低强度脉冲超声经 PI3K/Akt 通路调控兔膝骨性关节炎软骨细胞凋亡的作用机制研究

摘要

Objective:To investigate the effects of low intensity pulsed ultrasound (LIPUS)on the apoptosis of chondrocytes in rabbits with osteoarthritis (OA)and explore the underlying mechanism.Methods:Thirty healthy New Zealand white rabbits were selected for this study.All the cultured cells were randomly divided into five groups:a normal control group (A group),an OA model group (B group),an OA model plus LIPUS group (C group),an OA model plus PI3K/Akt inhibitor group (D group),and an OA model plus PI3K/Akt inhibitor and LI-PUS group (E group).The OA model was made by knee anterior cruciate ligament transection.At the 6th week af-ter modeling,the rabbits were sacrificed by air embolism method and pathologic changes were assessed on articular surface of femoral condyles.Meanwhile chondrocytes were isolated and cultured in vitro ,and cultured cells were i-dentified by immuohistochemistry.The expression of collagen protein type Ⅱ,MMP-13,Akt,pAkt,P53,and Bcl-2 was detected by Western blotting.Results:As compared with the A group,the expression of collagen type Ⅱ, pAkt and Bcl-2 was significantly reduced in the B group,but that of MMP-13 and P53 was significantly increased in the B group (P <0.05 ).As compared with the B group,the expression of collagen type Ⅱ,pAkt and Bcl-2 was significantly increased,and that of MMP-13 and P53 was significantly reduced in the C group (P <0.05);the ex-pression of collagen type Ⅱ,pAkt and Bcl-2 was significantly reduced (P <0.05),and that of MMP-13 and P53 was significantly increased in the D group (P <0.05).Although the expression of collagen type Ⅱ,MMP-13,pA-kt,P53,and Bcl-2 showed no significant difference be-tween the E group and the B group,the expression of collagen type Ⅱ, MMP-13, pAkt, P53 and Bcl-2 showed significant difference between C group and D group (P <0.05).There was no significant difference in the expression of Akt among all groups.Conclusions:The LIPUS can obviously decrease the apoptosis rate of chondrocytes in the knee joints of OA rabbits via activating PI3K/Akt pathway,and the underlying mechanisms are closely related to upregulation of Bcl-2 expression and downregulation of P53 expression.The LIPUS can accelerate the recovery of cartilage for the purpose of treatment of OA.%目的：：探讨低强度脉冲超声(LIPUS)对兔膝骨性关节炎(OA)软骨细胞凋亡的影响及有关作用机制。方法：共选取30只新西兰大白兔,随机分为正常对照组(A 组)、OA 模型组(B 组)、OA+ LIPUS 组(C 组)、OA+LY294002(PI3K/Akt 抑制剂)组(D 组)、OA+LY294002+LIPUS 组(E 组),每组各6只。采用右后膝前交叉韧带切断术(ACLT)造模。于造模后第6周时采用空气栓塞法处死实验兔,切取软骨组织进行组织学观察,并进行Mankin 评分；提取软骨细胞进行体外培养并采用免疫组化染色法鉴定；应用 western blot 检测各组软骨细胞中Ⅱ型胶原(COL2)、MMP-13、Akt、pAkt、P53、Bcl-2蛋白表达情况。结果：B 组 COL2、pAkt、Bcl-2蛋白含量较 A 组降低,MMP-13、P53蛋白含量较 A 组增高(P <0.05)。与 B 组相比,C 组 COL2、pAkt、Bcl-2蛋白含量增高,MMP-13、P53蛋白含量下降(P <0.05)；D 组 COL2、Bcl-2、pAkt 蛋白含量下降,MMP-13、P53蛋白含量升高(P <0.05),E 组COL2、MMP-13、P53、Bcl-2、pAkt 蛋白含量无明显变化,但与 C 组、D 组相比差异显著(P <0.05)。各组 Akt 蛋白表达无明显差异。结论：LIPUS 能通过 PI3K/Akt 通路降低兔膝骨性关节炎软骨细胞凋亡率,促进抗凋亡基因 Bcl-2表达,下调促凋亡基因 P53水平,促进关节软骨损伤修复。