Zhang Qingbin, Lai Shixiang, Hou Xunyao, Cao Wei, Zhang Ying, Zhang Zhaoqiang
Temporomandibular Joint Department, Stomatology Hospital of Guangzhou Medical UniversityGuangzhou 510140, Guangdong, China.
Key Laboratory of Oral Medicine, Guangzhou Institute of Oral DiseaseGuangzhou 510140, Guangdong, China.
Am J Transl Res. 2018 Mar 15;10(3):762-770. eCollection 2018.
As the major reason for limb dysfunction, osteoarthritis (OA) is closely correlated with the level of cellular autophagy. PI3K/Akt is a classical signaling pathway which regulates autophagy, but with unclear roles in OA related cartilage injury. Studying PI3K/Akt induced autophagy in rat knee joint cartilage injury and possible functional mechanism is of critical importance for clinical treatment. This study established a rat knee joint cartilage injury model, in which Akt agonist IGF-1 or autophagy inducer Rapamycin was administrated. Western blot was used to detect the expression level of AKT, phosphorylated AKT, Beclin-1 and LC3-II/I. Formation of autophagosome and lysosome was assessed by transmission electron microscopy. HE, safranin O and toluidine blue staining was used to evaluate cartilage injury. TUNEL staining was performed to measure cell apoptosis. Real-time quantitative PCR measured the expression of cartilage injury indexes such as Aggrecan, Collagen II and MMP13. Compared with normal group, iodacetic acid treatment group showed cartilage injury, whereas AKT activation and autophagy induction groups had significant improvement. mRNA analysis showed enhanced degradation of Aggrecan and Collagen II in AKT activation and autophagy groups with decreased MMP13 mRNA level (P<0.05). Western blot results showed that after AKT activation and autophagy induction, protein levels of Beclin-1 and LC3-II/I were remarkably elevated (P<0.05). TUNEL assay showed significant inhibition of cell apoptosis. In conclusion, activation of PI3K/Akt signal pathway can improve iodacetic acid induced rat knee joint cartilage injury through inducing cell autophagy.
作为肢体功能障碍的主要原因，骨关节炎（OA）与细胞自噬水平密切相关。PI3K/Akt是一条调节自噬的经典信号通路，但在OA相关软骨损伤中的作用尚不清楚。研究PI3K/Akt诱导的大鼠膝关节软骨损伤自噬及其可能的功能机制对临床治疗至关重要。本研究建立了大鼠膝关节软骨损伤模型，给予Akt激动剂IGF-1或自噬诱导剂雷帕霉素。采用蛋白质免疫印迹法检测AKT、磷酸化AKT、Beclin-1和LC3-II/I的表达水平。通过透射电子显微镜评估自噬体和溶酶体的形成。采用苏木精-伊红（HE）、番红O和甲苯胺蓝染色评估软骨损伤。进行TUNEL染色检测细胞凋亡。实时定量PCR检测软骨损伤指标如聚集蛋白聚糖、Ⅱ型胶原和基质金属蛋白酶13（MMP13）的表达。与正常组相比，碘乙酸处理组出现软骨损伤，而AKT激活组和自噬诱导组有明显改善。mRNA分析显示，AKT激活组和自噬组中聚集蛋白聚糖和Ⅱ型胶原的降解增强，MMP13 mRNA水平降低（P<0.05）。蛋白质免疫印迹结果显示，AKT激活和自噬诱导后，Beclin-1和LC3-II/I的蛋白水平显著升高（P<0.05）。TUNEL检测显示细胞凋亡受到显著抑制。综上所述，PI3K/Akt信号通路的激活可通过诱导细胞自噬改善碘乙酸诱导的大鼠膝关节软骨损伤。
