Application of a Traditional Chinese Medicine Composition in the Preparation of a Treatment for Postmenopausal Osteoporosis

Abstract

The present invention relates to the technical field of traditional Chinese medicine, and provides an application of a traditional Chinese medicine composition in the preparation of treating postmenopausal osteoporosis. The traditional Chinese medicine composition is prepared from the following components in parts by weight: 25-35 parts of Glossy Privet Fruit, 10-20 parts of Morinda officinalis How, 10-20 parts of Drynaria fortunei (Kunze) J. Sm., and 5-15 parts of Alisma orientale (Sam.) Juz. Among them, Glossy Privet Fruit is the monarch drug, with a mild nature, being good at nourishing the liver and kidney, and replenishing yin and generating essence; Morinda officinalis How and Drynaria fortunei (Kunze) J. Sm. together serve as the ministerial drugs, and Alisma orientale (Sam.) Juz. serves as the assistant and guiding drug. The whole formula can achieve the effects of tonifying both yin and yang, tonifying without stagnation, and dispelling pathogenic factors and strengthening healthy qi.

Description

[0001] This application is a divisional application. The original application has the application number 202310850911.2, the application date is July 12, 2023, and the invention title is: A traditional Chinese medicine for treating postmenopausal osteoporosis and its preparation method. Technical Field

[0002] This invention relates to the field of traditional Chinese medicine technology, and in particular to the application of a traditional Chinese medicine composition in the preparation of a treatment for postmenopausal osteoporosis. Background Technology

[0003] Postmenopausal osteoporosis (PMOP) is a metabolic bone disease caused by the decline in ovarian hormone synthesis after menopause in women. It is characterized by systemic bone loss and changes in bone microstructure, leading to increased bone fragility and a high susceptibility to fractures. Osteoporosis has become a serious public health problem, with a persistently high incidence. Fractures are the most common complication of osteoporosis, frequently occurring in the forearm, wrist, vertebrae, and hip, with hip fractures being the most severe. Patients with hip fractures experience significant mobility issues, and 12% to 40% may die from various complications within one year, severely impacting their quality of life. The enormous medical and nursing costs further burden patients financially. In early menopause, anti-resorption drugs alone can achieve bone protection, but in late menopause, long-term treatment with osteoproliferative drugs is necessary to partially alleviate bone loss.

[0004] Treatment guidelines for postmenopausal osteoporosis primarily recommend exercise therapy, physical therapy, and medication. Medication mainly includes drugs that inhibit bone resorption, such as estrogen, calcitonin, and diazepam; and drugs that promote bone formation, such as fluoride, growth hormone, and vitamin K. Research on traditional Chinese medicine (TCM) treatment of postmenopausal osteoporosis has been widely reported, summarizing effective treatment formulas based on clinical experience, which is currently an important approach to TCM treatment of osteoporosis. Clinically, common treatment principles for postmenopausal osteoporosis include tonifying the liver and kidneys and strengthening tendons and bones, generally focusing on tonifying kidney yin and yang, often neglecting the issue of kidney turbidity retention caused by deficiency leading to excess.

[0005] Based on this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a traditional Chinese medicine composition for the preparation of a treatment for postmenopausal osteoporosis. The whole formula can achieve the effects of tonifying both Yin and Yang, tonifying without stagnation, and eliminating pathogens and supporting the body's resistance.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a traditional Chinese medicine for treating postmenopausal osteoporosis, prepared from the following components in parts by weight: 25-35 parts of Ligustrum lucidum, 10-20 parts of Morinda officinalis, 10-20 parts of Drynaria fortunei, and 5-15 parts of Alisma plantago-aquatica.

[0009] This invention also provides a method for preparing a traditional Chinese medicine for treating postmenopausal osteoporosis, comprising the following steps:

[0010] (1) Extract the following herbs by reflux with ethanol solution: Ligustrum lucidum, Morinda officinalis, Drynaria fortunei, and Alisma plantago-aquatica.

[0011] (2) The extract from step (1) is collected and concentrated under reduced pressure to obtain the Chinese medicine.

[0012] Preferably, the total mass ratio of Ligustrum lucidum, Morinda officinalis, Drynaria fortunei, and Alisma plantago-aquatica in step (1) to the ethanol solution is 1:9-11; and the volume fraction of the ethanol solution is 30-70%.

[0013] Preferably, the reflux extraction in step (1) is performed 1 to 3 times; the extraction time for each extraction is 0.5 to 1.5 hours, and the extraction temperature for each extraction is 70 to 90 degrees Celsius.

[0014] Preferably, the pressure of the vacuum concentration in step (2) is 0.1 to 0.2 MPa; and the temperature of the vacuum concentration is 60 to 70°C.

[0015] Preferably, the relative density of the extract obtained by concentration in step (2) is 1.25 to 1.30 g / mL.

[0016] Preferably, the traditional Chinese medicine is mixed with excipients to form a soft material, which is then sieved, granulated, and dried under reduced pressure to produce traditional Chinese medicine granules.

[0017] Preferably, the excipient is dextrin; the mass ratio of the traditional Chinese medicine to dextrin is 1:0.8-1.1.

[0018] Preferably, the mesh size of the sieve is 10 to 14 mesh.

[0019] Preferably, the specific operation of the reduced pressure drying is as follows: drying at 0.1-0.2 MPa and 45-55°C for 35-37 hours.

