Liuwei Dihuang Pill regulate osteoclast membrane fusion to improve postmenopausal osteoporosis in mice: the CLCF1/NFATc1 signaling axis as a therapeutic target

OBJECTIVES: To explore the mechanism of Liuwei Dihuang Pill (LWDHW) for improving postmenopausal osteoporosis (PMOP). METHODS: Thirty female C57BL/6 mice were randomized equally into sham-operated group, PMOP model group, and LWDHW treatment group. The mice in the latter two groups underwent ovariectomy to simulate PMOP, followed by gavage of normal saline and LWDHW (twice daily) for 60 days, respectively. Bone mineral density (BMD) of the mice was detected, and TRAP staining was used to observe osteoclast and osteoclast maturation in the tibia tissue. In RANKL-induced RAW264.7 cells with or without lentivirus-mediated CLCF1 knockdown, the effects of incubation with normal rat serum or LWDHW-medicated rat serum on cell membrane fusion rate and F-actin loop formation were observed using immunofluorescence staining with phalloidin peptide; the mRNA and protein expressions of CLCF1, NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2 were detected using RT-qPCR and Western blotting. RESULTS: In PMOP mice, LWDHW treatment significantly increased BMD, reduced osteoclast maturation, osteoclast membrane fusion rate, and F-actin ring formation, upregulated the mRNA and protein expressions of CLCF1 in tibial tissues, and lowered the expressions of NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2. In RANKL-induced RAW264.7 cells with CLCF1 knockdown, treatment with LWDHW-medicated rat serum significantly decreased osteoclast membrane fusion rate, F-actin ring formation, and the expressions of NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2. CONCLUSIONS: LWDHW improves PMOP in mice by regulating osteoclast membrane fusion through upregulating CLCF1 expression and downregulating NFATc1, DC-STAMP, OC-STAMP and ATP6V0D2 expressions.