Investigation Of Cadmium Pollution In Swine Farms Around Harbin And Effects Of Cadmium Poisoning On Ileum Injury Of Swine

Cadmium was one of the harmful heavy metals in soil.Cadmium exposure had been proved to induce different diseases in human and animals.Acute cadmium poisoning could lead to headache,sore throat,cough,bronchitis,pneumonia,pulmonary edema and death due to respiratory failure.Chronic cadmium poisoning could lead to emphysema,pulmonary fibrosis,nephritis,and renal failure,as well as osteoporosis,fracture and other skeletal diseases.With the rapid development of livestock and poultry industry,people paying more and more attention to the food safety of livestock and poultry,so it is necessary to study the cadmium pollution in swine farms.In order to explore this situation and the damage effect of cadmium exposure on swine intestines,this paper carried out the following four parts of the study.(1)Firstly,six standardized swine farms around Harbin were selected.Five pregnant sows and five reserve swines were randomly selected from each swine farm.The content of cadmium in feces and blood of swines were detected by inductively coupled plasma mass spectrometry(ICP-MS).(2)Then 10 healthy 6-week-old piglets were randomly divided into control group and cadmium treatment group.They were fed with basal diet and high cadmium diet(basal diet+20 mg/kg cadmium chloride).The experiment lasted for 40 d.The model of cadmium exposure in swines was established,the development(mental state and body weight)and small intestine structure(microstructure and ultrastructure)of swines were observed.The swines were tested oxidative stress(contents of ROS and MDA,activities of T-AOC and SOD),calcium homeostasis(expressions of ATP2A,PMCA,RyR1,RyR3,IP3R and CAMK?),endoplasmic reticulum stress(ER)(expressions of GRP78,CHOP,XBP1,LAMP and ATF6),programmed necroptosis(expressions of FADD,FAS,MLKL,RIPK1,RIPK3 and TNF),and inflammatory(expressions of NF-?B?IL-1??IL-2?IL-7?IFN-??IL-4?IL-10?iNOS?COX-2 and HO-1),heat shock proteins(expressions of HSP27,HSP40,HSP60,HSP70 and HSP90).The purpose of this study was to explore the damage effect and potential mechanism of cadmium poisoning on small intestine of swines.(3)The IPEC-J2 small intestinal epithelial cell line was used as the test object,Added 0 ?M(control group),5 ?M(low dose group),15 ?M(medium dose group)and 25 ?M(high dose group)of cadmium chloride to culture medium for 6 h,established cadmium poisoning cell model,observe the damage(morphology and activity)of IPEC-J2 cells,Oxidative stress(contents of ROS and MDA,activities of T-AOC and SOD),calcium homeostasis(calcium concentration,expressions of ATP2A,PMCA,RYR1,RYR3,IP3R and CAMK?),ER(expressions of GRP78,CHOP,XBP1,LAMP and ATF6),necroptosis(FADD,FAS,MLKL,RIPK1,RIPK3 and TNF expression levels),inflammation(expressions of NF-?B?IL-1??IL-2?IL-7?IFN-??IL-4?IL-10?iNOS?COX-2 and HO-1),heat shock proteins(expressions of HSP27,HSP40,HSP60,HSP70 and HSP90).To confirm the damage effect and mechanism of cadmium poisoning on swine intestinal epithelial cells,and analyze the dose-response relationship between cadmium and swine intestinal epithelial cells damage,and screen the appropriate exposure dose of cadmium in IPEC-J2 cells(the cadmium dose with the lowest calcium concentration).(4)Finally,according to the IPEC-J2 cell activity test,the appropriate intervention dose of GSK1016790A was selected,using 0.5 ?M GSK1016790A joint 25 ?M cadmium chloride treated IPEC-J2 cells,The experiment were divided into control group(0 ?M GSK1016790A+0 ?M cadmium chloride),cadmium chloride poisoning group(25 ?M cadmium chloride),GSK1016790A intervention group(0 ?M GSK1016790A+25 ?M cadmium chloride),GSK1016790A intervention group(0.5 ?M GSK1016790A),GSK1016790A treated with cadmium chloride for 6 h and GSK1016790A for 20 min.In GSK1016790A intervention group,GSK1016790A acted on the cells first.The results might screen out the target of cadmium induced intestinal injury in swines,and provide a theoretical basis for the prevention and treatment of cadmium induced intestinal toxicity.The results are as follows:(1)Investigation results of cadmium pollution in swine farm in HarbinThe investigation results of cadmium concentration in feces and blood of six large-scale swine farms around Harbin showed that there were different degrees of cadmium pollution in swine farms.