Pharmaceutical composition and application thereof in preparation of medicine for treating postmenopausal osteoporosis

Abstract

The invention discloses the application of the Chinese wolfberry fruit-glossy privet fruit and calcitriol composition in the medicine for treating postmenopausal osteoporosis for the first time, and through three-layer research of'in vitro cell-animal model-molecular pathway ', it is clear that the composition can bidirectionally regulate and control kidney vitamin D metabolic enzyme by activating'PI3K-AKT' signal pathway for the first time, so that the curative effect on postmenopausal osteoporosis is achieved. Further, the serum 25 hydroxyl vitamin D and blood calcium level are improved, and the bone mineral density is improved. The mechanism provides a modern molecular biology support for the traditional Chinese medicine theory of'kidney dominating bone ', and also establishes a new path of'regulating kidney metabolic enzyme to treat osteoporosis'. By optimizing the metabolic bone protection effect of the vitamin D, bone loss of ovariectomized mice is effectively delayed, meanwhile, the vitamin D and calcitriol play a synergistic effect, and the vitamin D has a multi-target bone protection effect in postmenopausal PMOP, has anti-osteoporosis activity and can be used for treating the PMOP.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, specifically relating to a pharmaceutical composition and its application in the preparation of a drug for treating postmenopausal osteoporosis. Background Technology

[0002] Postmenopausal osteoporosis (PMOP) is a systemic bone metabolic disease closely related to age and a sharp decline in estrogen levels, and is a common type of primary osteoporosis. Its core pathological mechanism lies in the decline of ovarian function after menopause, leading to a sharp drop in endogenous estrogen levels (especially 17β-estradiol). This weakens the protective effect of estrogen on bones, resulting in excessive activation of osteoclast activity and differentiation, while osteoblast function is relatively insufficient. Consequently, the rate of bone resorption far exceeds the rate of bone formation, disrupting the balance of bone remodeling and ultimately leading to a significant decrease in bone mineral density (BMD), weakened bone biomechanical strength, and a significantly increased risk of fractures. Currently, Western medicine clinical drug treatment mainly includes: promoting bone formation, inhibiting bone resorption, and basic treatment drugs (active vitamin D analogs, such as calcitriol). Calcitriol has some efficacy in clinical applications, but it has limitations: 1. It is difficult to effectively repair bone structure; 2. Calcitriol accelerates its own degradation by activating CYP24A1, reducing its efficacy; 3. It increases the risk of hypercalcemia. Osteogenesis-promoting drugs (such as teriparatide) are superior to anti-resorption drugs in improving bone mineral density and microstructure, but cost and side effects limit their clinical application and duration. Combination or sequential treatment strategies can enhance efficacy and reduce side effects; in particular, the synergistic effect of targeting CYP24A1 inhibition and VDR activation is significant and superior to monotherapy.

[0003] According to Traditional Chinese Medicine (TCM) theory, PMOP falls under the categories of "bone atrophy," "bone stagnation," and "bone arthralgia," following the TCM theory that "the kidney governs bones and produces marrow." Its core pathogenesis is kidney essence deficiency: the kidney is the foundation of innate constitution, governing bone and producing marrow. Sufficient kidney essence provides a source for bone marrow production, nourishing and strengthening the bones; deficiency of kidney essence leads to insufficient bone marrow and malnourishment of the bones, resulting in weak and fragile bones, manifesting as "bone atrophy." The liver and kidney share a common origin for essence and blood, mutually nourishing each other; prolonged kidney essence deficiency inevitably leads to insufficient liver blood. Therefore, the root cause of PMOP is "liver and kidney deficiency."

[0004] The "Goji Berry-Ligustrum lucidum" herbal combination primarily nourishes kidney yin, replenishes essence and blood. Goji berries, sweet and neutral in nature, enter the liver and kidney meridians, excelling at nourishing the liver and kidneys, and replenishing essence and blood. Ligustrum lucidum, sweet, bitter, and cool in nature, also enters the liver and kidney meridians, excelling at nourishing the liver and kidneys, clearing heat and improving eyesight. The two herbs work synergistically, nourishing water to nourish wood and tonifying the kidneys to nourish the liver, achieving the combined effect of tonifying the liver and kidneys and strengthening bones and muscles. Their active ingredients (such as oleanolic acid and goji berry polysaccharides) reduce bone resorption by inhibiting the NF-κB pathway, promote osteoblast differentiation by activating the Wnt / β-catenin and BMP / Smad signaling pathways, and regulate bone metabolism-related genes (RUNX2, OPG / RANKL), significantly increasing bone mineral density and improving bone microstructure in PMOP model rats, thus exerting an osteoprotective effect. In the prior art, there is no known technology for the use of the wolfberry-privet fruit composition in PMOP, nor is there any known application of the wolfberry-privet fruit and calcitriol drug composition in drugs for the treatment of postmenopausal osteoporosis. Summary of the Invention

[0005] The purpose of this invention is to provide a pharmaceutical composition and its application in the preparation of a drug for treating postmenopausal osteoporosis, so as to make up for the shortcomings of existing drugs for treating postmenopausal osteoporosis.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0007] A pharmaceutical composition comprising a traditional Chinese medicine component and calcitriol, wherein the traditional Chinese medicine component is composed of the following raw materials in parts by weight: 7-15 parts of Lycium barbarum and 6-12 parts of Ligustrum lucidum, and the mass ratio of the traditional Chinese medicine component to calcitriol is (2.6 × 10⁻⁶). 7 ~5.4×10 7 ):1.

[0008] Preferably, the traditional Chinese medicine components are made from the following raw materials in parts by weight: 10-15 parts of wolfberry and 9-12 parts of privet fruit.

[0009] Preferably, the traditional Chinese medicine components are made from the following raw materials in parts by weight: 10 parts of wolfberry and 9 parts of privet fruit.

[0010] The use of any of the above-described pharmaceutical compositions in the preparation of a drug for treating postmenopausal osteoporosis.

