Pharmaceutical composition for preventing and treating non-traumatic femoral head necrosis as well as preparation method and application thereof

Abstract

The invention provides an application of an active ingredient targeting miR-365a-3p or a preparation product containing the active ingredient in preparation of a medicine for preventing and treating non-traumatic femoral head necrosis disease. The active ingredient is a medicine composition for dredging arthralgia and generating bone and / or beta-achyransterone. According to the application, experiments prove that the arthralgia-dredging and bone-growing soup and beta-achyranthes bidentata sterone can promote angiogenesis and osteogenesis by improving the expression of miR-365a-3p, so that the arthralgia-dredging and bone-growing soup and the beta-achyranthes bidentata sterone play a positive role in treating NONFH; on a constructed SONFH rabbit model, it is verified that the arthralgia-relieving bone-generating soup can promote bone tissue repair, inhibit fat formation and effectively adjust bone fat metabolism steady state unbalance to achieve bone fat metabolism balance; the optimal dosage of the arthralgia-dredging and bone-generating soup is explored, and it is proved that the arthralgia-dredging and bone-generating soup achieves the effect of regulating and controlling the dynamic balance of the bone fat through a multi-target-point synergistic mechanism of regulating and controlling lipid metabolism, promoting osteogenesis and improving the microenvironment.

Description

Technical Field

[0001] The present application relates to the field of biopharmaceutical technology, and specifically to a pharmaceutical composition for preventing and treating non-traumatic femoral head necrosis, a preparation method thereof, and an application thereof. Background Art

[0002] Non-traumatic osteonecrosis of the femoral head (NONFH) is a particularly challenging orthopedic condition to treat. Its root causes are a variety of non-traumatic factors, including steroid-induced osteonecrosis of the femoral head due to improper hormone use, alcoholic osteonecrosis of the femoral head due to long-term excessive drinking, and idiopathic osteonecrosis of the femoral head of unknown etiology. The course of this disease is relatively short, but the disease progresses extremely rapidly. When the disease progresses to ARCO stage III, femoral head collapse is almost inevitable, and currently no treatment has been found in the medical field that can effectively reverse the progression of this disease. NONFH causes the disability rate of patients to remain at a high level, causing extremely serious adverse effects on patients' ability to work and daily quality of life, while also placing an extremely heavy burden on individual patients, their families, and the entire social system. According to incomplete retrospective statistics, NONFH accounts for 86.3% of osteonecrosis cases in my country. Currently, MRI technology is primarily used to diagnose femoral head necrosis at an early stage. However, further research is needed to effectively treat the disease early and prevent femoral head necrosis and collapse. The pathological mechanism of femoral head necrosis is still unclear. Theories of its pathogenesis include osteoporosis, microcirculation disorders, and lipid metabolism disorders. The most critical of these theories is the insufficient osteoblast differentiation capacity within the femoral head and the obstruction of microvascular circulation, which leads to nutritional impairment. Summary of the Invention

[0003] In response to the above-mentioned technical limitations, the present application proposes a pharmaceutical composition for preventing and treating non-traumatic femoral head necrosis, as well as a preparation method and application thereof; it overcomes the deficiencies and defects mentioned in the background technology.

[0004] To achieve the above objectives, this application adopts the following technical solutions:

[0005] The invention of this application is to provide an active ingredient targeting miR-365a-3p or a preparation product containing the active ingredient in the preparation of a drug for preventing and treating non-traumatic femoral head necrosis.

[0006] Optionally, in the above application, the active ingredient targeting miR-365a-3p includes an agonist that promotes miR-365a-3p expression.

[0007] Optionally, in the above application, the agonist that promotes miR-365a-3p expression includes a Tongbi bone-forming pharmaceutical composition and / or β-oxanthellate sterol.

[0008] Optionally, in the above-mentioned application, the tonifying and bone-forming medicinal composition is prepared from the following raw materials in parts by weight: 10-20 parts of raw rehmannia, 10-20 parts of cyathula, 8-12 parts of prepared rehmannia, 8-12 parts of angelica, 8-12 parts of millettia reticulata, 8-12 parts of astragalus, 8-12 parts of epimedium, 8-10 parts of epimedium, 8-10 parts of psoralea corylifolia, 8-10 parts of eucommia, 8-10 parts of dipsaccharum officinale, 8-10 parts of drynaria, 8-10 parts of chuanxiong, 8-10 parts of peach kernel, 8-10 parts of safflower, 8-10 parts of white peony root, 8-10 parts of gentiana macrophylla, and 8-10 parts of licorice.

