中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
4期
612-614
,共3页
姜雨刚%王文见%王冕%张志辉%黄楷%张远起%王深明
薑雨剛%王文見%王冕%張誌輝%黃楷%張遠起%王深明
강우강%왕문견%왕면%장지휘%황해%장원기%왕심명
下肢动脉硬化性闭塞症%血清淀粉样蛋白A%炎性体%Nod样受体家族包含pyrin结构域蛋白3%白细胞介素-1β
下肢動脈硬化性閉塞癥%血清澱粉樣蛋白A%炎性體%Nod樣受體傢族包含pyrin結構域蛋白3%白細胞介素-1β
하지동맥경화성폐새증%혈청정분양단백A%염성체%Nod양수체가족포함pyrin결구역단백3%백세포개소-1β
Arteriosclerosis obliterans%Serum amyloid A%Irfflammasome%NLR family,pyrin domain containing 3%Interleukin-1β
目的 观察血清淀粉样蛋白A( SAA)、Nod样受体家族包含pyrin结构域蛋白3( NLRP3)、白细胞介素-1β(IL-1β)在人下肢闭塞性动脉硬化症(ASO)中的表达,并通过SAA激活NLRP3炎性体的体外实验分析在ASO中NLRP3炎性体激活的可能机制.方法 免疫组织化学方法检测SAA、NLRP3、IL-1β在ASO血管壁中的表达.SAA处理巨噬细胞,Western blot检测成熟型IL-1β和裂解型Caspase-1,检测细胞中IL-1β前体蛋白、Caspase-1前体蛋白的表达.RNA干扰技术下调单核细胞(THP-1)细胞中NLRP3的表达.免疫荧光技术检测SAA对人巨噬细胞中核因子(NF) -κB p65入核的影响.结果 ASO下肢血管壁中SAA、NLRP3、IL-1β的表达较正常动脉壁上调倍数分别为10.19 ±0.29、7.11 ±0.14、5.06 ±0.15,与正常动脉比较差异有统计学意义(P＜0.01),各蛋白表达呈现出共区域化；SAA处理巨噬细胞,SAA各剂量组(1、2、5、10 mg/L)较对照组比较,NLRP3上调倍数相应为(2.49±0.34、3.19 ±0.22、3.61 ±0.29、4.46±0.20,P＜0.01),Caspase-1p20上调倍数相应为(1.32 ±0.11、4.59±0.28、17.55±1.40、25.69±1.71,P＜0.01),IL-1β p17上调倍数相应为(1.24±0.09、2.48 ±0.22、15.59±0.96、23.57±0.80,P＜0.01),下调THP-1巨噬细胞中的NLRP3表达后,SAA诱导的IL-1β分泌明显减低.SAA可以上调人巨噬细胞中IL-1β前体蛋白的表达,而且被NF-κB特异性阻滞剂Bay1 1-7082所抑制,上调倍数分别为17.64 ±2.07、6.45±1.78,两组比较差异有统计学意义(P＜0.01).免疫荧光显示SAA促进巨噬细胞中NF-κB p65入核.结论 SAA在ASO血管壁中广泛沉积,并上调了NLRP3和IL-1β的表达.SAA可以激活NLRP3炎性体,引起IL-1β的分泌.SAA可以上调IL-1β前体蛋白的表达,从而起到NLRP3炎性体激活致IL-1β分泌的预启动功能.因此在ASO中SAA可能通过激活NLRP3炎性体而促进动脉硬化炎症的发生、发展.