[0011] Preferably, the pharmaceutical composition exerts its therapeutic effect through the following means:

[0012] In HK-2 renal epithelial cells, the expression of vitamin D-degrading enzyme CYP24A1 was inhibited, while the expression of CYP27B1, VDR, and Klotho was upregulated, and the activity of vitamin D was reduced. Degradation and optimization of vitamin D metabolism;

[0013] Alternatively, it can enhance the alkaline phosphatase activity and mineralization capacity of osteoblast progenitor cells MC3T3-E1.

[0014] Alternatively, it can improve bone density and bone microstructure in the body after oophorectomy;

[0015] Alternatively, it can improve bone volume fraction, trabecular bone number, and bone strength in the body after oophorectomy.

[0016] Alternatively, it can help restore the body's vitamin D metabolic balance after oophorectomy.

[0017] Preferably, the medication for treating postmenopausal osteoporosis contains the pharmaceutical composition and one or more pharmaceutically acceptable excipients.

[0018] Preferably, the excipient is at least one of the following: excipient, sustained-release agent, filler, binder, wetting agent, disintegrant, absorption promoter, surfactant, antibacterial agent, fragrance, antioxidant, pH adjuster, protectant, diluent, lubricant, solvent, matrix or carrier material.

[0019] Preferably, the dosage form of the drug for treating postmenopausal osteoporosis is oral granules, tablets, pills, capsules, or oral liquid.

[0020] The present invention adopts the above technical solution, and its beneficial effects are as follows:

[0021] This invention discloses for the first time the application of a combination of Lycium barbarum, Ligustrum lucidum, and calcitriol in the treatment of PMOP. Through a three-tiered study involving in vitro cells, animal models, and molecular pathways, the core mechanism of the Lycium barbarum-Ligustrum lucidum combination in treating PMOP was clearly identified for the first time: this combination can effectively restore and regulate renal vitamin D metabolic enzymes (upregulating 1α-hydroxylase and downregulating 24-hydroxylase) by activating the PI3K-AKT signaling pathway, thereby increasing serum 25-hydroxyvitamin D and blood calcium levels, ultimately achieving increased bone mineral density (the lumbar spine bone mineral density in the intervention group mice increased by 16.4% compared to the model group). This mechanism not only provides modern molecular biological support for the traditional Chinese medicine theory of "the kidney governs the bones" but also establishes a new pathway for "regulating renal metabolic enzymes to treat osteoporosis," providing a theoretical basis for subsequent related research. The wolfberry-privet fruit composition of this invention effectively delays bone loss in ovariectomized mice by optimizing vitamin D metabolism and bone protection. It also works synergistically with calcitriol. Compared to using calcitriol or the wolfberry-privet fruit composition alone, it significantly improves the therapeutic effect. The combined use of calcitriol and the wolfberry-privet fruit composition exhibits a synergistic effect exceeding that of their individual components. It has multi-target bone protection in the prevention and treatment of PMOP, and possesses anti-osteoporosis activity, making it suitable for the treatment of osteoporosis, particularly PMOP. Attached Figure Description

[0022] Figure 1 The effect of serum containing different concentrations of Lycium barbarum-Ligustrum lucidum combination on the activity of HK-2 cells.

[0023] Figure 2 The effect of drug-containing serum on alkaline phosphatase activity in MC3T3-E1 cells.

[0024] Figure 3 The effect of drug-containing serum on the mineralization capacity of MC3T3-E1 cells.

[0025] Figure 4 The effect of drug-containing serum on the expression of CYP24A1, CYP27B1, VDR and Klotho genes in HK-2 cells was investigated.

[0026] Figure 5 The effect of drug-containing serum on the expression of CYP24A1, CYP27B1, VDR and Klotho proteins in HK-2 cells was investigated.

[0027] Figure 6 HE staining results of bone tissue from ovariectomized osteoporotic mice.

[0028] Figure 7 Micro-CT bone microstructure of the L4-L5 lumbar vertebrae in ovariectomized osteoporotic mice.

[0029] Figure 8 Micro-CT analysis of bone microstructure parameters in the L4-L5 lumbar vertebrae of ovariectomized osteoporotic mice.

[0030] Figure 9 Analysis of the three-point bending mechanical properties of the right femur in ovariectomized osteoporotic mice.

[0031] Figure 10 PCR analysis of CYP24A1, CYP27B1, VDR and Klotho gene expression in the kidneys of ovariectomized osteoporotic mice.

[0032] Figure 11 Western blot analysis of CYP24A1, CYP27B1, VDR and Klotho protein expression in the kidneys of ovariectomized osteoporotic mice. Detailed Implementation

[0033] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other. The described embodiments are merely some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention. The terminology used herein is for the purpose of describing specific embodiments only and is not restrictive, as the scope of this invention is limited only by the appended claims and their equivalents. The technical solutions of this invention will be further described below with reference to the accompanying drawings of the embodiments of this invention. This invention is not limited to the following specific embodiments.

[0034] The following analysis, using drug composition intervention combined with in vitro cell culture, animal models, and molecular pathways, examines the osteoprotective effects and molecular mechanisms of the drug composition in a PMOP mouse model. This provides experimental evidence for the precise intervention of PMOP by the Lycium barbarum-Ligustrum lucidum drug pair based on the "kidney governs bone" theory, and provides experimental evidence for the synergistic effect of the Lycium barbarum-Ligustrum lucidum composition and calcitriol, demonstrating multi-target osteoprotective effects and anti-osteoporosis activity in the prevention and treatment of PMOP.

[0035] Experiment Example 1: In vitro cell experiments

[0036] 1.1 Materials and Methods

[0037] 1.1.1 Cell Culture and Treatment

[0038] The experiment used mouse osteoblast precursor cells MC3T3-E1 (catalog number: SCSP-5218, Chinese Academy of Sciences Type Culture Collection Cell Bank) and human kidney epithelial cells HK-2 (catalog number: ZQ0313, Zhong Qiao Xinzhou). Four treatment conditions were set up: saline-containing serum group (NG), wolfberry-privet fruit drug pair group (LLG), whole calamine group (CG), and wolfberry-privet fruit drug pair combined with whole calamine group (LLCG).

