2.7. Experimental Methods

After 1 week of adaptive feeding, rats underwent bilateral ovarian resection under anesthesia (3% pentobarbital sodium 30 mg/kg intraperitoneal injection) to create postmenopausal osteoporosis (PMOP) model. On the 7th day after the operation, the rats began to administer the medicine by gavage daily. The sham operation group and the model group were intragastrically administered with 10 mL/kg of normal saline. The rats in the LDD group were gavaged with LDD 6.75 g crude drug/kg. The rats in the positive control group were gavaged with alendronate sodium 0.1 mg/mL.

Under anesthesia with 3% pentobarbital sodium 30 mg/kg, blood was collected from the abdominal aorta. After the blood was placed at room temperature for 1 hour, the blood was centrifuged at 3 500 r/min for 15 minutes. The serum was separated and stored in a refrigerator at −80°C. The right femur of the rat was stored at −20°C for bone density detection and Quantitative Real-time PCR (qRT-PCR). The left femoral head of the rat was fixed with 4% paraformaldehyde for HE staining.

Serum E2 and ALP were detected by ELISA. BMD was measured by a dual-energy X-ray bone densitometer.

The left femoral head was fixed with a volume fraction of 4% paraformaldehyde for 72 h and then decalcified with 10% EDTA decalcification solution, and the decalcification solution was changed every 3 days. After decalcification was completed, it was rinsed with PBS, dehydrated with gradient ethanol, cleared with xylene, embedded in paraffin, and sliced for HE staining. The histopathological changes in the femoral head were observed under a light microscope.

Blood RNA was extracted and reverse-transcribed into cDNA for qRT-PCR detection. Reaction system was as follows: PCR forward primer (2 μM) 2 μL; PCR reverse primer (2 μM) 2 μL; 2X All-in-One qPCR Mix 10 μL; Template 2 μL; 50X Rox Reference Dye 0.4 μL; ddH20 3.6 μL; total 20 μL. The reaction conditions were set as follows: predenaturation: 95°C 10 min, 1 cycle; amplification reaction: 95°C 10 S, 62°C 20 s, 72°C 15 S, 45 cycles; draw melting curve: 95°C 10 S, 25°C 30 s. Each sample was repeated 3 times. For data analysis, the 2^−ΔCt method was used to calculate the difference in Wnt3a and β-catenin gene mRNA transcription levels. The gene sequence was searched in the NCBI database, and primers were designed, as shown in Table 2.

Primers.