[0020] Postmenopausal osteoporosis is rooted in kidney deficiency, compounded by the stagnation of turbidity within the kidneys. This deficiency leads to excess, causing the disease to linger. Treatment should consider both yin and yang tonification, combining tonification and purgation. This invention proposes a treatment principle of tonifying the kidneys and purging turbidity. The invented Zhenrong Decoction consists of Ligustrum lucidum, Morinda officinalis, Drynaria fortunei, and Alisma plantago-aquatica. The *Shennong Bencao Jing Shu* records that Ligustrum lucidum enters the Kidney Meridian of Foot-Shaoyin and is an essential medicine for clearing heat and replenishing essence. The *Bencao Bian Du* states that Ligustrum lucidum "possesses the function of sweetness, coolness, and purity, entering the kidneys to benefit yin." Morinda officinalis is slightly warm and not drying, nourishing primordial yang and entering the Kidney Meridian of Foot-Shaoyin, making it an essential medicine for kidney deficiency. The *Bencao Tongxuan* calls Morinda officinalis "a blood-regulating medicine for the Kidney Meridian," capable of strengthening muscles and bones, treating impotence, replenishing essence, and stopping seminal emission. The *Compendium of Materia Medica* records that *Drynaria fortunei* "is suitable because it enters the Kidney Meridian of Foot-Shaoyin and treats diseases between bones." The *Miscellaneous Materia Medica* states that it "is bitter and strengthens the kidneys, warms and promotes blood circulation, heals injuries and fractures, and treats bone weakness." *Alisma plantago-aquatica* enters the Kidney Meridian and has the functions of dispelling dampness, promoting urination, and purging turbidity. The *Handbook of Materia Medica* records that "Alisma plantago-aquatica is sweet, bland, salty, and cold; it enters the Kidney and Bladder to guide water and dampness from the lower burner." Naturally, damp heat is eliminated, ministerial fire is lowered, and pathogenic factors are removed, thus the body's vital energy benefits. Therefore, it is often used in combination with other kidney-tonifying herbs. In this invention, *Ligustrum lucidum* is the principal herb, with a mild nature, excelling at nourishing the liver and kidneys, and benefiting Yin and generating essence; *Morinda officinalis* and *Drynaria fortunei* are the assistant herbs, and *Alisma plantago-aquatica* is the adjuvant herb. The entire formula achieves the effect of simultaneously tonifying Yin and Yang, tonifying without stagnation, and dispelling pathogenic factors while supporting the body's vital energy. This invention, from the perspective of tonifying the kidneys and purging turbidity, considers simultaneously tonifying both Kidney Yin and Kidney Yang, tonifying without stagnation, guiding pathogenic factors outward, thereby restoring the body's internal balance.

[0021] Animal and cell experiments further verified that the traditional Chinese medicine prepared in this invention has a good therapeutic effect on the prevention and treatment of postmenopausal osteoporosis. The traditional Chinese medicine prepared in this invention can promote the expression of alkaline phosphatase in osteoblasts, increase calcified nodules, and increase the gene and protein expression levels of osteoblast-related cytokines RUNX2, ALP, and Osterix. It can also increase the OPG / RANKL ratio at both the molecular and protein levels. After administration, it was found that osteoclast-related transcription factors decreased, and the activation of RANKL-induced NFATc1 and c-Fos was inhibited. The phosphorylation level of PI3K / Akt decreased after the formulation intervention. The formulation treatment can improve bone loss and bone microstructure disorder in OVX rats, thereby delaying the disease progression of postmenopausal osteoporosis. Further mechanistic studies revealed that the formulation may upregulate OPG protein expression, increase the OPG / RANKL ratio, exert a bone-protective effect, downregulate the expression of downstream bone resorption-related factors in the RANKL / RANK signaling pathway, participate in regulating bone metabolism balance, and achieve the therapeutic effect of preventing and treating PMOP. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 The cell growth status during the primary osteoblast culture and identification process in Experiment Example 1;

[0024] Figure 2 This study examines the effects of different concentrations of Zhenrong Decoction on osteoblast activity at different time points in Experiment Example 1.

[0025] Figure 3 In Experiment 1, osteoblasts were treated with serum containing different concentrations of Zhenrong Decoction for 21 days. The formation of calcium nodules was observed by Alizarin Red staining (A), and the calcium nodules were dissolved for semi-quantitative analysis (B). *P<0.05.

[0026] Figure 4 The expression levels of Runx2, ALP, Osterix, and OPG / RANKL mRNA in cranial osteoblasts after 72 hours of intervention with different concentrations of drug-containing serum in Experiment Example 1 were compared with those in the control group. **p<0.01.

[0027] Figure 5 The expression levels of ALP, Osterix, Runx2, and OPG / RANKL proteins were detected (A) and semi-quantitatively analyzed (B) after different concentrations of drug-containing serum intervened in cranial osteoblasts for 72 hours in Experiment Example 1. Compared with the control group, *p<0.05, **p<0.01.

[0028] Figure 6 The results of RAW264.7 cells induced with 50 ng / mL RANKL in Experiment 1 and the results of TRAP staining are 100X.

[0029] Figure 7 The effect of different concentrations of Zhenrong Decoction on the viability of RANKL-induced RAW264.7 cells at different time points (24h, 48h, 72h, 96h) in Experiment Example 1 was compared with the control group. *p<0.05, **p<0.01.

[0030] Figure 8The changes in the expression levels of c-fos, NFATc1, CTSK, Trap, Integrinαvβ3 and MMP-9 mRNA in RAW264.7 cells after 72 h of intervention with serum containing different concentrations of Zhenrong Decoction under RANKL 50 ng / mL induction were compared with the control group (***p < 0.001).

[0031] Figure 9 The expression levels of TRAF6, c-fos, NFATc1, and MMP-9 proteins were detected in the serum containing Zhenrong Decoction 72 h after intervention.

[0032] Figure 10 The expression level of PI3K / AKT protein was detected in the serum containing Zhenrong Decoction 72 h after intervention.

[0033] Figure 11 Masson staining results (100X) of tibial bone samples from rats with osteoporosis treated with Zhenrong Decoction for 12 weeks in Experiment Example 1;

[0034] Figure 12 In Experiment 1, microCT scans were used to observe the microstructure of the tibial metaphysis after the bone tissue of the tibia was fixed.