[0039] 1.1.2 Preparation of drug-containing serum

[0040] Forty SPF-grade male SD rats, weighing approximately 200g, were randomly selected. The clinically equivalent dose was calculated based on the "Equivalent Dose Ratio Table for Humans and Rats Based on Body Surface Area Conversion." Rats were divided into four groups: saline group, herb pair group, whole herb group (containing calcitriol), and herb pair combined with whole herb group (n=10 per group). Each rat was administered herb via gavage twice daily: 1. Saline group (NG): Saline administered via gavage; 2. Ligustrum lucidum-Lycium barbarum herb pair group (LLG): Ligustrum lucidum-Lycium barbarum herb pair aqueous extract administered via gavage; 3. Whole herb group (CG): Calcitriol administered via gavage; 4. Combined group (LLCG): Herb pair + calcitriol. Specifically, the dosage of the herbal medicine for rats (200g) and humans (70kg) was converted according to "Experimental Animal Pharmacology" (coefficient of 9.1, combined with mouse-to-rat body surface area conversion), determining the dosage of Ligustrum lucidum and Lycium barbarum decoction to be 0.136g / kg, with a gavage dose of 5mL / kg. The total dose of roxarsone for rats was 0.5 μg / kg, with roxarsone completely dissolved in corn oil. When administered via gavage in combination with a decoction of privet fruit and wolfberry, the gavage volume was 5 mL / kg, with roxarsone completely dissolved in the decoction. 2 mL was administered per dose for 3 consecutive days. Blood was drawn within 2 hours of the last gavage, centrifuged, and the supernatant serum was collected, inactivated, and stored at -80°C for later use.

[0041] 1.1.3 Cell Culture

[0042] Cell resuscitation: MC3T3-E1 and HK-2 cells were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. They were then seeded into α-MEM (MC3T3-E1) or DMEM / F12 (HK-2) medium containing 10% FBS and cultured in a 37°C, 5% CO2 incubator. When cell confluence reached 80-90%, the cells were digested with 0.25% trypsin and passaged to passages 3-5 for experimental use. The HK-2 cell density was adjusted to... HK-2 cells were seeded at a density of 100 μL / well in 96-well or 2 mL / well in 6-well plates for subsequent experiments. Drug-containing serum (NG, LLG, CG, and LLCG groups) was added to DMEM / F12 medium at a 25% (v / v) ratio. After 24 hours of seeding, the original medium was discarded, and drug-containing serum medium was added (100 μL / well for 96-well plates and 2 mL / well for 6-well plates). Treatment time for each group was 48 hours, with a control group included.

[0043] 1.1.4 Cell proliferation detection (CCK-8 assay)

[0044] After treatment, discard the culture medium and add 100 μL of DMEM / F12 medium containing 10% CCK-8 reagent to each well. Incubate at 37°C for 2 hours, and measure the absorbance (OD value) at 450 nm using a microplate reader. Calculate cell viability: Viability = (OD experimental group - OD blank) / (OD control - OD blank) × 100%.

[0045] 1.1.5 Osteogenic Induction Differentiation

[0046] Logarithmic growth phase MC3T3-E1 cells were seeded into 24-well plates (500 μL of basal medium per well) and cultured in a 37°C, 5% CO2 incubator. After 24 hours, when the cell confluence reached 80%, the original medium was discarded, and preheated osteogenic induction medium (500 μL per well) was slowly added along the well wall. Fresh induction medium was then added every 2-3 days thereafter, and the cells were cultured until the target time point (ALP staining detection at 7 days of induction, Alizarin Red staining detection at 14 days of induction). During this period, cell morphological changes were observed regularly (osteogenic differentiation characteristics: cells become rounder, cytoplasmic refractive index increases, and brown deposits appear in the matrix mineralization area).

[0047] 1.1.6 Alkaline phosphatase (ALP) staining and alizarin red staining identification

[0048] After 7 days of induction culture, the culture medium was aspirated, and the cells were gently washed twice with PBS. 500 μL of 4% paraformaldehyde fixative was added to each well and the cells were fixed at room temperature for 15 min. After washing three times with PBS, 500 μL of BCIP / NBT chromogenic working solution (containing 0.1% BCIP and 0.1% NBT) was added to each well to cover the cell layer and incubated at 37°C in the dark for 30-60 min (until a blue-purple precipitate was visible). The staining solution was aspirated, and the reaction was terminated by washing three times with PBS. An appropriate amount of PBS was added for hydration, and the cells were observed and photographed under a microscope. ALP-positive areas showed blue-purple granular deposits.

[0049] After 14 days of induction culture, the culture medium was discarded, and the wells were gently washed twice with PBS. 4% paraformaldehyde fixative (500 μL per well) was added and fixed at room temperature for 15 min. After washing three times with PBS, 0.5% Alizarin Red S staining solution (pH 4.2, 500 μL per well) was added and stained at room temperature for 30 min. The staining solution was discarded, and the wells were gently washed 3-5 times with deionized water (avoiding the pipette tip from touching the bottom of the well; wash to remove non-specific precipitates). A suitable amount of PBS was added to cover the bottom of the wells, and the wells were observed and photographed under a microscope. Calcium nodule-positive areas appeared as deep orange-red mineralized patches.

[0050] 1.1.7 Quantitative Reverse Transcription PCR

[0051] Total RNA was extracted from tissues using the EZB RNA Extraction Kit (catalog number B0004DP or EZB-RN4, EZBioscience), and RNA content was determined using a micro-volume UV-Vis spectrophotometer (Denovix, USA, model: DS-1). 1 μg of RNA was reverse transcribed into cDNA using a first-strand cDNA synthesis kit (Shanghai Beyotime Biotechnology Co., Ltd.). Amplification was performed using a Bio-ad quantitative PCR instrument (Bio-Rad Biomedical, UK, model: CFX Connect) with the following cycling conditions: 95℃ for 30 seconds, 95℃ for 5 seconds, and 60℃ for 34 seconds, for a total of 40 cycles. GAPDH was used as the reference gene for human cell mRNA, and Gapdh was used as the reference gene for mouse cell mRNA. The primer sequences used in this study are as follows:

[0052] Table 1 Primer sequences of human genes

[0053]

