Study On Apoptosis Of Epithelial Cells In Small Intestine Of Pigs Caused By High Zinc-induced Autophagy

High zinc(Zn)is widely used in pigs feeds because of its growth-promoting effects and anti-diarrhea in weaned piglets.High doses of Zn will not only accumulate in the body organs of pigs,it but also influence the function of small intestinal epithelial cells.High proportion of the Zn is not absorbed and removed with the excrement of feces.This will not only affect the pig health and pork quality,it but also causes environmental pollution and the waste of resources.Therefore,it is important to reveal the mechanism of high Zn in porcine small intestinal epithelial cells.Also it is of great significance for the rational application of Zn in the pig production and the prevention of environmental Zn pollution.Autophagy and apoptosis are involved in the proliferation and differentiation of cells and other cells processes,but the role of autophagy and apoptosis in high Zn-treated porcine small intestinal epithelial cells has not been reported.This study investigated the current status of the use of Zn in pigs in large-scale pig farms.The effects of high Zn diets on the histomorphology of small intestinal epithelium and the expression of apoptosis-related genes were studied using Bama miniature pigs.Then,the mechanism of autophagy in high Zn-induced intestinal epithelial cells damage was studied by using porcine small intestinal epithelial cells(IPEC-J2).The main researches were as following:1.Concentrations of Zn in feeds and feces in large scale pig farmsTo investigate the present status of Zn in feeds and feces in large scale pig farms,the feed and feces samples of growing and fattening pigs,nursery pigs and weaned pigs were collected from 19 intensive pig farms.The concentrations of Zn in feeds and feces samples were detected and the correlation between feeds and fecal Zn concentrations was analyzed.The results showed that the average concentration of Zn in all pig feeds was 658.9 mg/kg,ranging from 69.50 to 2654 mg/kg.The highest concentration of Zn in the pig compound feeds was 150 mg/kg as the standard,the exceeding rate of feeds was 71.9%.Also the average Zn concentration of all pig feces was 1197 mg/kg,ranging from 135.6 to 4283 mg/kg.The limit value of Zn in agricultural sludge control standard(GB 4284-1984)was 1000 mg/kg as standard,the exceeding rate of feces was 29.8%.The concentrations of Zn in the pig feces of various stages were higher than that in the corresponding feeds(p<0.05).except the growing and fattening pigs from No.3,No.12 and No.14 pig farms and the nursery pigs from No.1,No.2,No.4,No.5,No.12 and No.14 pig farms.The concentrations of Zn in feed samples of weaned pigs were higher than growing and fattening pigs and nursery pigs(p<0.05),except No.4 and No.15 pig farms.There was significantly positive correlation between the Zn concentration in feeds and the Zn concentration in feces,and the correlation coefficient was 0.9594.The correlation coefficients were 0.5342,0.1761 and 0.8488 in growing and fattening pigs,nursery pigs and weaned pigs respectively.The linear regression slope was 1.806 between feeds and feces,and 1.220,0.7219 and 1.494 for growing and fattening pigs,nursery pigs and weaned pigs respectively.The results indicate that high Zn diet is widely used in the pig production and the problems of excessive and residual Zn are serious.2.The effect of high Zn diets on the morphology of small intestine and the expression of apoptosis-related genes in epithelial cells of Bama miniature PigsIn the previous survey,the average Zn concentration of pig feeds was 658.9 mg/kg and much higher than the upper limit of feed standards in China.It shows that the average Zn concentration of the pig feeds was much higher than the recommended amount and high Zn feeds were widely used.The small intestine is the main contact and absorption organ of high Zn diets.The effects of high Zn diets on small intestinal epithelium are not clear.The aim of this study is to investigate the effects of high Zn diets on small intestinal morphology and small intestinal epithelial cells.Eighteen six-month-old male Bama miniature pigs were randomly allocated into three groups and treated with three levels of Zn(Control,T1 and T2,diet supplemented with 0,50 and 1500 mg/kg Zn respectively,as zinc sulfate)for 38 days.The tissue of small intestine was excised.The results showed that Zn concentrations of small intestine,serum and feces in T2 group were higher than those in the Control and T1 group(p<0.05).In T2 group,high Zn diets treatment caused marked damages to duodenal and jejunal epithelium.The expression of Bax,cleaved caspase-3 and caspase-8 increased in duodenum and jejunum of T2 group compared with the Control(p<0.05).The mRNA transcript level of BAX,CYCS and CASP3 genes was up-regulated in duodenum and jejunum of T2 group compared with the Control.It indicated that high Zn diets increased the accumulation of Zn and the expression of apoptosis-regulated genes,which might activate the apoptosis and lead to the marked injury of porcine small intestinal epithelium.3.The effect of high Zn diets on apoptosis and autophagy in porcine intestinal epithelial cells(IPEC-J2)Previous studies have found that high Zn diets damaged duodenal