OPG-induced Pyroptosis Of Osteoclasts In Mice

Osteoprotegerin(OPG)is a new member of the tumor necrosis factor(TNF)receptor superfamily.It acts as a bait receptor for the nuclear factor kappa B receptor activator ligand(RANKL).OPG can inhibit the differentiation and activity of osteoclasts by binding to nuclear factor kappa B receptor activator(RANK)competitively with nuclear factor kappa B receptor activator ligand(RANKL).The previous experiments found that osteoprotegerin can induce apoptosis of mature osteoclasts in vitro,and this apoptosis can be one of the ways to inhibit the activity of mature osteoclasts,thereby exerting its role in protecting bone tissue.Pyroptosis is a new type of cell death that is different from apoptosis.Pyroptosis is characterized by the rupture of cell membranes and the release of inflammatory factors.It is not clear that whether osteoprotegerin can induce mature osteoclasts pyroptosis and thereby playing its role in protecting bone tissue.In this experiment,the primary isolated mouse bone marrow mononuclear cells(BMMs)were induced to differentiate into mature osteoclasts as the research object.OPG was added with different concentrations,and through flow cytometry,real-time fluorescent quantitative PCR(qRT PCR),Western Blot and other technical methods to explore the problems above.So we can reveal the influences of osteoprotegerin on osteoclasts’ activity and the expressions of pyroptosis related genes and proteins and preliminarily clarify the molecular mechanism of osteoclasts pyroptosis induced by OPG,thereby providing theoretical basis and new ideas for the research and development of related drugs.1.Osteoprotegerin induces pyroptosis of mouse osteoclastsIn this study,4-week-old Balb/c mouse bone marrow mononuclear cells were used with macrophage colony stimulating factor(M-CSF)and nuclear factor kappa B receptor activator ligand(RANKL)to induce the differentiation of mature osteoclasts in vitro.Different concentrations of OPG(0、40、80、100 ng/ml)were added to this cell model and cultured for 12 hours.CCK8 kit,LDH cytotoxicity kit,flow cytometry were used to detect the changes in the death rate of cells,ELISA kits were used to detect the release of related inflammatory factors,Western blot was used to detect the expression levels of pyroptosis related proteins.The results showed that compared with the control group,the survival rate of osteoclasts in the osteoprotegerin treatment group was significantly reduced(p<0.05),and it was extremely significantly reduced at 40ng/ml and 100ng/ml(p<0.01);The contents of IL-1β and IL-18 in the cell culture supernatant both increased,and showed a extrem significant difference at 80ng/ml and 100ng/ml(p<0.01);the activity of LDH in the culture supernatant was no significant change on 40ng/ml OPG treatment(p>0.05),but it showed a dose-dependent increase with the increase of OPG dosage;a large number of osteoclast plasma membranes were observed to rupture in bright field,and their morphology were lost.Subsequently,the protein expression level of the key protein GSDMD-N was measured.The results showed that compared with the control group,the protein expression of GSDMD-N in the osteoclasts of the experimental group was extremely significantly increased(p<0.01).The above results indicate that osteoprotegerin can rupture the cell membrane of osteoclasts and induce pyroptosis of osteoclasts accompanying the realease of inflammatory factors related to the pyroptosis.2.The molecular mechanism of osteoprotegerin-induced pyro ptosis of mouse osteoclastsIn order to explore the related pathways of osteoprotegerin-induced pyroptosis of osteoclasts,4-week-old Balb/c mouse bone marrow mononuclear cells were used with macrophage colony stimulating factor(M-CSF)and nuclear factor kappa B receptor activator ligand(RANKL)to induce the differentiation of mature osteoclasts in vitro.Different concentrations of OPG(0,40,80,100 ng/ml)were added to this cell model and cultured for 12 hours,and then we used qRT PCR and Western blot to detect the transcription levels and protein expression levels of genes related to the classic pathway of pyroptosis.The results showed that compared with the control group,in the osteoprotegerin treatment group,the transcription levels of genes related to the classical pathway of pyroptosis,such as ASC,NLRP3,caspase 1,GSDMD were significantly increased(p<0.05 or p<0.01)and the proteins expressed by these genes were also increased significantly(p<0.05 or p<0.01).In addition,the gene transcription levels of pyroptosis related inflammatory factors such as IL-1β and IL-18 were significantly increased(p<0.05 or p<0.01),and the protein expression levels of IL-1β was significantly increased(p<0.05 orp<0.01).These results indicate that osteoprotegerin can induce osteoclasts to undergo pyroptosis through classical pathway.In summary,OPG can induce osteoclast pyroptosis and its mechanism is related to the expression levels of ASC,NLRP3,caspase 1 and GSDMD which were included in the classical pathway of pyroptosis.