The Study Of GGCX Inhibits Chondrocyte Apoptosis And Inflammation In Osteoarthritis By Targeting The NF-κB Signaling Pathway

Background and Objective:Osteoarthritis(OA)is a clinically very common chronic joint degenerative disease,and it is one of the diseases most affecting the quality of life of middle-aged and elderly people in my country.Because the specific etiology and pathogenesis of OA are still unclear,so far there is no exact and effective treatment for its etiology.Therefore,exploring the effective molecular targets in the occurrence and development of OA is a current research hotspot.Studies have shown thatγ-glutamylcarboxylase(GGCX)can regulate the vascular inflammation in patients with type 2 diabetes through the NF-κB signaling pathway.However,there are few studies on the role of GGCX in OA and its specific mechanism of action.In our previous studies,we found that GGCX is related to the inflammation and apoptosis of osteoarthritis,and the NF-κB signaling pathway is also one of the classic pathways that regulate the proliferation and apoptosis of osteoarthritis.Therefore,we suspect that GGCX can reduce the apoptosis and inflammation of osteoarthritis chondrocytes through the NF-κB signaling pathway.This study aims to explore the effects of GGCX on OA apoptosis and inflammation and its related mechanisms,and provide a theoretical basis for exploring new treatment methods for OA.Method:1.Separate the knee articular cartilage of 5-week-old SD rats,digest them with trypsin and type II collagenase,and centrifuge to obtain primary SD rat articular cartilage cells,which are then cultured in an incubator.2.After the cultured primary SD rat chondrocytes were passaged to the first generation,the obtained cells were identified by toluidine blue staining,alcian blue staining and type II collagen immunocytochemical staining.3.Use IL-1β(10ng/ml)to construct osteoarthritis chondrocyte model;use the overexpression plasmid to up-regulate the expression of GGCX in articular chondrocytes and divide the experiment into 4 groups: control group(ctrl group);GGCX overexpression group;IL-1β group;GGCX+IL-1β group.4.Observe the morphological changes of the cells in each group under a microscope,monitor the cell density,and draw the cell growth curve;use the EDU method to determine the proliferation of each group of cells..5.Flow cytometry was used to detect the apoptosis level of each group of cells,and Western blotting was used to detect the expression of apoptosis-related proteins BAX,BCL-2 and Caspase-3 in each group.6.The levels of IL-1β,IL-6 and TNF-α in each group of cells were detected by enzyme-linked immunosorbent assay,and the expression of IL-1β and TNF-α protein in each group of chondrocytes was detected by Western blotting.7.Western blotting was used to detect the expression changes of NF-κB P65,p-NF-κB P65 and P-IκBα proteins in each group of NF-κB pathway to determine the activation of NF-κB pathway.Result:1.The isolated 5-week-old SD rat knee joint chondrocytes adhered slowly.After24 hours of culture,they began to adhere to the wall,and a large number of cells adhered to the wall after 48 hours.After passaging,the cell adhesion time and proliferation speed increase significantly,and it can be passed down again in about 72 hours.The cells are mostly triangular or polygonal.2.Identification of chondrocytes: Toluidine blue staining shows that the cytoplasm is blue and the nucleus is dark blue;Alcian blue staining shows that the cytoplasm is light blue;immunocytochemical staining of type II collagen shows that the cytoplasm is brown and the nucleus is Dark brown.The above three chondrocyte identification methods are all positive.3.abnormal cell morphology,which is a long spindle,flat,and very sparse;GGCX+IL-1β The morphology of the cells in the group returned to normal,triangular,polygonal,and relatively dense;the EDU results showed that the proliferation of chondrocytes in the IL-1β group was significantly lower than that in the control group and the GGCX overexpression group(**P < 0.01),and the proliferation ability of chondrocytes in the GGCX+IL-1β group was increased relative to that in the IL-1βgroup(** P<0.01).4.There was no significant difference in the apoptosis rate between the control group and the GGCX overexpression group,and the IL-1β group’s apoptosis rate was significantly higher than that of the control group and the GGCX overexpression group(**P < 0.01).Compared with the IL-1β group,the apoptosis rate of the GGCX+IL-1β group was significantly decreased(**P<0.01);WB showed that the BAX and Caspase-3 protein levels in the IL-1β group were significantly higher than those in the control group and the GGCX overexpression group,BCL-2 protein level was significantly lower than the control group and GGCX overexpression group(**P<0.01).Compared with the IL-1β group,the BAX and Caspase-3 protein levels in the GGCX+IL-1β group were significantly reduced,and the BCL-2 protein levels were significantly increased(*P<0.05).5.ELISA indicated that the levels of IL-1β,IL-6 and TNF-α in the IL-1β group were significantly higher than those in the control group and the GGCX overexpression group(**P < 0.01).The levels of IL-1β,IL-6 and TNF-α in the GGCX+IL-1β group were significantly lower than those in the IL-1β group(*P<0.05);the protein levels of TNF-α and IL-1β in the IL-1β group were significantly higher than those in the control group and GGCX overexpression group(*P<0.05).Compared with IL-1β group,TNF-α and IL-1β protein levels in GGCX+IL-1β group were significantly reduced(*P<0.05).6.The p-P65/P65 of IL-1β group was significantly higher than that of control group and GGCX group(** P<0.01).The p-P65/P65 of GGCX+IL-1β group was significantly lower than that of IL-1β group(**P<0.01).The expression of P-IκBαprotein in IL-1β group was significantly higher than that in the control group and GGCX group(**P<0.01),while the expression of P-IκBα protein in GGCX+IL-1βgroup was lower than that in IL-1β group(**P< 0.05).Conclusion:1.Overexpression of GGCX can reduce IL-1β-induced increase in the phosphorylation level of P65 protein in SD rat OA chondrocytes,and may partially inhibit the activation of NF-κB pathway by inhibiting phosphorylation of P-IκBαprotein.2.Overexpression of GGCX can reduce the apoptosis and inflammation of osteoarthritis chondrocytes through the NF-κB pathway.