N-AcetyI-L-cysteine and pyrrolidine dithiocarbamate inhibited nuclear factor-κB activation in alveolar macrophages by different mechanisms

Aim:To study the effects of N-acetyl-L-cysteine(NAC)and pyrrolidine dithio- carbamate(PDTC)on the phosphorylation of IκB kinase(IKK)β,IKKα,and IλBα in alveolar macrophages(AM),and to explore the pharmacological mechanisms of NAC and PDTC as inhibitors of NF-κB activation.Methods:AM were collected from bronchoalveolar lavage fluid from the patients with chronic obstructive pul- monary disease.The AM were incubated for 1.5 h with NAC and PDTC,and then stimulated for 90 min by either tumor necrosis factor(TNF)-α or interleukin(IL)-1. Western blotting was used to detect the protein phosphorylation levels of IKKβ, IKKα,and IκBα.NF-κB activity was analyzed by using an electrophoretic mobil- ity shift assay.Results:NAC inhibited the phosphorylation of IKKβ,IKKα,and IκBα induced by TNF-α,but had no effect on the phosphorylation of IKKβ,IKKα and IκBα induced by IL-1.PDTC did not inhibit the phosphorylation of IκBα induced by TNF-α or IL-1.Similarly,NAC inhibited the activation of NF-κB induced by TNF-α,but had no effect on the activation of NF-κB induced by IL-1. PDTC significantly inhibited the activation of NF-κB induced by TNF-α and IL-1. The electrophoretic mobility shift assay also showed that PDTC and NAC do not directly inhibit NF-κB DNA binding activity in vitro.Conclusion:PDTC prevents the degradation of IκBα via the ubiquitylation-proteasome proteolytic pathway. NAC can inhibit the processes upstream of IKK activation induced by TNF-α, which results in the decline of NF-κB activity.