Wenshen Tongluo Zhitong Recipe Improves the Osteogenic Differentiation of BMSC and Bone Loss in Senile Osteoporosis Model Mice by Inhibiting Macrophage Senescence

  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.