| Gossypol is the main anti-nutritional factor present in cottonseed meal and has toxic effects on animals.This growth experiment firstly investigated the effect of gossypol on the growth performance and intestinal absorptive capacity of grass carp.Next,in vivo and vitro experiment,we explored the effect of gossypol on the intestinal histological structure,antioxidation enzyme activities and genes expression,Nrf2 related signaling molecule gene and protein expression,DNA fragmentation,caspases activities as well as the expression of apoptosis related genes and relevant signaling molecules,which explored the influence of gossypol on the intestinal cell structural integrity and its mechanism;Thirdly,in vivo and vitro experiments,we investigated the effect of gossypol on the gene expression of tight junction protein as well as related signaling molecules of fish intestine,which explored the influence of gossypol on the intercellular structural integrity and its possible mechanism in fish intestine.Fourthly,in the challenge test of Aerobacterium hydrophila,we investigated the effect of gossypol on inflammatory response,immunologic substance content and the gene expression of inflammatory cytokine as well as related signaling molecules of fish intestine,which explored the influence of gossypol on immune barrier function as well as its possible underlying mechanism in fish intestine.The main contents and results are presented as follow:1.The effect of gossypol on the growth performance and intestinal absorption capacity of young grass carpA total of 540 grass carp(230.93±0.50 g)were randomly distributed into six groups of each three replicates.The fish were fed six diets containing different levels of free gossypol(0,121.38,243.94,363.89,759.93 and 1162.06 mg/kg diet)from gossypol-acetic acid for 60 days.This study was conducted to evaluate the effect of gossypol on the growth,intestinal histology,intestinal free amino acid contents and amino acid and peptide transporter genes expression in intestines of young grass carp(Ctenopharyngodon idella).Results indicated that gossypol could reduce the growth of young grass carp.Interestingly,the gossypol modulated the absorptive abilities of various AAs and mechanisms are not unified in fish.Firstly,increasing gossypol level could significantly decrease the contents of tryptophan(Trp),threonine(Thr),valine(Val),phenylalanine(Phe),glycine(Gly),serine(Ser)and tyrosine(Tyr)in the three intestinal segments of fish partly associated with down-regulating the mRNA levels of SLC6A19b,SLC7A5,SLC7A8,SLC1A5,SLC38A2 and PepTl in the intestines.Secondly,gossypol reduced the contents of methionine(Met),isoleucine(Ile),proline(Pro),cysteine(Cys)and arginine(Arg)in the proximal intestine(PI)and mid intestine(MI)of fish partly related to down-regulating the mRNA levels of SLC7A7,SLC7A6,SLC7A1,SLC6A19b,SLC7A5,SLC7A8,SLC1A5 and SLC38A2 and PepTl in the PI and MI.Thirdly,gossypol inhibited the aspartate(Asp)and taurine(Tau)contents in the PI and MI of fish partly related to down-regulating the mRNA levels of SLC1A2a,SLC1A3,PepTl and SLC6A6 in the PI and MI.Fourthly,gossypol suppressed the Lysine(Lys)and Histidine(His)contents in the MI and distal intestine(DI)of fish partly related to down-regulating the mRNA levels of SLC7A7,SLC7A5 and SLC38A2 in the MI,and SLC7A6.SLC7A1,SLC7A8 and PepTl in the MI and DI.Fifthly,gossypol significantly suppressed the gene expression and protein phosphorylation of TOR in the intestines of grass carp.Besides,based on PWG and FE,the control dose of gossypol in the diet for young grass carp growth should be less than 179 mg/kg diet.In summary,gossypol depressed the intestinal AAs absorption capacity of young grass carp partly relate to down-regulating the mRNA levels of AA and peptide transporters by inhibiting the TOR signaling pathway.2.The effect of gossypol on the oxidative damage and cell apoptosis in the intestines of grass carp2.1 The effect of gossypol on the intestinal anti-oxidative damage capacity of grass carp2.1.1 The effect of gossypol on the oxidative damage,activity of anti-oxidase and related signaling molecules genes expression in the