青藤碱对炎性刺激下体外培养关节软骨细胞的保护作用

Effect of sinomenine on inflammatory response induced by lipopolysaccharide in chondrocytes in vitro

目的体外培养脂多糖（LPS）诱导的初生小牛关节软骨细胞炎症损伤模型并以青藤碱（sN）干预，从而研究SN对关节软骨细胞的保护作用。方法无菌分离并体外培养初生小牛的膝关节软骨细胞，以不同浓度组LPS刺激细胞诱导炎症损伤模型，比色法检测培养液上清中的一氧化氮（NO）生产量，MTY法检测不同浓度SN对正常培养细胞活力的影响，从而选择合理的造模及给药剂量；以低、中、高3种剂量的SN干预LPS诱导的软骨细胞炎症损伤模型，并设立模型对照组、空白对照组，检测各组细胞培养液上清中的NO、丙二醛（MDA）产生量，检测一氧化氮合成酶（NOS）、超氧化物歧化酶（SOD）活性，分析sN对细胞炎症损伤模型的干预作用。结果各浓度LPS均能剂量依赖性地促进软骨细胞产生NO，依次为（11．0±0．4）、（16．7±0．5）、（21．0±0．7）、（26．3±0．6）、（42．4±0．5）、（52．4±0．9）、（72．3±0．5）μmol／L，分别与空白组比较，差异均有统计学意义（P值均〈0．01）。MTT法检测各浓度SN作用下软骨细胞相对活力，OD值分别为0．444-0．01、0．43±0．02、0．444-0．0l、0．43±0．02、0．43±O．02，分别与空白组差异均无统计学意义（P值均〉0．1）。未加SN干预LPS组与对照组NO、MDA、NOS、SOD比较差异有统计学意义，t值分别为-135．0、-16．1、-150．4、32．3，P值均〈0．0l。加入各浓度SN的LPS组分别与不加SN的LPS组比较NOS释放量SN组较低，t值分别为-74．7、-33．5、43．4，P值均〈0．01；分别比较NO释放量SN组较低，t值分别为-11．4、-7．1、-5．5，P值均〈0．01；分别比较SOD活力SN组较高，t值分别为-105．5、40．3、-20．5，P值均〈0．01；分别比较MDA产生量SN组较低，t值分别为21．5、10．7、17．7，P值均〈0．01，差异有统计学意义，且呈一定的剂量依赖性。结论sN能对抗LPS诱导的脂质过氧化及炎性损伤，提高软骨细胞的超氧化物歧化酶活性，提高氧自由基清除水平，对软骨细胞具有保护作用。

Objective To observe the protective effects of sinomenine（SN） on lipopolysaccharide（LPS） in- duced chondrocytes. Methods Bovine chondrocytes from newborn cattle were prepared and cultured in vitro. The inflammatory responses in chondrocytes were induced by LPS of seven concentrations, and nitricoxide（NO） contents in the nutrient solution were measured. A MTT assay was set for cell proliferation cultured in the nutrient medium with SN of five concentrations. The LPS induced inflammatory responses in chondrocytes were treated with/without SN of high, normal, and low dosage, then the NO and MDA content in the nutrient solution, and the NOS and SOD activity were measured. Results The NO products[ （11.0±0.4） , （16.7±0.5）, （21.0±0.7）, （26.3±0.6）, （ 42.4±0.5 ）, （ 52.4 ± 0.9 ）, （ 72.3± 0.5 ） μmol/L ] of chondrocytes with a certain dose dependent （ P 〈 0.01 respectively）. The MTY assays showed each dosage of SN made no differences to the cell proliferation respectively compared with the control group, P 〈0.01 respectivelyl. The NO, MDA, NOS and SOD in the nutrient solution of the cells induced with LPS had differences significantly compared with the control group （t =-135. 0, t =-16. 1, t = 150.4, t =32.3 ; each P 〈0.01 ）. The NOS level of each LPS group treated with SN of the dosages of was respectively lower compared with the untreated LPS groups （ t = -74.7, t = -33.5, t = -43.4, respectively ; each P 〈 0.01 ）.Meanwhile, the excess of NO of each treated groups was respectively lower significantly compared with the untreated （t =-11.4, t =-7.1, t =-5.5, respectively; each P 〈 0.01 ）, and the SOD level of each treated groups was higher significantly compared with the untreated （ t = -105.5, t= -40.3, t = -20.5, respectively; each P 〈 0.01 ）, and the release of MDA of each treated group was respectively lower compared with the untreated （t =21.5, t =-10.7, t = 17.7, respectively; each P 〈 0.01 ）. Conclusion LPS damaged bovine chondrocytes in vitro by increasing the level of NO and oxyradical. SN antagonized the lipid peroxidation and inflammatory response induced by lipopolysac- charide in bovine chondrocytes, heightened the SOD activity of chondrocytes and the clearance of oxyradical, and nrotected the chnndrocvtes against LPS induced inflammatory damage.