靶向siRNA沉默长正五聚蛋白3对主动脉瓣膜间质细胞表达炎症细胞因子的影响

Gene Silencing of PTX3 by siRNA Inhibits Expression of Inflammatory Factors in Aortic Valve Interstitial Cells

目的通过观察脂多糖(lipopolysaccharide,LPS)对主动脉瓣膜间质细胞(aortic valve interstitial cells,AVICs)炎症细胞因子表达的影响,以及利用小干扰RNA(small interfering RNA,siRNA)靶向沉默长正五聚蛋白3(pentraxin 3,PTX3)后AVICs炎症细胞因子白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、单核细胞趋化因子-1(MCP-1)表达的变化,探讨PTX3在钙化性主动脉瓣膜病(calcific aortic valve disease,CAVD)发病机制中的作用。方法收集诊断为CAVD的瓣膜标本12例为实验组,并以心脏移植术中无病变的主动脉瓣膜标本12例为对照组,行免疫组织化学染色;体外培养AVICs,以不同浓度LPS(0、50、100、200 ng/mL)干预AVICs 24 h,利用Real-time PCR检测炎症细胞因子IL-6、IL-8、MCP-1的表达水平;利用PTX3靶向小干扰RNA(PTX3 siRNA)转染AVICs 48 h后,Western blot检测PTX3的沉默效果;转染PTX3 siRNA后的AVICs再以LPS(100 ng/mL)干预24 h,Real-time PCR检测其炎症细胞因子IL-6、IL-8、MCP-1的表达水平;结果①与对照组比较,实验组瓣膜PTX3表达水平明显增加;②不同浓度LPS干预后,AVICs表达IL-6、IL-8、MCP-1的水平均呈浓度依赖性增加,LPS浓度达100 ng/mL时,IL-6、IL-8、MCP-1的表达较对照组均显著增加(P<0.05,P<0.01);③PTX3 siRNA可有效沉默PTX3的表达(P<0.01);④与对照组(Scramble siRNA组)比较,在100 ng/mL LPS干预后,LPS+PTX3 siRNA组AVICs表达IL-6、IL-8、MCP-1均明显减少(均P<0.05)。结论 CAVD患者瓣膜表达PTX3较对照组明显增加;LPS促进AVICs表达炎症细胞因子IL-6、IL-8、MCP-1,但靶向沉默PTX3可明显抑制AVICs在LPS刺激下IL-6、IL-8、MCP-1的表达水平。PTX3对炎症细胞因子表达的调节可能在CAVD的发生机制中起重要作用。

Objective To explore the role of lipopolysaccharide(LPS)in the pathogenesis of calcific aortic valve disease(CAVD)by detecting the expression of inflammatory factors in aortic valve interstitial cells(AVICs),and the expression of interleukin-6(IL-6),interleukin-8(IL-8),monocyte chemoattractant protein-1(MCP-1)after silencing pentraxin 3(PTX3)gene,to test the effect of PTX3 on the pathophysiological process of CAVD.Methods We obtained aortic valves for immunohistochemistry staining from 12 patients with CAVD,and from 12 patients without aortic valve disease receiving heart transplant operation as control.AVICs were cultured in vitro,different concentrations of LPS(0,50,100,200 ng/mL)treated cells for 24 h,and then the expression levels of IL-6,IL-8 and MCP-1 were analyzed by real-time polymerase chain reaction(real-time PCR).After AVICs were transfected with PTX3 siRNA for 48 h to knockdown the expression of PTX3 protein,PTX3 was tested by Western blotting.The levels of IL-6,IL-8 and MCP-1 were detected by real-time PCR 24 h after treatment with LPS(100 ng/mL)in AVICs with PTX3 siRNA transfection. Results The calcific aortic valves significantly expressed PTX3 as compared with control.LPS dose-dependently increased IL-6,IL-8 and MCP-1 in AVICs.Compared with control groups,100 ng/mL LPS significantly increased IL-6,IL-8 and MCP-1 mRNA(P<0.05,P<0.01).PTX3 siRNA markedly decreased the levels of PTX3 protein compared with control groups(P<0.01).Levels of IL-6,IL-8 and MCP-1 were significantly reduced in LPS plus PTX3 siRNA group compared with controls(all P<0.05).Conclusion The calcific aortic valves have a higher level of PTX3 than control valves.LPS stimulates the expression of IL-6,IL-8 and MCP-1 in AVICs,but silencing PTX3 gene significantly inhibits LPS-stimulated expression of IL-6,IL-8 and MCP-1.PTX3 may play a role in CAVD pathogenesis by regulating expression of inflammatory factors.