Protective Effects And Mechanisms Of The Intrabody Against Prolyl Hydroxylase 2(PHD2) On Acetaminophen-induced Liver Injury

Liver injury caused by viral infection,hepatotoxins,alcohol ingestion or drugs has become a significant human health problem worldwide with increasing incidence.At present,there is no effective treatment except liver transplantation for acute liver failure and end-stage chronic liver injury.However,due to the limited sources of donor tissues and the high cost,treatment of liver transplantation is severely restricted.Therefore,there is a pressing need to explore new and effective prevention and treatment strategies.Acetaminophen(4' hydroxyacetanilide,paracetamol,APAP)overdose has become the most common cause of drug-induced acute liver failure.The studies of APAP-induced liver injury(AILI)showed that angiogenesis and redox homeostasis play important roles in liver protection and repair of AILI.Hypoxia-inducible factor-1(HIF-1)is a transcription factor that plays a key role in regulating the expression of genes related to angiogenesis,redox homeostasis,and energy balance.For acute tissue injury including acute liver injury,HIF-1 is the most sensitive factor for acute hypoxic stress and the up-regulation of HIF-1 is the key to promoting angiogenesis and tissue damage repair.Intracellular prolyl hydroxylases(PHDs)promote the degradation of HIF-? ubiquitin-proteasome pathway by catalyzing the hydroxylation of HIF-? proline residues,and are rate-limiting enzymes that regulate the stability of HIF-?.In the PHDs family,PHD2 is a key rate-limiting enzyme that catalyzes HIF-1? proline hydroxylation.Studies have shown that inhibiting the activity of PHD2 leads to the stabilization of HIF-1?,thereby enhancing the induction of a large number of downstream target genes of HIF-1,which regulate a wide range of hypoxic responses such as angiogenesis,energy compensation,redox,pH homeostasis,and many other processes,ultimately improving tissue damage during acute and chronic hypoxia.Therefore,PHD2 has become a potential target for promoting angiogenesis and tissue damage repair.In view of the lack of specificity and targeting of PHD inhibitors at present and a large number of toxic side effects,so how to obtain highly effective and specific inhibitors of PHD2 activity has attracted much attention.Because of high specificity and affinity,antibodies have attracted much attention in recent years.With the development of antibody engineering technology,the derived intrabody technology can achieve the phenotypic knockout of important target molecules in the cell through appropriate modification of antibody molecules.Hence,an intracellular single-chain variable fragment(scFv)antibody against PHD2 was prepared using intrabody technology,and its activity and role in AILI were analyzed.In our previous research,we have used recombinant human PHD2 protein as the antigen to screen a scFv antibody against PHD2 from the phage library.Through soluble expression,purification and ELISA analysis,we have found that the scFv can specifically bind to recombinant PHD2.In order to target PHD2 in cells,in this study,an intrabody against PHD2(ER-INP)was prepared using the anti-PHD2 scFv gene as a template and modifying it with the endoplasmic reticulum(ER)retention signal sequence.Then we detected whether ER-INP was successfully expressed in cells and analyzed its biological activity.Finally,the role and possible mechanisms of ER-INP in AILI in mice were explored.The following results were obtained:1.The endoplasmic reticulum-retained intrabody against PHD2(ER-INP)was successfully constructed.Using the anti-PHD2 human scFv gene as a template,the intracellular localization signal to retain scFv antibody in the endoplasmic reticulum and E-tag sequence were introduced by PCR to successfully prepare the endoplasmic reticulum-retained intrabody against PHD2 gene(ER-INP).ER-INP was cloned into a eukaryotic expression vector pcDNA3.1 to construct the recombinant eukaryotic expression vector of ER-INP,pER-INP.pER-INP was transfected into cells and experiments of RT-PCR,immunofluorescence,and Western blot were performed.The results showed that the anti-PHD2 intrabody gene with the endoplasmic reticulum retention signal and E-tag sequence could efficiently express and bind to PHD2 proteins,at the same time localize in the endoplasmic reticulum.2.The intrabody against PHD2(ER-INP)specifically bound to intracellular PHD2 and inhibited its prolyl hydroxylase activity.After pER-INP was transfected into cells,co-immunoprecipitation assay,Western blot and immunofluorescence were used to analyze the bliological functions of intrabody.The results showed that ER-INP intrabody could recognize and bind to the PHD2 protein in the cells.ER-INP dramatically reduced the levels of hydroxylated HIF-1? at Pro564 and Pro402 and notably elevated HIF-1? protein levels,which