中药封包外治法对雌兔膝关节软骨细胞的DNA氧化损伤及凋亡的影响

Impacts of the External Therapy with Enveloped Herbal Medicine on DNA Oxidative Damage and Chondrocyte Apoptosis in the Knee Joints of Female Rabbits

目的观察中药封包外治法(温阳通络外治法)对雌兔膝关节骨关节炎模型软骨细胞的DNA氧化损伤及凋亡的影响,进而探讨其治疗的可能作用机制。方法 28只新西兰雌性兔随机等分为空白组、模型组、中药封包外治法组、玻璃酸钠组。除空白组外,其余雌兔均作卵巢去势处理。造模1周后,空白组和模型组不作任何处理;中药封包外治法组2次/d熨烫兔膝处,连用5周;玻璃酸钠组于右膝关节内注射玻璃酸钠注射液0.15 ml/kg,1次/周,连用5周;造模6周后分别进行雌二醇(E_2)含量测定、DNA提取、8-羟基脱氧鸟甙检测、苏木精-伊红染色观察胫骨软骨形态,检测胫骨软骨细胞凋亡率,并做统计学处理。结果雌兔E_2水平测定空白组术前1周与术后1周、6周比较差异均无统计学意义(P>0.05),模型组、中药封包外治法组、玻璃酸钠组术前1周与术后1周、6周分别作比较差异均有统计学意义(P<0.05);造模12周后8-羟基脱氧鸟苷含量测定空白组与模型组比较,差异有统计学意义(P<0.05),模型组与中药封包外治法组比较,差异有统计学意义(P<0.05),模型组与玻璃酸钠组比较,差异有统计学意义(P<0.05),中药封包外治法组与玻璃酸钠组比较,差异无统计学意义(P>0.05),同时两组中8-羟基脱氧鸟苷含量比空白组、模型组明显降低;造模6周后取雌兔右膝关节内侧胫骨平台软骨组织,光镜下见空白组、模型组检测软骨组织有明显退行性变,而中药封包外治法组、玻璃酸钠组稍有退变;免疫组化观察胫骨平台软骨细胞凋亡率空白组与模型组比较,差异有统计学意义(P<0.05),模型组与中药封包外治法组比较,差异有统计学意义(P<0.05),模型组与玻璃酸钠组比较,差异有统计学意义(P<0.05),中药封包外治法组与玻璃酸钠组比较,差异无统计学意义(P>0.05)。结论 (1)本实验雌兔卵巢去势造模很成功。(2)中药封包外治法组与玻璃酸钠注射组对膝关节骨性关节炎都有效,且两者治疗效果无明显差异,可能对减轻氧自由基软骨细胞DNA损伤有一定的作用。(3)中药封包外治法组与玻璃酸钠注射组对软骨细胞凋亡有抑制作用,并且两者的治疗作用相当。

Objective To observe the impacts of the external therapy with enveloped herbal medicine on DNA oxidative damage and apoptosis of chondrocytes in the knee joints of female rabbits and explore its effect mechanism.Methods Twenty-eight New Zealand female rabbits were randomized equally into a blank group,a model group,an herbal medicine group and a sodium hyaluronate group.Except the blank group,the ovarian castration was done in the rest groups.In 1 week of modeling,no any treatment was applied in the blank group and the model group.In the herbal medicine group,the external therapy with the enveloped herbal medicine was used to iron the knees of rabbits,twice a day,continuously for 5 weeks.In the sodium hyaluronate group,the sodium hyaluronate was injected to the right knee joint,0.15 ml/kg,once a week,continuouslyfor 5 weeks.In 6 weeks of modeling,E2 and 8-hydroxy-2 deoxyguanosine（8-OHDG）were determined,DNA was extracted and HE staining was used to observe the apoptosis of tibia chondrocytes.The statistical management was done.Results The difference was not significant statistically in E2 in comparison of 1 week before operation,1 week and 6 weeks after operation in the blank group（P〉0.05）.The difference was significant statistically in comparison of 1 week before operation,1 week and 6 weeks after operation in the model group,the herbal medicine group and the sodium hyaluronate group separately（P〈0.05）.In 12 weeks of modeling,the level of 8-OHDG was different significantly between the blank group and the model group（P〈0.05）,between the model group and the herbal medicine group（P〈0.05）,and between the model group and the sodium hyaluronate group（P〈0.05）.The difference was not significant between the herbal medicine group and the sodium hyaluronate group（P〉0.05）,8-OHDG in these two groups was reduced apparently as compared with the blank group and the model group.In 6 weeks of modeling,the cartilage tissue was collected from the medial tibia of the right knee joint.It was found under optic microscope that the cartilage tissue was obviously degenerated in the blank group and the model group and it was slightly degenerated in the herbal medicine group and the sodium hyaluronate group.IHC observed that the chondrocyte apoptosis was different significantly between the blank group and the model group（P〈0.05）,between the model group and the herbal medicine group（P〈0.05）and between the model group and the sodium hyaluronate group（P〈0.05）.The difference was not significant between the herbal medicine group and the sodium hyaluronate group（P〉0.05）.Conclusion 1.The ovarian castration of female rabbits is modeled successfully.2.The treatment in the herbal medicine group and the sodium hyaluronate group is effective on osteoarthritis of knee joint and the therapeutic effects are not different between the two groups.These two therapies alleviate chondrocyte DNA damage of oxygen free radicals.3.The chondrocyte apoptosis is inhibited in the herbal medicine group and the sodium hyaluronate group and the effects of the two groups are similar.