| Glycinin is a major protein component and antinutritional factor in soybean meal.Current studies have found that glycinin can reduce the growth performance of some fish and the digestion and absorption capacity of the intestinal tract,but there are few studies on its effect on the structural integrity of fish intestines,and the involved mechanisms are still unclear.This study first investigated the effects of glycinin on the growth performance,intestinal morphology,and structural integrity of grass carp,and explored the effects of glycinin on the intestinal oxidative damage,apoptosis and tight junctions of grass carp;second,this study explored the digestive metabolism of glycinin on grass carp through in vivo and in vitro digestion experiments,and explored the effect of glycinin digestion products at different times on the structural integrity of intestinal epithelial cells of grass carp.The most negative products were separated with molecular sieve,and the influence of different separation peaks on the structural integrity of the intestinal epithelial cells of grass carp was explored,and the possible effectors in the digestion products were found;third,this study explored the impact of glycinin and its digestion product DP3 on ROS production and clearance in intestinal epithelial cell of grass carp and related mechanisms;fourth,this study explored the effects of glycinin and its digestion product DP3 on apoptosis status of intestinal epithelial cell of grass carp and related signals.The main research contents and results are as follows:1.The effect of glycinin on the growth performance and intestinal structural integrity of juvenile grass carp360 juvenile grass carp(13.75 ± 0.07g)were selected and randomly divided into three treatment groups,including the control group,the 8% glycinin group,and the 8% glycinin + glutamine relief group.Each treatment was repeated three times.A 49-day growth test was carried out,and the test results are as follows:1.1 The effect of glycinin on growth performance,intestinal health and intestinal structural integrity of juvenile grass carpCompared with the control group,8% glycinin significantly reduced the feed intake,end weight,daily gain,specific growth rate,feed coefficient and feed efficiency of juvenile grass carp(P<0.05),caused the nucleus migration in the midgut of young grass carp and the proliferation of lymphocytes in the hindgut,significantly increased the serum diamine oxidase activity and D-lactic acid content of juvenile grass carp(P<0.05).The results showed that glycinin reduced the growth performance of juvenile grass carp and destroyed the structural integrity and health status of the intestinal tract of grass carp.1.2 The effect of glycinin on intestinal oxidative damage and ROS signaling of juvenile grass carpCompared with the control group,the glycinin treatment group significantly increased the ROS content in the midgut and hindgut of the grass carp,as well as the content of oxidative damage products PC and MDA(P<0.05);compared with the control group,glycinin significantly decreased NOX activity of foregut(P<0.05),increased NOX activity of midgut and hindgut(P<0.05),reduced XOD activity of foregut(P<0.05),and increased XOD activity of hindgut(P<0.05),but had no significant effect on XOD activity of midgut(P>0.05),significantly increased the protein abundances of NOX2 in the midgut of grass carp(P<0.05),and significantly increased protein abundances of NOX1 in the midgut and hindgut(P<0.05),significantly reduced the m RNA levels of NOX1,NOXO1 a,NOXO1b,NOXA1,p22 phox,NOX2,p47 phox,p67phox,DUOX and DUOXA in the foregut of grass carp(P<0.05),and significantly increased NOX1,NOXO1 b,NOXA1,NOX2,p47 phox and p67 phox m RNA levels in the midgut(P<0.05),significantly increased NOX1,NOXO1 b,NOXA1 and p22 phox m RNA levels of hindgut(P<0.05);compared with the control group,glycinin treatment significantly reduces the activities of T-AOC,ASA,T-SOD,Cu Zn-SOD,Mn-SOD and CAT in the foregut of grass carp(P<0.05),and significantly reduces the T-AOC,ASA,AHR,T-SOD,Cu Zn-SOD,CAT,GPx,GST,GR activity and GSH content in the midgut(P<0.05),significantly reduced the activity of T-AOC,ASA,AHR,T-SOD,Cu