Fig. 3: SIRT2-KO^hep prevents OVX-induced bone loss through upregulating LRG1 expression in hepatocytes.

a, Venn diagram showing the overlap numbers of SIRT2-KO^hep-regulated plasma proteins by mass spectra (MS) and SIRT2-KO^hep-regulated hepatic mRNAs by RNA-seq in aged mice (18 months of age). b, LRG1 mRNA expression in the livers of aged LoxP and SIRT2-KO^hep mice measured by real-time PCR. One technical replicate of 10 (LoxP mice) or 12 (SIRT2-KO^hep mice) biological replicates for each group. c,d, Western blot analysis of LRG1 protein expression in the primary hepatocytes and supernatant-derived sEVs of aged female (c) and male (d) LoxP and SIRT2-KO^hep mice; n = 3 mice, one technical replicate of three biological replicates for each group. e,g, Represented in situ immunofluorescence images of murine femurs in aged female (e) and male (g) LoxP and SIRT2-KO^hep mice (18 months of age) (scale bar, 50 µm). DAPI, 4,6-diamidino-2-phenylindole. f,h, Quantitation of ratio of LRG1 and CTSK double-positive area to CTSK-positive area on bone sections of the aged female (f) or male (h) LoxP (n = 3) and SIRT2-KO^hep (n = 3) mice, was measured by ImageJ. (e–h, one technical replicate of three biological replicates for each group). i, Schematic view of enrichment of H4K16ac on LRG1 promoter region from ChIP-seq data from Cistrome DB Toolkit. j, ChIP analysis showing enrichment of H4K16ac at the LRG1 proximal promoter region in NC and shSIRT2-AML12 hepatocytes using the primers p1, p2 and p3; n = 3 biologically independent experiments. k, The experimental procedure for hepatocyte-specific LRG1 knockdown by AAV8 virus. LoxP and SIRT2-KO^hep mice (12 weeks of age) were given tail injections of 2 × 10¹¹ viral particles of either AAV8-shLRG1 or Ctrl vector 7 d before OVX to maximize the viral expression and knockdown efficiency. The real-time observation of gene expression was performed by BLI 14 d after viral injection. Mice were killed 5 weeks after OVX for CTX-1 and bone mass test. l, Plasma CTX-1 was detected by ELISA. m,n, Represented images of 3D restoration and quantification of trabecular BV/TV, Tb.N, Tb.Sp and Tb.Th of distal femurs of the indicated group mice, as measured by μ-CT (n = 7, Sham-LoxP-Ctrl mice; n = 7, Sham-SIRT2-KO^hep-Ctrl mice; n = 8, OVX-LoxP-Ctrl mice; n = 8, OVX-SIRT2-KO^hep-Ctrl mice and n = 8, OVX-SIRT2-KO^hep-shLRG1 mice). (l–n, one technical replicate of seven (Sham-LoxP-Ctrl mice); seven (Sham-SIRT2-KO^hep-Ctrl mice); eight (OVX-LoxP-Ctrl mice); eight (OVX-SIRT2-KO^hep-Ctrl mice) and eight (OVX-SIRT2-KO^hep-shLRG1 mice) biological replicates for each group). Data are presented as mean ± s.d., with biologically individual data points shown. P values were determined by unpaired two-tailed Student’s t-test (b,f,h), one-way ANOVA followed by Tukey’s test (l,n) and two-way ANOVA followed by Tukey’s test (j).

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