[0035] Figure 13 Microscopic parameters BMD (B), BV / TV (C), Tb.N (D), and Tb.Th (E) were obtained by microCT scanning analysis of the tibial metaphysis bone tissue after fixation in Experiment Example 1. (Note: a: Compared with OVX, *P<0.05, **P<0.01, **P<0.001; b: Compared with SHAM group, ##P<0.01.)

[0036] Figure 14 The results of the maximum tibial load measurement in ovariectomized rats in Experiment Example 1 are shown below (Note: a: *P<0.05 compared with OVX; b: #P<0.05 compared with SHAM group).

[0037] Figure 15 The effect of Zhenrong Decoction on blood biochemical indicators of ovariectomized rats in Experiment Example 1 (Note: a: compared with OVX, *P<0.05, **P<0.01; b: compared with SHAM group, ##P<0.01);

[0038] Figure 16 The effect of Zhenrong Decoction on osteoblast formation-related indicators in ovariectomized rats in Experiment Example 1 (Note: a: compared with OVX, *P<0.05, **P<0.01, ***P<0.001; b: compared with SHAM group, ##P<0.01);

[0039] Figure 17 The effect of Zhenrong Decoction on the level of bone formation-related factor protein in the femur of castrated rats in Experiment 1 (Note: a: compared with OVX, *P<0.05, **P<0.01; b: compared with SHAM group, #P<0.05, ##P<0.01);

[0040] Figure 18 To detect the changes in the mRNA levels of osteoclast formation and differentiation-related factors of Zhenrong Decoction in castrated rats in Experiment Example 1 (Note: a: compared with OVX, *P<0.05, **P<0.01; b: compared with SHAM group, ##P<0.01);

[0041] Figure 19 The experiment in Example 1 investigated the changes in the levels of osteoclast-related factor proteins in castrated rats treated with Zhenrong Decoction (Note: a: compared with OVX, *P<0.05, **P<0.01; b: compared with SHAM group, ##P<0.01). Detailed Implementation

[0042] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0043] Example 1

[0044] A method for preparing Zhenrong Granules, a traditional Chinese medicine granule for treating postmenopausal osteoporosis, includes the following steps:

[0045] 25g of Ligustrum lucidum, 10g of Morinda officinalis, 10g of Drynaria fortunei, and 5g of Alisma plantago-aquatica were placed in a round-bottom flask. A 30% ethanol solution was added at a mass ratio of 1:9. The mixture was refluxed at **℃ for 0.5h. The extract was then concentrated at 0.1MPa and 60℃ to a relative density of 1.25g / mL to obtain the traditional Chinese medicine. The traditional Chinese medicine was mixed with dextrin at a mass ratio of 1:0.8 to form a soft mass. The mass was then granulated through a 10-mesh sieve and dried at 0.1MPa and 45℃ for 35h. The granules were then passed through a 9-mesh sieve to obtain the traditional Chinese medicine granule—Zhenrong granules.

[0046] Example 2

[0047] A method for preparing Zhenrong Granules, a traditional Chinese medicine granule for treating postmenopausal osteoporosis, includes the following steps:

[0048] 35g of Ligustrum lucidum, 20g of Morinda officinalis, 20g of Drynaria fortunei, and 15g of Alisma plantago-aquatica were placed in a round-bottom flask. A 70% ethanol solution was added at a mass ratio of 1:11, and the mixture was refluxed at **℃ for 3 times, 1.5h each time. After extraction, the extract was concentrated at 0.2MPa and 70℃ to a relative density of 1.30g / mL to obtain the traditional Chinese medicine. The traditional Chinese medicine was mixed with dextrin at a mass ratio of 1:1.1 to form a soft mass. The mass was then granulated through a 14-mesh sieve, dried at 0.2MPa and 55℃ for 37h, and passed through a 12-mesh sieve to obtain the traditional Chinese medicine granule—Zhenrong granules.

[0049] Example 3

[0050] A method for preparing Zhenrong Granules, a traditional Chinese medicine granule for treating postmenopausal osteoporosis, includes the following steps:

[0051] 30g of Ligustrum lucidum, 15g of Morinda officinalis, 15g of Drynaria fortunei, and 10g of Alisma plantago-aquatica were placed in a round-bottom flask. A 50% ethanol solution was added at a mass ratio of 1:10. The mixture was refluxed twice at **℃ for 1 hour each time. After extraction, the extract was concentrated at 0.1MPa and 65℃ to a relative density of 1.28g / mL to obtain the Chinese herbal medicine. The Chinese herbal medicine was mixed with dextrin at a mass ratio of 1:1 to form a soft mass. The mass was then granulated through a 12-mesh sieve, dried at 0.1MPa and 50℃ for 36 hours, and passed through a 10-mesh sieve to obtain the Chinese herbal medicine granules—Zhenrong granules.

[0052] Experimental Example 1

[0053] 1. Preparation of drug-containing serum

[0054] Take 200g of the Chinese herbal granules obtained in Example 3, add 1.5L of 50% ethanol and reflux extract twice (the extraction time for each extraction is 1h). Combine the filtrates obtained from the two extractions and concentrate to prepare a gavage solution with a relative density of 0.9g / mL—Zhenrong Decoction (calculated according to the clinical dose for rats, the gavage dose is 2.2g / (kg·d) per day).

[0055] Ten 6-week-old male SD rats were administered Zhenrong Decoction by gavage at a dose of 2.2 g / kg for 7 consecutive days. Samples were collected 15 minutes after the last administration on day 7. After anesthesia with isoflurane, blood was collected from the abdominal aorta into additive-free blood collection tubes, with a blood volume of 5 mL. The blood was centrifuged (4℃, 3000 rpm, 10 min) after standing at room temperature for 1 hour, and the supernatant serum containing the drug was transferred to cryovials and stored at -80℃ for later use. Another 10 rats were administered blank saline by gavage according to the same Zhenrong Decoction administration protocol. Blank serum was separated and stored at -80℃ for later use.