Zn-SOD,Mn-SOD,CAT,GPx,GST,GR and GSH content of hindgut(P<0.05),significantly reduced the m RNA levels of Cu Zn-SOD,Mn-SOD and GR in the foregut of grass carp,GSTO1,GST02,GSTR,GPx1 a,GPx1b,GPx4 a and GPx4 b m RNA levels of midgut,Mn-SOD,GSTO1,GSTO2,GSTR,GPx1 a,GPx1b,GPx4 a and GPx4 b m RNA levels in the hindgut(P<0.05),significantly reduced the protein levels of total Nrf2 and nuclear Nrf2 in the midgut and hindgut of grass carp(P<0.05),significantly increased the protein level of cytoplastic Nrf2 in the midgut and hindgut of the grass carp(P<0.05).The above results indicate that glycinin significantly increased ROS content and caused oxidative damage in the midgut and hindgut of young grass carp,the increased ROS-generated signals NOX(midgut NOX1 and NOX2 and hindgut NOX1)and XOD(hindgut)while decreased antioxidant capacity is related to the inhibition of Nrf2.1.3 The effect of glycinin on intestinal apoptotic status of juvenile grass carpCompared with the control group,glycinin aggravated the DNA fragmentation in the midgut and hindgut of grass carp,significantly increased the activity of caspase-3,8 and 9 in the midgut and hindgut,and significantly increased m RNA level of caspase-3 in the midgut,,the m RNA level of caspase-3,8 and 9 in the hindgut(P<0.05),significantly increased the protein level of cytochrome c in the cytoplasm(exclude mitochondria)of in the midgut and hindgut of grass carp(P<0.05),and decreased Bax and Bcl-2 m RNA levels in the foregut,Bcl-2 m RNA levels of midgut,Bcl-2,Mcl-1 and IAP-1 m RNA abundance of hindgut,increased TNF-α,Bax,Mcl-1 and Apaf-1 m RNA levels of midgut,TNF-α,Fas L,Bax and Apaf-1 m RNA levels of hindgut(P <0.05).The above results indicate that glycinin significantly aggravated the mitochondrial pathway apoptosis and death receptor pathway apoptosis in the midgut and hindgut of juvenile grass carp.1.4 The effect of glycinin on the m RNA abundance of tight junction proteins in the intestine of juvenile grass carpCompared with the control group,the glycinin treatment significantly reduced the m RNA level of claudin-c in the foregut of grass carp,ZO-1,ZO-2b,Occludin,claudin-7a,claudin-12 and ZO-1 of midgut,the m RNA levels of ZO-2b,Occludin,claudin-3c,claudin-7a,claudin-7b,claudin-11 and claudin-12 of hindgut(P<0.05),and significantly increased claudin-15 a and claudin-15 b m RNA levels in the midgut and hindgut of grass carp(P<0.05).The above results indicate that glycinin significantly disrupted the tight junction of intestinal cells in the midgut and hindgut of grass carp.2.The effect of glycinin digestive products on the structural integrity of grass carp intestinal cells2.1 The digestive metabolism of glycinin in grass carp in vivo and in vitroIn the in vivo digestion test,the acidic subunits of glycinin were quickly degraded 0.5 hours after the initial meal,and new digestion products appeared,approximately in the bands of 30 Kda and 6-15Kda;0.5-6h,the acidic subunits were almost completely degraded,the newly emerged peptides are slowly degrading but not completely disappeared.The undigested products always remain in the feces of fish fed with soybean glycinin,including basic subunits and newly degraded peptides of 5-15Kda;the in vitro digestion results are basically the same as in vivo digestion experiments.2.2 The effect of glycinin and the digestive products of glycinin at different times on the structural integrity of the intestinal epithelial cells of grass carpCompared with the control group,glycinin at doses of 6 mg/m L and above can significantly up-regulate the ROS content of grass carp intestinal epithelial cells(P<0.05),and glycinin at doses of 8 mg/m L and above can significantly up-regulate malondialdehyde,protein carbonyl and lactate dehydrogenase activity in the culture medium of intestinal epithelial cells of grass carp(P<0.05);compared with the control group,the 0.5h,1h and 2h glycinin digestion products at doses of 6mg/m L and above can significantly up-regulate the ROS content of intestinal epithelial cells of grass carp(P<0.05),and 4h of glycinin digestion products can significantly up-regulate the ROS content of grass carp intestinal epithelial cells at a dose of 4 mg/m L and