[0054] 1.1.8 Western blot for protein immunoblotting

[0055] Total protein was extracted from cells using RIPA lysis buffer (Catalog No.: P0013C, Beyotime), and protein concentration was determined using a BCA protein quantification kit (Catalog No.: P0011, Beyotime). Protein samples were separated by 10% SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked in QuickBlock™ protein-free Western blocking buffer (Catalog No.: P0240, Beyotime) at room temperature for 25 minutes, followed by overnight incubation at 4°C with the following primary antibodies: rabbit anti-CYP24A1 antibody (Catalog No.: HA500196, Huaan Biotech, 1:5000), rabbit anti-CYP27B1 antibody (Catalog No.: ER1907-01, Huaan Biotech, 1:5000), and rabbit anti-Klotho antibody (Catalog No.: ET170). 5-88, Huaan Biotechnology, 1:5000), rabbit anti-VDR antibody (catalog number ET1704-09, Huaan Biotechnology, 1:5000) and mouse anti-β-actin antibody (catalog number AA128, Beyotime, 1:1000); then, the membrane was thoroughly washed with TBST and incubated at room temperature for 1 hour with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (catalog number: A0208, Beyotime, 1:1000) and goat anti-mouse secondary antibody (catalog number A0216_, Beyotime, 1:1000); finally, the membrane was developed using an ECL chemiluminescence kit (Thermo Fisher Scientific, USA) and the protein band grayscale values ​​were analyzed using ImageJ software (NIH, USA).

[0056] 1.1.9 Statistical Analysis

[0057] All data are expressed as mean ± standard deviation (Mean ± SD) and analyzed using R4.5.1 software. One-way ANOVA was used for comparisons among multiple groups, and Tukey's post-hoc test was used to determine differences between groups. Significance criteria: *P < 0.05, **P < 0.01, ***P < 0.001. Graphs were created using GraphPad Prism 10.1.2.

[0058] 1.2 Results

[0059] 1.2.1 Effects of Lycium barbarum-Ligustrum lucidum herb pair on the activities of 1α-hydroxylase and 24-hydroxylase in human proximal renal tubular epithelial cells by serum containing the herb pair.

[0060] The results of the CCK-8 experiment show (see...) Figure 1 In serum containing 25% Lycium barbarum-Ligustrum lucidum (LLG) drug pair, the survival rate of human proximal renal tubular epithelial cells (HK-2) reached 1.17±0.06%, significantly higher than 1% (0.64±0.06), 5% (0.69±0.04), 10% (0.86±0.04), 30% (0.96±0.06), and 35% (0.86±0.04) (P<0.05), but not significantly different from 15% (0.99±0.06) and 20% (1.07±0.09) (P>0.05). This indicates that LLG at a 25% concentration has the best cell viability and biocompatibility. Future studies will use a 25% concentration for intervention.

[0061] 1.2.2 Effects of drug-containing serum on osteogenic differentiation of MC3T3-E1 cells

[0062] ALP and alizarin red staining results showed (see...) Figure 2 and Figure 3 LLG and CG significantly enhanced the alkaline phosphatase activity and mineralization capacity of MC3T3-E1 cells, indicating that LLG directly improves bone formation capacity by promoting osteoblast differentiation and matrix mineralization. LLG enhanced the alkaline phosphatase activity and mineralization capacity of MC3T3-E1 cells better than LLG and CG alone, indicating that the superiority of the combined LLG and calcitriol therapy stems from the synergistic effect of the drug pair with calcitriol, which enhances osteoblast activity. This cellular-level osteoprotective effect provides direct evidence for the potential application of the drug pair in postmenopausal osteoporosis (PMOP).

[0063] 1.2.3 Expression of CYP24A1 and other vitamin D metabolism-related genes in HK-2 cells

[0064] PCR analysis showed (see) Figure 4LLG was slightly superior to CG in terms of cyp27b1 and VDR expression in HK-2 cells (P<0.05), suggesting that the drug pair optimizes vitamin D metabolic homeostasis by enhancing 1,25-(OH)2D3 synthesis and VDR signaling efficiency. LLG significantly downregulated CYP24A1 expression in HK-2 cells while upregulating cyp27b1, VDR and klotho expression (P<0.05), which was superior to LLG and CG, showing the best effect in improving vitamin D metabolism. The synergistic effect of LLG combined with CG treatment indicates that LLG reduces vitamin D degradation by inhibiting CYP24A1 and enhances VD receptor function together with calcitriol.

[0065] 1.2.4 Expression of CYP24A1 and other vitamin D metabolism-related proteins in HK-2 cells

[0066] Western blot results (see...) Figure 5 The LLG and CG groups showed superior expression of Klotho (P<0.05 vs. NG), CYP27B1 (P<0.05 vs. NG), and VDR (P<0.001 vs. NG) compared to the NG group. However, CYP24A1 expression was significantly lower in the LLG group than in the NG group (P<0.01), while there was no significant difference in the CG group (P>0.05 vs. NG). This indicates that the drug pair inhibits CYP24A1, activates the Klotho-VDR axis, prolongs the action time of 1,25-(OH)2D3, and enhances signal transduction efficiency. The LLG group showed the lowest CYP24A1 expression in cells (P<0.001 vs. NG, P<0.01 vs. LLG / CG), while Klotho, CYP27B1, and VDR expression were the highest (P<0.05 vs. NG; CYP27B1, P<0.01 vs. NG; VDR, P<0.001 vs. NG). The results showed that the combined treatment of LLG and CG was superior to that of the LLG and CG groups, indicating that the combined treatment of LLG and CG effectively enhanced the balance of vitamin D metabolism. The superior performance of the LLG group (VDR P<0.05 vs. CG; CYP24A1 P<0.01 vs. LLG, <0.01 vs. CG) further verified its synergistic role in cellular regulation.

