Increased TNN in Knee Osteoarthritis Accelerates Cartilage Damage via the Negative Regulation of AMPK‐PPARγ Signalling

In this study, we aimed to get a better understanding of which genes are involved in cartilage damage in KOA and the pathological mechanisms. RNA-seq for cartilage tissues obtained from Normal rats and KOA rats, for IL-1β-stimulated chondrocytes and IL-1β-stimulated chondrocytes treated with TNN recombinant protein, was conducted respectively. The degree of cartilage injury was evaluated by HE and Safranin O-fast green staining, and the expression abundance of TNN and p-AMPK in cartilage tissue or chondrocytes was observed by immunofluorescence. TNN was inhibited in vivo and in vitro by lentivirus and siRNA, respectively. The gene and protein levels of protease MMP3, MMP13, ADAMTS4, and ADAMTS5 and AMPK/PPAR-γ pathway-related factors -AMPK, AMPK, PPAR-γ, PGC-1α, mTOR were detected by PCR and WB, respectively. The mitochondrial membrane potential of chondrocytes was evaluated by JC1 probe, and the oxygen level of chondrocytes was evaluated by ROS immunofluorescence. RNA-seq revealed TNN was significantly up-regulated in the KOA group, and DEGs were mainly enriched in 'extracellular matrix'. Subsequently, TNN inhibition could reduce the expression of MMPs, ADAMTSs were demonstrated in vivo and in vitro. Further on, RNA-seq on IL-1β-stimulated chondrocytes and chondrocytes treated with TNN recombinant protein after IL-1β stimulation confirmed that AMPK-PPARγ signalling might be the downstream pathway of TNN, and the negative regulation of TNN on AMPK-PPARγ signalling was observed in vivo and in vitro. This study innovatively unveils the increased TNN in KOA accelerates cartilage damage, and this damage-promoting effect is achieved by negative regulation of AMPK-PPARγ signalling.