higher(P<0.05),6h of soybean glycinin digestion products at a dose of 2mg/m L and higher can significantly up-regulate the ROS content of grass carp intestinal epithelial cells(P<0.05);compared with the control group,0.5h,1h and 2h glycinin digestion products can significantly up-regulate the activity of lactate dehydrogenase in the culture medium of intestinal epithelial cell of grass carp(P<0.05)at doses of 8mg/m L and higher,and 4h glycinin digestion products at doses of 8 mg/m L and higher,the activity of lactate dehydrogenase in the culture medium of grass carp intestinal epithelial cells can be significantly increased(P<0.05),and the 6-hour glycinin digestion product at 4 mg/m L and higher can significantly up-regulate the activity of lactate dehydrogenase in the culture medium of intestinal epithelial cells of grass carp(P<0.05).The above results indicate that with the increase of digestion time,the damaging effect of glycinin digestion products on the structural integrity of the intestinal epithelial cells of grass carp gradually increases.2.3 Isolation of 6h glycinin digestive product components and their effects on the structural integrity of grass carp intestinal epithelial cellsThe 6h digestion product with the most significant negative effect was separated by molecular sieve and passed through a Sephacryl S-100 chromatographic column.A total of 8 digestion products were separated.According to the separation peak pair sequence,they were named DP1-DP8 respectively;according to the standard curve,DP1 was greater than 200 k Da,DP2 and DP3 are about 16 and 10 k Da respectively,while DP4-DP6 is about 5-6k Da,DP7-8 is 3-5k Da;DP1 accounts for the highest proportion at 57.65%,and the second largest component is DP3,accounting for 21.46%,followed by DP8,accounting for 10.33%,and the digestion products of the remaining separated peaks accounted for less than 5%;DP1 was detected by SDS-PAGE and found to be composed of the basic subunits of glycinin and the new product composition produced by digestion.Compared with the control group,the digestion products of DP1,DP4 and DP6 can significantly up-regulate the ROS content of grass carp intestinal epithelial cells at a dose of 2 mg/m L and higher(P<0.05),while the digestion products of DP3 at 1 mg/m L and higher doses can significantly increase the ROS content of grass carp intestinal epithelial cells(P<0.05),and the remaining digestion products have no significant effect on the ROS content of grass carp intestinal epithelial cell culture medium;compared with the control group,DP1,DP4 And DP6 digestion products can significantly up-regulate the activity of lactic dehydrogenase in the culture medium of grass carp intestinal epithelial cells at doses of 4 mg/m L and higher(P<0.05),while DP3 digestion products can significantly increase the activity of lactate dehydrogenase in the intestinal epithelial cell culture medium at dose of 1 mg/m L and higher(P<0.05).The remaining digestion products have no significant effect on the activity of lactate dehydrogenase in the intestinal epithelial cell culture medium of grass carp.The above results indicate that DP1,DP3,DP4,and DP6 may be the effectors in the 6h glycinin digestion products that disrupted the structural integrity of the intestinal epithelial cells of grass carp,and DP3 has the strongest negative effect.3.The effect of soybean glycinin and its digestive products on the oxidative damage of intestinal epithelial cells of grass carp and involved mechanism3.1 The effect of glycinin on the production of ROS in the intestinal epithelial cells of grass carp and involved mechanismCompared with the control group,8 mg/m L glycinin can significantly increase the content of ROS,malondialdehyde and protein carbonyl in the intestinal epithelial cells of grass carp(P<0.05),and significantly increase NOX activity and mitochondrial ROS content(P<0.05),but has no significant effect on the XOD activity of intestinal epithelial cells.Compared with glycinin supplementation group,glycinin plus NOX inhibitor Apocynin can significantly reduce ROS,protein carbonyl,malondialdehyde,NOX activity and mitochondrial reactive oxygen content of intestinal epithelial cells