骨关节炎Ⅰ号方预防氢化可的松对兔软骨细胞的损伤作用

背景骨关节炎Ⅰ号方中的补肾活血祛风湿中药具有治疗骨关节炎、预防糖皮质激素副作用的作用.目的建立体外兔关节软骨细胞培养体系,观察骨关节炎Ⅰ号方预防氢化可的松对软骨细胞的损伤作用.设计完全随机设计,对照实验.单位福建省第二人民医院风湿免疫科.材料实验于2003-11/2004-03在福建中医学院骨伤系分子实验室完成.关节炎Ⅰ号方由仙灵脾、巴戟天、川芎、三七、祖师麻组成.用胶原酶Ⅱ消化兔关节软骨,建立软骨细胞体外培养体系,在软骨细胞培养体系中分别加入不同浓度的骨关节炎Ⅰ号方与氢化可的松的混合液,根据加入的药物浓度不同随机分成2组,一组加入1.25g/L氢化可的松和/或150g/L骨关节炎Ⅰ号方,另一组加入2.50 g/L氢化可的松和/或300 g/L骨关节炎Ⅰ号方.每组再分为4个亚组,各组再分别分为对照组、氢化可的松组、氢化可的松+关节炎Ⅰ号方组、关节炎Ⅰ号方组,每组5孔.方法①1.25g/L氢化可的松+150g/L关节炎J号方组每孔分别加入1.25 g/L氢化可的松和150g/L骨关节炎Ⅰ号方混合液0.1 mL.②1.25g/L氢化可的松组每孔分别加入磷酸盐缓冲液和1.25 g/L氢化可的松的混合液0.1 mL.③150g/L骨关节炎Ⅰ号方组每孔分别加入150g/L骨关节炎Ⅰ号方药液0.1 mL.④对照组1每孔分别加入磷酸盐缓冲液0.1 mL.⑤2.50g/L氢化可的松+300g/L骨关节炎Ⅰ号方组每孔分别加入2.50g/L氢化可的松和300 g/L骨关节炎Ⅰ号方的混合液0.1 mL.⑥2.50g/L氢化可的松组每孔分别加入磷酸盐缓冲液和2.50g/L氢化可的松的混合液0.1 mL.⑦300 g/L骨关节炎Ⅰ号方组每孔分别加入300g/L骨关节炎Ⅰ号方药液0.1 mL.⑧对照组2每孔分别加入磷酸盐缓冲液0.1 mL.⑨上述各组加药培养24h后,用倒置显微镜观察各组软骨细胞形态变化.④采用四氮唑蓝比色法检测各孔软骨细胞的A值,以反映软骨细胞增殖情况.主要观察指标骨关节炎Ⅰ号方与氢化可的松联合用药对兔软骨细胞增殖的影响.结果①软骨细胞形态倒置显微镜观察见对照组软骨细胞呈多角形或梭形贴壁生长,1.25和2.50 g/L氢化可的松浓度培养24 h的软骨细胞呈固缩或漂浮状态,各浓度氢化可的松+骨关节炎Ⅰ号方组、骨关节炎Ⅰ号方组的软骨细胞呈多角形或梭形贴壁生长与对照组无差异.②软骨细胞A值1.25 g/L氢化可的松组明显低于对照组1(0.007 6±0.001 8,0.015 2±0.002 6,t=5.374 0,P＜0.01);1.25 g/L氢化可的松+150 g/L关节炎Ⅰ号方组和150g/L关节炎Ⅰ号方组明显高于1.25g/L氢化可的松组(0.065 6±0.016 9,0.086 6±0.019 0,t=7.630 9,9.255 9,P＜0.01);2.50 g/L氢化可的松组明显低于对照组2(0.0053±0.0014,0.0153±0.0023,t=8.304 5,P＜0.01);2.50 g/L氢化可的松+300 g/L关节炎Ⅰ号方组和300 g/L关节炎Ⅰ号方组明显高于2.50 g/L氢化可的松组(0.0858±0.015 0,0.092 6±0.016 8,t=11.948 3,11.579 4,＜0.01).结论高浓度氢化可的松可导致软骨细胞已严重损伤或死亡,而氢化可的松加中药培养的软骨细胞生长良好,骨关节炎Ⅰ号方有预防氢化可的松对软骨细胞的损伤作用.

