Effects Of Sulforaphane On Secreting TNF-α In The Alveolar Macrophage Of Pulmonary

Objective:To explore the effects of Sulforaphane on secreting TNF-α in the alveolar macrophage of pulmonary emphysema rats.Methods:1.The pulmonary emphysema rat model Construction: 18 male Wistar rats, specific pathogen free animal(SPF), were randomly divided into control group(n=6) and model group(n=12): The pulmonary emphysema rat model was constructed by two intratracheal instillation of lipopolysa- ccharide(d1,d14) and cigarette smoking inhalation for 8 weeks(except d1 and d14), The control group with normal saline insteading of lipopolysacch- aride and fresh air.After 8 weeks, Take the posterior lobe of the right lung in rat for the pathological slices and HE dyeing observation to identify model; 2. The primary alveolar macrophages extraction, purification and intervention:After succeedly model induction, all the rats were performed broncho alveolar lavage.The total white blood cells and AM percentage were counted in the broncho alveolar lavage fluid(BALF).Alveolar macrophages were collected and purified from BALF.Different concentrations of SFN(0 μmol/L;5μmol/L;10μmol/L;20 μmol/L)intervented in AM of pulmonary emphysema rats for 16 h.According to treatment methods, AM were divided into five groups:control group;SFN0 group;SFN5group;SFN10 group;SFN20 group; 3. Laboratory detection: The content of TNF-α was measured by enzyme-linked immunosor- bent assay(ELISA). The protein expression level of Nrf2,NQO1 and HO1 were measured by Western blot, respectively.The protein expression level of NF-κB was measured by Western blot, and NF-κB m RNA by real time RT-PCR( q RT-PCR).Results:1. Before model Constructed, the weight of the rats in control group were no statistical difference with the model group(211.97 ± 8.71 g VS 206.32 ± 6.52 g, p >0.05). After 6 weeks smoking, the weight of model rats were less than control group(567.48 ± 24.38 g VS 358.86 ± 20.75 g,P<0.01); 2. The total amount of cells and AM percentage in BALF of pulmonary emphysema rats were significantly higher than control group, respectively(2.79±0.42)x106/m L,(98.7±1.0)% and(0.77±0.16)x106/m L,(90.0±2.5) %, all P < 0.001; 3. In the SFN groups, content of TNF-α was higher than control group(control: 12.2±2.7pg/m L; SFN0: 150.7±11.0pg/m L; SFN5: 133.1±13.7pg/m L; SFN10: 65.3±14.8pg/m L;SFN20: 48.2±11.1pg/m L).With SFN concentration increasing, TNF-α content decreased accordingly(all P < 0.05); 4. Compared with control group, Nrf2 protein expression in nucleus was less, and more in cytoplasm in SFN0 group. its expression accordingly increased with SFN concentration increasing(all P < 0.05); 5. Compared with control group, both the expression of NQO1 and HO1 were less in SFN0 group, and accordingly increased with SFN concentration increasing(all P< 0.05).which were negatively correlated with the content of TNF-α, respectively(r=-0.918, P<0.01; r =-0.939, P<0.01); 6. Compared with control group, NF-κB protein expression in nucleus was increased significantly and less in cytoplasm in SFN0 group(all P<0.05). its expression of cell nucleus decreased with SFN concentration increasing,and exprssiong of cell cytoplasm increased accordingly(all P<0.05), which indicated poor nuclear translocation; 7. Compared with control group, NF-κB m RNA expression level in every SFN group was higher(P<0.05), but there were no statistical difference among all SFN concentration goups( P>0.05).Conclusion:1. Sulforaphane could inhibit secretion of TNF-α of alveolar macrophage of pulmonary emphysema rats; 2. Sulforaphane could increase expression of Nrf2 downstream target genes(NQO1, HO1) by promoting Nrf2 to translocate to nucleus,which lead to poor secretion of TNF-α of alveolar macrophage; 3. Sulforaphane could also inhibit NF-κB to translocate to nucleus, which lead to poor secretion of TNF-α of alveolar macrophage.