of grass carp(P<0.05);Compared with the glycinin supplementation group,soybean glycinin plus mitochondrial ROS inhibitor mitoq can significantly reduce intestinal epithelial cell ROS,protein carbonyl,malondialdehyde and mitochondrial ROS content(P<0.05).8mg/m L glycinin can significantly increase the protein and m RNA abundance of NOX2 in intestinal epithelial cells and the m RNA levels of its ligands p47 phox and p67phox(P<0.05).Compared with the glycinin supplementation group,glycinin plus NOX2 inhibitor or NOX2-si RNA can significantly reduce the ROS,protein carbonyl,malondialdehyde,NOX activity and mitochondrial ROS content of grass carp intestinal epithelial cells(P<0.05).The above results indicate that soybean glycinin may promote the production of ROS in the intestinal epithelial cells of grass carp through NOX2 signaling.3.2 The effect of soybean glycinin digestive product DP3 on the production of ROS in the intestinal epithelial cells of grass carp and involved mechanismCompared with the control,DP3 can significantly up-regulate the ROS content of grass carp intestinal epithelial cells at a dose of 0.5 mg/m L and above(P<0.05),and DP3 can significantly up-regulate the content of malondialdehyde and protein carbonyl and lactate dehydrogenase activity in the medium(P<0.05)in the grass carp intestinal epithelial cells at a dose of 1 mg/m L and higher.Compared with the control group,1mg/m L DP3 can significantly increase the NOX activity of intestinal epithelial cells and the ROS content of mitochondria(P<0.05),but it has no significant effect on the XOD activity of intestinal epithelial cells.Compared with the DP3 supplementation group,DP3 plus the NOX inhibitor Apocynin can significantly reduce the ROS,protein carbonyl,malondialdehyde,NOX activity and mitochondrial ROS content of the intestinal epithelial cells of grass carp(P<0.05);compared with the DP3 supplementation group,DP3 plus mitochondrial ROS inhibitor mitoq can significantly reduce the content of reactive oxygen species,protein carbonyls,malondialdehyde and mitochondrial reactive oxygen species in intestinal epithelial cells(P<0.05).Compared with the control group,1mg/m L DP3 can significantly increase the protein and m RNA abundance of NOX1 in intestinal epithelial cells and the m RNA levels of its ligands NOXO1 b and NOXA1(P<0.05).Compared with the DP3 supplementation group,DP3 plus NOX1 inhibitor and NOX1-si RNA can significantly reduce the ROS,protein carbonyl,malondialdehyde,NOX activity and mitochondrial ROS content of grass carp intestinal epithelial cells(P<0.05).The above results indicate that the digestion product DP3 may promote the production of reactive oxygen species in the intestinal epithelial cells of grass carp through the pathway of NOX1.3.3 The effect of soybean glycinin on the scavenging capacity of ROS in the intestinal epithelial cells of grass carp and invovled mechanismCompared with the control group,8mg/m L glycinin treatment can significantly increase the ROS,malondialdehyde and protein carbonyl content of the intestinal epithelial cells of grass carp,and significantly reduce the enzyme activities of T-SOD,CAT,Gpx and GR(P<0.05),significantly reduced the m RNA abundance of Cu Zn SOD,Mn SOD,CAT,Gpx1 a and GR in the intestinal epithelial cells of grass carp(P<0.05),and significantly reduced the protein abundance of Nrf2 in the nucleus of grass carp intestinal epithelial cells(P< 0.05).Compared with the glycinin group,the glycinin+keap1a treatment group had no significant effect on the ROS,malondialdehyde and protein carbonyl content of the intestinal epithelial cells of grass carp,and the enzyme activities of T-SOD,CAT,Gpx and GR,no significant effect on the m RNA abundance of Cu Zn SOD,Mn SOD,CAT,Gpx1 a and GR in the intestinal epithelial cells of grass carp.The glycinin + keap1 a treatment group has no significant effect on the protein abundance of Nrf2 in the nucleus of the intestinal epithelial cells of grass carp.Compared with the glycinin group,the glycinin+keap1b treatment group significantly reduced the ROS,malondialdehyde and protein carbonyl content of the intestinal epithelial cells of grass carp,and significantly increased the enzyme