Effect Of The Human Parvovirus B19VP1Unique Protein On Myocardiocytes And The Underlying Mechanism

【Background】Human parvovirus B19(B19) is an isometric non-enveloped viruscontaining a single-stranded DNA. It is a common human pathogen that causes avariety of diseases with outcomes ranging from asymptomatic diseases to death,such as spontaneous abortion, fetal anomaly, aplastic crises, glomerular nephritis,and arthritis. High positive rate of B19infection had been found in cases withheart malformation, affirmed by detecting B19DNA in fetal heart autopsyspecimens with heart malformation and in myocardial tissue samples ofcongenital heart disease, and this finding was first reported by our lab. Therealso are some researches indicate that B19infection has close relationship withmyocarditis, Kawasaki disease, coronary artery disease and other cardiovasculardisease.B19can not separate culture in conventional method or set experimentalanimal models, and the mechanism of B19infection has not been clarified yet.The DNA of B19encodes two structural proteins VP1and VP2. VP2is involvedin the C-terminal region of VP1, and VP1contains an additional227aminoacids at its N-terminal end called VP1unique (VP1u) region. Previous studiesregard VP1u protein as one of candidates for subunit vaccine research. But in our study, we found that VP1u protein alone is sufficient to elicit the myocardialinjury. The myocardium pathological changes include Henle’s fissuresbroadening, myofilament dissolving, vacuolization of the chondriosomes and soon. So it is necessary to re-examine the function of B19VP1u protein, andanswer those questions list below. What is the mechanism of the myocardiumpathological changes induced by B19VP1u protein? Whether the myocardiumpathological change is caused directly by the toxic effect of viral protein orindirectly through autoantibodies? What are the signaling pathways that involvein the myocardium pathological changes? Whether the myocardium pathologicalchange induced by B19VP1u protein has relationship with fetal heart edema,abortion and heart malformation? Whether it mightily indicate that B19VP1uprotein can not use for vaccine research? In this research, purified B19VP1uprotein and mAb had been used to treat myocardiocytes in vivo and in vitro.Then, histopathology, molecular biology, flow cytometry and signal pathwayassay were used to detect the changing of structure, apoptosis, cell-surfacemarkers, cytokines, cell adhesion factors and signaling pathways. Through thisstudy, we hope to propose some new findings about the toxicity, immunologicinjury and signaling pathways of B19viral protein in myocardium pathologicalchanges, providing meaningful, referential data for further works on vaccine,diagnosis and treatment research.【Aims】1. Expression of B19VP1u protein and preparation of VP1u specific mAbfor further research.2. Based on the histopathology, apoptosis, cytokines, cell surface markersand signaling pathways changing of myocardiocytes after treated by B19VP1uprotein in vivo and in vitro, then investigating the mechanism of myocardiumpathological changes in the toxicity of viral protein, immunologic injury andsignaling pathways.3. Studying the impact of B19VP1u protein on the fetal heart weight, structure, apoptosis, cell surface markers, signaling pathways and fetal miceabortion, then discussing the relationship between B19VP1u protein with heartmalformation and fetal abortion.【Methods】1. Purifying B19VP1u protein and preparing VP1u-specific mAb⑴According to the VP1u protein expression of purification system whichhad been built, and got B19VP1u protein.⑵The mice were immunized with purified VP1u protein, and thencell-fusion and clone selection of hybridoma cells were used to obtainVP1u-specific mAbs.2. Detecting the pathological changes of myocardiocytes in vitroinduced by B19VP1u protein⑴To acquire purified myocardiocytes in vitro, enzymatic digestion,differential velocity adherence and5-Brdu inhibition were used.⑵Myocardiocytes were primarily cultured and treated by VP1u proteinand/or VP1u-specific mAb. Firstly, analyzed the ratio of VP1u protein into thecytoplasm. Secondly, detected structure changing by light microscope andelectron microscope. Thirdly, detected the function enzymes of myocardium bydry chemical analysis and cytokines quantitated by ELISA.