The Effects Of NF-κB/TNF-α Signaling Pathway On The Invasive Pulmonary Aspergillosis

Objective: To study the roles that early expressions of nucleus NF-κB and cytoplasmic TNF-αin the lung of normal mice and IPA model mice infected with Aspergilus fumigatus played on the anti-infection of Aspergilus fumigatus and their effects on the pathogenesis of invasive pulmonary aspergillosis.Methods:1 The establishment of IPA model mice: 160 BALB/c mice were divided into 4 groups (40mice each group). Group A: Healthy group; Group B: immunosuppressive group injected intraperitoneally with CY; Group C: Healthy group infected with Aspergilus fumigatus.; Group D: IPA model mice group infected with Aspergilus fumigatus following the administration of CY i.p. The 5 mice each group were sacrificed at 8h,24h,48h and 72h post infection for the collection of blood, lung tissues, thymus and spleen. The white blood cells counts, MPO values in lung, thymus index, spleen index, counts of Af colonies in cluture medium and observation of pathological alteration of lung tissue were used evaluate the establishment of IPA model mice.2 Semi-quantification of nucleus NF–κB p65 and cytoplasmic TNF-αexpression level in murine lung tissue with Western blot: The nucleus proteins and cellular proteins abstracted from the lung tissue of each mice groups were separated respectively via SDS-PAGE. The separated proteins transferred electrically into fibre membrane that were reacted with first antibodies respectively and second antibodies for the detection of nucleus NF–κB p65 and cytoplasmic TNF-αandβ-actin as intern control. The reacted membranes were exposed to Fuji film after being luminescence with ECL kit. The NF–κB p65 protein band, TNF-αband andβ-actin band were scanned and semi-quantitatively analyzed with image analysis software.3 The determination of MPO value in lung tissue: 100mg lung tissue was minced in 2mL 0.5%CTAB solution. The organelles were smashed using ultrasound method and centrifuged at 12000g for 20min at 4°C. 0.1mL supernatant mixed with 2.9mL reaction solution completely was scanned with spectrophotometer at a 460 nm wavelength for 2min at 25°C. 1 unit of MPO activity (light absorbance from 30 second to 90 second) was determined using H2O2 as substrate.ResuLts:1 The establishment of IPA mice model: At 8h~72h 4 time points post infection of Aspergilus fumigatus, the white blood cells counts, MPO value, thymus index and spleen index were significantly in immunosuppressive group injected intraperitoneally with CY lower than in healthy group (P<0.05)。The colonies of lung tissue cuLture were in immunosuppressive group significantly more than in healthy group (P<0.05)。The observation of pathological slide showed that conidia grew into hypha that invaded the pulmonary tissue, causing the necrosis of alveolar as the result of acute lung injury. The data suggested the IPA mice model was established efficiently. 2 Semi-quantification of nucleus NF–κB p65 and cytoplasmic TNF-αexpression level in murine lung tissue with Western blot: At 8h~72h 4 time points post infection of Aspergilus fumigatus, nucleus NF–κB p65 and cytoplasmic TNF-αexpression level of lung tissue in immunosuppressive group were significantly lower than in healthy group(P<0.05). At 8h~72h 4 time points, nucleus NF–κB p65 and cytoplasmic TNF-αexpression level of lung tissue in healthy group with infection and IPA model mice group were significantly lower than in healthy group(P<0.05), suggesting that the infection Af conidia could trigger the pathway of NF–κB /TNF-αin mice lung.3 The determination of MPO value in lung tissue: At 8h~72h 4 time points post infection of Aspergilus fumigatus, the MPO values in immunosuppressive group were significantly lower than in healthy group(P<0.05). The MPO values in IPA model mice group were significantly lower than in healthy group with infection (P<0.05). The MPO values in IPA model mice group and healthy group were significantly higher than in immunosuppressive group and healthy group(P<0.05), meaning the infection of conidia could induce the registration of neutrophils into lung.Conclusion1 The immunosuppressive mice were establishment efficiently. 2 The IPA mice model with typical acute lung injury was established successfully.3 The early infection of Aspergilus fumigatus conidia could activate the cytoplasmic NF-кB, upreguLate the level of nucleus NF-кBp65 and enhance the expression level of TNF-αin murine lung tissue.4 This study revealed that injection of CY i.p. couLd significantly decrease the physiological level of nucleus NF-кBp65 and cytoplasmic TNF-αin mice pulmonary tissue, as part of molecular mechanisms leading to the progression of conidia infection into IPA.