低强度脉冲超声对兔膝骨性关节炎骨软骨连接血管形成与侵袭的影响

Pulsed ultrasound regulates angiogenesis and vascular invasion of the osteochondral junction in arthritic knee cartilage

目的观察低强度脉冲超声（LIPUS）对早期兔膝骨性关节炎骨软骨连接血管形成与侵袭的影响。方法18只新西兰大白兔按随机数字表法分为对照组、模型组和治疗组，每组6只，模型组和治疗组接受前交叉韧带切断法手术，4周后建立早期骨性关节炎疾病模型，而对照组仅接受假手术处理。治疗组接受LIPUS辐射治疗4周，模型组和对照组仅接受LIPUS假辐射。采用番红O-固绿染色比较各组膝关节骨软骨连接血管形成与侵袭情况；采用免疫印迹（Westernblot）技术检测各组的整合素β1、磷酸化黏着斑激酶（p-FAK）、磷酸化p38、磷酸化c-Jun氨基末端激酶（p-JNK）、血管内皮生长因子（VEGF）及软骨调节素（CHM）-1蛋白表达水平。结果模型组和治疗组与对照组比较，骨软骨连接血管形成明显增多，潮线出现漂移、重复，结构紊乱，甚至消失，部分血管突破潮线，侵入非钙化软骨层，而治疗组较模型组病理改变明显减轻。模型组的整合素β1、p-FAK和VEGF表达水平[（0．92±0．07）、（0．83±0．09）和（1．02±0．10）]较对照组[（0．45±0．04）、（0．18±0．03）和（0．35±0．02）]明显增高，且组间差异有统计学意义（P〈0．05）；模型组的p38和JNK磷酸化水平[（0．99±0．11）和（1．86±0．06）]亦较对照组[（0．31±0．04）和（0，38±0．03）]明显增高（P〈0．05）；而模型组的ChM-1表达（0．61±0．04）较对照组（1．91±0．08）下降，且差异有统计学意义（P〈0．05）。治疗组与模型组比较，治疗组的整合索β1和p-FAK表达[（1．10±0．09）和（1．09±0．08）]亦较模型组明显增高，组间差异有统计学意义（P〈0．05）；但治疗组的VEGF表达（0．58±0．06）较模型组明显降低（P〈0．05），治疗组的p38和JNK磷酸化水平[（0．54±0．09）和（1．29±0．08）]亦较模型组降低，且差异有统计学意义（P〈0．05）；但治疗组的ChM-1表达（1．09±0．12）较模型组明显升高，差异有统计学意义（P〈O．05）。结论LIPUS可以抑制骨性关节炎骨软骨连接血管形成与侵袭，其作用机制可能与LIPUS通过整合素β1-FAK—p38／JNK—VEGF／ChM-1通路调控血管形成相关因子VEGF和ChM-1的表达有关。

Objective To observe the effect of low-intensity pulsed ultrasound （LIPUS） on angiogenesis and vascular invasion in the osteochondral junctions of rabbits with osteoarthritis. Methods Eighteen 2 to 2.5 kg New Zealand rabbits were randomly divided into an early control group （EC） , an early osteoarthritis group （EO） and an osteoarthritis plus LIPUS treatment group （ET） , each of 6. The rabbits of the EO and ET groups received anterior erueiate ligament resection at their left hind limbs, while those of the EC group were sham-operated. The ET group was exposed to LIPUS radiation four weeks later when osteoarthritis had been established, while the EO and EC groups were exposed to fake LIPUS radiation. After four weeks of treatment the rabbits were sacrificed and pathological changes in the articular surface of the femoral condyle were assessed using Safranin O-fast green cartilage staining. Western blotting was used to examine the expression of integrln β1, p-FAK, p-JNK, p-p38, VEGF and chondromod- ulin-1（ChM-1）. Results Compared with the EC group, significantly more angiogenesis and vascular invasion was observed in the osteochondral junctions of the EO and ET groups, with more vessels at the osteochondral junction and marked disorder of the tidal line broken up by vessels. The expression of integrin β1, p-FAK, VEGF, p-p38 andp-JNK was significantly higher in the EO group than in the EC group, while ChM-1 expression was significantly lower. Compared with the EO group, the expression of integrinβ1, p-FAK and ChM-1 in the ET group was significantly higher, while that of VEGF, p38 and p-JNK was significantly lower. Conclusions LIPUS inhibited angiogenesis and vascular invasion in the osteochondral junctions of the osteoarthritie rabbits through activating an integrinβ1- FAK-p38/JNK-VEGF/ChM- 1 pathway.