[0056] Serum sample preparation: Accurately aspirate 2 mL of drug-containing serum stored at -80℃ into a centrifuge tube, add 10 mL of methanol, vortex for 2 min, centrifuge at 10000 rpm for 10 min, collect the supernatant, dry with nitrogen at room temperature, and reconstitute the residue with 500 μL of 50% ethanol by sonication (500 W for 5 min), then centrifuge at 10000 rpm for 10 min, collect the supernatant, which is the Zhenrong Decoction-containing serum test sample. Prepare blank serum test samples by processing blank serum in the same way.

[0057] 5% drug-containing serum: Take 50 μL of drug-containing serum sample and add 250 μL of methanol for alcohol precipitation. Centrifuge and collect the supernatant. Dry it under nitrogen and reconstitute it with 1 mL of α-MEM medium or 1 mL of DMEM medium.

[0058] 10% drug-containing serum: Take 100 μL of drug-containing serum sample and add 250 μL of methanol for alcohol precipitation. Centrifuge and collect the supernatant. Dry it under nitrogen and reconstitute it with 1 mL of α-MEM medium or 1 mL of DMEM medium.

[0059] 20% drug-containing serum: Take 200 μL of drug-containing serum sample and add 250 μL of methanol for alcohol precipitation. Centrifuge and collect the supernatant. Dry it under nitrogen and reconstitute it with 1 mL of α-MEM medium or 1 mL of DMEM medium.

[0060] Primary osteoblasts were treated with 1 mL of drug-containing serum reconstituted in α-MEM medium, while RAW264.7 cells were treated with 1 mL of drug-containing serum reconstituted in DMEM medium.

[0061] 2. Primary osteoblast assay

[0062] 2.1 Primary osteoblast culture and identification

[0063] Osteoblasts were extracted from the skulls of 72-hour-old SD mice. After rapid euthanasia by cervical dislocation, the skull was sterilized with 75% ethanol, transferred to a biosafety cabinet, the neck was cut off, and the skull was separated. The skull was then immersed in 7.4M PBS. The scissors and forceps were flammed over an alcohol lamp, cooled for 30 seconds, and the surface tissue of the skull was thoroughly cleaned. The skull was transferred to a new culture dish containing PBS, and the skull was chopped as finely as possible. The PBS was discarded, and 6 mL of 0.25% trypsin was added. The mixture was incubated at 37°C for 10 minutes. After digestion, the mixture was pipetted and discarded. 6 mL of 0.2% type I collagenase was added, and the mixture was incubated again at 37°C for 60 minutes, mixing every 10 minutes. After 60 minutes, the collagenase solution was collected in centrifuge tubes and centrifuged at 12000 rpm for 3 minutes. A precipitate was observed after centrifugation. Discard the supernatant, resuspend in α-MEM (purchased from Gibco, catalog number 41061037, with added 10% FBS + 1% penicillin and streptomycin), and incubate at 37°C for 12 hours. If... Figure 1 As shown in Figure A, the adherent cells are spindle-shaped, cone-shaped, and polygonal. After culturing for another 12 hours, a few adherent cells are still visible. The medium is then changed, the cells are washed with PBS, and α-MEM is added. The cells are then cultured at 37°C until the third day. Figure 1 As shown in Figure B, the number of cells increases and they gradually merge; by the fifth day of culture, as... Figure 1 As shown in Figure C, the cells gradually exhibit a "paving stone" growth pattern, with a cell confluence rate exceeding 90%, indicating that they are ready for passage. By the third generation, as... Figure 1 As shown in Figure D, cells in this state were stained with Alizarin Red, as shown in Figure D. Figure 1 As shown in Figure E, nodules are visible in cells stained with Alizarin Red; when the cells are cultured for 10 days, they are stained again, as shown... Figure 1 As shown in Figure F, the cells exhibit obvious calcified nodules.

[0064] 2.2. CCK8 assay to determine the serum intervention concentration of the drug in each group.

[0065] The effect of Zhenrong Decoction on the activity of cranial osteoblasts was detected using a CCK8 assay kit. The cranial osteoblasts (adherent cells from the third passage) were cultured in Corning T25cm flasks at 2.1°C. 2 Add 5 ml of the blank serum test sample (0%), 5%, 10%, and 20% drug-containing serum prepared according to Part 1 to the culture medium, respectively, and incubate in induction differentiation medium (purchased from Haixing Biotechnology) at 37℃ and 5% CO2 for 96 h. Results are as follows: Figure 2 As shown, 5% and 10% of the solution showed no significant toxicity to cells at different time points (24h, 48h, 72h, 96h). Under this premise, 5% and 10% were selected for subsequent osteoblast stimulation induction experiments.

[0066] 2.3. Detection of alkaline phosphatase content in osteoblast culture supernatant using the ALP method.

[0067] Primary osteoblasts were seeded in 24-well plates. When cell confluence reached 80%, serum-free medium was added for starvation for 12 hours. The medium was then discarded, and physiological saline blank serum, 5% and 10% drug-containing serum (prepared in Part 1) were added respectively. Cells were cultured for another 72 hours. The cell supernatant was collected, centrifuged at 1300 rpm for 10 minutes, and the alkaline phosphatase (ALP) content was measured. (The corresponding reagents were prepared according to the ALP kit (A059-2, purchased from Nanjing Jiancheng Bioengineering Institute), and the ALP content was calculated using the formula: ALP (King's units / 100 mL) = (sample OD / standard OD) × phenol content in the standard × (100 mL / 0.05 mL) (repeated 5 times).) The results are shown in Table 1. After 72 hours of culture, the ALP content in the 5% Zhenrong decoction-containing serum group began to be higher than that in the physiological saline blank serum group, and the ALP content in the 10% Zhenrong decoction-containing serum group was significantly higher (P<0.05). This indicates that the Zhenrong Decoction of the present invention can promote the expression of alkaline phosphatase in osteoblasts.

