Study On The Function Of Zinc Finger Protein ZNF446Homologous LT-7for MAPK Pathway Of THP1Macrophages Infected With Brucella RB51

Object：Brucellosis is a zoonosis caused by Brucella species. Macrophage was one of important targetcells for its present and replicates, Brucella intracellular survival is associated with inhibition of host cellapoptosis, and brucella outer membrane protein OMP25plays an important role in virulence and parasiticcell. Our research group to obtained42T7peptides by Phage Display Techniques. The bioinformaticsanalysis showed that the identity between LT-7C peptides and zinc finger protein peptide ZNF446aminoacid sequence was100%. This study to established the model of human monocyte THP-1cells infected byBrucella abortus strain RB51, research the influence of MAPK pathway at different stages by LT-7Cpeptides, and then discuss the impact on the cell apoptosis.Methods：In this study, heptapeptide incubated with the human monocytic cell line THP-1. The toxicitytest was determined by MTT method, and the right concentration of peptide was selected. Both infectiongroups and uninfected groups, including uninfected control groups, uninfected linear peptide group,uninfected cyclic peptide group, THP-1infected with RB51(infected control group), THP-1infected withBrucella abortus strain RB51before+linear peptide LT-7C(infected linear peptide group), THP-1infected with RB51infected before+cyclic peptide(infected cyclic peptide group), were designed. Withthe multiplicity of infection (MOI)50:1to established the model of THP-1cells by the Brucella abortusstrain RB51, which did not obtained the O-polysaccharide (OPS) of B.abortus. Cells were collected afterTHP-1cells had been infected at4,8,12,24and48hours. To analyze its effect on apoptosis of THP-1cellswere infected with B.abortus RB51at different stages by flow cytometry; The human monocytic cell lineTHP-1were infected at4and48hours, and intracellular bacteria were counted after infected cells dealedwith0.1%Triton X-100. The MAPK signaling pathway of ERK1/2, JNK1/2, P38of mRNA and proteinexpression were analyzed by qRT-PCR and Western blot at different stages.Results: i) No toxic effect was found in the cyclic heptapeptide; ii) Flow cytometry detection showed thatuninfected linar peptide group and uninfected cyclic peptide group compared with uninfected control groupthe apoptosis rate as the change of time is on the rise, statistical analysis there was no statisticallysignificant difference between groups(P>0.05); infected cyclic peptide group and infected linar peptidegroup compared with infected control group the apoptosis rate were on the rise before12h,after12h appeared lower, at4h infected cyclic peptide group is higher than the control group, the difference wasstatistically significant (P <0.05),48h infected cyclic peptide group is lower than the control group, thedifference was statistically significant (P <0.05).iii)Intracellular CFU count results show that infectedcyclic peptide group at4h lower than the control group, and the difference was statistically significant (P <0.05), linear peptide group was higher than infection control group, there was no statistically significantdifference; There was no statistically significant difference between groups during the48h. iv) The resultsof qRT-PCR showed that the copies of gene erk1in infection linear heptapeptide groups and infectioncyclic heptapeptide groups descended compared with being infected with Brucella RB51at4h, the copiesof erk2, p38, jnk1and jnk2in other groups had no changes, uninfected groups also had no changes.Compared with the control infection group, the gene jnk1/2’s copies in cyclic heptapeptide of infectiongroups had descended; the copies of erk1, erk2and p38in other groups had no changes, uninfected groupsalso had no changes. v）the results were consistent between Western blot assay and qRT–PCR detection.Conclusion:i) Infected cyclic peptide group, With the multiplicity of infection (MOI)50:1, we found thatcyclic heptapeptide could inhibit RB51into the cell at the4th hour after infection, but there is no effectionat the48th hour; ii) The infected cyclic heptapeptide group ERK1/2lower expression associated with theincreased rate of apoptosis at the4th hour,48h JNK1/2lower expression related to apoptosis rate reduce.