Although factors such as age, genetics, mechanical stress, trauma and metabolism are associated with OA progression, the cellular signaling and metabolic changes that occur in chondrocytes are not fully understood. In this study, we found that the expression of P2Y12 increased during the progression of OA, and P2Y12 regulated apoptosis and ECM degradation by regulating the PI3K/AKT signaling pathway, thus changing the homeostasis of cartilage. In terms of mechanism, we found that P2Y12 inhibits the binding of Bad and Bcl2 through the Pi3k/Akt pathway, thus inhibiting apoptosis. Then, we further applied the OA animal model to verify the potential therapeutic effect of P2Y12 in the progression of OA. The P2Y receptor is a G-coupled transmembrane protein that exists in almost any cell type and ligands are purine and pyrimidine nucleotides. From the phylogenetic and structural perspective, two P2Y receptor subgroups with high structural differences were identified. The first subgroup includes Gq-coupled subgroups (P2Y1, P2Y2, P2Y4, P2Y6 and P2Y11). The second subgroup includes Gi coupling subtypes (P2Y12, P2Y13, and P2Y14) . P2y12-like receptors in apoptosis and inflammation have been reported to be involved in platelets and microglia [16–18] chemotaxis and phagocytosis. Although facet arthritis and microglial inflammation share many similar features of joint injury, they represent different etiologies and pathologic processes. Whether P2Y12 regulates Cartilage steady-state in the pathogenesis of OA has not been studied in detail. Our data suggest that P2Y12 expression level is associated with OA severity.
Considering the correlation between the expression of P2Y12 and OA conditions, we induced OA by injecting MIA to establish a rat articular process FJOA model, and MIA has been widely used to study the pathogenesis of OA [19–20]. The lumbar facet joints were then taken and divided into non-operative and operative groups. Immunohistochemical staining showed significantly increased P2Y12 expression in articular cartilage of severely degenerative FJOA. In vitro Western blot analysis showed that P2Y12 and Bax and Caspase-3 expression were significantly increased after 36 h of IL-1β stimulation on SW1353 cells. Il-1 β is a key pro-inflammatory cytokine in the pathogenesis of OA and is usually used to stimulate chondrocytes to simulate OA conditions in vitro studies [21–23]. In addition, co-localization of P2Y12/ Cleaved caspase-3 in chondrocyte apoptosis models was detected by double-labeled immunofluorescence staining. SiRNA silencing P2Y12 increased il-1 β -induced Bax and Caspase-3 cleavage expression, suggesting that P2Y12 negatively regulates chondrocyte apoptosis through caspase-dependent signaling pathways. Flow cytometry showed that inhibition of P2Y12 significantly enhanced IL-1β -induced apoptosis of SW1353 cells. In order to investigate the mechanism of inhibiting chondrocyte P2Y12 function and increasing IL-1 β induced chondrocyte apoptosis, we analyzed signaling molecules in the PI3K/AKT signaling pathway by Western blot. We treated chondrocytes with the specific PI3K inhibitor LY294002 in the presence of IL-1β. Western blot analysis showed that Cleaved Caspase 3 and Bcl2 expression were consistent with control group after siPP2Y12 transfected chondrocytes treated with LY294002. TUNEL staining analysis showed that the number of apoptotic cells was up-regulated after siPP2Y12 transfected with LY294002. Based on our data, we conclude that P2Y12 may play a key role in inhibiting the apoptosis of articular chondrocytes in rats.
In the lumbar region, facet joints play the most important role in maintaining the stability of spinal movement. FJOA is a degenerative joint disease characterized by chondrocyte degeneration  and chondrocyte apoptosis . However, the exact molecular mechanism of chondrocyte apoptosis remains unclear. In this study, we focused on the role of P2Y12 in chondrocyte apoptosis in lumbar facet joint osteoarthritis. First, we established a rat articular process FJOA model, and then took the lumbar facet joints and divided them into non-surgical and surgical group. Immunohistochemical staining showed that the expression of P2Y12 in the articular cartilage of severely degenerated FJOA was significantly increased. In vitro, Western blot analysis showed that P2Y12 was significantly increased 36 h after IL-1β stimulated SW1353 cells, and caspase-3 was also up-regulated. In addition, the co-localization of P2Y12/cleaved caspase-3 was detected in the cell chondrocyte apoptosis model by doubl-labeled immunofluorescence staining. Silencing of P2Y12 by siRNA can increase the expression of caspase-3 cleavage induced by IL-1β, indicating that P2Y12 negatively regulates chondrocyte apoptosis through a caspase-dependent signaling pathway. Flow cytometry showed that P2Y12 knockdown significantly enhanced IL-1β induced apoptosis of SW1353 cells. Based on our data, we conclude that P2Y12 may play a key role in inhibiting the apoptosis of rat articular chondrocytes through the PI3K/AKT signaling pathway.
As a G protein coupled receptor, P2Y12 has the typical characteristics of 7 hydrophobic transmembrane (TM) regions, which are connected by 3 extracellular loops (EL) and 3 intracellular loops . The human P2Y12 receptor consists of 342 amino acid residues . Like most G protein coupled receptors, P2Y12 receptors are in the extracellular N-terminal (Cys17), the first extracellular ring (Cys97), the second extracellular ring (CYS175) and the third extracellular ring (Cys270) Has 4 extracellular hormone residues . The P2Y12 receptor is coupled to the Gαi2 subunit . After stimulation, Gα and Gβγ subunits dissociate to activate various signal transduction pathways . The Gβγ subunit stimulates the activity of phosphatidylinositol 3 kinase (PI3K), which leads to the late accumulation of phosphatidylinositol 3,4-bisphosphate [PtdIns (3,4) P2] and phosphatidylinositol 3,4,5- Triphosphate [PtdIns (3,4) rapid transient accumulation, 5) P3] [27–29]. The PI3K pathway also activates Rap1b and Akt . Gβγ dimers can activate inward rectifier potassium channels (GIRKs) that mediate Src tyrosine kinase G protein-gated inward rectifier potassium channels (GIRKs) . The P2Y12 receptor inhibits apoptosis by activating the PI3K/Akt pathway: P2Y12 activation induces Akt and Bad phosphorylation. After being phosphorylated by Akt, Bad is sequestered in the cytoplasm by the adaptor protein 14-3-3, thereby preventing the binding of Bad to Bcl-xL. As a result, the free Bcl-xL heterodimer with Bak protein prevents the dimerization of Bak in the mitochondria, thereby antagonizing its pro-apoptotic activity. Bcl-xL indirectly inactivates Bax by inhibiting its translocation to mitochondria .
P2Y12 receptors are mainly expressed in platelets and neuronal tissues . It is worth noting that few studies have reported the function of P2Y12 in the chondrocytes of the lumbar facet joints. In our research, we comprehensively show that P2Y12 is up-regulated in cartilage and participates in the development of FJOA. In normal cartilage, the expression of P2Y12 is weak, while in the joint cartilage of the most severe surgical group, the expression of P2Y12 is higher. A positive correlation was obtained between the expression of P2Y12 and the severity of FJOA. We also found the expression of P2Y12. Our hypothesis P2Y12 plays a vital role in FJOA and may be one of the key factors in inhibiting chondrocyte apoptosis.