Pecchi Emilie, Priam Sabrina, Gosset Marjolaine, Pigenet Audrey, Sudre Laure, Laiguillon Marie-Charlotte, Berenbaum Francis, Houard Xavier
Arthritis Res Ther. 2014 Jan 20;16(1):R16. doi: 10.1186/ar4443.
Nerve growth factor (NGF) level is increased in osteoarthritis (OA) joints and is involved in pain associated with OA. Stimuli responsible for NGF stimulation in chondrocytes are unknown. We investigated whether mechanical stress and proinflammatory cytokines may influence NGF synthesis by chondrocytes.
Primary cultures of human OA chondrocytes, newborn mouse articular chondrocytes or cartilage explants were stimulated by increasing amounts of IL-1β, prostaglandin E₂ (PGE₂), visfatin/nicotinamide phosphoribosyltransferase (NAMPT) or by cyclic mechanical compression (0.5 Hz, 1 MPa). Before stimulation, chondrocytes were pretreated with indomethacin, Apo866, a specific inhibitor of NAMPT enzymatic activity, or transfected by siRNA targeting visfatin/NAMPT. mRNA NGF levels were assessed by real-time quantitative PCR and NGF released into media was determined by ELISA.
Unstimulated human and mouse articular chondrocytes expressed low levels of NGF (19.2 ± 8.7 pg/mL, 13.5 ± 1.0 pg/mL and 4.4 ± 0.8 pg/mL/mg tissue for human and mouse articular chondrocytes and costal explants, respectively). Mechanical stress induced NGF release in conditioned media. When stimulated by IL-1β or visfatin/NAMPT, a proinflammatory adipokine produced by chondocytes in response to IL-1β, a dose-dependent increase in NGF mRNA expression and NGF release in both human and mouse chondrocyte conditioned media was observed. Visfatin/NAMPT is also an intracellular enzyme acting as the rate-limiting enzyme of the generation of NAD. The expression of NGF induced by visfatin/NAMPT was inhibited by Apo866, whereas IL-1β-mediated NGF expression was not modified by siRNA targeting visfatin/NAMPT. Interestingly, PGE₂, which is produced by chondrocytes in response to IL-1β and visfatin/NAMPT, did not stimulate NGF production. Consistently, indomethacin, a cyclooxygenase inhibitor, did not counteract IL-1β-induced NGF production.
These results show that mechanical stress, IL-1β and extracellular visfatin/NAMPT, all stimulated the expression and release of NGF by chondrocytes and thus suggest that the overexpression of visfatin/NAMPT and IL-1β in the OA joint and the increased mechanical loading of cartilage may mediate OA pain via the stimulation of NGF expression and release by chondrocytes.
骨关节炎（OA）关节中神经生长因子（NGF）水平升高，且与OA相关疼痛有关。软骨细胞中负责刺激NGF的刺激因素尚不清楚。我们研究了机械应力和促炎细胞因子是否会影响软骨细胞合成NGF。
通过增加白细胞介素-1β（IL-1β）、前列腺素E₂（PGE₂）、内脏脂肪素/烟酰胺磷酸核糖转移酶（NAMPT）的量，或通过循环机械压缩（0.5赫兹，1兆帕）刺激人OA软骨细胞、新生小鼠关节软骨细胞或软骨外植体的原代培养物。在刺激前，用吲哚美辛、Apo866（NAMPT酶活性的特异性抑制剂）预处理软骨细胞，或用靶向内脏脂肪素/NAMPT的小干扰RNA（siRNA）转染软骨细胞。通过实时定量聚合酶链反应评估NGF mRNA水平，并通过酶联免疫吸附测定法测定释放到培养基中的NGF。
未受刺激的人和小鼠关节软骨细胞表达低水平的NGF（人关节软骨细胞、小鼠关节软骨细胞和肋外植体分别为19.2±8.7皮克/毫升、13.5±1.0皮克/毫升和4.4±0.8皮克/毫升/毫克组织）。机械应力诱导条件培养基中NGF释放。当受到IL-1β或内脏脂肪素/NAMPT（软骨细胞对IL-1β产生的一种促炎脂肪因子）刺激时，在人和小鼠软骨细胞条件培养基中均观察到NGF mRNA表达和NGF释放呈剂量依赖性增加。内脏脂肪素/NAMPT也是一种细胞内酶，作为烟酰胺腺嘌呤二核苷酸生成的限速酶。Apo866抑制内脏脂肪素/NAMPT诱导的NGF表达，而靶向内脏脂肪素/NAMPT的siRNA不改变IL-1β介导的NGF表达。有趣的是，软骨细胞对IL-1β和内脏脂肪素/NAMPT产生的PGE₂不刺激NGF产生。同样，环氧化酶抑制剂吲哚美辛不抵消IL-1β诱导的NGF产生。
这些结果表明，机械应力、IL-1β和细胞外内脏脂肪素/NAMPT均刺激软骨细胞表达和释放NGF，因此提示OA关节中内脏脂肪素/NAMPT和IL-1β过表达以及软骨机械负荷增加可能通过刺激软骨细胞表达和释放NGF介导OA疼痛。