[0067] Experiment Example 2: In vivo animal experiments

[0068] 2.1 Materials and Methods

[0069] 2.1.1 Construction of experimental animals and osteoporosis models

[0070] SPF-grade female C57BL / 6J mice at 12 weeks of age (purchased from Vital River Laboratories Co., Ltd., license number: SCXK (Zhe) 2021-0006), weighing 20±2 g, were housed in an environment with a constant temperature of (22±1)°C, humidity of 50±10%, and a 12-hour light-dark cycle. They had free access to food and water. The animal ethics number was: PZSHUTCM250523003 (Shanghai University of Traditional Chinese Medicine). The postmenopausal osteoporosis model was established by bilateral ovariectomy (OVX): After the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (40 mg / kg), the ovaries were exposed through a dorsal incision, and the ovarian tissue was completely removed; in the sham operation group, only an equal volume of adipose tissue adjacent to the ovaries was removed. Penicillin (50,000 U / kg) was injected subcutaneously for 3 consecutive days after the operation to prevent infection.

[0071] Grouping and drug intervention

[0072] Fifty successfully modeled mice were randomly divided into 5 groups (n = 10): 1. Sham operation group (SHAM): gavaged with normal saline (0.1 mL / 10 g / d); 2. Model group (OVX): OVX surgery + gavaged with normal saline (0.1 mL / 10 g / d); 3. Lycii fructus-Cornus officinalis pair group (LLG): OVX surgery + gavaged with the water extract of Lycii fructus-Cornus officinalis pair (0.1 mL / 10 g / d); 4. Rocaltrol group (CG): OVX surgery + gavaged with calcitriol (1 μg / kg / d, Rocaltrol dissolved in corn oil); 5. Combined group (LLCG): OVX surgery + pair + calcitriol (0.1 mL / 10 g / d + 1 μg / kg / d, Rocaltrol dissolved in the water decoction).

[0073] According to the Chinese Pharmacopoeia (2015 edition), the conventional doses of the Chinese medicinal materials in the Lycii fructus-Cornus officinalis composition are 5-15 g / day and 6-12 g / day. Taking the common dosage, 10 g of Lycii fructus and 9 g of Cornus officinalis were used to determine the gavage dose of the Lycii fructus-Cornus officinalis pair for mice. Specifically, the dosage of the Chinese medicinal composition in mice (20 g) and humans (70 kg) was converted according to "Experimental Animal Pharmacology" (coefficient 9.1). The dose of the water decoction of the Lycii fructus-Cornus officinalis composition was determined to be 0.2715 g / kg, and the gavage dose was 0.1 mL / 10 g.

[0074] Micro-CT scanning (lumbar vertebrae L4-L5)

[0075] Lumbar spine samples were fixed in 4% paraformaldehyde for 24 hours, washed for 2 hours, and then tested. Six L4 vertebrae were selected from each group, and a 3D model excluding accessory structures such as transverse processes and spinous processes was constructed. Analysis was performed using Micro-CT (Scanco Medical AG, Switzerland, model: vivaCT40) to directly acquire parameters such as bone mineral density (BMD), bone volume fraction (BV / TV), number of trabeculae (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp).

[0076] 2.1.4 Three-point bending test (right femur)

[0077] The right femur was immersed in saline solution, and its bone mechanical properties were evaluated using a three-point bending test within 48 hours. The tibia was placed on two support points with a span of 5.71 mm, and a load was applied to the mid-shaft of the femur at a constant displacement rate of 1 mm / min until fracture. The maximum load and stiffness parameters were extracted from the resulting load-displacement curves.

[0078] 2.1.5 HE staining of bone tissue

[0079] Tibiae (or other designated bones) were fixed in 4% paraformaldehyde for 24 hours, then decalcified and dehydrated in EDTA solution, embedded in paraffin, and cut into 5-micrometer thick sections. The sections were stained with hematoxylin and eosin (H&E) for routine histological assessment of bone structure, cell distribution, and bone marrow composition.

[0080] 2.1.6 Quantitative Reverse Transcription PCR

[0081] Total RNA was extracted from mouse kidneys using the EZB RNA Extraction Kit (Catalog No. EZB-RN4, EZBioscience), and RNA content was determined using a micro-volume UV-Vis spectrophotometer (Denovix, USA, Model: DS-1). 1 μg of RNA was reverse transcribed into cDNA using a first-strand cDNA synthesis kit (Shanghai Beyotime Biotechnology Co., Ltd.). Amplification was performed using a Bio-ad quantitative PCR instrument (Bio-Ad Biomedical, UK, Model: CFX Connect) with the following cycling conditions: 95℃ for 30 seconds, 95℃ for 5 seconds, and 60℃ for 34 seconds, for a total of 40 cycles. Gapdh was used as the reference gene for mouse mRNA. The primer sequences used in this study are as follows:

[0082] Table 2 Primer sequences of mouse-derived genes

[0083]

[0084] 2.1.7 Western blot for protein immunoblotting

[0085] Add two grinding beads to each EP tube and prepare a lysis buffer mixture (phosphatase inhibitor: protease inhibitor: lysis buffer = 1:1:100). Pre-cool the grinder and centrifuge beforehand. Take the tissue out of the -80°C freezer and operate on ice. Cut out sesame seed-sized pieces of renal tissue with a blade and place them in the EP tube. Place the tube on ice and add 220 μL of lysis buffer to each EP tube. Pre-cool the grinder beforehand. After pre-cooling, select the corresponding tissue grinding program on the circulation interface. For example, for kidneys, select 70 Hz, run for 15 seconds, stop for 30 seconds, and let stand for 15 minutes. After standing, centrifuge at 12,000-14,000 rpm for 15 minutes at 4°C and collect the supernatant into another labeled new EP tube. The remaining steps and antibody are the same as in 1.1.8.

[0086] 2.1.8 Statistical Analysis

[0087] All data are expressed as mean ± standard deviation (Mean ± SD) and analyzed using R4.5.1 software. One-way ANOVA was used for comparisons among multiple groups, and Tukey's post-hoc test was used to determine differences between groups. Significance criteria: *P < 0.05, **P < 0.01, ***P < 0.001. Graphs were created using GraphPad Prism 10.1.2.

[0088] 2.2 Results

[0089] 2.2.1 HE staining of tibia in ovariectomized osteoporotic mice

[0090] HE staining showed (see) Figure 6 Ovariectomy (OVX) significantly disrupted bone microstructure in mice, validating the successful establishment of the PMOP model. Both LLG and CG improved bone microstructure, with LLG showing the most significant effect, approaching the level of the sham-operated group. This indicates that LLG enhances bone strength by repairing trabecular bone structure, and the combination of LLG and calcitriol further amplifies this effect.

