OBJECTIVE  To study the intervention effects of Wenshen Tongluo Zhitong Recipe(WTZR) on macrophage senescence and senile osteoporosis.

  METHODS  The senescent macrophage model was established using hydrogen peroxide (H₂O₂) and subsequently divided into four groups: control, model, low-dose drug-treated serum, and high-dose drug-treated serum.β-galactosidase staining, Western blot and qPCR were employed to evaluate the mRNA expression of senescence markers p21 and p53. ROS staining and JC-1 staining were applied to assess mitochondrial function in macrophages. The mRNA levels of Interleukin(IL)-6, IL-10, CD206, and inducible nitric oxide synthase (iNOS) were determined by qPCR analysis. Immunofluorescence was used to evaluate arginase(ARG1) and iNOS protein expressions for assessing the impact of drug-containing serum on macrophage polarization. qPCR analysis was conducted to measure osteocalcin(OCN), collagen type Ⅰ alpha 1(Col1a1), runt-related transcription factor 2(Runx2) mRNA levels as osteoblast-related markers; ALP staining along with alizarin red staining were performed to evaluate the effect of macrophage conditioned medium treated with drug-containing serum on bone marrow mesenchymal stromal cell(BMSC)osteogenic differentiation. C57BL/6J mice were randomly allocated into four groups: control group, model group, WTZR low-dose group, and WTZR high-dose group. The senile osteoporosis (SOP) mouse model was established by D-galactose. Micro-CT scanning analyzed femur microstructure while HE staining detected pathological changes in femur bone tissue samples collected from each experimental condition. Furthermore, Western blot was used to detect the senescence-related molecules p21 and p53 and the osteogenesis-related markers OCN and Runx2, qPCR analysis measured tibial expression levels of senescence-related molecules (p21, p53) as well as macrophage polarisation-related molecules (IL-6, iNOS, CD206, and IL-10) to assess the effect of this compound on a mouse model simulating SOP.

  RESULTS  Following intervention with serum containing WTZR, there was a significant decrease in the number of senescent positive cells compared to the model group. Additionally, there was a notable decrease in p21 and p53 mRNA and proteins expression (P < 0.05, P < 0.01, P < 0.001). Furthermore, drug-containing WTZR effectively inhibited ROS production induced by H₂O₂ and mitigated mitochondrial membrane potential reduction in macrophages (P < 0.05, P < 0.001). Treatment with drug-containing WTZR resulted in down-regulated mRNA expression of M1-related gene iNOS (P < 0.05) while up-regulating mRNA expression of M2-related genes CD163 and CD206 (P < 0.05). The drug-containing WTZR significantly reduced fluorescence intensity for iNOS (P < 0.01) while increasing ARG1 (P < 0.05) fluorescence intensity. Moreover, conditioned medium from macrophages treated with drug-containing serum increased ALP positive cell count (P < 0.01, P < 0.001), alizarin red positive area (P < 0.05), as well as Col1a1, Runx2 and OCN mRNA expression levels (P < 0.05, P < 0.01). The Tb.N, BMD, and BV/TV were significantly higher in the WTZR group compared to the model group (P < 0.05, P < 0.01, P < 0.001); meanwhile, Tb.Sp was notably lower than that observed in the model group (P < 0.05, P < 0.01); bone trabeculae were significantly improved, increased in number and widened. Additionally, the compound could significantly inhibit the D-galactose induced up-regulation of tibial senescence-related genes and proteins p21 and p53(P < 0.05, P < 0.001, P < 0.0001), promote the expression of osteogenesis-related markers OCN and Runx2 protein(P < 0.01, P < 0.0001), promote the down-regulation of M1 related genes IL-6 and iNOS (P < 0.05), and promote the expression of M2 related genes IL-10 and CD206 (P < 0.05, P < 0.01).

  CONCLUSION  Wenshen Tongluo Zhitong Recipe may play an anti-osteoporosis effect by inhibiting macrophage senescence and promoting the osteogenic differentiation of BMSC.