intestines of grass carpThis study investigated the effect of gossypol on the histological structure,antioxidative damage,activity of anti-oxidase,GSH content,antioxidant enzyme and Nrf2 signaling pathway related genes expression in the intestines of young grass carp to explore the effect of gossypol on the structural integrity of intestines and the related mechanism in fish.Results showed that(1)dietary gossypol damage the intestinal structure of grass carp,(2)gossypol increased the reactive oxygen species(ROS),malondialdehyde(MDA)and protein carbonyl(PC)contents(P<0.05),and decreased the activities of antioxidant enzymes and GSH content(P<0.05)in the three intestinal segments of young grass carp,(3)gossypol down-regulated the mRNA levels of CuZnSOD,MnSOD,CAT,GPxla,GPx4a,GPx4b,GSTR,GSTP1,GSTP2,GSTO2 and GR(except DI)in the three intestinal segments of fish(P<0.05),(4)gossypol down-regulated the mRNA and protein levels of Nrf2,and up-regulated the Keapla mRNA level in the three intestinal segments of young grass carp(P<0.05).The above results showed that gossypol caused the intestinal structural damage in grass carp,which may be related to inhibiting the Nrf2 signaling pathway and reducing the ability of antioxidation in fish intestines2.1.2 The effect of gossypol on the oxidative damage,antioxidant enzymes activity and nuclear Nrf2 protein level in enterocytes of grass carpThe results of animal trial showed that gossypol could reduce the antioxidant damage ability in the intestines of grass carp.In order to reveal the detail mechanisms,we used in vitro trial to further investigate the effect of gossypol on the oxidative damage and Nrf2 signaling pathway in enterocytes of grass carp.The experiment in this part includes two in vitro trials.Firstly,in order to investigate the optimal concentration of gossypol to induce the oxidant damage in enterocytes of grass carp,the first experiment has six treatments with four replicates each.The enterocytes were treatment with graded levels of gossypol(0,10,20,30,40 and 50 ?mol/L)for 6 h.The results showed that 40 ?mol/L or higher gossypol could significantly increase the content of MDA and ROS in enterocytes(P<0.05),suggesting that gossypol at 40 ?mol/L could effectively induce oxidative damage to enterocytes of grass carpFour treatments were designed in experiment 2,each with three replicates:control group(Gossypol-),gossypol group(Gossypol+),Nrf2 activator group(TBHQ+)and gossypol+activator group(Gossypol+TBHQ+).The aim of this trial was to investigate whether gossypol-induced oxidative damage can be reduced by activating Nrf2.The results showed that(1)compared with the control group,the activation of Nrf2 could significantly reduce the content of ROS,MDA and PC in enterocytes of grass carp induced by gossypol(P<0.05),(2)Compared with the control group,the activities of CAT,GPx and SOD,and GSH content were significantly decreased after gossypol stress(P<0.05),while the activation of Nrf2 could significantly increase the activities of CAT,GPx and SOD,and GSH content,which were inhibited by gossypol(P<0.05),(3)compared with the control group,could significantly increase the expression of nuclear Nrf2 protein inhibited by gossypol(P<0.05).The above results showed that gossypol caused the oxidative damage in enterocytes of fish by inhibiting Nrf2 signaling pathway,down-regulating the expression of antioxidant enzymes related genes and decreasing the activity of antioxidant enzymes.2.2 The effect of gossypol on the intestinal cell apoptosis of grass carp2.2.1 The effect of gossypol on the intestinal DNA fragmentation,apoptotic factors and related signaling molecules gene expression of grass carpThis studygossypol could significantly reduce the nuclear Nrf2 protein level(P<0.05),while the TBHQ investigated the effect of gossypol on the DNA fragmentation,apoptotic factors and related signaling molecules gene expression in the intestines of young grass carp to explore the effect of gossypol on the cell apoptosis of intestine and the related mechanism in fish.The results observed that(1)compared with the control group,with the increase of gossypol level in diet to 