[0091] 2.2.2 Micro-CT analysis of the L4-L5 lumbar spine microstructure in ovariectomized osteoporotic mice

[0092] Table 3 Micro-CT bone parameter analysis of lumbar spine L4-L5

[0093] Group Bone volume fraction VOX-BV / TV Connectivity density (Conn-Den). Trabecular bone number DT-Tb.N Trabecular thickness DT-Tb.Th Trabecular separation DT-Tb.Sp Mean 1 (mg HA / ccm) of average bone mineral density SHAM (Sham Surgery Group) 0.3306 ± 0.0396 134.49 ± 28.57 4.8523 ± 0.4946 0.0647 ± 0.0048 0.1973 ± 0.0246 207.83 ± 28.24 OVX (model group) 0.2065 ± 0.0253 138.27 ± 21.84 4.2488 ± 0.3043 0.0523 ± 0.0035 0.2336 ± 0.0190 113.87 ± 15.82 LLG (Drug Pair Group) 0.2398 ± 0.0118 133.69 ± 8.79 4.3616 ± 0.1657 0.0553 ± 0.0020 0.2249 ± 0.0088 139.85 ± 10.22 CG (Roggai Group) 0.2772 ± 0.0179 135.37 ± 14.05 4.7113 ± 0.1874 0.0575 ± 0.0028 0.2035 ± 0.0098 162.91 ± 12.39 LLCG (Joint Group) 0.3356 ± 0.0358 144.83 ± 37.05 4.9073 ± 0.5995 0.0645 ± 0.0040 0.1963 ± 0.0237 209.63 ± 29.46

[0094] Micro-CT three-dimensional bone microstructure and parameter analysis showed (see Table 3) Figure 7 and Figure 8The improvement in the LLG group was better than that in the model group (BV / TV P=0.021, Mean1 P<0.01) but lower than that in the CG group, showing a certain bone-protective effect. The LLG group performed best in bone volume fraction (BV / TV, P=0.816 vs. SHAM), number of trabeculae (P=0.862 vs. SG), trabecular thickness (0.0645 ± 0.0040, P=0.940 vs. SHAM), and bone mineral density (209.63 ± 29.46 mg HA / ccm, P=0.915 vs. SHAM), approaching or exceeding that in the sham surgery group, and was significantly higher than that in the CG group (BV / TV, P<0.05; Mean1, P<0.05), indicating that the synergistic effect of combined LLG treatment was significantly better than that of single intervention (P<0.05 vs. CG). This improvement in bone microstructure and density suggests that the Lycium barbarum-Ligustrum lucidum drug pair overcomes the limitations of monotherapy by enhancing the osteoprotective effect of calcitriol, providing a more effective strategy for the treatment of postmenopausal osteoporosis.

[0095] 2.2.3 Mechanical properties of the right femur in ovariectomized osteoporotic mice under three-point bending test

[0096] Table 4. Analysis of biomechanical parameters of the right femur at three points of bending.

[0097] index SHAM (Sham Surgery Group) OVX (model group) LLG (Drug Pair Group) CG (Roggai Group) LLCG (Joint Group) N (load, N) 14.1317 ± 0.6042 8.9267 ± 0.8181 11.1583 ± 0.4543 11.0167 ± 0.6685 14.0450 ± 0.3870 Displacement (mm) 0.3509 ± 0.0623 0.3765 ± 0.1801 0.2782 ± 0.0512 0.3977 ± 0.1611 0.6277 ± 0.3331 σ (stress, MPa) 70.7063 ± 6.5873 46.2133 ± 3.7282 58.6545 ± 3.1659 54.9110 ± 6.8967 70.3397 ± 2.8225 E (Young's modulus, GPa) 6719.3047 ± 565.3302 5229.6178 ± 2407.2963 6305.6609 ± 1383.3702 5602.8623 ± 2437.2672 5860.4341 ± 3562.5378 ε (strain) 0.0106 ± 0.0016 0.0114 ± 0.0061 0.0098 ± 0.0023 0.0119 ± 0.0057 0.0183 ± 0.0109

[0098] Three-point bending mechanical parameter analysis shows (see Table 4 and...) Figure 9 Of the treatments, the LLCG group showed the best biomechanical performance, approaching that of SHAM. Its load (P=0.891 vs. SHAM) and stress (P=0.928 vs. SHAM) were almost equivalent to SHAM, significantly superior to CG (P<0.05) and LLG (P<0.05), and significantly improved compared to OVX (P<0.01). LLCG's Young's modulus (5860.4341±3562.5378 GPa) was superior to OVX (P<0.05), with higher displacement and strain (P<0.05). OVX performed the worst, validating the influence of the disease model. The superior performance of LLCG demonstrates a synergistic effect, enhancing bone strength, surpassing single treatments, and approaching that of normal bone, indicating that LLCG reduces fracture risk by enhancing the osteoprotective effect of calcitriol.

[0099] 2.2.4 Reverse transcription quantitative PCR analysis of vitamin D metabolism-related genes in the kidneys of ovariectomized osteoporotic mice

[0100] Animal experiment PCR results showed (see) Figure 10Both the LLG and CG groups were superior to the model group (P<0.05), but the effects of single treatment were limited. This indicates that the drug pair inhibits CYP24A1, activates the Klotho-VDR axis, prolongs the action time of 1,25-(OH)2D3, and enhances signal transduction efficiency. The LLG group showed similar levels of renal cyp24a1, cyp27b1, VDR, and klotho expression to the sham-operated group (P>0.05), which was superior to the LLG and CG groups. This suggests that the combined treatment of LLG effectively restores vitamin D metabolic balance. The excellent performance of the combined treatment further validates its synergistic effect in the regulation of the renal-bone axis.