⑶To analyze the apoptosis of myocardiocytes in vitro treated by VP1uprotein and/or VP1u-specific mAb, TUNEL assay and flow cytometry wereused.⑷Immunocytochemical method was used to detect the cell adhesionmolecules changing of myocardiocytes in vitro after treated by VP1u proteinand/or VP1u-specific mAb.⑸NF-κB signaling pathway and p38MAPK signaling pathway weredetected by Western blot, after myocardiocytes were treated by VP1u proteinand/or VP1u-specific mAb in vitro. 3. Observing pathological changes of myocardiocytes in vivo inducedby B19VP1u protein⑴Observed histopathology changings by light microscope and electronmicroscope, detected serum myocardial enzymes by dry chemistry test anddetected cytokines by ELISA after mice were treated by B19VP1u proteinand/or VP1u-specific mAb.⑵To analyze the apoptosis of myocardiocytes in vivo treated by VP1uprotein and/or VP1u-specific mAb, TUNEL assay and flow cytometry wereused.⑶Immunohistochemistry was used to find the cell adhesion moleculeschanging of myocardiocytes in vivo treated by VP1u protein and/orVP1u-specific mAb.⑷Molecular hybridization technique was used to find the NF-κB, PKC-αand PDGF-β changings in myocardiocytes in vivo treated by VP1u proteinand/or VP1u-specific mAb.⑸Proposing signaling pathways of pathological changes ofmyocardiocytes induced by B19VP1u protein.4. The effect of B19VP1u protein on the fetal mice heart⑴Observed the abortion, weight and histopathology changings of fetalmice heart after pregnant mice were treated by B19VP1u protein and/orVP1u-specific mAb.⑵To analyze the apoptosis of myocardiocytes in fetal mice treated byVP1u protein and/or VP1u-specific mAb, TUNEL and flow cytometry wereused.⑶Immunohistochemistry was used to find the cell adhesion moleculeschanging of myocardiocytes in fetal mice treated by VP1u protein and/orVP1u-specific mAb.⑷Molecular hybridization technique was used to find the NF-κB, PKC-α and PDGF-β changing in myocardiocytes in fetal mice treated by VP1u proteinand/or VP1u-specific mAb.【Results】1. Purifying the B19VP1u protein and preparing VP1u-specific mAb⑴B19VP1u protein was obtained. The molecular weight is22kDa,concentration is1.2mg/mL and purity is96.7%. Two VP1u-specific mAbsnamed4-H-5and5-B-9were prepared by hybridoma techniques. The isotypesof the two mAbs were determined by a two-side amplified ELISA, and weredetermined as IgG2a. While the dilution of the mAb was1:16000, the result waspositive. The antibody secreted by4-H-5has much higher titer. Theconcentration of4-H-5is900μg/mL, and5-B-9is1110μg/mL.2. Detecting the pathological changes of myocardiocytes in vitroinduced by B19VP1u protein⑴Through enzymatic digestion, differential velocity adherence and5-Brduinhibition, acquired purified myocardiocytes in vitro. After6~8h, themyocardiocytes adherented. After12~16h, irregular impulse was found, and therate of impulse was40~80beat.min-1. The adherented myocardiocytes were withspindle shape, nucleolus were seen in the core of the cells, and some pustutesstretched out of the cells. The cell survival rate of myocardiocytes was82%.After5~6d, functional symplasts were found. At this moment, the impulse ofmyocardiocytes was regular and the impulse rate was52.6±18.6beat.min-1.⑵Autophagy was seen under electron microscope in VP1u protein and/ormAb group.⑶The quantity of VP1u protein into the cytoplasm of myocardiocytes was0.036~0.468μg through BCA method detecting. The difference of dosage groupsin different time points was statistically insignificant, and the difference ofdosage groups was statistically insignificant too. The quantity of VP1u proteininto the cytoplasm of myocardiocytes was0.012~0.076μg through ELISAdetecting. The difference of dosage groups in different time points was statistically insignificant, and the difference of dosage groups was statisticallyinsignificant too.⑷There was no significant difference in the function enzymes ofmyocardium between experiment groups and control group in vitro.⑸There was no significant difference in the cytokines between experimentgroups and control group in vitro.⑹In the vitro research, we first demonstrated that VP1u protein couldinduce myocardiocytes apoptosis through TUNEL and flow cytometry methods.The apoptosis rate of VP1u protein group was5.4%, the apoptosis rate of VP1uprotein and mAb group was7.7%and the apoptosis rate of VP1u mAb groupwas15.5%.