(2)Experimental results of cadmium treatment on swines1)The weight of piglets in cadmium treatment group was significantly lower than that of control group(P<0.05),indicating that cadmium exposure inhibited the growth and development of piglets.2)The microstructure and ultrastructure of ileum in cadmium treated group were obviously damaged.3)The contents of ROS and MDA in ileum of piglets in cadmium treatment group were significantly higher than those in control group(P<0.05),and the activities of T-AOC and SOD were significantly lower than those in control group(P<0.05),suggesting that cadmium could cause oxidative stress in ileum.4)The mRNA and protein expression levels of ATP2A and PMCA in ileum of piglets in cadmium treatment group were significantly higher than those in control group(P<0.01),but the mRNA and protein expression levels of RyR1?RyR3?IP3R?CAMKII in cadmium treatment group were significantly lower than those in control group(P<0.05);3D PCA analysis showed that ATP2A was positively correlated with PMCA,GRP78,CHOP,XBP1,LAMP and ATF6,and negatively correlated with RyR1,RyR3,IP3R and CAMK;These results indicated that cadmium damages the calcium homeostasis and causes ER in ileum.5)The mRNA and protein expression levels of FADD,RIPK1,RIPK3,MLKL and FAS were significantly increased in cadmium treated ileum(P<0.05),suggesting that cadmium exposure triggered RIPK3 dependent necroptosis.6)The mRNA and protein expression levels of HO-1?IL-1 ??iNOS and COX2 in the Cd group were significantly higher than those in the control group(P<0.05),suggesting that cadmium exposure could induce ileal inflammation in swines;The mRNA and protein expressions of IL-4,IL-6 and IL-10 in the experimental group were significantly higher than those in the control group(P<0.05),the expression of IFN-y in the Cd group was significantly lower than that in the control group(P<0.05),suggesting that cadmium exposure aggravates inflammatory reaction.7)The mRNA expressions of HSP40 and HSP90 in cadmium treated ileum were significantly higher than those in control group(P<0.05),The protein expressions of HSP60,HSP70 and HSP90 in Cd group were significantly higher than those in the control group(P<0.05),suggesting that cadmium exposure may lead to the damage of ileum in swines.(3)Results of cadmium chloride on IPEC-J2 small intestinal epithelial cells1)The contents of ROS and MDA in IPEC-J2 cells treated with cadmium were significantly higher than those in the control group(P<0.05),and the activities of T-AOC and SOD in IPEC-J2 cells treated with cadmium were significantly lower than those in the control group(P<0.05),These results suggested that cadmium couuld induce ROS accumulation and oxidative stress in IPEC-J2 cells.2)The concentration of Ca²⁺in IPEC-J2 cells treated with cadmium was significantly lower than that in the control group(P<0.05),the mRNA and protein expressions of ATP2A and PMCA were significantly higher than those of the control group(P<0.01),though the mRNA levels of RYR1,RYR3,IP3R and CAMKII were significantly lower than those of the control group(P<0.05),The mRNA and protein expressions of GRP78 and LAMP were significantly higher than those in the control group(P<0.01),suggesting that cadmium damaged the calcium homeostasis of IPEC-J2 cells and caused endoplasmic reticulum stress.GSK1016790A significantly alleviated the increase of ATP2A and PMCA expression and the decrease of RYR3,IP3R and CaMK? expression induced by cadmium(P<0.05),these results indicated that GSK1016790A could inhibit the imbalance of calcium homeostasis and endoplasmic reticulum stress induced by cadmium in IPEC-J2 cells.3)The mRNA and protein expression levels of FAS,RIPK1,RIPK3 and TNF in IPEC-J2 cells treated with cadmium were significantly increased(P<0.05),suggesting that cadmium exposure triggered RIPK3 dependent necroptosis in IPEC-J2 cells.4)The mRNA and protein expressions of iNOS and COX-2 in IPEC-J2 cells treated with cadmium were significantly higher than those in the control group(P<0.05),the expression of IFN-? was significantly decreased(P<0.05),IFN-? The protein expression level was significantly decreased(P<0.05),suggesting that cadmium exposure aggravates inflammatory reaction.5)The mRNA and protein expressions of HSP27,HSP70 and HSP90 in IPEC-J2 cells treated with cadmium were significantly higher than those in the control group(P<0.05),the protein expressions of HSP60,HSP70 and HSP90 were significantly higher than those in the control group(P<0.05),suggesting that cadmium exposure could cause damage to IPEC-J2 cells.Comprehensive analysis of the experimental results showed that there were different degrees of cadmium pollution in swine farms around Harbin.Cadmium exposure led to oxidative stress in swine ileum,imbalance of calcium homeostasis,ER and heat shock protein activation,triggering necroptosis and inflammatory reaction,and eventually lead to injury of swine ileum.The results could provide new ideas for scientific prevention and control of cadmium enteric toxicity,and also provide reference for comparative medicine and public he alth safety evaluation.