Miao Daoyi, Zhang Lingzhou
Department of Orthopedic Surgery, The Third Affiliated Hospital of Wenzhou Medical University, Ruian, Zhejiang 325200, P.R. China.
Mol Med Rep. 2015 Aug;12(2):1761-8. doi: 10.3892/mmr.2015.3646. Epub 2015 Apr 20.
Obesity has been demonstrated to be involved in the progress of intervertebral disc degeneration (IDD). However, the associated mechanisms remain to be elucidated. The purpose the present study was to examine the effect of leptin on the expression of degeneration-associated genes in rat nucleus pulposus (NP) cells, and determine the possible mechanism. Normal NP cells, obtained from Sprague Dawley rats, were identified using immunocytochemistry for the expression of collagen II and CA125, and treated with leptin and/or interleukin (IL)-β. Subsequently, the mRNA expression levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, aggrecan and COL2A1 were detected by reverse transcription-quantitative polymerase chain reaction (RT-q-PCR). Alcian staining and immunocytochemistry were used to examine the expression levels of proteoglycan and collagen II. The pathway activation was investigated using western blotting, and inhibitors of the pathways were used to reveal the effect of these pathways on the NP cells. The results of the RT-qPCR demonstrated that leptin alone upregulated the mRNA expression levels of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5 and COL2A1. Synergy of leptin and IL-β was found in the increased expression levels of MMP-1, MMP-3 and ADAMTS-5. The leptin-treated NP cells exhibited decreased expression of collagen II. The mitrogen-activated protein kinase (MAPK) pathway (c-Jun-N-terminal kinase, phosphorylated extracellular signal-regulated kinase and p38), phosphatidylinositol 3-kinase (PI3K)/Akt pathway and Janus kinase (JAK)2/signal transducer and activator of transcription 3 pathway were all activated by leptin, however, inhibitors of all the pathways, with the exception of the PI3K/Akt pathway, reversed the expression levels of MMP-1 and MMP-13. These results suggested that leptin promoted catabolic metabolism in the rat NP cells via the MAPK and JAK2/STAT3 pathways, which may be the mechanism mediating the association between obesity and IDD.
肥胖已被证明与椎间盘退变（IDD）的进展有关。然而，相关机制仍有待阐明。本研究的目的是检测瘦素对大鼠髓核（NP）细胞中退变相关基因表达的影响，并确定其可能的机制。从Sprague Dawley大鼠获取的正常NP细胞，通过免疫细胞化学检测Ⅱ型胶原蛋白和CA125的表达进行鉴定，并用瘦素和/或白细胞介素（IL）-β处理。随后，通过逆转录-定量聚合酶链反应（RT-q-PCR）检测基质金属蛋白酶（MMP）-1、MMP-3、MMP-9、MMP-13、含血小板反应蛋白基序的解聚素和金属蛋白酶（ADAMTS）-4、ADAMTS-5、聚集蛋白聚糖和COL2A1的mRNA表达水平。采用阿尔新蓝染色和免疫细胞化学检测蛋白聚糖和Ⅱ型胶原蛋白的表达水平。使用蛋白质印迹法研究信号通路的激活情况，并使用这些信号通路的抑制剂来揭示这些信号通路对NP细胞的影响。RT-qPCR结果表明，单独使用瘦素可上调MMP-1、MMP-13、ADAMTS-4、ADAMTS-5和COL2A1的mRNA表达水平。在MMP-1、MMP-3和ADAMTS-5表达水平升高方面发现了瘦素与IL-β的协同作用。经瘦素处理的NP细胞Ⅱ型胶原蛋白表达降低。瘦素激活了丝裂原活化蛋白激酶（MAPK）通路（c-Jun氨基末端激酶、磷酸化细胞外信号调节激酶和p38）、磷脂酰肌醇3-激酶（PI3K）/Akt通路和Janus激酶（JAK）2/信号转导和转录激活因子3通路，然而，除PI3K/Akt通路外，所有信号通路的抑制剂均逆转了MMP-1和MMP-13的表达水平。这些结果表明，瘦素通过MAPK和JAK2/STAT3通路促进大鼠NP细胞的分解代谢，这可能是介导肥胖与IDD之间关联的机制。