[0101] Experiment Example 3: Molecular Biological Analysis

[0102] 3.1 Materials and Methods

[0103] 3.1.2 Western blot for protein immunoblotting

[0104] Total protein was extracted from tissues / cells using RIPA lysis buffer (Catalog No.: P0013C, Beyotime), and protein concentration was determined using a BCA protein quantification kit (Catalog No.: P0011, Beyotime). Protein samples were separated by 10% SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were blocked in QuickBlock™ protein-free Western blocking buffer (Catalog No.: P0240, Beyotime) at room temperature for 25 minutes, followed by overnight incubation at 4°C with the following primary antibodies: rabbit anti-CYP24A1 antibody (Catalog No.: HA500196, Huaan Biotech, 1:5000), rabbit anti-CYP27B1 antibody (Catalog No.: ER1907-01, Huaan Biotech, 1:5000), and rabbit anti-Klotho antibody (Catalog No.: ET170). 5-88, Huaan Biotechnology, 1:5000), rabbit anti-VDR antibody (catalog number ET1704-09, Huaan Biotechnology, 1:5000) and mouse anti-β-actin antibody (catalog number AA128, Beyotime, 1:1000); then, the membrane was thoroughly washed with TBST and incubated at room temperature for 1 hour with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody (catalog number: A0208, Beyotime, 1:1000) and goat anti-mouse secondary antibody (catalog number A0216_, Beyotime, 1:1000); finally, the membrane was developed using an ECL chemiluminescence kit (Thermo Fisher Scientific, USA) and the protein band grayscale values ​​were analyzed using ImageJ software (NIH, USA).

[0105] 3. Results

[0106] Western blot results (see...) Figure 11The LLG and CG groups showed superior expression of Klotho (P<0.001 vs. Model), CYP27B1 (LLG P<0.05, CG P>0.05 vs. Model), and VDR (P<0.05 vs. Model) compared to the model group, while CYP24A1 expression was lower in the LLG group (CG P<0.01). However, the effects of single treatment were limited, indicating that the drug pair prolonged the action time of 1,25-(OH)2D3 and enhanced signal transduction efficiency by inhibiting CYP24A1 and activating the Klotho-VDR axis. The LLG group showed renal Klotho, CYP27B1, and VDR expression similar to the sham-operated group (Klotho, P>0.05; CYP27B, P>0.05; VDR, P>0.05), with the lowest CYP24A1 expression (P<0.01 vs. Model), which was superior to the LLG and CG groups, suggesting that combined treatment effectively restored vitamin D metabolic balance. The superior performance of the LLCG group (Klotho P < 0.01 vs. LLG; VDR P < 0.05 vs. LLG; CYP24A1 P < 0.05 vs. LLG) further validates its synergistic role in the regulation of the kidney-bone axis, providing molecular support for the traditional Chinese medicine theory of "the kidney governs the bones".

[0107] In summary, by analyzing the effects of the Lycium barbarum-Ligustrum lucidum drug pair (LLG) and its combination with calcitriol (LLCG) on bone phenotype and vitamin D metabolism-related molecular mechanisms in PMOP model mice, animal experiments verified that LLG significantly improved bone volume fraction (BV / TV), trabecular bone number, and bone strength in ovariectomized osteoporosis (PMOP) mice. The LLCG group was close to the level of the sham-operated group. The results indicate that the Lycium barbarum-Ligustrum lucidum drug pair (LLG) and its combination with calcitriol (LLCG) further support the homeostasis of vitamin D metabolism in the kidney-bone axis by inhibiting renal CYP24A1 and enhancing Klotho and VDR expression.

[0108] Mechanistically, the Lycium barbarum-Ligustrum lucidum drug pair inhibited the expression of vitamin D-degrading enzyme CYP24A1 and upregulated the expression of Klotho and vitamin D receptor (VDR). The combined treatment with calcitriol resulted in even more significant effects. In vitro experiments showed that serum containing the Lycium barbarum-Ligustrum lucidum drug pair (LLG) significantly enhanced the activity of alkaloid phosphatase (ALP) and mineralization capacity in MC3T3-E1 cells, promoting osteogenic differentiation by activating the Wnt / β-catenin and BMP / Smad pathways. Simultaneously, in HK-2 cells, it downregulated CYP24A1 expression, reduced the degradation of active vitamin D (1,25-((OH)2D3), and upregulated the expression of CYP27B1, VDR, and Klotho, optimizing vitamin D metabolism. The combined effect of calcitriol (LLCG) was superior to that of calcitriol (CG) alone.

[0109] In vitro and in vivo studies have demonstrated that LLG promotes osteogenic differentiation and improves bone phenotype through both cellular and metabolic mechanisms. When combined with calcitriol (LLCG), it exhibits a synergistic effect that is significantly superior to single therapy. This provides molecular mechanism support for the traditional Chinese medicine theory of "kidney governs bone," indicating that the combination of LLG and calcitriol is a highly effective, safe, and cost-efficient strategy for treating PMOP, with significant potential for clinical translation.

[0110] The superior performance of LLCG in bone phenotype, molecular expression, and cellular function demonstrates that combination therapy, by integrating the CYP24A1 inhibition and Klotho-VDR activation effects of LLCG, is significantly superior to monotherapy. LLCG improved bone mineral density compared to the CG group, showing a cost-effective advantage over expensive bone-forming agents (such as teriparatide), and based on the conventional use of drug pairs, it has a low safety risk.

[0111] This study established that the Lycium barbarum-Ligustrum lucidum drug pair enhances the osteoprotective effect of vitamin D in postmenopausal osteoporosis by inhibiting CYP24A1 and upregulating Klotho and VDR, and has synergistic benefits when combined with calcitriol. This discovery provides a novel, evidence-based strategy for PMOP management by addressing the metabolic limitations of calcitriol and enhancing its osteogenic effects. This invention discloses for the first time the application of a Lycium barbarum-Ligustrum lucidum and calcitriol composition in the treatment of postmenopausal osteoporosis. The Lycium barbarum-Ligustrum lucidum composition described in this invention has multi-target osteoprotective effects in the prevention and treatment of postmenopausal osteoporosis, exhibiting anti-osteoporosis activity. In vitro experiments simultaneously verified that the composition promotes osteoblast differentiation and mineralization, inhibits CYP24A1, activates Klotho expression, enhances the osteoprotective effect of vitamin D, and effectively delays bone loss in ovariectomized mice. It can be used for the treatment of osteoporosis, especially postmenopausal osteoporosis, and the effect is more significant when treated synergistically with calcitriol.