⑺The cell adhesion molecules were much higher in VP1u protein and/orVP1u-specific mAb groups through immunocytochemical method.⑻PKC/p38MAPK signaling pathway and NF-κB signaling pathway werefound in the cell apoptosis.3. Observing pathological changes of myocardiocytes in vivo inducedby B19VP1u protein⑴The Henle’s fissures broaden, nuclear swelling, increased karyoplasmicratio were found under light microscope in experiment groups. Chondriosomesconcentration, karyopyknosis, compact chromatin near karyotheca were foundunder electron microscope.⑵There was significant difference in the function enzymes of myocardiumbetween experiment groups and control group after LSD-t test.⑶There was significant difference in the cytokines between experimentgroups and control groups after LSD-t test.⑷In the vivo research, we first demonstrated that VP1u protein cloudinduce myocardiocytes apoptosis through TUNEL, immunohistochemistry andflow cytometry methods. The apoptosis rate of VP1u mAb group was the highest (15.5%). In this apoptosis changing, Bcl-2which is an anti-apoptosisfactor was found.⑸The cell adhesion molecules were much higher in VP1u protein and/orVP1u-specific mAb groups through immunohistochemistry.⑹The NF-κB, PKC-α and PDGF-β were found in VP1u protein and/orVP1u-specific mAb groups through molecular hybridization technique.4. The effect of B19VP1u protein on the fetal mice heart⑴In this part,23mice got pregnant in the experimental session, andabortion was not found in experiment groups or control group. There was nosignificant difference in the number of newborn mice between experimentgroups and control group. The difference of newborn mice heart weight betweenexperiment groups and control group was insignificant.⑵In the research on the heart of fetal mice, the Henle’s fissures broaden,kytoplasm lighten and some fatty degeneration were found under lightmicroscope in experiment groups. Under electron microscope, the Henle’sfissures broaden, vacuole under cellular membrane and secondary lysosomeswere found in VP1u protein group. Chondriosomes concentration andvacuolization were found in VP1u protein and mAb group or mAb group.⑶In the research on the heart of fetal mice, we first demonstrated thatVP1u protein cloud induce myocardiocytes apoptosis through TUNEL andimmunohistochemistry methods.⑷In the research on the heart of fetal mice, the cell adhesion moleculeswere a little bit higher in VP1u protein and/or VP1u-specific mAb groupsthrough immunohistochemistry method.⑸In the research on the heart of fetal mice, the NF-κB, PKC-α andPDGF-β were found in VP1u protein and/or VP1u-specific mAb groups throughmolecular hybridization technique.【Conclusions】 ⑴After obtaining purified VP1u protein and two VP1u-specific mAbs, itmade the foundation for further research on B19VP1u protein.⑵Through ELISA directly detected and BCA indirectly detected thequantity of VP1u protein, the results indicated that VP1u protein doesn’t enterthe cytoplasm of myocardiocytes.⑶The function enzymes of myocardium of experiment groups were normalin vitro, but the function enzymes of myocardium of experiment groups werehigher than control group in vivo. It indicates that VP1u protein doesn’t breakup myocardiocytess in vitro. The myocardiocytes injured by VP1u proteinperhaps have relationship with autoantibodies that are produced by molecularmimicry of VP1u protein.⑷We first demonstrated the pathogenic effect of VP1u protein onmyocardium through inducing apoptosis on myocardiocytes.⑸Cell apoptosis inhibitor molecules such as ICAM-1, VCAM-1,E-selectin and Bcl-2were higher in treated group when myocardiocytes hadapoptosis changing. It indicates that both apoptosis and anti-apoptosis occurredin the pathological process. To clarify the interrelationship, we need to docorrelation analysis based on different time.⑹We demonstrated that PKC/p38MAPK signaling pathway and NF-κBsignaling pathway are include in cell apoptosis signaling pathway. In furtherresearch, we will use inhibitors to find whether those signaling pathways are themain signaling pathways.⑺B19VP1u protein has relationship with heart edema of fetal mice, andhas no relationship with fetal mice death, abortion or heart malformation.