Expression of serum amyloid A and NLR family, pyrin domain containing 3 in arteriosclerosis obliterans of the lower extremity and activation of the NLR family, pyrin domain containing 3 inflamma some by serum amyloid A

Abstract

Objective To investigate the expression of serum amyloid A (SAA),NLR family,pyrin domain containing 3 (NLRP3) and interleukin (IL) -1β in arteriosclerosis obliterans (ASO) of the lower extremity.To identify the mechanism of NLRP3 inflammasome activation by SAA in vitro experiment.Methods The expression of SAA,NLRP3 and IL-1β in ASO artery tissues were detected by immunohistochemical (IHC) technique.Macrophages were incubated with SAA for 12 h,IL-1β-p17 in supernatants and caspase-1,pro-IL-1 β in cell extracts were detected by immunoblot.THP-1 macrophages were treated with NLRP3-targeted small interfering RNA (siRNA) to diminished NLRP3 mRNA,to detect the IL-1β-p17 and cleaved caspase-1 by immunoblot.To observe the entry of nuclear factor-κB (NF-κB) p65 from human macrophages cellular cytoplasm into nucleus after treated with SAA by immunofluorescence.Results The expressions of SAA,NLRP3 and IL-1β in ASO artery tissues were upregulated,fold of comparing with normal artery tissues is 2.49 ±0.34,3.19 ±0.22,3.61 ±0.29 and 4.46 ±0.20 respectively.In ASO artery tissues,both NLRP3 and IL-1β frequently colocalized with SAA.A significant,dose-dependent release of cleaved IL-1β ( fold of control were 1.24 ± 0.09,2.48 ± 0.22,15.59 ± 0.96,23.57 ± 0.80 respectively)and cleaved caspase-1 (fold of control were 1.32 ± 0.11,4.59 ± 0.28,17.55 ± 1.40,25.69 ± 1.71 respectively) was induced by SAA(dose of SAA were 1,2,5,10 mg/L respectively).The release of IL-1β was diminished after knockdown the NLRP3 mRNA.The expression of pro-IL-1β was upregulated while human macrophages were treated with SAA,and the upregulation of pro-IL-1β can be suppressed by NF-κB p65 inhibitor ( Bayl 1-7082).Conclusion SAA is deposited in atherosclerotic lesions of the lower extremity with a higher frequency.Expression of SAA was positively correlated with NLRP3 and IL-1β in ASO artery tissues.SAA can activate the release of IL-1β in a NLRP3 inflammasome-dependent manner.Enhanced expression of NLRP3 is necessary for NLRP3 inflammasome activation caused by SAA.SAA can prime macrophages for the NLRP3 inflammasome activation by upregulaling the expression of pro-IL-1β through NF-κB pathway. Key words: Arteriosclerosis obliterans; Serum amyloid A; Irfflammasome; NLR family,pyrin domain containing 3; Interleukin-1β