骨碎补总黄酮对膝骨关节炎模型兔HIF-1α和VEGF表达的影响

Effects of Total Flavonoids of Drynariae rhizoma on the Expression of HIF-1α and VEGF in Knee Osteoarthritis Model Rabbits

目的:研究骨碎补总黄酮对膝骨关节炎(KOA)模型兔滑膜组织及软骨组织/细胞中低氧诱导因子1α(HIF-1α)、血管内皮生长因子(VEGF)表达的影响。方法:将30只家兔随机分为正常组、模型组和骨碎补总黄酮低、中、高剂量组(30、60、100 mg/kg),每组6只。除正常组外,其余各组家兔均切开左膝关节,剪断内侧副韧带和前后交叉韧带,完整切除内侧半月板,复制KOA模型。术后5周,各给药组家兔灌胃相应剂量的骨碎补总黄酮,正常组和模型组家兔灌胃等容生理盐水,每天3次,连续14 d。末次给药后,采用酶联免疫吸附测定法检测家兔滑膜组织中HIF-1α和VEGF的含量,并考察两者的相关性;以苏木精-伊红染色观察家兔滑膜组织的形态学变化;采用免疫组化法检测家兔软骨组织中HIF-1α和VEGF阳性表达的情况;采用蛋白质印迹法检测家兔软骨细胞中HIF-1α和VEGF蛋白表达的情况。结果:与正常组比较,模型组家兔滑膜组织中HIF-1α和VEGF的含量均显著升高,差异均有统计学意义(P<0.01);滑膜表层出现裂隙,细胞层次不清晰且排列无序;软骨组织中HIF-1α和VEGF阳性细胞数增加,软骨细胞中HIF-1α和VEGF蛋白的相对表达量均显著升高,差异均有统计学意义(P<0.01)。与模型组比较,骨碎补总黄酮各剂量组家兔滑膜组织中HIF-1α和VEGF的含量均显著下降,差异均有统计学意义(P<0.05或P<0.01),且两者呈正相关(r=0.923,P<0.01);滑膜表面裂隙减少,细胞排列较为均匀,损伤程度减轻;软骨组织中HIF-1α和VEGF阳性细胞数减少,软骨细胞中HIF-1α和VEGF蛋白的相对表达量均显著下降,差异均有统计学意义(P<0.05或P<0.01)。结论:骨碎补总黄酮可减轻KOA模型兔滑膜及软骨的损伤程度,这种作用可能与下调滑膜组织及软骨组织/细胞中HIF-1α和VEGF的表达有关。

OBJECTIVE:To study the effects of total flavonoids of Drynariae rhizoma on the expression of HIF-1αand VEGF in synovial tissues and cartilage tissues/cells of knee osteoarthritis(KOA)model rabbits.METHODS:Totally 30 rabbits were randomly divided into normal group,model group and total flavonoids of D.rhizoma low-dose,medium-dose and high-dose groups(30,60,100 mg/kg),with 6 rabbits in each group.Except for normal group,KOA model of rabbits was induced in other groups by cutting the left knee joint,shearing off the medial collateral ligament,anterior and posterior cruciate ligaments,and completely removing the medial meniscus.Five weeks after surgery,administration groups were given relevant dose of total flavonoids of D.rhizoma intragastrically;normal group and model group were given constant volume of normal saline intragastrically,3 times a day,for consecutive 14 d.After last medication,the contents of HIF-1αand VEGF in synovial tissues of rabbits were detected by ELISA,and the relationship of them was investigated.HE staining was used to observe the morphology of the synovial tissues;immunohistochemical staining was used to detect the positive expression of HIF-1αand VEGF in cartilage tissues;protein expression of HIF-1αand VEGF in cartilage cells was detected by Western blot method.RESULTS:Compared with normal group,the contents of HIF-1αand VEGF in synovial tissues of rabbits in model group were increased significantly,with statistical significance(P<0.01);synovial tissues surface appeared fissures,and the cell layers were not clear and arranged in disorder;the number of positive cells of HIF-1αand VEGF in cartilage tissues was increased,and the relative expression of HIF-1αand VEGF in cartilage cells was increased significantly,with statistical significance(P<0.01).Compared with model group,the contents of HIF-1αand VEGF in synovial tissues of rabbits were decreased significantly in total flavonoids of D.rhizoma groups,with statistical significance(P<0.05 or P<0.01),showing positive relationship(r=0.923,P<0.01);the fissures were reduced,and the cell arrangement was more uniform,the degree of synovium injury was reduced;the number of positive cells of HIF-1αand VEGF in cartilage tissues was decreased,and the relative expression of HIF-1αand VEGF in cartilage cells was decreased significantly,with statistical significance(P<0.05 or P<0.01).CONCLUSIONS:Total flavonoids of D.rhizoma can relieve synovium and cartilage injury in KOA model rabbits,the effect of which may be associated with regulating the expression of HIF-1αand VEGF in synovial tissues and cartilage tissues/cells.