低强度脉冲超声波对兔膝骨性关节炎软骨整合素-FAK-MAPKs信号通路蛋白表达的影响

Effects of low-intensity pulsed ultrasound on integrin-FAK-MAPKs mechanochemical transduction pathway in rabbit knee osteoarthritis cartilage

目的:观察低强度脉冲超声波(low-intensity pulsed ultrasound,LIPUS)对兔膝骨性关节炎(osteoarthritis,OA)软骨中整合素-FAK-MAPKs力化学细胞信号转导通路相关蛋白表达的影响。方法:18只成年新西兰大白兔随机平均分成3组,分为正常对照组(normal control,NC),OA模型组(OA group,OA),OA模型照射组(O+L)。OA组接受右侧后肢前交叉韧带切断(ACLT)处理术后4周接受LIPUS假辐射,O+L组同样手术处理,术后4周接受LIPUS辐射,NC组仅切开关节囊。LIPUS作用6周后,处死实验动物,取右后肢,采用HE染色行改良Mankin评分比较各组胫骨平台关节表面病理学改变。同时,采用Western blot技术检测Ⅱ型胶原,MMP-13,整合素β1的蛋白表达水平以及FAK、MAPKs家族蛋白(p38、ERK1/2、JNK)磷酸化水平。结果:1组织学观察及Mankin评分:LIPUS干预后,OA软骨表层轻微不平整,染色轻度缺失,可见软骨细胞增殖;Mankin评分,NC组:4.67±0.57;OA组:10.57±2.55;O+L组:7.66±1.74。与NC组相比,OA组、O+L组Mankin评分明显增高,但OA组增高更为明显(P<0.05);与OA组相比,O+L组Mankin评分明显降低(P<0.05)。2Ⅱ型胶原、MMP-13含量:LIPUS干预后,II型胶原含量较NC组升高,MMP-13含量有明显下降。3整合素β1-FAK-MAPKs信号通路相关蛋白表达:LIPUS干预使整合素β1、磷酸化FAK表达增高;同时MAPKs信号通路相关蛋白ERK1/2、p38磷酸化水平明显下降,而JNK磷酸化水平无明显变化。结论:LIPUS可以减轻骨性关节炎软骨ECM损伤程度,与LIPUS产生机械应力使关节软骨中细胞表面应力受体之一整合素β1及其下游分子黏着斑激酶FAK磷酸化表达增高,进一步下游的磷酸化p38,ERK1/2表达下调有关。LIPUS的机械效应可经力化学转导途径作用于OA关节软骨,为治疗骨性关节炎提供一种新的思路。

Objective： To observe the effect of low-intensity pulsed ultrasound（LIPUS） on integrin-FAK-MAPKs mechano- chemical transduction pathway related protein expression in rabbit knee osteoarthritis cartilage. Method： Eighteen 2--2.5kg New Zealand rabbits were chosen and randomly divided into three groups, includ- ing normal control group（NC）, OA model group（OA） and OA model plus LIPUS group（O＋L）. Twelve of these rabbits underwent anterior cruciate ligament transaction（ACLT） at the right hind limbs, the rest were taken fake operation. Six of operated rabbits, O＋L group, were exposed to LIPUS radiation four weeks after OA models established. The other six operated rabbits, OA group, were exposed to fake LIPUS radiation. The rest of rabbits acted as normal control. After six weeks LIPUS radiation, the rabbits were sacrificed and pathological changes of articular surface of femoral condyle were assessed by Mankin Scores. Also, protein expressions of collagen type II, MMP-13, integrin[31, FAK, ERK1/2, JNK, p38, p-FAK, p-ERK1/2, p-JNK, p-p38 were mea- sured with Western blot analysis. Result： ~collagen type II： Compared with NC group, the expression of collagen type II was significantly low- er in OA and O＋L group, with more significant decrease in OA group（P〈0.05）; Compared with OA group, col- lagen type II expression was significantly higher in O＋L group（P〈0.05）. @MMP-13： Compared with NC group, the expression of MMP-13 was significantly higher in OA and O＋L group, with more significant in- crease in OA group（P〈0.05）; Compared with OA group, MMP-13 expression was significantly lower in O＋L group（P〈0.05）. @integrin [31 and p-FAK： Compared with NC group, the expressions of integrin [31 and phos- phorylated FAK were significantly higher in OA and O＋L group, with more significant increase in O＋L group （P〈0.05）. Compared with OA group, expressions of integrin[31 and phosphorylated FAK were significantly high- er in O＋L group （P〈0.05）. @p-ERK1/2 and p-p38： Compared with NC group, the ratio of p-ERK/ERK, p-p38/ p-38 were significantly higher in OA group and O＋L group （P〈0.05）. Compared with OA group, expressions of these two ratios decreased significantly in O＋L group （P〈0.05）. @p-JNK： Compared with NC group, the ra- tio of p-JNK was significantly higher in OA group and O＋L group （P〈0.05）. Compared with OA group, there was no significant p-JNK expression change in O＋L group （P〈0.05）. Conclusion： LIPUS inhibits the ECM degradation in rabbit knee osteoarthritis cartilage by activating integrin- FAK-MAPKs mechanochemical transduction pathway. Integrin^l, as one of the stress receptors on the surface of chondrocytes, was activated by mechanical loading produced by LIPUS and induced the phosphorylation of FAK and MAPKs. The results show that LIPUS might repair the ECM of OA via integrin-FAK-MAPKs mecha- nochemical transduction pathway, providing a new possibility for OA treatment.