activities of T-SOD,CAT,Gpx and GR(P<0.05),significantly increased the m RNA abundance of Cu Zn SOD,Mn SOD,CAT,Gpx1 a and GR in the intestinal epithelial cells of grass carp(P<0.05),and significantly increased the abundance of Nrf2 protein in the nucleus of the intestinal epithelial cells of grass carp(P<0.05).The above results indicate that glycinin may inhibit the ROS scavenging ability of grass carp intestinal epithelial cells through Keap1b-Nrf2 rather than Keap1a-Nrf2 signals.3.4 The effect of glycinin digestive product DP3 on the scavenging ability of reactive oxygen species in the intestinal epithelial cells of grass carp and involved mechanismCompared with the control group,treatment with 1 mg/m L soybean glycinin digestion product DP3 can significantly increase the ROS,malondialdehyde and protein carbonyl content of the intestinal epithelial cells of grass carp,and significantly reduce the enzyme activities of T-SOD,CAT,Gpx and GR(P<0.05),significantly reduced the m RNA abundance of Cu Zn SOD,Mn SOD,CAT,Gpx1 a and GR in the intestinal epithelial cells of grass carp(P<0.05),and significantly reduced the abundance of Nrf2 protein in the nucleus of the intestinal epithelial cells of grass carp(P<0.05).Compared with the DP3 group,the DP3+keap1a treatment group significantly reduced the ROS,malondialdehyde and protein carbonyl content of the intestinal epithelial cells of grass carp,and significantly increased the enzyme activities of T-SOD,CAT,Gpx and GR(P<0.05)),significantly increased the m RNA abundance of Cu Zn SOD,Mn SOD,CAT,Gpx1 a and GR in the intestinal epithelial cells of grass carp(P<0.05),and significantly increased the abundance of Nrf2 protein in the nucleus of the intestinal epithelial cells of grass carp(P<0.05).Compared with DP3,the treatment group of DP3+keap1b had no significant effect on the ROS,malondialdehyde and protein carbonyl content of the intestinal epithelial cells of grass carp,and the enzyme activities of T-SOD,CAT,Gpx and GR.It had no significant effect on the m RNA abundance of Cu Zn SOD,Mn SOD,CAT,Gpx1 a and GR and nuclear protein abundance of Nrf2 on the intestinal epithelial cells of grass carp.The above results indicate that DP3,the product of glycinin digestion,may inhibit the ROS scavenging ability of grass carp intestinal epithelial cells through the signal of Keap1a-Nrf2 rather than Keap1b-Nrf2.4.The effect of glycinin and its digestive products on the apoptosis of intestinal epithelial cells of grass carp and involved mechanism4.1 The effect of glycinin on apoptosis and apoptotic signals of intestinal epithelial cell and related mechanismsCompared with the control group,treatment with glycinin at 8 mg/m L and above can significantly promote the DNA fragmentation of the intestinal epithelial cells of grass carp.Compared with the control group,treatment with glycinin at 8 mg/m L significantly increased the cytochrome c protein released by mitochondria(P <0.05),significantly reduced the m RNA abundance of IAP,Mcl-1,and Bcl-2,and significantly increased the m RNA abundance of Apaf-1 and Bax while has no significant effect on the m RNA abundance of Fas L,TNF-α,and JNK,and significantly increasing the permeability of mitochondria(P<0.05),and significantly increasing the activity of caspase-3 and caspase-9(P<0.05)and the m RNA level of caspase-3(P<0.05)significantly increased the ratio of P-p38-MAPK/T-p38-MAPK(P <0.05),but has no significant effect on the ratio of P-JNK1/2/T-JNK1/2.Compared with the glycinin group,glycinin + SB202190(p38-MAPK phosphorylation inhibitor)significantly reduced DNA fragmentation and the release of cytochrome c from mitochondria(P<0.05),and significantly reduced the the mitochondrial permeability of epithelial cells(P <0.05),significantly increased the IAP,Mcl-1,and Bcl-2 m RNA abundance of grass carp intestinal epithelial cells(P <0.05),and significantly reduced the m RNA abundance of Apaf-1 and Bax in the intestinal epithelial cells of grass carp(P <0.05),significantly reduced caspase-3 and caspase-9 activities in the intestinal epithelial cells of grass carp(P <0.05).The above results indicate that glycinin may promote the mitochondrial apoptosis of grass carp intestinal