[0068] Table 1. Effects of Zhenrong Decoction on ALP activity in osteoblasts (n=6, )

[0069] Group ALP content (mg / mL) physiological saline blank serum 0.027±0.0093 Zhenrong Decoction contains 5% of the patient's serum. 0.1213±0.02* Zhenrong Decoction contains 10% of the patient's serum. 0.1507±0.06*

[0070] 2.4. Zhenrong Decoction contains serum that promotes the maturation and differentiation of osteoblasts.

[0071] Figure 1 In the late osteogenic differentiation stage of cells in the middle ferrite group, mineral deposition (calcium nodules) was observed. To detect the formation of calcium nodules, after osteogenic induction in section 2.2, cells from the blank serum group (control), 5% (ZRD-5), 10% (ZRD-10), and drug-containing serum group were cultured for 21 days. The medium was changed every 3 days during culture, for a total of seven times. After culture, alizarin red staining was performed, and semi-quantitative analysis was conducted. The results are as follows: Figure 3 As shown, serum containing Zhenrong Decoction promoted the formation of osteoblast calcified nodules. The levels of calcified nodules were initially higher in the 5% Zhenrong Decoction-containing serum group than in the saline blank serum group, and were even more pronounced in the 10% Zhenrong Decoction-containing serum group (P<0.05). This is consistent with the results of the ALP assay in section 2.3 (the increase in alkaline phosphatase and the formation of calcified nodules both indicate that the drug has the ability to promote osteogenic formation), and Zhenrong Decoction can also increase calcified nodules.

[0072] 2.5 Results of qPCR detection of bone formation-related factors RUNX2, Osterix, ALP, and OPG / RANKL mRNA expression

[0073] Following the results in section 2.2, after osteoblasts were treated with 5% and 10% drug-containing serum for 72 hours, the mRNA expression of bone formation-related factors RUNX2, Osterix, ALP, and OPG / RANKL in the control, ZRD-5, and ZRD-10 groups was detected by qPCR. The results are as follows: Figure 4 As shown, compared with the saline blank serum (control), the expression levels of RUNX2, Osterix, ALP, and OPG / RANKL mRNA, which are related to osteoblast formation, were increased in the ZRD-5 and ZRD-10 groups. It is speculated that the serum containing Zhenrong Decoction exerts its effect on osteoblast proliferation by regulating the above factors.

[0074] 2.6 Results of Western Blot analysis of bone formation-related factors RUNX2, Osterix, ALP, OPG, and RANKL protein expression.

[0075] (1) Following the results in 2.2, osteoblasts were seeded into 6-well plates (1×10⁻⁶ cells per well). 5 Cells were cultured with 0%, 5%, and 10% drug-containing serum, 2 ml per well. After 72 h of intervention, the α-MEM medium (containing 10% FBS + 1% PS) was discarded, and 300 μL of RIPA lysis buffer + 10 μL of LPMSF was added to each well. Cells were lysed on ice, centrifuged at 14000 rpm and 4 °C for 30 min, and proteins were extracted. After measuring the protein concentration, subsequent experiments were performed.

[0076] (2) Preparation and loading of SDS-PAGE gels: Prepare the separating gel and stacking gel of the required concentration separately in a fume hood. Slowly add the separating gel between clean glass plates with a thickness of 1.0 mm. Press the interface of the separating gel with anhydrous ethanol. After the separating gel has completely solidified, discard the anhydrous ethanol, blot dry, and slowly add 5% stacking gel. Gently and quickly insert the sample comb into the upper gel and keep it still without moving it until the gel solidifies. Fix the gel plate to the electrophoresis clamp, add electrophoresis buffer, vertically remove the sample comb, observe the condition of the sample wells, add 3 μL of protein pre-stained marker to one end, and load 20 μg of protein.

[0077] (3) Electrophoresis: Electrophoresis at 75V for 30 minutes to ensure that the sample is neatly electrophoresed into the stacking gel. After electrophoresis at 140V for 40 minutes, observe carefully to ensure that the target protein is clearly separated and then stop electrophoresis.

[0078] (4) Transfer: Near the end of electrophoresis, pour the transfer buffer (stored at 4℃) into an enamel tray. Remove the glass gel plate. Carefully pry up the short glass plate near the glass gel plate with a spatula to cut off the gel containing the target protein. Discard the rest. Cut a 0.22µm PVDF membrane slightly larger than the gel area. Activate the membrane in methanol for 30 seconds and then place it in the transfer buffer, keeping it moist throughout. Take the sandwich transfer clamp. Soak the filter paper in the transfer buffer and align it in the following order: white clamp - sponge pad - filter paper - membrane - gel - filter paper - sponge pad - black clamp. Ensure there are no air bubbles between the gel and the membrane. Use a roller to remove air bubbles and then close the transfer clamp. Insert the transfer clamp into the transfer tank with the positive and negative electrodes aligned. Add pre-cooled transfer buffer. To prevent excessive heat generation, place ice cubes outside the transfer tank for rapid cooling. Set the voltage to 100V and the time to 30 minutes, which can be adjusted according to the molecular weight.

[0079] (5) Sealing: After the transfer is completed, take out the PVDF membrane, place it in the sealing solution incubation box, and seal it on a shaker (50 rpm) for 2 hours.

[0080] (6) Primary and secondary antibody immune response: After blocking, add diluted primary antibody. Incubate overnight at 4°C on a shaker (60 rpm). The next day, recover the primary antibody and wash the PVDF membrane with TBST 4 times for 5 min on a shaker (90 rpm). Add the corresponding secondary antibody at a ratio of 1:10000 according to the species of the primary antibody. Incubate at room temperature on a shaker (60 rpm) for 90 min. After removing the secondary antibody, wash the membrane with TBST 10 min at a time on a shaker (90 rpm) for 3 times.

[0081] The relevant information for primary and secondary antibodies is shown in Tables 2 and 3:

[0082] Table 2. Relevant information about primary antibodies.

