The Study Of The Effect Of FAAH On Osteoclast Differentiation And Function And Its Mechanism

Objective:1.To study the effect of fatty amide hydrolase(FAAH)on osteoclast differentiation and function in vitro.2.To explore the specific molecular mechanism of FAAH regulating osteoclast differentiation and function.3.To observe the changes of bone tissue microstructure and related serum indexes in ovariectomy(OVX)-induced osteoporosis model mice treated with PF-04457845(PF),a specific small molecule inhibitor of FAAH.Methods:1.BMMs were induced by RANKL to be differentiated as osteoclast,and the expression of FAAH was detected by qPCR,Western blot,and immunofluorescence assay.2.In the process of osteoclast differentiation,PF and FAAH-shRNA lentivirus were used to interfere with BMMs.TRAP staining was performed to detect osteoclast formation,F-actin ring staining and bone plate absorption assay were used to measure osteoclast bone absorption function.The expression levels of osteoclast marker genes and the activation of MAPK and NF-κB pathways were investigated by qPCR and Western blot assay.3.An OVX mouse model was established to simulate postmenstrual osteoporosis.After model establishment,the mice were given solvent or PF intragastric administration 5 times a week.Two months later,bilateral femurs and lumbar vertebrae were collected for Micro-CT scanning and reconstruction.Bone sections were stained with HE,TRAP and IHC.Serum was taken by enucleation of the eyeball for ELISA detection.4.During osteoclast differentiation,PF and FAAH-shRNA lentivirus were used to interfere with BMMs,qPCR and Western blot assay to detect the expression of cannabinoid receptors CB1 and CB2.BMMs were interfered with PF,and CB1 and CB2 were selectively blocked by AM251 and AM630,respectively.Osteoclast formation and expression of osteoclast marker genes were detected.5.Whole genome RNA sequencing(RNA-seq)was performed on BMMs stimulated by RANKL for 3 days with or without PF intervention to explore the downstream genes and signaling pathways that might be affected by FAAH inhibition.6.Recombinant mouse-derived IL17 protein was used to rescue the inhibition of osteoclast differentiation caused by PF intervention,the formation and function of osteoclasts,the expression of osteoclast marker genes,and activation of MAPK and NF-κB signaling pathways were detected.7.Osteogenic precursor cell line MC3T3-E1 cells were cultured in an osteogenic induction medium.Alizarin red staining and alkaline phosphatase(ALP)staining were used to evaluate mineralization and osteogenic differentiation capability.The levels of mRNA and protein expression of the osteoblast markers genes were measured by qPCR and Western blot assay.Results:1.In the process of osteoclast differentiation,the expression of FAAH at mRNA and protein levels increased significantly,which was further confirmed using immunofluorescence detection.2.Both FAAH-shRNA lentivirus and PF could significantly inhibit osteoclast formation and bone resorption,and down-regulate the expression of osteoclast marker genes and the activation of NF-κB and MAPK signal pathways.3.Micro-CT,HE and TRAP staining of bone tissue section showed that PF could reduce OVX-induced bone loss and osteoclast formation in mice.IHC staining and ELISA detection confirmed that PF could inhibit the increase of IL17 expression induced by OVX.4.FAAH-shRNA lentivirus and PF had no significant effect on the expression of CB1 and CB2 at mRNA and protein levels.When PF was used to interfere with BMMs,antagonists AM251 and AM630 could not save the inhibitory effect of PF on osteoclast differentiation.5.RNA-seq showed that the IL17 signal pathway was significantly inhibited after PF intervention during osteoclast differentiation.The supplement of recombinant mouse-derived IL17 protein can partially save the inhibition of osteoclast differentiation and the activation of NF-κB signal pathway induced by PF intervention,indicating that IL17 signal pathway is involved in the regulation of FAAH-mediated osteoclast differentiation.6.The inhibition of FAAH had no significant effect on osteoblast differentiation and the expression of osteoblast marker genes.Conclusions:FAAH plays an important role in RANKL-induced osteoclast formation and bone resorption.In vitro,FAAH inhibition partially inhibits RANKL-induced osteoclast differentiation by blocking IL17/NF-κB signaling cascades.In vivo,PF can also alleviate OVX-induced bone loss by inhibiting the formation of osteoclasts.At the same time,PF intervention had no significant effect on osteoblast differentiation.This study suggests that targeting FAAH is a new potential therapeutic strategy for osteoporosis and other osteoclast-related diseases.