ObjectiveTo explore the mechanism of Tibetan medicine Ershiwuwei Guijiuwan against osteoporosis in ovariectomized rats through the classical Wnt/β-catenin signaling pathway. MethodForty-eight 3-month-old SD female rats were randomized into model group （equivalent volume of normal saline）， sham operation group （equivalent volume of normal saline）， estradiol group （0.1 mg·kg^-1 estradiol valerate）， and high-， medium-， low-dose Ershiwuwei Guijiuwan groups （800， 400， 200 mg·kg^-1， respectively）. For the modeling， some adipose tissue near the ovaries was removed in the sham operation group， and ovaries were excised in other groups. Administration （ig， once a day） started two weeks after the operation and lasted 12 weeks. After sampling， based on hematoxylin and eosin （HE） staining， the morphological changes of the right femur and lumbar spine of the rats were observed. The enzyme-linked immunosorbent assay （ELISA） was performed to detect the serum levels of rat tartrate-resistant acid phosphatase 5b （TRAP5b）， collagen type Ⅰ C-terminal peptide （CTX-Ⅰ）， bone alkaline phosphatase （BALP）， amino-terminal propeptide of type Ⅰ procollagen （PINP）， and bone Gla-protein （BGP）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was performed to examine the mRNA expression of β-catenin and Runt-related transcription factor 2 （Runx2） in femur， and Western blot to detect the protein expression of β-catenin， Runx2， and low-density lipoprotein receptor-related protein-5 （Lrp-5）. ResultCompared with the sham operation group， the model group showed disordered and sparse bone trabecula with fewer connections， decrease in serum levels of BALP， BGP， and PINP （P<0.01）， increase in levels of CTX-Ⅰ and TRAP5b （P<0.01）， reduction in mRNA expression of β-catenin and Runx2 in femoral tissue （P<0.01） and protein expression of Lrp-5， β-catenin， and Runx2 （P<0.01）. Compared with the model group， estradiol and each dose of Ershiwuwei Guijiuwan increased the volume of bone trabecula， made thebone trabecula closely arranged， reduced the loss of the trabecular meshwork， raised the serum levels of BALP and BGP （P<0.01）， and lowered TRAP5b level （P<0.01）. PINP level was significantly increased in the high-dose Ershiwuwei Guijiuwan group and estradiol group （P<0.01） and CTX-Ⅰ level was significantly decreased in the high-dose Erwenwuwei Guijiuwan group and the estradiol group （P<0.01） compared with those in the model group. The mRNA expression of β-catenin in femoral tissue and protein expression of Lrp-5 and β-catenin in estradiol group and three Ershiwuwei Guijiuwan groups were significantly increased （P<0.05， P<0.01）， and the levels of Runx2 mRNA and protein were significantly increased in the high-dose Ershiwuwei Guijiuwan group and the estradiol group （P<0.01） compared with those in the model group. ConclusionTibetan medicine Ershiwuwei Guijiuwan is effective for the osteoporosis in ovariectomized rats， which may be related to the classic Wnt/β-catenin signaling pathway. It affects bone metabolism by up-regulating the expression of osteogenesis-related genes in the signaling pathway.

ObjectiveTo investigate the mechanism of Ershiwuwei Guijiu pill in preventing and treating postmenopausal osteoporosis （PMOP） by activating the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/mammalian target of rapamycin （mTOR） signaling pathway and inhibiting excessive autophagy. MethodFemale SD rats were ovariectomized and randomly divided into the sham operation group （Sham）， the operation group （OVX）， the Ershiwuwei Guijiu pill （GJ） group， and the raloxifene hydrochloride （RLX） group， with 10 rats in each group. Enzyme-linked immunosorbent assay （ELISA） and colorimetric methods were used to detect the levels of estrogen， bone metabolism markers in serum， and total superoxide dismutase （T-SOD）， malondialdehyde （MDA）， and glutathione peroxidase （GSH-Px） in tibial tissue. Flow cytometry was used to detect reactive oxygen species （ROS） levels in bone marrow mesenchymal stem cells. Masson staining was used to observe pathological changes in the proximal tibia， and micro-computed tomography （Micro-CT） was used to observe changes in tibial microstructural parameters. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were used to detect the expression of autophagy-related proteins Beclin1， microtubule-associated protein 1A/1B-light chain 3 （LC3）， autophagy-related 5 （Atg5）， as well as PI3K， Akt， and mTOR in tibial tissue. ResultCompared with the Sham group， the OVX group showed a significant decrease in serum levels of estradiol （E₂） and calcium ion （Ca²⁺）， and T-SOD， GSH-Px， PI3K， Akt， and mTOR mRNA levels in bone tissue （P<0.05， P<0.01）， significantly reduced bone mineral density （BMD）， bone surface/bone volume （BS/BV）， trabecular thickness （Tb.Th）， trabecular number （Tb.N）， and trabecular connectivity （Con） in the tibia （P<0.05， P<0.01）， thinner epiphyseal growth plate， and the bone marrow cavity filled with fat vacuoles. Moreover， the levels of phosphorus （P）， MDA， ROS， and mRNA and protein expression of Beclin1， LC3， and Atg5， as well as trabecular separation （Tb.Sp） were significantly elevated （P<0.05， P<0.01）. Compared with the OVX group， the GJ and RLX groups showed significant increases in serum E₂ and Ca²⁺， and bone tissue levels of SOD， GSH-Px， and the mRNA levels of PI3K， Akt， and mTOR （P<0.05， P<0.01）， significantly increased BMD， BS/BV， Tb.Th， Tb.N， and Con in the tibia， thickened epiphyseal growth plate， and significantly reduced fat vacuoles in the bone marrow cavity （P<0.05， P<0.01）. Additionally， the levels of P， MDA， ROS， Beclin1， LC3， Atg5 mRNA and proteins， and Tb.Sp were significantly decreased （P<0.05， P<0.01）. The ratios of p-PI3K/PI3K， p-Akt/Akt， and p-mTOR/mTOR， which were significantly reduced in the OVX group （P<0.01）， were significantly increased in the GJ and RLX groups （P<0.01）. ConclusionThe Ershiwuwei Guijiu pill reduces oxidative stress and inhibits autophagy， thereby preventing and treating postmenopausal osteoporosis. Its mechanism may be related to the activation of the PI3K/Akt/mTOR signaling pathway， which inhibits autophagy.