Study Of The Mechanism Of NLRP3 Effect On The Skeleton Of De-ovulated Mice

Objective Postmenopausal osteoporosis caused by estrogen deficiency affects millions of women worldwide.An intracellular multiprotein complex called NODlike receptor thermoprotein structural domain-associated protein 3(NLRP3)controls the maturation and secretion of the Caspase-1-dependent proinflammatory cytokines interleukin-1β(IL-1β)and interleukin-18(IL-18),which are responsible for mediating inflammation.Aberrant activation of NLRP3 inflammasomes can also lead to osteoblast pyroptosis through upregulation of caspase-1 and gasdermin D(GSDMD)expression,which in turn releases inflammatory factors.Additionally,through regulating the development of osteoblasts and osteoclasts,NLRP3 inflammatory vesicles play a significant role in the etiology of osteoporosis.The purpose of this work was to examine the mechanism by which NLRP3 contributes to osteoporosis brought on by estrogen shortage.It was demonstrated that NLRP3 causes osteoblast pyroptosis and subsequently an inflammatory response,which prevents osteogenic differentiation in de-ovulated mice.Methods Four sets of eight-week-old C57BL/6 female mice were chosen and created:control(Control,n=15),de-ovulatory group(OVX,n=15),NLRP3 knockout group(NK,n=15)and de-ovulatory + NLRP3 knockout group(OVX+NK,n=15).The control group underwent sham surgery,and the de-ovulatory and deovulatory + NLRP3 knockout groups underwent bilateral oophorectomy under pentobarbital sodium(60 mg/kg)anesthesia.Tumour necrosis factor-α(TNF-α),IL-1β and IL-18,osteogenic-specific transcription factor 2(Runx2),osteocalcin(OCN),osteopontin(OPN),in femoral tissues were detected by immunohistochemistry and Western Blotting;HE staining was used to observe the femoral bone structure in mice.Four groups of mouse femoral bone marrow stem cells were isolated and used for cellular experiments,and Caspase-1 and GSDMD,a key actuator of cell pyroptosis,IL-1β and IL-18,markers of inflammatory response,and Runx2 and OCN,markers of osteogenesis,were detected by Western Blotting and Real-Time PCR.The LDH release assay kit and Hoechst 33342/PI double staining were used to further determine the level of cellular pyroptosis.ResultsⅠ.Animal experimentsCompared with Control group,after OVX modeling,immunohistochemical results showed that TNF-α,IL-1β and IL-18 contents were significantly increased in OVX group;OCN,Runx2 and OPN protein contents were significantly decreased,and Western Blotting results of mouse femoral tissue showed that TNF-α,IL-1β and IL-18 content was significantly increased(P<0.05);OCN,Runx2,OPN content was significantly decreased(P<0.05).HE staining analysis showed that the femoral trabeculae in the OVX group were unevenly distributed,sparsely arranged and with a low number of trabeculae.TNF-α,IL-1β,and IL-18 protein expression in NLRP3 knockout mice femoral tissues was significantly lower in the NK group compared to the OVX group,whereas OCN,Runx2,and OPN protein expression was significantly greater;Western Blotting results of mouse femoral tissues showed that TNF-α,IL-1β and IL-18 were significantly lower in the NK group(P<0.05);OCN,Runx2 and OPN were significantly increased(P<0.05).The femoral trabeculae in the NK group had a large quantity,were uniformly distributed,and were closely aligned,according to the HE data.Compared with the OVX group,after OVX+NLRP3 knockdown,immunohistochemical results showed that TNF-α,IL-1β and IL-18 protein expression was lower in the OVX+NK group;OCN,Runx2 and OPN protein expression was higher;Western Blotting of mouse femoral tissue showed that TNF-α,IL-1β and IL-18 content was lower in the OVX+NK group;OCN,Runx2 and OPN content was higher in the OVX+NK group(P<0.05).HE staining showed that the distribution of bone trabeculae in OVX+NK group was more uniform,tightly arranged and the number of bone trabeculae was higher.Ⅱ.Cellular experimentsCompared with the Control group,the Western Blotting and Real-Time PCR results showed a significant increase in GSDMD-N,Caspase-1,IL-1β and IL-18 expression and a significant decrease in OCN and Runx2 expression after osteoblast induction of osteogenic differentiation in the OVX group(P<0.05);lactate dehydrogenase(LDH)activity was significantly increased(P<0.05).Hoechst 33342/PI double staining results showed high PI fluorescence intensity.In comparison to the OVX group,the NK group’s Western Blotting and RealTime PCR data revealed considerably overexpression of OCN and Runx2 and markedly elevated protein of GSDMD-N,Caspase-1,IL-1β,and IL-18;LDH activity was reduced considerably(P<0.05).The PI fluorescence intensity was low in the Hoechst 33342/PI double staining data.Compared with the OVX group,Western Blotting and Real-Time PCR results in the OVX+NK group showed lower expression of GSDMD-N,Caspase-1,IL-1β and IL-18(P<0.05)and superior expression of OCN and Runx2(P<0.05);lower LDH activity(P<0.05).Hoechst 33342/PI double staining showed low PI fluorescence intensity.ConclusionsNLRP3 promoted the pyroptosis of osteoblasts in de-ovalized mice,promoted the inflammatory response and inhibited the osteogenic differentiation of osteoblasts,thus participating in the development of osteoporosis.