[0083] Primary Antibody Product Number Manufacturer ALP antibody sc-271431 Santacruz Osterix antibody sc-393325 Santacruz RANKL antibody sc-377079 Santacruz RUNX2 antibody 12556S CST OPG antibody sc-390518 Santacruz NFATc1 antibody A00340-1 Boster TRAF6 antibody ab137452 abcam MMP9 antibody 10375-2-AP proteintech c-FOS antibody 66590-1-Ig proteintech GAPDH antibody 60004-1-Ig proteintech

[0084] Table 3. Relevant information about secondary antibodies.

[0085] Secondary antibody Product Number Manufacturer Secondary antibody against mice (donkey-resistant mice) SA00001-8 proteintech Rabbit anti-rabbit (donkey anti-rabbit) SA00001-9 proteintech

[0086] (7) Chemiluminescence imaging: The chemiluminescence imager is preheated. The imagelab 6.0 software is opened. The developer solutions A and B are mixed evenly in a 1:1 ratio. The PVDF membrane is placed in the developing chamber. The developer solution is added evenly to completely cover the membrane surface. The surface air bubbles are removed. The dark door is closed. The software is set to the exposure time. The instrument is run to perform imaging and the experimental results are analyzed.

[0087] Changes in osteogenic-related proteins among groups were detected using Western blotting. Results are as follows: Figure 5As shown, compared with the Control (CON) group, the expression levels of Osterix, RUNX2, and ALP proteins in the serum containing Zhenrong Decoction were significantly increased, and the OPG / RANKL ratio was also increased. Figure 5 (E).

[0088] 3. Serum containing Zhenrong Decoction inhibits RANKL-induced osteoclast differentiation.

[0089] 3.1 Culture of bone marrow mononuclear macrophages

[0090] RAW264.7 cells (purchased from Wuhan Biotechnology Co., Ltd., catalog number: CL-0190) are small in size and nearly round, settling gently to the bottom of the flask. During cell culture, trypsin digestion is not required; handle the cells gently and avoid vigorous pipetting. Use a 1×10⁻⁶ folding motion. 6 Inoculate at a concentration of 25cm 2 Add 5 mL of 10% FBSDMEM culture medium to the culture flask and incubate at 37°C for 1 day. Observe the color of the culture medium and the cell growth status daily, and change the medium in time to prevent differentiation.

[0091] 3.2 Determine the concentration of drug-containing serum in each group of RAW264.7 cells and the results of TRAP staining. RAW264.7 is an osteoclast precursor. The cells are small in size, round or irregular in shape, and may have pseudopodia. The nuclei are small and round, and the vast majority are mononuclear, with occasional 2-3 nuclei.

[0092] RAW264.7 cells were induced on day 1 with 50 ng / mL RANKL. Figure 6 As shown in Figure A, the cells begin to enlarge, with more pseudopodia, and the nuclei become larger and oval-shaped; after RANKL induction for 3 days, as shown... Figure 6 As shown in Figure B, cells begin to fuse into large cells with more than three nuclei; after 5 days of RANKL induction, as... Figure 6 As shown in Figure C, numerous multinucleated giant cells appeared, ranging in size from tens to hundreds of micrometers, with irregular shapes. These cells were induced by RANKL on day 6. Figure 6 As shown in Figure D, multinucleated cells are clearly visible in the center of the cell, the cavity gradually enlarges, and there are numerous pseudopodia, reaching up to dozens of nuclei. On day 7 of RANKL induction, as... Figure 6 As shown in Figure E, as cells continue to fuse, the cavity within the multinucleated giant cells increases in size and the boundaries become more distinct; TRAP staining of cells on day 7 of RANKL induction... Figure 6 As shown in Figure F, the staining is positive, indicating that osteoblasts have been successfully differentiated. The cells are wine-red or purplish-red, with deeper staining around the nucleus and under the cell membrane.

[0093] RAW264.7 cells were induced by adding different concentrations (5%, 10%, 20%) of Zhenrong Decoction-containing serum to RANKL-containing medium. Cell viability was measured using CCK-8 assay at 24h, 48h, 72h, and 96h of induction. The results are as follows: Figure 7 As shown. From Figure 7 It can be seen that when the serum concentration of Zhenrong Decoction is 5% and 10%, the activity of RAW264.7 cells is comparable to that of cells containing only RANKL medium. When the concentration reaches 20%, the activity of RAW264.7 cells is inhibited. Therefore, 5% and 10% concentrations were selected for the experiment.

[0094] 3.3. The inhibition of gene expression of bone resorption-related markers in serum containing Zhenrong Decoction.

[0095] The expression of osteoclast resorption-related factors c-fos, NFATc1, CTSK, Trap, Integrinαvβ3, and MMP-9 in cells treated with Zhenrong Decoction for 72 h in part 3.2 was detected by qPCR. The results are as follows: Figure 8 As shown, compared with the control group, serum containing 5% and 10% Zhenrong Decoction could effectively reduce the gene expression levels of c-fos, NFATc1, CTSK, Trap, Integrinαvβ3 and MMP-9, and inhibit bone resorption.

[0096] 3.4. Serum containing Zhenrong Decoction inhibits the protein expression of osteoclast-related bone resorption markers.

[0097] NFATc1 and c-fos are two transcription factors essential for precursor osteoclast fusion and for the transcriptional regulation of the expression of many precursor osteoclast-related genes.

[0098] The protein expression of related genes TRAF6, MMP-9, c-fos, and NFATc1 in cells treated with Zhenrong Decoction for 72 h were detected in section 3.2. The results are as follows: Figure 9 As shown, RANKL (50 ng / mL) induction and treatment with Zhenrong Decoction-containing serum for 72 h reduced TRAF6 expression, while also decreasing the protein expression levels of NFATc1 and c-Fos. The phosphorylation levels of PI3K and AKT in cells treated with Zhenrong Decoction for 72 h were detected in part 3.2, and the results are as follows. Figure 10 As shown, after administration of serum containing Zhenrong Decoction, the phosphorylation levels of P-PI3K and P-AKT were reduced, indicating that serum containing Zhenrong Decoction may affect the activation of downstream PI3K / AKT and inhibit osteoclast differentiation by inhibiting the activation of the RANKL / RANK signaling pathway.

