Gao Gui-Cheng, Cheng Xi-Gao, Wei Qiang-Qiang, Chen Wei-Cai, Huang Wen-Zhou
Department of Orthopedics, The Second Affiliated Hospital of Nanchang University, Nanchang, China.
J Cell Biochem. 2019 Oct;120(10):17167-17179. doi: 10.1002/jcb.28977. Epub 2019 May 20.
Proinflammatory cytokine such as interleukin (IL)-1β causes inflammation of articular cartilage. In this current study, we explored the chondroprotective effects of long noncoding RNA (lncRNA) MALAT-1 on cell proliferation, apoptosis, and matrix metabolism in IL-1β-induced inflammation in articular chondrocytes. Articular chondrocytes from knee joints of normal rats were isolated and cultured, followed by identification through observation of toluidine blue and COL II immunocytochemical stainings. The proliferation of chondrocytes at passage 2 was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The inflammatory chondrocytes induced by 10 ng/mL IL-1β were observed and identified by toluidine blue and COL II immunocytochemical stainings. pcDNA 3.1 and pcDNA-MALAT-1 were transfected in the chondrocytes. Ultrastructure of chondrocytes was observed by using a transmission electron microscope. The MTT assay was carried out to evaluate chondrocyte viability. Hoechst 33258 staining and flow cytometry were adopted to assess chondrocyte apoptosis. The chondrocytes at passage 2 with the biological characteristics of chondrocytes were used for subsequent experiments. In IL-1β-treated chondrocytes, the growth rate of chondrocytes slowed down, the cells became narrow and long, the vacuoles were seen in the cells, and the morphology of the chondrocytes was irregular. The toluidine blue staining and the immunohistochemical staining of COL II became weaker. In response to IL-1β induction, articular chondrocytes showed reduced MALAT-1 expression; moreover, obvious cartilage injury was observed with decreased chondrocyte viability and Col II expression and elevated chondrocyte apoptosis, MMP-13 expression, and p-JNK expression. With the treatment of pcDNA-MALAT-1, the cartilage injury was alleviated with increased chondrocyte viability and type II collagen (Col II) expression and reduced chondrocyte apoptosis, MMP-13 expression and p-JNK expression. Taken together these results, lncRNA MALAT-1 blocked the activation of the JNK signaling pathway; thereby, IL-1β-induced inflammation in articular chondrocytes was reduced with enhanced chondrocyte proliferation and suppressed chondrocyte apoptosis and extracellular matrix degradation.
白细胞介素（IL）-1β等促炎细胞因子会引发关节软骨炎症。在本研究中，我们探讨了长链非编码RNA（lncRNA）MALAT-1对IL-1β诱导的关节软骨细胞炎症中细胞增殖、凋亡及基质代谢的软骨保护作用。从正常大鼠膝关节分离并培养关节软骨细胞，通过甲苯胺蓝观察和II型胶原蛋白（COL II）免疫细胞化学染色进行鉴定。采用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-溴化四氮唑（MTT）法检测第2代软骨细胞的增殖情况。用10 ng/mL IL-1β诱导炎性软骨细胞，通过甲苯胺蓝和COL II免疫细胞化学染色进行观察和鉴定。将pcDNA 3.1和pcDNA-MALAT-1转染至软骨细胞中。使用透射电子显微镜观察软骨细胞的超微结构。进行MTT法评估软骨细胞活力。采用Hoechst 33258染色和流式细胞术评估软骨细胞凋亡。选用具有软骨细胞生物学特性的第2代软骨细胞进行后续实验。在IL-1β处理的软骨细胞中，软骨细胞生长速率减慢，细胞变得狭长，细胞内可见空泡，软骨细胞形态不规则。甲苯胺蓝染色和COL II免疫组化染色变弱。响应IL-1β诱导，关节软骨细胞MALAT-1表达降低；此外，观察到明显的软骨损伤，软骨细胞活力和Col II表达降低，软骨细胞凋亡、基质金属蛋白酶-13（MMP-13）表达及磷酸化c-Jun氨基末端激酶（p-JNK）表达升高。经pcDNA-MALAT-1处理后，软骨损伤减轻，软骨细胞活力和II型胶原蛋白（Col II）表达增加，软骨细胞凋亡、MMP-13表达及p-JNK表达降低。综上所述，lncRNA MALAT-1阻断了JNK信号通路的激活；从而减轻了IL-1β诱导的关节软骨细胞炎症，增强了软骨细胞增殖，抑制了软骨细胞凋亡和细胞外基质降解。
