摘要/Abstract
摘要： 目的·明确人参皂苷Re在改善椎间盘退行性变（intervertebral disc degeneration， IDD）中的作用并探讨其潜在机制。方法·10 ng/mL肿瘤坏死因子-α（tumor necrosis factor-α， TNF-α）诱导髓核细胞炎症；采用实时定量聚合酶链反应测定12.5～50 μmol/L人参皂苷Re对髓核细胞炎症及代谢相关基因，包括基质金属蛋白酶3（matrix metalloproteinase 3，Mmp 3）、血小板结合蛋白基序的解聚蛋白样金属蛋白酶5（a disintegrin and metalloproteinase with thrombospondin 5，Adamts 5）、聚集蛋白聚糖、Ⅱ型胶原a1（collagen typeⅡa1，Col2a1）表达量的影响；采用蛋白质印迹法检测在10 ng/mL TNF-α刺激下，加入或不加50 μmol/L人参皂苷Re时核因子κB（NF-κB）通路相关蛋白表达量。采用12只8周龄大鼠制作尾椎针刺退变模型（尾椎6/7作为假手术节段，尾椎7/8作为针刺节段）；实验组（n=6）腹腔注射50 μmol/L人参皂苷Re，对照组（n=6）注射磷酸缓冲盐溶液。1个月后通过X线检测椎间隙高度，通过番红固绿及苏木精-伊红染色观察大鼠椎间盘髓核组织变化并进行组织学退变分级，通过免疫荧光观察TNF-α、MMP 3、聚集蛋白聚糖、COL2A1的表达。结果·50 μmol/L人参皂苷Re可抑制炎症状态下髓核细胞Mmp 3、Adamts 5基因的表达，增加聚集蛋白聚糖及Col2a1基因的表达；与单用TNF-α刺激的髓核细胞比较，差异均有统计学意义（均P=0.000）。蛋白质印迹法结果显示，与单用TNF-α刺激的髓核细胞比较，加入人参皂苷Re显著降低了髓核细胞p-p65和p-IκBα蛋白表达量（均P=0.000）。X线检测结果显示，与对照组大鼠尾椎针刺节段相比，注射人参皂苷Re的实验组大鼠针刺节段椎间隙高度较高（P=0.004），组织学退变分级较低（P=0.000）；免疫荧光结果显示，大鼠针刺节段尾椎椎间盘聚集蛋白聚糖和COL2A1表达量较高（P=0.000）、TNF-α和MMP 3表达量较低（P=0.000）。结论·人参皂苷Re可通过抑制NF-κB通路降低髓核细胞相关炎症因子的表达，维持椎间隙高度，有效减轻大鼠IDD程度。
关键词: 人参皂苷Re, 椎间盘退行性变, 髓核, 大鼠
Abstract:
Objective · To clarify the therapeutic effect of ginsenoside Re on intervertebral disc degeneration (IDD) and the potential underlying mechanism. Methods · 10 ng/mL tumor necrosis factor-α (TNF-α) was used to stimulate nucleus pulposus (NP) cells. The expression of inflammation and cartilage-related genes, including matrix metalloproteinase 3 (Mmp 3), a disintegrin and metalloproteinase with thrombospondin 5 (Adamts 5), aggrecan and collagen typeⅡa1 (Col2a1), were measured and compared by real-time quantitative polymerase chain reaction with ginsenoside Re concentration varying from 12.5-50 μmol/L. After stimulation of NP cells with 10 ng/mL TNF-α, the amounts of nuclear factor-κB (NF-κB) pathway-related proteins were measured by using Western blotting with or without 50 μmol/L ginsenoside Re. Twelve 8-week-old rats were used to make coccyx degeneration models (coccygeal vertebrae 6/7 as sham, coccygeal vertebrae 7/8 as puncture), intraperitoneal injection of 50 μmol/L ginsenoside Re as treatment group (n=6) and phosphate buffered saline solution (PBS) as control group (n=6). One month later, the heights of the intervertebral discs were detected by X-ray. The rats' intervertebral disc NP tissues were observed by safranin O-fast green staining and hematoxylin-eosin staining, and histological grade was evaluated. By immunofluorescence, the expressions of TNF-α, MMP 3, aggrecan and COL2A1 were observed. Results · The expressions of inflammatory factors in the NP cells including Mmp 3 and Adamts 5 decreased and the expressions of aggrecan and Col2a1 increased under the treatment of 50 μmol/L ginsenoside Re (all P=0.000). Western blotting showed that compared with the NP cells only stimulated by TNF-α, the amounts of protein p-p65 and p-IκBα decreased significantly under the treatment of ginsenoside Re (both P=0.000). X-ray showed that compared with the puncture segment of PBS injection group, the puncture segment of ginsenoside Re injection group had better disc height indices (P=0.004) and degeneration grades (P=0.000). Immunofluorescence showed ginsenoside Re injection group expressed more aggrecan and COL2A1 (P=0.000), and less TNF-α and MMP 3 (P=0.000). Conclusion · GinsenosideRe can reduce the expressions of inflammatory factors by inhibiting NF-κB pathway in NP cells as well as maintain the disc heights and effectively alleviate the IDD level of rats.
Key words: ginsenoside Re, intervertebral disc degeneration (IDD), nucleus pulposus (NP), rat
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