[0099] 4. The intervention effect of Zhenrong Decoction on ovariectomized rats

[0100] 4.1 Establishment of PMOP model in female SD rats and animal grouping

[0101] Ninety SPF-grade female SD rats, three months old, were acclimatized and fed (normal diet and water) for 7 days. They were divided into two groups: the SHAM (sham operation) group (n=18) and the OVX (model group) group (n=72). In the OVX group, both ovaries were removed, while in the sham operation group, only the fat around the ovaries was removed. After surgery, each rat was given 8 IU of penicillin for 3 consecutive days and routine anti-infection treatment was performed. There were no rat deaths. Animal groups were as follows: 18 animals in the sham-operated group (SHAM) (physiological saline) (1 mL / 100 g), 18 animals in the model group (OVX) (physiological saline) (1 mL / 100 g), 18 animals in the positive control group (OVX-EV) (0.1 mg / (kg·d)), 18 animals in the low-dose Zhenrong Decoction group (OVX-ZRDL) (2.2 g / (kg·d)), and 18 animals in the high-dose Zhenrong Decoction group (OVX-ZRDH) (4.4 g / (kg·d). The administration volume was 1 mL / 100 g. After 12 weeks of administration, bone morphology, bone strength, and other indicators were observed. The positive control group received estradiol valerate tablets (EV) 1 mg / tablet (batch number: 558A).

[0102] 4.2 Observation of bone tissue morphology

[0103] Tibial bone samples from rats in groups treated in section 4.1 for 12 weeks were subjected to Masson staining, as shown below. Figure 11 As shown in Table 4, the relative area ratio of trabecular bone in rat tibias stained with Masson staining was statistically analyzed. Figure 11 As shown in Table 4, at 12 weeks, the trabeculae in the SHAM group were tightly arranged, evenly distributed, and connected in a network with small gaps and no breaks. In the OVX group, after 12 weeks of oophorectomy, the number of trabeculae decreased, the distribution was scattered and irregular, the number of broken trabeculae increased, and the width between trabeculae increased. Compared with the OVX group, the OVX-ZRDH group had better trabecular and structural integrity, more trabeculae, smaller gaps, and fewer breaks (P<0.01). The OVX-ZRDL group had slightly worse efficacy than the OVX-ZRDH group, but compared with the OVX group, it significantly improved the sparseness of trabeculae. All of these differences were significant (P<0.05).

[0104] Table 4. Statistical results of relative trabecular area ratio in rat tibia Masson staining ( n=6)

[0105]

[0106] Note: a: Compared with OVX, *P<0.05, **P<0.01; b: Compared with SHAM group, ##P<0.01.

[0107] 4.3 Results of microCT scan measurement of bone mineral density in rat tibial metaphysis

[0108] The changes in the microstructure of the tibia in section 4.2 were observed and analyzed using micro-CT three-dimensional reconstructed images, and the changes in micro parameters were obtained, such as... Figure 12 , 13 As shown, changes were found in the tibial trabecular structure in the OVX model group, while both OVX-ZRDH and OVX-ZRDL reduced the structural changes caused by OVX.

[0109] Figure 13 Bone mineral density (BMD), bone volume fraction (BV / TV), number of trabeculae (Tb.N), and trabeculae thickness (Tb.Th) were significantly increased.

[0110] 4.4 Results of rat bone strength measurement

[0111] The maximum tibial load was measured in each group, as shown in Table 5. Figure 15 As shown, after 12 weeks of ovariectomy in rats, the maximum tibial load decreased. There was no significant difference between the SHAM group and the OVX group. However, the maximum tibial load was significantly increased in the Zhenrong Decoction-treated groups (OVX-ZRDL, OVX-ZRDH) compared with the SHAM group and the OVX group.

[0112] Table 5 Maximum Tibial Load (Unit: Kn) (n=6, )

[0113] time SHAM OVX OVX-EV OVX-ZRDL OVX-ZRDH 12W 0.060±0.026 0.053±0.007 <![CDATA[0.082±0.03 *# ]]> 0.069±0.006 <![CDATA[0.082±0.005 *# ]]>

[0114] Note: a: *P < 0.05 compared to OVX; b: #P < 0.05 compared to the SHAM group.

[0115] 4.5 Changes in serum metabolic markers in rats of different groups

[0116] Measuring the levels of serum bone metabolism biochemical indicators in each group can reflect the status of bone homeostasis. These include bone-specific alkaline phosphatase BALP, which is related to bone formation; tartrate-resistant acid phosphatases TRAP5b, CTK, and β-CTX, which are related to bone resorption; IL-6, IL-1β, and TNF-α; and estrogen E2.

[0117] The results are shown in Table 6 and Figure 15As shown, after ovariectomy (OVX), the concentration of bone alkaline phosphatase (BALP) decreased significantly (P<0.01), while the concentration of tartrate-resistant acid phosphatase 5b (TRAP5b) increased significantly (P<0.01). This indicates that in castrated rats, the inhibition of osteoclast activity lessens with the decrease in estrogen levels, resulting in bone resorption exceeding bone formation and an imbalance in bone homeostasis. After administration of Zhenrong Decoction, the serum levels of bone phosphatase increased (P<0.01) and decreased the serum levels of tartrate-resistant acid phosphatase 5b (P<0.01), maintaining bone homeostasis. CTK and β-CTX are factors related to osteoclast maturation and function. After administration of Zhenrong Decoction, the levels of CTK and β-CTX decreased (P<0.01), indicating that Zhenrong Decoction inhibits osteoclast maturation and reduces bone resorption. Furthermore, Zhenrong Decoction can reduce the levels of serum inflammatory factors TNFα, IL-6, and LI-1β.

