| Objective: Staphylococcus aureus, an important human pathogen, can cause a series ofinfections. The strains producing Panton-Valentine leukocidin(PVL) which contributeto the virulence, are widespread and cause a series of severe community associatedinfections. Compelling epidemiological data point to PVL as an important virulencefactor in S.aureus necrotizing infections, but its pathogenic mechanisms are notcompletely known. Therefore, in the present study, from animal experiment studies, westudy the mechanism of recombinant PVL (rPVL)-induced lung inflammation andinjury. To explore the role of polymorphonuclear leukocytes（PMNs）, nucleartranscription factor-κB (NF-κB) protein and cytokines in Panton-Valentine leukcocidin（PVL）–induced lung inflammation and injury. All these studies may establish thebasis for the further studies of its pathogenic mechanism and shed light on the treatmentof PVL-associated lung injury.Methods:(1) Establishment of animal model.45New Zealand white rabbits weredivided randomly into three groups, fifteen rabbits in each group. They were the controlgroup, rPVL treatment group and VCR+rPVL treatment group (vinblastine-treated).Then control group was treated with PBS, the other two groups were directly treatedwith endotracheal instillation of rPVL.（2） Study on the counts of PMN,apoptosis/necrosis, release of reactive oxygen species(ROS), LDH activity and lungpermeability index(LPI) in BALF. After9h post-infection, bronchoalveolar lavagefluid (BALF) was collected for counting PMNs, PMNs apoptosis/necrosis and therelease of ROS were analysed by flow cytometry(FCM), LDH activity was measured by colorimetric method, and LPI was detected by BCA.（3）Study on W/D ratio andhistopathology. After9h post-infection, the lung was collected from the rabbits fordetermine W/D ratio and H&E-stained.（4）Study on nuclear transcription factor-κB(NF-κB) protein and cytokines in lung injury. The lung was collected at3,6, or9hpostinfection, each time period had five rabbits. ELISA was performed to evaluate thelevels of expression of IL-6, IL-8, TNF-α和IL-10. NF-κB p65protein of the lung tissuewas assessed by immunohistochemistry and Western blotting method.Results:(1) Compared with control group, the counts of PMNs in BALF in rPVLtreatment group were significantly higher, which was (3.01±0.02)×106/ml vs (0.57±0.01)×106/ml. PMNs were dominated by late apoptosis, the apoptosis rate of PMNswas significantly higher, which was (63.56±3.53)%vs (0.95±0.33)%. The release ofROS was significantly higher, MFI was（1.56±0.39） vs（0.41±0.03）. LDH activity andLPI were all significantly higher. All inflammatory indicators in VCR+rPVL treatmentgroup were no significantly higher.(2) Compared with control group, W/D ratio inrPVL treatment group was significantly higher, which was (6.45±0.35) vs (3.51±0.17),histopathology study showed diffuse infiltration of inflammatory cells, hemorrhage,edema and other manifestations of lung injury. W/D ratio in VCR+rPVL treatmentgroup was no significantly higher, no inflammatory changes in lung.(3) The levels ofIL-6, IL-8and TNF-α in lung tissue in rPVL treatment group were increased gradually,and the level of IL-10was increased at9h postinfection. No cytokine in VCR+rPVLtreatment group was increased. The results of immunocytochemistry method showedthat in rPVL treatment group, increased NF-κB activity with nuclear localization in atime-dependent manner was observed. Western blotting method showed that expressionof NF-κB in nucleus in rPVL treatment group was increased gradually.Conclusion:(1) From animal experiment studies, these results indicate that rPVL caninduce lung inflammation and injury, independent of bacterial survival and replicationin the lung.(2) rPVL-induced lung inflammation and injury could be the result of recruitment and subsequent lysis of PMNs, which could damage the lung by releasingthe contents of cytotoxic granules and reactive oxygen metabolites.(3) After rPVLinfection, the expression of NF-κB and pro-inflammatory IL-6, IL-8, TNF-α in lungtissue were increased gradually, and lately IL-10was increased. We concluded rPVLcan induced the high expression of inflammatory cytokines by activating NF-κB protein,leading to lung tissue injury. |
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