and jejunal epithelial cells and increased the expression of apoptosis-related genes.It indicated that high Zn diets might induce the apoptosis in duodenal and jejunal epithelial cells.In order to reveal the effect of high Zn on porcine small intestinal epithelial cells,this study aims to investigate the effect of Zn on IPEC-J2 cells and determine the molecular mechanism underlying this effect.IPEC-J2 cells are a jejunal porcine epithelial cell line,and the Zn source is zinc sulfate.The MTT assay was used to assess the inhibitory effect of Zn on IPEC-J2 cells.Apoptosis,cell cycle and mitochondrial membrane potential measurements were analyzed using flow cytometry.The expression of protein and mRNA of related genes were determined by using fluoro-immunoassay and quantitative real-time polymerase chain reaction(RT-PCR).The protein expression of cleaved caspase-3,P62 and LC3 II was detected through western blotting.Results showed that Zn inhibits the proliferation of IPEC-J2 cells and induces apoptosis in a dose-dependent manner.The results of flow cytometry showed that the mitochondrial membrane potential of IPEC-J2 cells significantly decreased after 1000 μM Zn treatment compared with 0 μM Zn treatment,and the apoptosis level increased and the G2 phase of cell cycle arrest was observed.The results of fluorescence immunoassay showed that IPEC-J2 cells increased the protein expression of LC3 II after 1000 μM Zn treatment compared with 0 μM Zn treatment.The results of western blotting showed that the protein expression of cleaved caspase-3 and LC3 II increased,but the protein expression of P62 decreased in IPEC-J2 cells after 1000 μM Zn treatment compared with 0 μM Zn treatment.The results of RT-PCR showed an up-regulation of BAX,CASP3,CASP8,CYCS,MDM2,CYCLINB and LC3 Ⅱ in IPEC-J2 cells after 1000 μM Zn treatment compared with 0 μM Zn treatment.These results suggested that the cytotoxicity mechanism of high Zn in IPEC-J2 cells involved the mitochondrial apoptosis and autophagy.4.The role of autophagy inhibitor in high Zn-induced apoptosis in porcine intestinal epithelial cells(IPEC-J2)Previous studies revealed that high Zn induced apoptosis and autophagy in IPEC-J2 cells,but the interaction mechanism of apoptosis and autophagy was unclear.Consequently,this study aims to investigate the interaction mechanism of high Zn-induced apoptosis and autophagy.Apoptosis,cell cycle and mitochondrial membrane potential measurements were analyzed using flow cytometry.The protein and mRNA expression of related gene were determined using fluoro-immunoassay and quantitative RT-PCR.The protein expression of cleaved caspase-3,P62 and LC3 Ⅱ was detected through western blotting.3-MA,autophagy inhibitor,inhibited the expression of LC3 Ⅱ and increased the expression of P62,which means inhibited autophagy.Suppression of autophagy inhibited high Zn-induced increase of expression of cleaved caspase-3 protein,the degradation of P62,the increase level in apoptosis,the increase in the number of G2 phase cells and the decrease in mitochondrial membrane potential.The results of RT-PCR showed that inhibition of autophagy could partially prohibit high Zn-induced genes upregulation such as BAX,CASP3,CYCS,CDC2,MDM2,CYCLIN B1 and LC3 Ⅱ.These results showed that high Zn could induce mitochondrial apoptosis by increasing autophagy level in IPEC-J2 cells.5.The effect of high Zn on autophagy-related differential expression genes in intestinal porcine epithelium cells(IPEC-J2)Previous studies revealed that high Zn induced mitochondrial apoptosis by increasing autophagy level in IPEC-J2 cells and caused the damage of jejunal epithelial cells.But the mechanism of high Zn-induced autophagy in small intestinal epithelial cells is not clear.Sequencing of mRNA transcripts(RNA-Seq)allows the identification of differentially expressed genes under various physiological and pathological conditions to elucidate the molecular pathways involved.We used RNA-seq to examine genes differentially expressed during zinc-induced autophagy in IPEC-J2 cells to reveal the mechanism of high Zn induced autophagy.After the treatments of the control and the zinc treatment groups,RNA was extracted and sequencing performed to identify novel transcripts up-or down-regulated in the zinc treatment groups and constructing protein-protein interaction networks.A total of 15446 genes and 1723 new genes and 457 differentially expressed genes were identified in the control and treatment groups.Among them,42.01%were upregulated and 57.99%were downregulated.Initiation of autophagy by zinc was associated with upregulation of INSR,UBC,MAPK10,CMC4,MPV17L2,CCNG2,GADD45B,ATG9B,DDIT3,DUSP1,FOS,JUN and NR4A1,and downregulation of RICTOR,FLNC,MAP3K2,MYC,MET,SACS,PDP1,SERPINE1,SOGA1,BIRC3,IL1A and IL1RAP.Many other differentially expressed genes were associated with cell signaling pathways(including ’mTOR signaling pathway’,’MAPK signaling pathway’s and ’p53 signaling pathway’).In IPEC-J2 cells,high Zn affects many cellular signals and metabolic-related genes,which are also associated with apoptosis.The results indicated that,after the treatment of high zinc,IPEC-J2 cells were up-regulated ATG9 and JNK of MAPK signaling pathway,down-regulated RICTOR of mTOR signaling pathway,and induced the occurrence of autophagy.