363.89,363.89 and 759.93 mg/kg,the visualized DNA fragmentation appeared in the PI,MI and DI of young grass carp,(2)gossypol significantly up-regulated the caspase-2,-3,-7,-8,-9,FasL,Apafl,Bax,JNK and p38MAPK mRNA levels,and down-regulated the Bcl-2,Mcl-1(only PI)and IAP(except DI)mRNA levels in the three intestinal segments of young grass carp.These results indicated that gossypol may activate the expression of apoptotic factors by activating JNK and p38MAPK signaling pathways,and aggravate the cell apoptosis in fish intestine2.2.2 The possible action pathway of gossypol aggravating the cell apoptosis in fish intestineThe results of animal trial indicated that gossypol could aggravate the cell apoptosis in the intestines of young grass carp.In order to reveal the detail mechanisms,we used in vitro trial to further investigate the effect of gossypol on the DNA fragmentation,caspases activity and the protein levels of JNK and p38MAPK in enterocytes of grass carp.The experiment in this part includes three in vitro trials.The first experiment has six treatments with four replicates each.The enterocytes were treatment with graded levels of gossypol(0-50 ?mol/L)for 6 h,which used to investigate the effect of gossypol on the apoptosis of enterocytes and screen out the appropriate gossypol concentration.The results showed that the gossypol concentration at 40 ?mol/L could significantly up-regulate the caspase-3,caspase-8 and caspase-9 mRNA levels in enterocytes(P<0.05),suggesting that gossypol at 40 ?mol/L could effectively induce apoptosis in enterocytes of grass carpFour treatments were designed in experiment 2,each with three replicates:control group(Gossypol-),gossypol group(Gossypol+),JNK inhibitor group(SP600125+)and gossypol+inhibitor group(Gossypol+SP600125+).To explore whether gossypol regulates apoptosis through the JNK signaling pathway.The results showed that(1)the inhibition of JNK could reduce the DNA fragmentation in enterocytes induced by gossypol treatment,(2)compared with the control group,the activities of caspase-3 and caspase-9 were significantly increased under gossypol treatment(P<0.05),while the SP600125 could significantly decrease the activities of caspase-3(P<0.05)and caspase-9(P<0.05)activated by gossypolFour treatments were designed in experiment 3,each with three replicates:control group(Gossypol-),gossypol group(Gossypol+),p38MAPK inhibitor group(SB202190+)and gossypol+inhibitor group(Gossypol+SB202190+).The aim of this study was conduct to explore whether gossypol regulates apoptosis through the p38MAPK signaling pathway The results showed that(1)the inhibition of p38MAPK could reduce the DNA fragmentation in enterocytes induced by gossypol treatment,(2)gossypol could significantly increase the phosphorylation levels of p38MAPK protein in enterocytes of grass carp(P<0.05),while the SB202190 could reduce the p38MAPK protein phosphorylation activated by gossypol(P<0.05),(3)compared with the control group,the activities of caspase-3,caspase-8 and caspase-9 were significantly increased under gossypol treatment(P<0.05),while the SB202190 could significantly decrease the activities of caspase-3,caspase-8 and caspase-9(P<0.05)activated by gossypol.The above results indicated that gossypol could aggravate the apoptosis in fish enterocytes by activating JNK and p38MAPK signaling pathways3.The effect of gossypol on the intestinal tight-j unction structure of grass carp3.1 The effect of gossypol on the tight junction proteins(TJs)and signal molecule genes expression in the intestine of grass carpThis study investigated the effect of gossypol on the TJs and related signaling molecules genes expression in the intestines of young grass carp to explore the effect of gossypol on the intestinal tight junction structure and the related mechanism in fish.The results observed that(1)gossypol significantly down-regulated the ZO-1,ZO-2,occluding,occludin-b,claudin-3c and claudin-11 mRNA levels in the three intestinal segments of young grass carp,(2)gossypol significantly up-regulated the claudin-12,claudin-15b and MLCK mRNA levels in the PI,MI and DI of young grass carp.These results showed that