Fig. 4: Hepatocyte-derived sEV-LRG1 mediates the protection of SIRT2-KO^hep against osteoclastogenesis and bone loss.

a, Schema of BMDM treatment with sEVs. The sEVs were purified from the supernatant of shSIRT2-AML12 cells infected with Ctrl or shLRG1 lentiviral vectors. sEV-LRG1 protein expression was analyzed by western blot. The isolated primary BMDMs were co-cultured with each set of transduced sEVs (4 µg ml⁻¹) and murine M-CSF/RANKL stimulation for 7 d to generate osteoclasts and followed TRAP staining and real-time PCR test. b, TRAP staining of osteoclasts treated with NC-sEVs or shSIRT2-sEVs or shSIRT2-shLRG1-sEVs (scale bar, 200 µm). c,d, Number and area of multi-nucleated TRAP⁺ cells with indicated treatment were measured. e, The mRNA expression of osteoclast-specific genes in the corresponding treated osteoclasts was measured by real-time PCR. (b–e, n = 3 biologically independent experiments). f, The experimental procedure for sEVs treatment in vivo. C57BL/6J mice were consecutively intravenously injected with the NC-sEVs, shSIRT2-sEVs, shSIRT2-shLRG1-sEVs and LRG1-sEVs (50 µg per mouse, every other day) 3 d after OVX. Micro-CT and TRAP staining were performed 6 weeks after the first injection. g, Representative biophotonic images of the tissue distribution of fluorescence signal in mice at 4 and 8 h after intravenous injection of PKH26-labeled sEVs isolated from the supernatant of AML12 cells. h,i, Represented images of 3D restoration (h) and quantification of trabecular BV/TV, Tb.N, Tb.Sp and Tb.Th of distal femurs of the indicated group mice (i), as measured by μ-CT (n = 7, sham + NC-sEVs; n = 7, OVX + NC-sEVs; n = 7, OVX + shSIRT2-sEVs; n = 7, OVX + shSIRT2-shLRG1-sEVs and n = 7, OVX + LRG1-sEVs). j, Plasma CTX-1 in each group was detected by ELISA. k, TRAP staining on paraffin-embedded femur sections in each group after corresponding sEV treatment (scale bar, 100 µm). l, Quantification of Oc.S/BS is shown on the right; (h–l, one technical replicate of seven biological replicates for each group). 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–e,i,j,l).

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