Study On The Mechanism Of NF-κB/Nrf2 Signal Pathway Mediated By Different C/N Bioflocs In The Regulation Of Immune Stress In Rhynchocypris Lagowskii Dybowski Exposed To Copper

This paper aims to explore the mechanism of NF-κB/Nrf2 signal pathway mediated by different C/N ratios formed bioflocs in the regulation of immune stress in Rhynchocypris lagowskii Dybowski exposed to copper.In this experiment,glucose was selected as the external carbon source,and four experimental groups were set up according to the difference of C/N: control group(C/N 10.8:1),II group(C/N 15:1),III group(C/N 20:1)and IV group(C/N 25:1).After 56 days of feeding,an acute copper exposure stress test was carried out for 96 h.From the point of view of nutritional regulation,enzyme-linked immunosorbent assay(Elisa),PCR and WB were used to reveal the mechanism of bioflocs in relieving immune stress of R.lagowskii.The main results and conclusions are as follows:1.Effects of different C/N ratios formed bioflocs on growth and digestive enzyme activity in R.lagowskii exposed to copper.Compared with the control group,the protein efficiency rate,WGR,SGR and SR in group III were improved remarkably(P<0.05),on the contrary,the feed conversion rate in group II,III and IV was declined remarkably(P<0.05).The activities of digestive enzymes in hepatopancreas and intestines of R.lagowskii exposed to copper increased(P<0.05)with the increase of C/N,and then tapering after reaching the peak value(III group).Compared with the control group,hepatopancreas PRS and AMS in the III and IV groups were remarkably aggrandized,and intestine AMS,PRS and LPS in II,III and IV groups were augmented(P<0.05)remarkably.(2)Effects of different C/N ratios formed bioflocs on immune enzymes,expression of genes and proteins related to NF-κBp65 signaling pathway in R.lagowskii exposed to copper.The results showed that compared with the control group,the levels of gill,kidney,gut,serum and hepatopancreas alkaline phosphatase(AKP),acid phosphatase(ACP),lysozyme(LSZ),nitric oxide synthase(NOS),complement C3,C4 and immunoglobulin M(Ig M)increased significantly in III and IV groups(P<0.05).The relative expression level of pro-inflammatory factors in hepatopancreas in groups II,III and IV was reduced(P<0.05)remarkably than that in the control group,while IL-10 and TGF-β were significantly increased(P<0.05).The protein expression of TLR4,My D88,p-NFκB and p-IκBα in the III group was remarkably descended(P<0.05)than that in the control,and the protein phosphorylation level of p-NFκB and p-I-κBα was significantly decreased in the III group.(3)Effects of different C/N ratios formed bioflocs on antioxidant enzymes,expression of genes related to Nrf2/Keap1 signaling pathway in R.lagowskii exposed to copper.Compared with the control group,the activities of superoxide dismutase,catalase,total antioxidant capacity,glutathione reductases,glutathione peroxidase and ascorbic acid in the gill,kidney,gut,serum and hepatopancreas of group III were significantly higher(P<0.05),while the activity of malondialdehyde was significantly lower(P<0.05).Compared with C/N 10.8:1 group,the relative expression of Keap1 in II,III and IV groups reduced significantly,and the lowest expression appeared in group III,and it had no statistical(P>0.05)difference between III group and IV group.Nevertheless,Nrf2,Maf and HO-1 expression appreciably highed(P<0.05),and reached the highest level in group III,and there was no statistical(P>0.05)difference between II group and IV group.(4)Effects of different C/N ratios formed bioflocs on bio-accumulation of R.lagowskii exposed to copper.Cu accumulation in the tissues and organs in group II,III and IV was reduced(P<0.05)remarkably,and group III was the lowest group.Also,the gill and kidney Cu-accumulation in group III was appreciably decliner(P<0.05)than that in group IV,but there was no significant difference in Cu-accumulation in brain,hepatopancreas,spleen,intestine and muscle between the two groups(P>0.05).At the same time,the accumulation of Cu in gill,brain,kidney,spleen,hepatopancreas,intestine and muscle of R.lagowskii increased gradually with the decrease of the ratio of C/N(20:1,15:1,10.8:1)in gill,brain,kidney,spleen,hepatopancreas,intestine and muscle(P<0.05).The tissue and organ with the largest accumulation of Cu was hepatopancreas(control group),while muscle tissue(III group)has the minimum Cu-accumulation.The order of Cu-accumulation in each tissue is hepatopancreas>gill> kidney> spleen> brain> intestine> muscle.(5)Effects of different C/N ratios formed bioflocs on the water quality of R.lagowskii.The dissolved oxygen in each group changed sharply at the beginning of culture,and decreased continuously in the first 21 days.II,III and IV in the experimental group were significantly lower(P<0.05)than those in the control group from 7 to 14 days,and then the dissolved oxygen content in each group remained between 5.0 and5.5 mg/L,and the difference was not significant(P>0.05).The contents of total ammonia nitrogen,nitrite and total phosphorus rose originally and thereafter decreased,reaching the highest on the 21 st day,14 th day and 21 st day,respectively,and then decreased rapidly and maintained in a relatively stable state.In the later stage of culture,the contents of II,III and IV group were reduced(P<0.05)remarkably.In the process of culture,the content of nitrate in water continue to rise,the higher the ratio of C/N,the slower the increase of nitrate content,while the difference among groups was not significant(P>0.05).The transparency decreased gradually,and there was no significant difference among groups(P>0.05).To sum up,the bioflocs formed under the condition of suitable C/N can effectively promote the growth performance of R.lagowskii,purify the water quality of cultured water,and alleviate the immune stress caused by copper exposure,which may be through the NF-κB/Nrf2 signal pathway.Among them,the most suitable C/N for the culture of R.lagowskii is 20:1.