人参皂苷Re对椎间盘退行性变的作用及机制

Effect and mechanism of ginsenoside Re on intervertebral disc degeneration

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12.5~50μmol/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50μmol/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50μmol/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·50μmol/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。

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 12.5-50μmol/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 50μmol/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 50μmol/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 50μmol/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-IκBαdecreased 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-αand MMP 3(P=0.000).Conclusion·Ginsenoside Re 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.