioxidant enzyme activity and the expression of apoptotic genes in intestinal tissues. |
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