4 Discussion
OA is a common clinical degenerative joint disease in elderly population, which is one of the main causes of chronic pain and joint disability. OA can severely reduce the quality of life of affected individuals. Current therapeutic goal of OA in clinical practice is to relieve the symptoms but cannot slow down the disease progression. Non-steroidal anti-inflammatory drugs (NSAIDs) are generally considered to be the first-line pharmacological treatments for OA. However, NSAIDs cannot achieve the long-term pain relief for OA patients, and they are associated with significant side efforts such as gastric ulceration, renal impairment, and cardiovascular accidents 14. At the end stage of OA, a replacement surgery remains to be the only treatment option. Thus, identification of emerging pharmaceutical therapies is urgently needed in the treatment of OA. Empagliflozin, a selective inhibitor of sodium-glucose cotransporter-2 (SGLT2), has been reported to show the anti-inflammatory properties in several conditions such as cardiovascular diseases, Alzheimer’s disease, liver injury, and chronic kidney disease 15-19. In this study, we discovered the protective effect of empagliflozin on cartilage degeneration via inhibition of NF-κB pathway.
Previously thought to simply be damaged from ”wear and tear”, OA is now understood as a complex cell-mediated process. Current research has shown that OA is characterized by progressive cartilage degradation, but also underlying bone remodeling, osteophyte formation, and synovial inflammation 3,20. A series of pathological factors such as ECM degradation, chondrocyte inflammation, oxidative stress, mitochondrial dysfunction, abnormal mechanical load, and senescent molecules contribute to the degeneration of chondrocytes, ultimately causing cartilage damage and OA development 21,22. Chondrocytes, as the only cellular component of cartilage, play a central role in the balance of ECM metabolism through the synthesis of cartilage matrix. However, the inflammatory factors like IL-1β can disrupt the balance of ECM metabolism by increasing the expression of catabolic enzymes including MMPs, causing the gradual loss of ECM. MMPs inhibitors could be promising agents for the treatment of OA 23. Our data revealed that IL-1β upregulated the expression of matrix-degrading enzyme (MMP9 and MMP13) and activated the inflammatory cascade reaction by the secretion of inflammatory cytokines (NO, PGE2, IL-6, COX2, and INOS) in mouse chondrocytes, whereas these effects could be offset by the administration of empagliflozin. These founds indicated that empagliflozin could protect mouse chondrocytes from IL-1β-induced ECM degradation and inflammatory reaction. What’s more, chondrocyte senescence is also involved in the pathological process of OA. Jeon et al found that senescent cells accumulated in the articular cartilage and synovium after anterior cruciate ligament transection, and local clearance of these senescent cells attenuated the development of post-traumatic OA 24. Therefore, the senescence analysis of chondrocytes was conducted in this study. Cellular senescence is generally regarded as a cell state characterized by an irreversible cell-cycle arrest 25. P21 and P53 are the key factors for promoting senescence. We found that empagliflozin could counteract the elevated expression of senescence markers (P21 and P53) induced by IL-1β. Empagliflozin could also decrease the SA-β-Gal-positive cells. Thus, empagliflozin could alleviate IL-1β-induced senescence in mouse chondrocytes in vitro. All these results displayed the antidegradation role of empagliflozin on chondrocytes in vitro. The effect of empagliflozin on cartilage damage in vivo was also observed in DMM-induced OA mice model. We found that empagliflozin could also protect mouse knee cartilage from wear and matrix degeneration.
Multiple signaling pathways are involved in OA such as NF-κB, Wnt/β-catenin, HIFs, TGFβ/ΒΜP and so on 26. NF-κB signaling participates in many OA-associated events, including chondrocyte catabolism, survival, and inflammation 27. NF-κB transcription factor has been considered as a disease-contributing factor of OA for a long time 27. When NF-κB exists in the cytoplasm, it is in an inactive state and cannot enter the nucleus to play its function because it binds to the inhibitory protein IκB. Once cells are stimulated by certain inductive factors, IκB is phosphorylated and the molecular conformation of IκB changes, resulting in the activation and nuclear translocation of NF-κB28,29. Activation of NF-κB ,on the one hand, can directly bind to the promoters of MMPs genes to promote the expression of matrix-degrading enzyme 30. On the other hand, activation of NF-κB regulates the transcription of many nuclear genes related to inflammation, including cyclooxygenase (COX2) and inducible nitric oxide synthase (iNOS) genes 31,32. COX2 and iNOS can promote the production and secretion of induce prostaglandin (PGE2) and nitric oxide (NO). Increased PGE2 and NO levels can upregulate the expression of MMPs, ultimately causing cartilage degeneration. NF-κB signaling pathway was focused in this study. Our results showed that empagliflozin inhibited the phosphorylation of IκB and P65, displaying the suppressive role on NF-κB signaling. Thus, the underlying mechanism of empagliflozin in mouse cartilage protective roles is related to the inhibition of NF-κB pathway.
However, there are still some limitations in our study. First at all, we confirmed the protective effect of empagliflozin on preventing cartilage degeneration via inhibiting the activation of NF-κB pathway. But whether NF-κB is the direct target of empagliflozin needs to be further identified. Secondly, we have only demonstrated the effects of empagliflozin on cellular inflammation and senescence in chondrocytes. The effects of empagliflozin on other aspects involving the onset and progression of OA deserve further investigation.