DDIT4 participates in high glucose-induced fibroblast-like synoviocytes overactivation and cartilage injury by regulating glycolysis.

Objective: More and more evidence show that diabetes is closely related to osteoarthritis (OA). However, the role and mechanism of DNA damage-inducible transcript 4 protein (DDIT4) in diabetic OA (DOA) have not been clarified. Methods: We collected OA patients and non-OA subjects who underwent total knee replacement surgery, and analyzed the DDIT4 expression in synovial samples using RT-qPCR. The cell viability of fibroblast-like synoviocytes (FLSs) was measured by CCK-8 assay. Annexin V-FITC/PI double staining was used to detect the cell apoptosis. Scratch and Transwell assays were used to determine cell migration and invasion, respectively. Results: The levels of cellular inflammatory factors (IL-1β, IL-6 and TNF-α), oxidative stress and glycolysis related indicators were detected by using kits. Western blot was used to determine the expression of DDIT4, Aggrecan, COL3A1, MMP3, MMP13, HK2, PFKP and PKM2 in FLSs or ATDC5 cells. The results showed that the expression level of DDIT4 was significantly reduced in the synovial samples of OA patients and primary FLSs. Functional studies showed that DDIT4 overexpression inhibited the overactivation, migration, and invasion of FLSs, as well as alleviated chondrocyte injury co-cultured with FLSs. Importantly, the expression of DDIT4 was down-regulated in patients with DOA and closely related to DOA. Further research found that high glucose (HG) promoted excessive activation, migration, and invasion of FLSs, and exacerbated the followed chondrocyte injury. Overexpression of DDIT4 alleviated HG-induced abnormal function of FLSs and injury to chondrocytes. Importantly, DDIT4 inhibited lactate synthesis, glucose uptake, LDH activity, extracellular acidification rate, oxygen consumption rate, and expression levels of glycolysis related protein (HK2, PFKP, PKM2) in HG-induced FLSs. And the glycolysis inhibitors (Cyto-B and 3BrPA) alleviated the injury of ATDC5 chondrocytes co-cultured with FLSs. Conclusions: DDIT4 participates in HG-induced FLSs overactivation and inflammation response, as well as chondrocyte injury and OA progression by regulating glycolysis processes.