Effect and mechanism of Gusong Qianggu Decoction on reducing bone loss in ovariectomized mice by targeting endoplasmic reticulum stress-induced apoptosis of osteocytes.

This study aims to investigate the role and mechanism of Gusong Qianggu Decoction (GSQG) in attenuating bone loss in ovariectomized mice by targeting the endoplasmic reticulum stress (ERS) -induced apoptosis of osteocytes. After the modeling of osteoporosis in mice with bilateral ovary removal (OVX), 60 mice were randomized by the random number method into six groups: sham, model, low-, medium-, and high-dose GSQG (GSQG-L, GSQG-M, and GSQG-H, respectively), and estradiol (E₂), with 10 mice in each group. The mice in each group were administrated with corresponding drugs by gavage one month after surgery and the administration lasted for 3 months. Enzyme-linked immunosorbent assay (ELISA) was employed to determine the serum levels of osteocalcin (OCN), procollagen type Ⅰ N-terminal propeptide (PINP), carboxy-terminal cross-linked telopeptide of type Ⅰ collagen (CTX), and anti-tartarte acid phosphatase 5b (TRAcP-5b). Micro-CT was employed to observe the changes in bone microstructure of the distal femur. Hematoxylin-eosin (HE) staining was employed to observe the morphology of the bone tissue. RT-qPCR was conducted to determine the m RNA levels of tibial stem osteogenesis-associated genes type Ⅰ collagen (Col-Ⅰ), alkaline phosphatase (ALP), Runtrelated transcription factor-2 (Runx2), bone sialoprotein (BSP), and OCN and bone-breaking related genes tartrate-resistant acid phosphatase (TRAP), nuclear factor-activated T cell 1 (NFATc1), and cathepsin K (CATK). TUNEL staining and immunohistochemistry were employed to detect the apoptosis of osteoblasts. Western blot was employed to measure the expression of ERS-related proteins glucose-regulated protein 78 (Grp78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), phosphorylated PERK (p-PERK), eukaryotic translation initiation factor 2 alpha (eIF2α), phosphorylated e IF2α (p-eIF2α), inositol-requiring enzyme 1 alpha (IRE1α), phosphorylated IRE1α (p-IRE1α), and activating transcription factor 6 (ATF6) in the proximal tibial bone tissue. The results showed that GSQG significantly recovered the levels of OCN, PINP, TRAc P-5b, and CTX in the serum of ovariectomized mice, and Micro-CT showed that GSQG improved the bone microstructure of distal femur in a dose-dependent manner. Compared with the model group, GSQG widened and increased the bone trabeculae, restored the reticular structure with neat arrangement and enlarged interstitial gaps, and reduced the number of TUNEL-positive cells (P<0. 05, P<0. 01). Furthermore, GSQG down-regulated the expression levels of cysteine aspartate protease-3 (caspase-3) and factor Bcl-2-associated X protein (Bax) (P< 0. 05, P<0. 01) and up-regulated the expression level of Bcl-2 (P<0. 05, P<0. 01). The GSQG groups showed up-regulated m RNA levels of Col-Ⅰ, ALP, Runx2, BSP, and OCN (P< 0. 01) and down-regulated m RNA levels of TRAP, NFATc1, and CATK (P< 0. 05, P<0. 01). In addition, GSQG, especially GSQG-H, down-regulated the protein levels of Grp78, p-PERK, p-eIF2, p-IRE1α, and ATF6 (P< 0. 05, P< 0. 01). In conclusion, GSQG can inhibit the apoptosis of osteocytes by inhibiting the Grp78/PERK/e IF2α/IRE1α/ATF6 signaling pathway in the proximal tibia tissue, thus reducing bone loss in ovariectomized mice.