gossypol might up-regulate the mRNA level of MLCK to down-regulate the mRNA levels of TJs genes in fish intestines,and ultimately destroy the intestinal intercellular structural integrity of fish3.2 The possible action pathway of gossypol destroying tight junctions in fish enterocytesIn this study,we used the in vitro trial to further investigate the effect of gossypol on the TJs and MLCK genes expression in enterocytes of grass carp and the possible mechanism Firstly,the fish enterocytes were treatment with graded levels of gossypol(0-50 ?mol/L)for 12 h,which used to investigate the effect of gossypol on the TJs genes expression in enterocytes and screen out the appropriate gossypol concentration.Secondly,four treatments were designed and each with three replicates:control group(Gossypol-),gossypol group(Gossypol+),MLCK inhibitor group(ML-7+)and gossypol+inhibitor group(Gossypol+ML-7+).The results indicated that(1)gossypol concentration up to 40,30,20 and 40 ?mol/L could significantly down-regulated the ZO-1,occludin and claudin-b mRNA levels,and increased lactic acid dehydrogenase(LDH)activity in the enterocytes of grass carp(P<0.05),respectively,(2)ML-7 could up-regulate the expression of ZO-1,occludin,claudin-b and claudin-f genes(P<0.05),and down-regulate the expression of claudin-12 and MLCK genes in enterocytes of grass carp induced by gossypol,respectively(P<0.05).The above results indicated that gossypol may damage the tight junction in fish enterocytes by activating the MLCK to down-regulate the expression of TJs genes4.The effect of gossypol on the intestinal immune barrier function of grass carpAt the end of the growth trial,15 fish of nearly average weight were selected and then challenged with Aeromonas hydrophila for 14 days to explore the effect of gossypol on the intestinal immune barrier function of young grass carp and the related mechanism.The results showed that dietary gossypol(1)could aggravate enteritis and damage the structure of intestinal epithelial cells,(2)decreased the lysozyme(LZ)and Acid phosphatase(ACP)activities,complement 3(C3),C4 and immunoglobulin M(IgM)contents,and it down-regulated the Hepcidin(except DI),immunoglobulin Z(IgZ),liver-expressed antimicrobial peptide(LEAP)-2B,Mucin2 and ?-defensin-1 mRNA levels in the PI,MI and DI(P<0.05),(3)up-regulated intestinal pro-inflammatory cytokines tumor necrosis factor a(TNF-a),interferon y2(IFN-y2),interleukin 1?(IL-1?),IL-6(only in PI),IL-8 and IL-12p35 mRNA levels partly related to nuclear factor kappa B(NF-?B)signalling,and(4)down-regulated the mRNA levels of anti-inflammatory cytokines such as transforming growth factor(TGF)-?1,TGF-?2,interleukin 4/13A(IL-4/13A)(except IL-4/13B),IL-10 and IL-11 partly relating to target of rapamycin(TOR)signalling in the intestines of young grass carp.Finally,based on the enteritis morbidity,the control dose of gossypol in the diet for young grass carp should be less than 117 mg/kg dietIn summary,gossypol reduced the growth and intestinal absorption capacity of young grass carp,which may be related to the down-regulation of amino acid and small peptide transporter gene expression by inhibiting TOR signaling pathway.Gossypol can inhibit the anti-oxidation ability of fish intestine,resulting in intestinal oxidative damage through inhibiting the Nrf2 signaling pathway;and activate the JNK and p38MAPK signaling pathways to induce apoptosis of fish enterocytes,leading to the disruption of the intestinal epithelium structural integrity of grass carp.Gossypol could down-regulate the gene expression of tight junction protein in the intestines of grass carp by activating the signal molecule MLCK,resulting in the damage to the structure and function of the intestinal physical barrier of grass carp.Gossypol reduced the contents of antimicrobial substances in fish intestines and up-regulated the gene expression of pro-inflammatory cytokines as well as down-regulated the expression of anti-inflammatory cytokines,which aggravated the intestinal inflammatory response of grass carp and reduced the intestinal immune barrier function of fish... |
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