[0112] Drug descriptions involved in the experimental examples of this application:

[0113] 1. Goji berries

[0114] Plant origin: The dried, ripe fruit of Lycium barbarum L., a plant belonging to the Solanaceae family. While Lycium chinense Mill. fruit is also available on the market, Ningxia Lycium barbarum is considered the best quality and is the authentic product listed in the Chinese Pharmacopoeia.

[0115] Latin name: Lycii Fructus

[0116] Other names: Gouqizi, Gouqi Hongshi, Tiancaizi, Xigouqi, Gounaizi, Hongqingjiao, Goutizi, Gouqiguo, Diguzi, Gouqieqie, Hongerzhui, Xuegouzi, Goudiyazi, Gouqidou, Xueqizi, Jin Gouqi, etc.

[0117] Medicinal part: dried, ripe fruit.

[0118] Properties and Channels Entered: Sweet, neutral. Enters the Liver and Kidney channels.

[0119] effect:

[0120] Nourishes the liver and kidneys: Used for symptoms such as soreness and weakness of the lower back and knees, dizziness, tinnitus, and seminal emission caused by liver and kidney yin deficiency;

[0121] Benefits essence and improves eyesight: It is especially effective in treating eye diseases such as decreased vision, blurred vision due to internal cataracts, and dry eyes caused by liver and kidney deficiency and deficiency of essence and blood. Long-term use has better effects.

[0122] It nourishes the lungs and can be used for coughs due to yin deficiency.

[0123] 2. Privet

[0124] Plant origin: The dried, mature fruit of Ligustrum lucidum Ait., a plant in the Oleaceae family.

[0125] Latin name: Ligustri Lucidi Fructus

[0126] Other names: Privet fruit, Holly fruit, Burdock fruit, White ash fruit, Mouse fruit.

[0127] Medicinal part: dried, ripe fruit.

[0128] Properties and Channels Entered: Sweet, bitter, cool. Enters the Liver and Kidney channels.

[0129] effect:

[0130] Nourishes the liver and kidneys: Used for symptoms such as soreness and weakness of the lower back and knees, dizziness and tinnitus, and premature graying of hair caused by liver and kidney yin deficiency;

[0131] Brightens eyes and darkens hair: Its liver and kidney tonifying effects can nourish the head and face, and it is used for blurred vision, decreased eyesight, and premature graying of hair;

[0132] Clearing deficiency heat: Because of its cool and moistening properties, it can reduce deficiency heat and can be used for tidal fever and irritability caused by yin deficiency and internal heat.

[0133] 3. Luo Gaiquan

[0134] Generic name: Calcitriol capsules

[0135] Active ingredient: Calcitriol

[0136] Chemical name: (5Z,7E)-9,10-open-cholesterol-5,7,10(19)-triene-1α,3β,25-triol

[0137] Manufacturer: Original manufacturer: Roche Pharma (Schweiz) Ltd. (Roche Pharmaceuticals Switzerland) / Manufacturing plant: Catalent Germany Eberbach GmbH

[0138] The main pharmacological effects include:

[0139] Promotes intestinal absorption of calcium: helps the body utilize ingested calcium more effectively.

[0140] Regulating bone mineralization: Promoting healthy bone formation and maintenance is crucial for treating diseases such as osteoporosis.

[0141] Obviously, the above embodiments and experimental examples of the present invention are merely illustrative of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, It is made from traditional Chinese medicine components and calcitriol. The traditional Chinese medicine components are made from the following raw materials in parts by weight: 7-15 parts of wolfberry and 6-12 parts of privet fruit. The mass ratio of the traditional Chinese medicine components to calcitriol is (2.6 × 10⁻⁶). 7 ~5.4×10 7 ):

1.

2. The pharmaceutical composition according to claim 1, characterized in that, The herbal ingredients are made from the following raw materials in parts by weight: 10-15 parts of wolfberry and 9-12 parts of privet fruit.

3. The pharmaceutical composition according to claim 1, characterized in that, The herbal ingredients are made from the following raw materials in parts by weight: 10 parts wolfberry and 9 parts privet fruit.

4. The use of the pharmaceutical composition according to any one of claims 1-3 in the preparation of a drug for treating postmenopausal osteoporosis.

5. The use of the pharmaceutical composition according to claim 4 in the preparation of a drug for treating postmenopausal osteoporosis, characterized in that, The pharmaceutical composition exerts its therapeutic effect through the following means: In HK-2 renal epithelial cells, the expression of vitamin D degrading enzyme CYP24A1 was inhibited, the expression of CYP27B1, VDR and Klotho was upregulated, the degradation of active vitamin D (1,25-(OH)2D3) was reduced, and vitamin D metabolism was optimized. Alternatively, it can enhance the alkaline phosphatase activity and mineralization capacity of osteoblast progenitor cells MC3T3-E1. Alternatively, it can improve bone density and bone microstructure in the body after oophorectomy; Alternatively, it can improve bone volume fraction, trabecular bone number, and bone strength in the body after oophorectomy. Alternatively, it can help restore the body's vitamin D metabolic balance after oophorectomy.

6. The use of the pharmaceutical composition according to claim 4 in the preparation of a drug for treating postmenopausal osteoporosis, characterized in that, The medication for treating postmenopausal osteoporosis contains the pharmaceutical composition and one or more pharmaceutically acceptable excipients.

7. The use of the pharmaceutical composition according to claim 6 in the preparation of a drug for treating postmenopausal osteoporosis, characterized in that, The excipients are at least one of the following: excipients, sustained-release agents, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, antibacterial agents, fragrances, antioxidants, pH adjusters, protectants, diluents, lubricants, solvents, matrix or carrier materials.

8. The use of the pharmaceutical composition according to claim 4 in the preparation of a drug for treating postmenopausal osteoporosis, characterized in that, The dosage form of the medication for treating postmenopausal osteoporosis is oral granules, tablets, pills, capsules, or oral liquid.

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