低强度脉冲超声通过p38/JNK-白细胞介素-6抑制类风湿性关节炎滑膜成纤维细胞增殖的研究

关键词：低强度脉冲超声  类风湿性关节炎  滑膜成纤维细胞  白细胞介素6  增殖

Keywords:low intensity pulsed ultrasound  rheumatoid arthritis  fibroblast-like synoviocytes  interleukin-6  proliferation

摘要：

      摘要 目的：探讨低强度脉冲超声（low-intensity pulsed ultrasound，LIPUS）对类风湿性关节炎滑膜成纤维细胞（fibroblast-like synoviocytes in rheumatoid arthritis， RA-FLS）异常细胞表型的抑制作用及可能机制。 方法：酶消化法分离滑膜细胞，显微镜下观察细胞形态，同时用免疫荧光检测Vimentin蛋白表达来鉴定RA-FLS。将细胞进行体外培养并分为4组：对照组、LIPUS组、肿瘤坏死因子-α（tumor necrosis factor， TNF-α）组和TNF-α+LIPUS组或3组：对照组、白细胞介素-6（interleukin-6， IL-6）组和IL-6+LIPUS组。CCK8和EDU实验分别检测LIPUS对RA-FLS细胞活性和增殖的作用， 划痕实验和Transwell迁移实验观察LIPUS对RA-FLS迁移能力的影响，RT-qPCR检测RA-FLS中重要的细胞因子、趋化因子和基质金属蛋白酶（matrix metalloproteinases， MMPs）的基因表达，ELISA进一步检测LIPUS对RA-FLS中关键效应分子IL-6蛋白水平的作用，Western Blot检测LIPUS对RA-FLS中丝裂原活化蛋白激酶（mitogen-activated protein kinase， MAPK）信号通路的影响。 结果：分离得到较纯净的RA-FLS。在体外培养的RA-FLS中，首先，LIPUS可以抑制TNF-α诱导的细胞活性（P<0.001）和增殖（P=0.007），但它对其迁移及迁移相关MMPs（MMP2和MMP9）转录水平的作用在组间无显著性差异（P>0.05）。在TNF-α诱导的炎性环境下，LIPUS能够抑制IL-6和白细胞介素-8（interleukin-8， IL-8）在mRNA水平上的高表达（P<0.001），但其对白细胞介素-1β （interleukin-1β， IL-1β）、MMP1和MMP13的抑制作用在组间无显著性差异（P>0.05）。与未处理组相比，LIPUS抑制TNF-α诱导的RA-FLS中IL-6的分泌（P<0.001），同时也抑制IL-6诱导的RA-FLS增殖（P=0.003）。LIPUS能够抑制MAPK信号通路中p38 MAPK（P=0.033）的磷酸化和c-Jun氨基末端激酶（c-Jun N-terminal kinase， JNK）的磷酸化（P=0.019），但其对细胞外信号调节激酶1/2（extracellular signal-regulated kinas 1/2， ERK1/2）蛋白的磷酸化则无显著作用（P>0.05）。 结论：LIPUS能够减少炎性状态下RA-FLS的异常增殖而不作用于其迁移，该作用可能与p38/JNK-IL-6信号通路的抑制有关。

Abstract:

      Abstract Objective： To explore the effect of low intensity pulsed ultrasound （LIPUS） on inhibiting the abnormal cell phenotype of fibroblast-like synoviocytes in rheumatoid arthritis（RA-FLS） and possible mechanism. Method： Synoviocytes were isolated by using enzyme digestion， and the morphology of cells was observed under microscope. At the same time，the expression of Vimentin protein was detected by immunofluorescence method to identify RA-FLS. Cells cultured in vitro were divided into four groups： control group， LIPUS group， tumor necrosis factor （TNF-α） group and TNF-α+LIPUS group or three groups： control group， interleukin-6 （IL-6） group and IL-6+LIPUS group. The effects of LIPUS on RA-FLS cell viability and proliferation were detected by CCK8 and EDU assay respectively， and the effects of LIPUS on RA-FLS migration were observed by scratch test and Transwell migration assay. RT-qPCR was used to detect the gene expression of important cytokines， chemokines and matrix metalloproteinases （MMPs） in RA-FLS. ELISA was used to further detect the effect of LIPUS on the expression of IL-6， a key effector of RA-FLS， and the effects of LIPUS on mitogen-activated protein kinase（MAPK）signaling pathway in RA-FLS were detected by Western Blot. Result： Purified RA-FLS were obtained. Firstly， LIPUS could suppress the cell activity （P<0.001） and proliferation （P=0.007） induced by TNF-α in RA-FLS cultured in vitro. However, the migration and the transcription levels of MMPs related to migration （MMP2 and MMP9）were not significantly different between groups （P>0.05）. LIPUS could inhibit the high expression of IL-6 and interleukin-8 （IL-8） at the mRNA level in the inflammatory environment induced by TNF-α （P<0.001）， but there was no significant difference in the suppression of interleukin-1β （IL-1β）， MMP1 and MMP13 （P>0.05）. In addition， compared with untreated group， LIPUS could inhibit the secretion of IL-6 in RA-FLS induced by TNF-α （P<0.001）， and also inhibited the proliferation of RA-FLS induced by IL-6 （P=0.003）. Finally， LIPUS could inhibit the phosphorylation of p38 MAPK and c-Jun N-terminal kinase （JNK） in MAPK signaling pathway （P=0.033）， but the effect on the phosphorylation of extracellular signal-regulated kinas 1/2 （ERK1/2） was not significantly （P>0.05）. Conclusion： LIPUS could reduce the abnormal proliferation of RA-FLS in inflammatory state without affecting its migration， which might be related to the inhibition of p38/JNK-IL-6 signaling pathway.