Fig. 7: Hepatocyte-derived shSIRT2-sEVs or human LRG1^high plasma sEVs inhibit human osteoclast differentiation and plasma sEV-LRG1 inversely correlates with bone resorption in patients.

a, Western blot analysis of LRG1 protein expression in the cytoplasm and sEVs derived from SIRT2-knockdown (shSIRT2) HepG2 human hepatocytes. b, Representative TRAP staining images of human PBMCs cultured with RANKL and sEVs (10 µg ml⁻¹) derived from the supernatant of control and shSIRT2-HepG2 cells (NC-sEVs, shSIRT2-1-sEVs, shSIRT2-2-sEVs) (scale bars, 200 µm). c, Number of multi-nucleated TRAP⁺ cells with indicated treatment was measured. d, The mRNA expression of osteoclast-specific genes in the corresponding treated osteoclasts was measured by real-time PCR. (a–d, n = 3 biologically independent experiments). e, Western blot analysis of LRG1 protein expression in sEVs derived from LRG1-high- or low-expression plasma. f, Representative TRAP staining images of human PBMCs cultured with RANKL and sEVs (20 µg ml⁻¹) derived from three LRG1-high-expression human plasma or three LRG1-low-expression human plasma (LRG1^low plasma sEVs, LRG1^high plasma sEVs) (scale bars, 200 µm). g,h, Number and area of multi-nucleated TRAP⁺ cells. i,The mRNA expression of osteoclast-specific genes measured by real-time PCR. (e–i, n = 3 biologically independent experiments). j, The inhibitory effect of sEVs and denosumab on osteoclast differentiation and the rebound effect after cessation of treatments. Representative TRAP staining images of PBMCs cultured with RANKL and sEV-LRG1 (10 µg ml⁻¹) or denosumab (500 ng ml⁻¹) (scale bars, 200 µm). The inhibitory experiments were performed by the administration of RANKL and sEVs or the commercial denosumab. After 10 d of treatment, half of PBMCs were stained by TRAP (top). At the same time, for the other half of PBMCs, both sEVs and denosumab administration were stopped, but PBMCs continued to be treated with RANKL for 4 d (bottom). k, Area of multi-nucleated TRAP⁺ cells with indicated treatment was measured. (j–k, n = 3 biologically independent experiments). l,m, The presented IHC images of SIRT2 expression levels in human liver tissues and association between SIRT2 expression levels and different ages are shown. LAG, low age groups (age <51 years, n = 54); HAG, high age groups (age >51 years, n = 60). n, Representative western blot analysis of protein expression of plasma sEV-LRG1 from female healthy control (BMD T score of lumbar spine is >−1, n = 28) and patients with osteoporosis (BMD T score of lumbar spine <−2.5, n = 25). o, Plots of protein expression of plasma sEV-LRG1 in female healthy control (n = 28) and osteoporotic patient group (n = 25). Association between human plasma sEV-LRG1 expression and BMD (p), bone resorption marker β-CTX (q) and bone formation markers PINP (r) and BALP (s) in 120 human participants of both sexes (females, n = 84 and males, n = 36). Data are presented as mean ± s.d., with biologically individual data points shown. P values were determined by one-way ANOVA followed by Tukey’s test (c,d), unpaired two-tailed Student’s t-test (g–i), unpaired two-tailed Mann–Whitney U-test (o), two-way ANOVA followed by Tukey’s test (k), two-tailed Spearman’s correlation test (m) and linear correlation and regression analyses (p–s).

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