Regulatory Mechanism Of GRP94 On Liver Cell Damage Of Pig Under Stress Condition

It is still unclear how glucose-regulated protein 94(GRP94)regulates liver injury in pig under stress conditions.This dissertation includes four experiments as following.EXP.1 Study on the expression of GRP94 and IGF-1 in growing pigs under chronic heat stressTwenty-seven growing Large White barrows with similar body weight(40.8±2.7 kg)were assigned into three treatments: 1)thermal-neutral group(TN),2)chronic heat stress group(HS),and 3)pair-fed in TN condition(PFTN).The experiment lasted for 21 days.The result turns out that: Compared with TN groups,the expression level of GRP94 and Insulin like growth factor 1(IGF-1)in the liver of HS group was significantly increased(P<0.05).A overall elevation of plasma IGF-1:IGFBP-3 ratio was detected in HS pigs compared with TN counterparts(P<0.01).The correlation coefficient between GRP94 protein expression and IGF-1 level under chronic high temperature was 0.69.The results showed that endoplasmic reticulum stress(ERS)was induced by chronic HS and IGF-1 was increased,and the IGF-1 content was strongly correlated with GRP94 expression.EXP.2 Modeling endoplasmic reticulum stress in porcine liver cellsPorcine hepatic stellate cell lines(HSC)were treated with tunicamycin(TM)at concentrations of 1,2,5,10 and 15 g/mL for 2 h,4 h,8 h,16 h,24 h and 36 h,respectively.According to the detection of cell inhibition rate,cell cycle,ERS related gene and protein expression,it was determined that the ERS model could be successfully established under treatment with 5 ?g/mL TM for 24 h.EXP.3 Effect of down-expression of GRP94 on cell damage,IGF-1 expression and ubiquitination in porcine hepatocytes under endoplasmic reticulum stressThe silencing lentiviruses with GRP94 gene were constructed,and was transfected into porcine HSC for 72 h,and then 5 ?g/mL TM was added to culture for 24 h.The cells were collected.The results showed that GRP94 silence expression significantly inhibited the protein expression of p-IRE1(P<0.01).Down-expression of GRP94 promoted apoptosis of stellate cells in pig liver under ERS.Moreover,GRP94 silence expression significantly decreased ubiquitin protein expression and IGF-1 expression under ERS(P<0.01).The results indicated that the inhibition of GRP94 expression induced cell damage and inhibited the expression of IGF-1 significantly.EXP.4 Establishment of endoplasmic reticulum stress model in piglets and detection of cell damage and ubiquitinationTwenty-one piglets with 35-day age and similar body weight were by intraperitoneal injection with low dosage TM(LD,0.1 mg/kg body weight TM),high dosage TM(HD,0.3 mg/kg body weight TM)or vehicle(DMSO)for 48 h.The results showed that hepatic ERS-related proteins and genes in LD and HD groups were significantly increased(P<0.01),and inflammatory cytokines and complements were significantly increased(P<0.05).Compared with the vehicle group,there were no significant difference in apoptosis rate,apoptosis-associated protein and gene expression in LD and HD group.Compared with the vehicle group,the ubiquitin protein was significantly up-regulated in LD liver(P=0.04),and the expression of ubiquitination-related genes was also significantly increased(P<0.05).The results showed that both doses of TM could successfully induce ERS in the liver of piglets,and ERS was more obvious in the HD group.EXP.5 Proteomics analysis of pig liver under endoplasmic reticulum stress and research on the relationship between GRP94 and IGF-1This test,using iTRAQ technology,analyzed effect of ERS on liver differential protein expression in piglets with administration of HD TM treatment.The results were as follows: this trial screened 311 differentially expressed proteins.It was found that the differential expressed proteins are mainly involved in inflammatory response and immune system regulation,and are concentrated in signaling pathways such as metabolic pathways,protein processing in endoplasmic reticulum,and complement and coagulation cascades via bioinformatics analysis.Both GRP94 and IGF-1 expression were significantly increased under ERS(P<0.05).Through String network analysis,it was found that GRP94 interacts with IGF-1 through PDIA3.In conclusion,IGF-1 changes positively with GRP94 under in vivo and in vitro stress.