自噬负性调节LPS诱导的猪主动脉瓣瓣膜间质细胞成骨样表型转换

该文主要研究自噬对脂多糖(lipopolysaccharide,LPS)诱导的瓣膜间质细胞成骨样表型转换的影响与机制。免疫组化检测钙化(3例)与正常人(3例)主动脉瓣膜标本中成骨、炎症、自噬相关指标的表达情况。LPS处理猪原代主动脉瓣膜间质细胞(porcine aortic valvular interstitial cells,pAVICs)(2~6代),Western blot检测成骨、炎症、自噬指标水平变化。自噬激动剂、抑制剂联合LPS处理pAVICs,Western blot检测自噬、成骨、炎症水平变化,转染GFP-LC3病毒以观察自噬激活水平。NF-κB抑制剂联合LPS处理pAVICs,Western blot检测炎症、成骨水平变化。结果显示,成骨指标(RUNX2、OPN)、炎症指标(p-NF-κB)、自噬指标(LC3、Beclin 1)在病变主动脉瓣膜标本中,表达水平皆高于正常对照组;Western blot显示,LPS处理可引起pAVICs的成骨、炎症、自噬水平上调;自噬抑制剂对LPS诱导的自噬激活有抑制作用,对LPS诱导的成骨水平上调有促进作用;自噬激动剂对LPS诱导的自噬激活有促进作用,可明显抑制LPS诱导的成骨水平上调;NF-κB抑制剂可抑制LPS诱导的炎症,成骨水平上调;自噬激动剂、抑制剂可分别抑制与促进LPS诱导的炎症水平上调。结果表明,自噬负性调节炎症刺激因素LPS诱导的瓣膜间质细胞成骨样表型转换,可能是通过抑制炎症通路NF-κB发挥调节作用。

Autophagy Negatively Regulates Osteogenic Phenotype Transformation of Porcine Aortic Valvular Interstitial Cells Induced by LPS

This article mainly investigated the effect and mechanism of autophagy on osteogenic phenotype transformation of valvular interstitial cells induced by LPS (lipopolysaccharide).The expression level of osteogenesis,inflammation and autophagy in calcific (3 cases) and normal (3 cases) aortic valve samples were detected by immunohistochemistry.LPS was used to treat pAVICs (porcine aortic valvular interstitial cells) (2-6 generations),and the protein levels of osteogenesis,inflammation and autophagy were detected using Western blot.Autophagy agonists and antagonists were combined with LPS to treat the primary pAVICs,and the changes of autophagy,osteogenesis and inflammation were detected by Western blot.Autophagy activation was detected by transfecting GFP-LC3.NF-κB antagonists combined with LPS to treat pAVICs,and the changes of inflammation and osteogenesis were detected by Western blot.Autophagy agonists and antagonists were combined with LPS to treat the primary pAVICs,and levels of inflammation were detected by Western blot.The results showed that the expression of the osteogenic index (RUNX2,OPN),inflammatory index (p-NF-κB) and autophagy index (LC3) in the lesion aortic valve specimens were higher than those in the normal control group.Western blot results showed an up-regulation of osteogenesis,inflammation and autophagy in primary pAVICs treated by LPS.Autophagy antagonists can inhibit autophagy activation and promote the up-regulation of osteogenesis induced by LPS.Autophagy agonists can promote autophagy activation and significantly inhibit the up-regulation of osteogenesis induced by LPS.NF-κB antagonists can inhibit inflammation and osteogenesis induced by LPS.Autophagy agonists and antagonists can inhibit and promote the up-regulation of inflammation induced by LPS,respectively.It is possible that autophagy negatively regulates the osteogenic phenotype transformation of VICs induced by LPS,which may play a regulatory role by inhibiting the inflammatory pathway,NF-κB.