[0118] Table 6. Effects of Zhenrong Decoction on blood biochemical parameters in ovariectomized rats (n=6, )

[0119] SHAM OVX OVX-EV OVX-ZRDL OVX-ZRDH BALP (ng / mL) 2.50±0.22 <![CDATA[1.60±0.20 ## ]]> 2.30±0.21* 2.0±0.12 2.20±0.13* TRAP5b(U / L) 5.10±0.32 <![CDATA[8.59±0.57 ## ]]> 5.11±0.10** 7.32±0.25* 5.27±0.17** CTK (pg / mL) 2.03±0.32 <![CDATA[3.76±0.15 ## ]]> 2.15±0.20** 3.15±0.11** 2.2±0.13** β-CTX (ng / mL) 12.13±101 <![CDATA[28.59±1.57 ## ]]> 13.15±0.99** 22.29±0.96* 15.37±1.52** TNF-α (pg / mL) 9.57±1.21 <![CDATA[18.46±2.05 ## ]]> 10.98±1.03** 15.05±0.66* 10.47±1.08** IL-6 (pg / mL) 39.9±3.22 <![CDATA[77.80±4.00 ## ]]> 45.65±5.51** 60.87±3.56* 51.14±2.44** IL-1β (pg / mL) 73.23±4.90 <![CDATA[98.80±6.39 ## ]]> 74.98±3.54** 91.72±2.43* 81.14±2.2**

[0120] Note: a: Compared with OVX, *P<0.05, **P<0.01; b: Compared with SHAM group, ##P<0.01.

[0121] 4.6. qPCR detection of the expression of osteoblast formation-related factors Runx2, RANKL, and OPG in rat femur.

[0122] Femoral tissue samples from each group of rats were collected to detect the expression of genes related to osteogenic formation, such as... Figure 16 As shown, compared with the SHAM group, the RANKL mRNA level was significantly increased in the OVX group, while the mRNA expression of OPG and Runx2 was significantly inhibited. After administration of Zhenrong Decoction, the mRNA expression levels of Runx2 and OPG increased, consistent with the OVE-EV group. RANKL mRNA expression was not affected. This indicates that Zhenrong Decoction may upregulate OPG protein expression, increase the OPG / RANKL ratio, and exert a bone-protective effect.

[0123] 4.7. Western Blot analysis of the expression of bone formation-related factors OPG, RANKL, and RUNX2 in the rat femur.

[0124] The expression of osteogenic-related proteins was detected in rat femurs, such as... Figure 17As shown, compared with the SHAM group, the OVX group showed increased RANKL protein expression (P < 0.05) and decreased OPG and RUNX2 protein expression (P < 0.01). After 12 weeks of administration of Zhenrong Decoction, the low-dose group began to show increased OPG and RUNX2 protein expression (P < 0.05), while the high-dose group showed a more significant increase in OPG protein expression (P < 0.01). RANKL protein expression was not affected. This indicates that Zhenrong Decoction can upregulate the OPG / RANKL protein ratio to exert a bone-protective effect.

[0125] 4.8. qPCR detection of expression levels of osteoclast formation and differentiation-related factors NFATc1 and c-Fos in rat femur.

[0126] In ovariectomized rats, the sharp drop in estrogen levels weakened its inhibition of osteoclast resorption, leading to increased osteoclast activity. The expression levels of osteoclast formation and differentiation-related factors NFATC1 and c-Fos in the rat femur were detected using qPCR. The results are as follows: Figure 18 As shown, the transcriptional levels of c-fos and NFATc1 were significantly increased in the OVX group (P < 0.01). In the Zhenrong Decoction group and the OVX-EV group, the expression of c-Fos and NFATc1 mRNA was significantly decreased. This indicates that Zhenrong Decoction can inhibit the expression of bone resorption-related factors.

[0127] 4.9. Western Blot analysis of the expression of osteoclast differentiation-related factors TRAF6, c-FOS, and NFATc1 in rat femur.

[0128] Based on 4.8, changes in RANKL / RANK-related pathway proteins during osteoclast differentiation were observed. For example... Figure 19 As shown, the OVX group exhibited increased osteoclast activity due to decreased estrogen levels, with increased expression of TRAF6, c-fos, and NFATc1 proteins. These levels significantly decreased in the OVX-EV group and after intervention with Zhenrong Decoction. This indicates that Zhenrong Decoction can inhibit the expression of bone resorption-related proteins.

[0129] As demonstrated in Experiment 1, Zhenrong Decoction of the present invention can promote the expression of alkaline phosphatase in osteoblasts, increase calcified nodules, and increase the gene and protein expression levels of osteoblast-related cytokines RUNX2, ALP, and Osterix. It can also increase the OPG / RANKL ratio at both the molecular and protein levels. After administration of Zhenrong Decoction, osteoclast-related transcription factors were reduced, and the activation of RANKL-induced NFATc1 and c-Fos was inhibited. PI3K / Akt phosphorylation levels decreased after Zhenrong Decoction intervention. Zhenrong Decoction treatment can improve bone loss and bone microstructure disorder in OVX rats, thereby delaying the progression of postmenopausal osteoporosis. Further mechanistic studies revealed that Zhenrong Decoction may upregulate OPG protein expression, increase the OPG / RANKL ratio, exert a osteoprotective effect, downregulate the expression of downstream bone resorption-related factors in the RANKL / RANK signaling pathway, participate in regulating bone metabolism balance, and achieve the therapeutic effect of preventing and treating PMOP.

[0130] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a traditional Chinese medicine composition in the preparation of a drug for treating postmenopausal osteoporosis, characterized in that, The traditional Chinese medicine composition is prepared from the following components in parts by weight: 25-35 parts of Ligustrum lucidum, 10-20 parts of Morinda officinalis, 10-20 parts of Drynaria fortunei, and 5-15 parts of Alisma plantago-aquatica.

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