Preventive effect of No. Ⅰ recipe for osteoarthritis on chondrocyte injury by hydrocortisone in rabbits

BACKGROUND:Kidney-nourishing and blood activating Chinese herbs in the No. Ⅰ recipe for ostooarthritis have the effects of treating osteoarthritis and preventing the side effect of glucocorticoid.OBJECTIVE: To build up rabbit chondrocyte culture system in vitro to investigate the effect of preventive effect of No. Ⅰ recipe for osteoarthritis on chondrocyte injury by hydrocortisone.DESIGN:A complete randomized design and controlled trial.SETTING: Department of Rheumatology and Immunology, Second Hospital of Fujian ProvinceMATERIALS:The experiment was conducted from November 2003 to March 2004 at the Molecular Laboratory of Department of Orthopedics &Grammatology, Fujian College of Traditional Chinese Medicine. No. Ⅰrecipe for osteoarthritis consisted of Herba Epimedii, Radix Morindae Officinalis, Rhizoma Ligustic Chuanxiong, Radix Notoginseng and Daphne geraldii Nitsche. Articular cartilage was digested with collagenase Ⅱ ,chondrocyte culture system in vitro was built up, then in the culture system the different concentrations of the mixture of No. Ⅰ recipe for osteoarthritis and hydrocortisone were added, according to the concentrations they were at random divided into two groups. One group was added 1.25 g/L hydrocortisone and/or 150 g/L No. Ⅰ recipe for osteoarthritis, the other group was added 2.50 g/L hydrocortisone and/or 300 g/L No. Ⅰ recipe for osteoarthritis. Each group was again subdivided as control, hydrocortisone, hydrocortisone + No. Ⅰ recipe for osteoarthritis and No. Ⅰ recipe for osteoarthritis groups, with 5 wells in each subgroup.teoarthritis group: In each well 0.1 mL mixture of 1.25 g/L hydrocortihydrocortisone group: In each well 0.1 mL mixture of phosphate buffer teoarthritis group: In each well No. 1 recipe for osteoarthritis 0.1mL was In each well 0.1 mL mixture of 2.50 g/L hydrocortisone and 300 g/L No.each well 0.1mL mixture of phosphate buffer and 2.50 g/L hydrocortisone after medicines were added, the morphological change of chondrocyte in all (A) value in each well was assayed with tetrazoline colorimetry as reflection of proliferation of the chondrocyte. MAIN OUTCOME MEASURES:Influence of No. Ⅰ recipe for osteoarthritis combined with hydrocortisone on chondrocyte proliferation in rabbits.croscope that the chondrocytes in the control group were mostly in multiangles or shuttle-like, and adhesive growth; those cultured for 24 hours with 1.25 and 2.50 g/L hydrocortisone were in pyknosis or flotation state.;those in the various concentrations of hydrocortisone +No. Ⅰ recipe for osteoarthritis, and No. Ⅰ recipe for osteoarthritis groups were mostly in multiangles or shuttle-like, and adhesive growth, without difference with those in 1.25 g/L hydrocortisone group than in the control group(0.007 6±0.0018,0.015 2±0.002 6,t=5.374 0,P＜0.01);it was obviously higher in the 1.25 g/L hydrocortisone +150 g/L .No. Ⅰ recipe for osteoarthritis and 150 g/L No. Ⅰ recipe for osteoarthritis groups than in the 1.25 g/L hydrocortisone group (0.065 6±0.016 9, 0.086 6±0.019 0, t=7.630 9, 9.255 9,P＜0.01); it was obviously lower in the 2.50g/L hydrocortisone group than in the control group 2(0.005 3±0.001 4,0.015 3±0.002 3 ,t=8.304 5,P＜0.01) ;it was obviously higher in the 2.50 g/L hydrocortisone +300 g/L No. Ⅰ recipe for osteoarthritis and 300 g/L No. Ⅰ recipe for osteoarthritis groups than in the 2.50 g/L hydrocortisone group (0.085 8±0.015 0,0.092 6 ±0.016 8, t=11.948 3,11.579 4, P ＜ 0.01).CONCLUSION :Hydrocortisone in higher concentration could cause serious injury or death of chondrocytes, however the chondrocyte grew well cultured in the mixture of hydrocortisone and traditional Chinese drug, No.Ⅰ recipe for osteoarthritis had the effect of preventing injury of chondrocyte from hydrocortisone.