目的 觀察血清澱粉樣蛋白A( SAA)、Nod樣受體傢族包含pyrin結構域蛋白3( NLRP3)、白細胞介素-1β(IL-1β)在人下肢閉塞性動脈硬化癥(ASO)中的錶達,併通過SAA激活NLRP3炎性體的體外實驗分析在ASO中NLRP3炎性體激活的可能機製.方法 免疫組織化學方法檢測SAA、NLRP3、IL-1β在ASO血管壁中的錶達.SAA處理巨噬細胞,Western blot檢測成熟型IL-1β和裂解型Caspase-1,檢測細胞中IL-1β前體蛋白、Caspase-1前體蛋白的錶達.RNA榦擾技術下調單覈細胞(THP-1)細胞中NLRP3的錶達.免疫熒光技術檢測SAA對人巨噬細胞中覈因子(NF) -κB p65入覈的影響.結果 ASO下肢血管壁中SAA、NLRP3、IL-1β的錶達較正常動脈壁上調倍數分彆為10.19 ±0.29、7.11 ±0.14、5.06 ±0.15,與正常動脈比較差異有統計學意義(P＜0.01),各蛋白錶達呈現齣共區域化；SAA處理巨噬細胞,SAA各劑量組(1、2、5、10 mg/L)較對照組比較,NLRP3上調倍數相應為(2.49±0.34、3.19 ±0.22、3.61 ±0.29、4.46±0.20,P＜0.01),Caspase-1p20上調倍數相應為(1.32 ±0.11、4.59±0.28、17.55±1.40、25.69±1.71,P＜0.01),IL-1β p17上調倍數相應為(1.24±0.09、2.48 ±0.22、15.59±0.96、23.57±0.80,P＜0.01),下調THP-1巨噬細胞中的NLRP3錶達後,SAA誘導的IL-1β分泌明顯減低.SAA可以上調人巨噬細胞中IL-1β前體蛋白的錶達,而且被NF-κB特異性阻滯劑Bay1 1-7082所抑製,上調倍數分彆為17.64 ±2.07、6.45±1.78,兩組比較差異有統計學意義(P＜0.01).免疫熒光顯示SAA促進巨噬細胞中NF-κB p65入覈.結論 SAA在ASO血管壁中廣汎沉積,併上調瞭NLRP3和IL-1β的錶達.SAA可以激活NLRP3炎性體,引起IL-1β的分泌.SAA可以上調IL-1β前體蛋白的錶達,從而起到NLRP3炎性體激活緻IL-1β分泌的預啟動功能.因此在ASO中SAA可能通過激活NLRP3炎性體而促進動脈硬化炎癥的髮生、髮展.
목적 관찰혈청정분양단백A( SAA)、Nod양수체가족포함pyrin결구역단백3( NLRP3)、백세포개소-1β(IL-1β)재인하지폐새성동맥경화증(ASO)중적표체,병통과SAA격활NLRP3염성체적체외실험분석재ASO중NLRP3염성체격활적가능궤제.방법 면역조직화학방법검측SAA、NLRP3、IL-1β재ASO혈관벽중적표체.SAA처리거서세포,Western blot검측성숙형IL-1β화렬해형Caspase-1,검측세포중IL-1β전체단백、Caspase-1전체단백적표체.RNA간우기술하조단핵세포(THP-1)세포중NLRP3적표체.면역형광기술검측SAA대인거서세포중핵인자(NF) -κB p65입핵적영향.결과 ASO하지혈관벽중SAA、NLRP3、IL-1β적표체교정상동맥벽상조배수분별위10.19 ±0.29、7.11 ±0.14、5.06 ±0.15,여정상동맥비교차이유통계학의의(P＜0.01),각단백표체정현출공구역화；SAA처리거서세포,SAA각제량조(1、2、5、10 mg/L)교대조조비교,NLRP3상조배수상응위(2.49±0.34、3.19 ±0.22、3.61 ±0.29、4.46±0.20,P＜0.01),Caspase-1p20상조배수상응위(1.32 ±0.11、4.59±0.28、17.55±1.40、25.69±1.71,P＜0.01),IL-1β p17상조배수상응위(1.24±0.09、2.48 ±0.22、15.59±0.96、23.57±0.80,P＜0.01),하조THP-1거서세포중적NLRP3표체후,SAA유도적IL-1β분비명현감저.SAA가이상조인거서세포중IL-1β전체단백적표체,이차피NF-κB특이성조체제Bay1 1-7082소억제,상조배수분별위17.64 ±2.07、6.45±1.78,량조비교차이유통계학의의(P＜0.01).면역형광현시SAA촉진거서세포중NF-κB p65입핵.결론 SAA재ASO혈관벽중엄범침적,병상조료NLRP3화IL-1β적표체.SAA가이격활NLRP3염성체,인기IL-1β적분비.SAA가이상조IL-1β전체단백적표체,종이기도NLRP3염성체격활치IL-1β분비적예계동공능.인차재ASO중SAA가능통과격활NLRP3염성체이촉진동맥경화염증적발생、발전.
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.
