Knockdown Of NLRP3 Improves Osteoporosis In Mice Through Inhibition Of PI3K/Akt/mTOR Signaling Pathway

ObjectiveTo investigate the effects of NLRP3 on osteoporosis and the PI3K/Akt/mTOR signaling pathway in mice.Methods1.Animal experiments: female C57BL/6 and NLRP3 knockout mice were randomly divided into blank control group(NC group),model group(S group),NLRP3 knockout group(KO group),and NLRP3 knockout osteoporosis group(KS group).The S and KS groups were established as de-ovalized mouse osteoporosis models.HE staining was done to observe structure of bone trabeculae.Immunohistochemistry was conducted to detect the expression of ALP,OCN,RUNX2,p-PI3 K,p-Akt and mTOR in each group.Western blotting was used to measure the protein expression of ALP,OCN,RUNX2,p-PI3K/PI3 K,p-Akt/Akt and mTOR in femurs of each group.2.Cell experiments: after conventional culture and passaged,mouse MC3T3-E1 cells were divided into five groups: routine group(C group),modeling group(Z group),NLRP3 inhibitor MCC950 group(M group),MCC950+PI3K inhibitor LY294002 group(M+L group),and LY294002 group(L group),with the exception of the routine control group,the other four groups were all stimulated with lipopolysaccharide(LPS)stimulation.OCN,IL-1β,and IL-18 were detected in cell supernatants using ELISA;Caspase-9,LC3 B,and Beclin1 mRNA expression was detected using PCR;and NLRP3,PI3 K,p-Akt,and m TOR protein expression levels were determined in each group using western blotting.Results1.Animal experiments: HE staining revealed that compared with NC group,the distribution of bone trabeculae was uneven,the arrangement was sparse,the number was significantly smaller and other pathological damage;KS group improved the distribution and arrangement of femoral bone trabeculae in osteoporotic mice compared with S group,and the damage of pathology was reduced.Immunohistochemical tests revealed a decrease in ALP,OCN and RUNX2 expression,while p-PI3 K,p-Akt and mTOR expression rose in the S group compared to the NC group.However,the KS group showed a significant reversal of the expressions of these assays as compared to the S group.The western blotting results showed that the levels of ALP,OCN and RUNX2 expression were obviously lower in the S group than in the NC group,while the expression levels of p-PI3K/PI3 K,p-Akt/Akt and mTOR were obviously higher in the S group than in the NC group.The mice in the KS group also displayed a significant reversal in the expression of the aforementioned assays as compared to the S group(P < 0.05).2.Cellular experiments: MCC950 significantly increased the supernatant OCN content of LPS-stimulated MC3T3-E1 cells,obviously decreased IL-1β and IL-18 levels,and obviously increased the LC3 B and Beclin1 expression,and obviously decreased the Caspase-9,NLRP3,PI3 K,p-Akt and m TOR expression(P < 0.05).ConclusionsThe inhibition of PI3K/Akt/mTOR signaling pathway may be the cause of the NLRP3 gene knockdown,which NLRP3 gene knockdown inhibits the development of osteoporosis.