indicated that ER-INP binds specifically to PHD2 and blocks the hydroxylation of HIF-1? at Pro564 and Pro402,therefore stabilizing HIF-1?.3.The intrabody against PHD2(ER-INP)showed significant proangiogenic activity in vitro and in vivo.In order to detect whether ER-INP can promote significant angiogenesis in vitro and in vivo,the wound healing assays were used to detect the migration ability of human umbilical vein endothelial cells(HUVEC),tube formation of HUVECs was determinded by the three-dimensional capillary tube formation assay,and chick chorioallantoic membranes(CAMs)was used to detect its angiogenesis.Furthermore,the expression of downstream target genes related to pro-angiogenesis of HIF-1 was analyzed using real-time quantitative PCR,Western blot and ELISA.The results showed that the expression of ER-INP significantly promoted the migration and tube formation of HUVEC,and enhanced the angiogenesis of chick embryo chorioallantoic membranes.In addition,real-time quantitative PCR,Western blot and ELISA results demonstrated that ER-INP promoted the expression of angiogenesis-related factors such as VEGF(vascular endothelial growth factor),ANGPTL-2(angiopoietin-like protein-2),MMP-2(matrix metalloproteinase-2),and MMP-13 in cells.4.The intrabody against PHD2 protected APAP-induced acute liver injury.The control plasmid with GFP was injected into the mice by tail vein.We found that the GFP protein was mainly distributed in the liver and the protein expression was detected from the first day to the third day after injection,reaching the highest level on the third day in the liver.Based on these results,we speculated that other proteins expressed by this plasmid,including ER-INP,possibly were mainly expressed in the liver and reached the highest level on the third day.Although ER-INP was expressed successfully in liver tissues of mice injected intravenously with pER-INP compared with the vector control.ER-INP did not alter liver histology or induce liver tissue damage leading to an increase in the serum ALT and AST levels.The mice were injected intraperitoneally with APAP(250 mg/kg),and sacrificed at 24 h,48 h,and 72 h after injection,respectively.It was found that the most serious injury was observed at 24 h,as evidenced by a significant increase in ALT and AST levels and apparent necrosis of liver lobular central area.However,compared with the control groups,pretreatment for mice with plasmids expressing ER-INP by tail vein injection for 48 h and then challenged with APAP could significantly inhibit the increase of serum ALT and AST and necrosis of liver lobular central area caused by APAP.5.Protective mechanisms of ER-INP on APAP-induced acute liver injury.On one hand,liver tissues in mice pretreated with ER-INP and challenged with APAP showed obvious angiogenesis effect.Immunohistochemical results showed that the expression of angiogenesis marker CD31 and HIF-1? in ER-INP-treated liver tissues was significantly up-regulated.Furthermore,the results of Western blot and ELISA of liver homogenates and sera demonstrated that the protein levels of VEGF and MMP-2,which are transactivated by HIF-1 and closely related to angiogenesis,were increased significantly in ER-INP-treated liver tissues compared with liver tissues from the control vector group.Real-time quantitative PCR results of HIF-1 target genes closely related to angiogenesis showed that VEGF,ANGPTL-2,and MMP-2 mRNA expression levels were upregulated in ER-INP-treated liver tissues compared with those in control tissues.On the other hand,liver tissues in mice pretreated with ER-INP and challenged with APAP showed obvious redox homeostasis.ER-INP pretreatment was beneficial to maintain the redox homeostasis of the liver tissues in mice challenged with APAP,which was manifested as a significant decrease of malondialdehyde(MDA)level,one of the markers of intracellular oxidative stress.Western blot results showed ER-INP pretreatment significantly inhibited APAP-induced increase of glucose regulated protein 78(GRP78),a marker of ER stress.However,ER-INP pretreatment significantly enhanced the activity of antioxidant enzyme,peroxiredoxin 4(PRDX4),and significantly inhibited the consumption of antioxidant molecule,L-glutathione(GSH).In conclusion,ER-INP may prevent APAP-induced liver injury by increasing angiogenesis and maintaining redox homeostasis.In summary,the intrabody against PHD2 can inhibit PHD2 activity in vivo and in vitro,improve the stability of HIF-1?,and promote the transcriptional activation of downstream target genes of HIF-1.The intrabody against PHD2 may protect the liver injury induced by APAP via promoting angiogenesis and maintaining redox homeostasis.These results will open a new way for the clinical treatment of tissue injury and ischemic diseases,providing a new idea and experimental basis for the development of new drugs in the future.