N-Acetyl-L-cysteine and pyrrolidine dithiocarbamate inhibited nuclear factor-kappaB activation in alveolar macrophages by different mechanisms.
OBJECTIVE
To study the effects of N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) on the phosphorylation of IkappaB kinase (IKK) beta, IKK alpha, and IkB alpha in alveolar macrophages (AM), and to explore the pharmacological mechanisms of NAC and PDTC as inhibitors of NF-kappaB activation.


METHODS
AM were collected from bronchoalveolar lavage fluid from the patients with chronic obstructive pulmonary 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)- alpha or interleukin (IL)-1. Western blotting was used to detect the protein phosphorylation levels of IKKbeta, IKK alpha, and IkappaB alpha. NF-kappaB activity was analyzed by using an electrophoretic mobility shift assay.


RESULTS
NAC inhibited the phosphorylation of IKKbeta, IKK alpha, and IkappaB alpha induced by TNF-a, but had no effect on the phosphorylation of IKKbeta, IKK alpha and IkappaB alpha induced by IL-1. PDTC did not inhibit the phosphorylation of IkappaB alpha induced by TNF- alpha or IL-1. Similarly, NAC inhibited the activation of NF-kB induced by TNF- alpha, but had no effect on the activation of NF-kappaB induced by IL-1. PDTC significantly inhibited the activation of NF-kappa B induced by TNF- alpha and IL-1. The electrophoretic mobility shift assay also showed that PDTC and NAC do not directly inhibit NF-kappa B DNA binding activity in vitro.


CONCLUSIONS
PDTC prevents the degradation of IkappaB alpha via the ubiquitylation-proteasome proteolytic pathway. NAC can inhibit the processes upstream of IKK activation induced by TNF- alpha, which results in the decline of NF-kappaB activity.
Li
Ya-qing
YQ
Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhang
Zhen-xiang
ZX
Xu
Yong-jian
YJ
Ni
Wang
W
Chen
Shi-xin
SX
Yang
Zhao
Z
Ma
Dan
D
D000111:Acetylcysteine / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D002478:Cells, Cultured; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism*; D020672:I-kappa B Proteins / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D029424:Pulmonary Disease, Chronic Obstructive / Q000473:pathology; D011759:Pyrrolidines / Q000494:pharmacology*; D013859:Thiocarbamates / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000975:Antioxidants; D020672:I-kappa B Proteins; D007375:Interleukin-1; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D011759:Pyrrolidines; D013859:Thiocarbamates; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; C020972:pyrrolidine dithiocarbamic acid; D051550:I-kappa B Kinase; D000111:Acetylcysteine