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

Low intensity pulsed ultrasound suppressed the proliferation of fibroblast-like synoviocytes in rheumatoid arthritis through p38/JNK-interleukin-6 trans-signaling pathway

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(fibroblastlike 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-q PCR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RAFLS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m RNA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 signalregulated 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。

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-αin 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.