epithelial cells through the p38-MAPK pathway rather than the JNK1/2 pathway.4.2 The effect of soybean glycinin digestion product DP3 on apoptosis and apoptotic signals of intestinal epithelial cell and related mechanismsCompared with the control group,treatment with the glycinin digestion product DP3 at a concentration of 1 mg/m L and above can significantly promote the DNA fragmentation of the intestinal epithelial cells of grass carp.Compared with the control group,treatment with 1 mg/m L of soybean glycinin digestion product DP3 can significantly aggravate the DNA fragmentation of the intestinal epithelial cells of grass carp,and significantly increase the cytochrome c protein released by mitochondria(P <0.05),and significantly increase the activity of caspase-3,caspase-8 and caspase-9(P <0.05),significantly reduced the m RNA abundance of IAP and Bcl-2,and significantly increased the m RNA abundance of TNF-α,JNK,Apaf-1 and Bax,but has no significant effect on the m RNA abundance of Fas L and Mcl-1(P <0.05), significantly increases the permeability of mitochondria(P <0.05),and significantly increases the ratio of P-JNK1/2/T-JNK1/2(P <0.05),but it has no significant effect on the ratio of P-p38-MAPK/T-p38-MAPK.Compared with the DP3 group,DP3+sp600125(JNK inhibitor)significantly reduced DNA fragmentation and the release of cytochrome c from mitochondria(P <0.05),and significantly reduced the activity of caspase-3,caspase-8 and caspase-9 in the intestinal epithelial cells of grass carp(P <0.05),significantly increased the m RNA abundance of Bcl-2 in the intestinal epithelial cells of grass carp(P <0.05),and significantly reduced the m RNA abundance of TNF-α,Apaf-1 and Bax in the intestinal epithelial cells of grass carp(P <0.05),significantly reduced the mitochondrial permeability of the intestinal epithelial cells of grass carp(P <0.05).The above results indicate that DP3,the digestion product of soybean glycinin,may promote TNF-α death receptor apoptosis and mitochondrial apoptosis of grass carp intestinal epithelial cells through the JNK1/2 pathway rather than the p38-MAPK pathway.In summary,glycinin can reduce the growth performance of juvenile grass carp,destroy the health and structural integrity of the intestinal tract of juveniale grass carp,damage the structural integrity of the intestine,which is related to caused oxidative damage,the destruction of the tight junctions and cell apoptosis in the midgut and hindgut of grass carp.Glutamine can partially relieve the growth inhibition and intestinal destruction effects of glycinin on grass carp.The possible reason is that glutamine can relieve the oxidative damage and apoptosis in the midgut of the grass carp;glycinin is difficult to be completely digested and degraded in the intestine of grass carp,and as its digestion time increases,the corresponding digestion products have a stronger effect on the structural integrity of grass carp intestinal epithelial cells,and the 6h glycinin digestion products after molecular sieve separation,a total of 4 products that destroy the structural integrity of grass carp intestinal epithelial cells were obtained,among which DP3 has the strongest negative effect;glycinin and its digestion product DP3 can both cause the increased ROS content and oxidative damage in the grass carp intestinal epithelial cells,glycinin mainly promotes the production of reactive oxygen in the intestinal epithelial cells of grass carp through the NOX2 pathway,and inhibits the reactive oxygen scavenging ability of the intestinal epithelial cells of grass carp by inhibiting the Keap1b-Nrf2 signal,and DP3 mainly through the NOX1 pathway to promote the production of ROS in the intestinal epithelial cells of grass carp,and inhibit the ROS scavenging ability of the intestinal epithelial cells of grass carp by inhibiting Keap1a-Nrf2 signal;glycinin may promote mitochondrial apoptosis of intestinal epithelial cells of grass carp through p38-MAPK signal.The glycinin digestion product DP3 may promote TNF-α death receptor apoptosis and mitochondrial apoptosis of grass carp intestinal epithelial cells through JNK1/2 signal. |
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