[0009] Preferably, the tonifying and bone-regulating pharmaceutical composition is prepared from the following raw materials in parts by weight: 15 parts of raw rehmannia, 15 parts of cyathula, 10 parts of prepared rehmannia, 10 parts of angelica, 10 parts of millettia reticulata, 10 parts of astragalus, 10 parts of epimedium, 9 parts of epimedium, 9 parts of psoralea corylifolia, 9 parts of eucommia, 9 parts of dipsaccharum officinale, 9 parts of drynaria, 9 parts of chuanxiong, 9 parts of peach kernel, 9 parts of safflower, 9 parts of white peony root, 9 parts of gentiana macrophylla, and 9 parts of liquorice.

[0010] Optionally, in the above application, the preparation product is selected from the following group: external preparations, oral preparations, and injections.

[0011] The second invention point of the present application is to provide a pharmaceutical composition for preventing and treating non-traumatic femoral head necrosis, which is prepared from the following raw materials in parts by weight: 10-20 parts of raw rehmannia, 10-20 parts of cyathula, 8-12 parts of prepared rehmannia, 8-12 parts of angelica, 8-12 parts of millettia reticulata, 8-12 parts of astragalus, 8-12 parts of epimedium, 8-10 parts of epimedium, 8-10 parts of psoralea corylifolia, 8-10 parts of eucommia bark, 8-10 parts of dipsaccharum officinale, 8-10 parts of drynaria, 8-10 parts of chuanxiong, 8-10 parts of peach kernel, 8-10 parts of safflower, 8-10 parts of white peony root, 8-10 parts of gentiana macrophylla, and 8-10 parts of licorice.

[0012] Preferably, the pharmaceutical composition is prepared from the following raw materials in parts by weight: 15 parts of raw rehmannia, 15 parts of cyathula, 10 parts of prepared rehmannia, 10 parts of angelica, 10 parts of millettia reticulata, 10 parts of astragalus, 10 parts of epimedium, 9 parts of epimedium, 9 parts of psoralea corylifolia, 9 parts of eucommia bark, 9 parts of dipsaccharum officinale, 9 parts of drynaria, 9 parts of chuanxiong, 9 parts of peach kernel, 9 parts of safflower, 9 parts of white peony root, 9 parts of gentiana macrophylla, and 9 parts of licorice.

[0013] King medicine:

[0014] Shengdi: It is sweet and cold in nature, with a cool and moist texture. It can nourish yin and blood, prevent drugs from damaging yin, and can "expel blood stasis and fill the bone marrow."

[0015] Sichuan Achyranthes: Enters the liver and kidney meridians, strengthens tendons and bones, and at the same time can activate blood circulation and dredge meridians, nourishing and promoting circulation.

[0016] Minister medicine:

[0017] Rehmannia root and Chinese angelica: nourish blood and yin, activate blood circulation and produce marrow.

[0018] Millettia reticulata: removes blood stasis, dredges meridians and relieves pain, replenishes blood without stagnation.

[0019] Astragalus: sweet and warm, it enters the spleen. Strengthening the spleen can stimulate the production of qi and blood, and nourish the tendons and bones.

[0020] Epimedium: enters the liver and kidney meridians, nourishes the kidneys and strengthens yang, dispels wind and dampness, promotes blood circulation and dredges menstruation.

[0021] Adjuvant:

[0022] Psoralea corylifolia, Eucommia ulmoides, Dipsacus asper, and Drynaria fortunei assist the main medicine in nourishing the kidneys and strengthening bones, benefiting the marrow and producing essence, and promoting blood circulation and relieving pain.

[0023] Chuanxiong, peach kernel, and safflower: promote blood circulation, remove blood stasis, promote qi and relieve pain.

[0024] White Peony Root: Nourishes the liver and strengthens the yin and tendons.

[0025] Qinjiao: Pungent and dispersing, bitter and purgative, moistening but not dry, it is the moistening agent among wind-clearing drugs, it can dispel pathogenic factors without harming the body, dispel rheumatism, relieve arthritis and pain, dredge blood vessels, relieve joint pain, and at the same time it can clear away dampness and heat.

[0026] Drug:

[0027] Licorice: harmonizes various medicines, relieves pain.

[0028] This prescription, developed based on clinical experience and guided by the pathogenesis theory of "liver and kidney deficiency, blood stasis blocking the collaterals," primarily promotes kidney and bone strengthening, replenishes blood and activates blood circulation, promotes qi and dissolves blood stasis, and relieves pain. Its core principles are promoting blood circulation and dissolving blood stasis, while strengthening the kidneys and bones. It effectively dispels blood stasis without damaging vital energy, achieving the goal of removing blood stasis and promoting new blood. Furthermore, the herbal compatibility within the prescription emphasizes regulating qi. Flowing qi promotes blood circulation, thereby promoting blood circulation throughout the body, nourishing tendons and veins and strengthening bones.

[0029] Optionally, the pharmaceutical composition is prepared in the form of capsules, granules or decoctions.

[0030] The third invention point of the present application is to provide a method for preparing the above-mentioned pharmaceutical composition, which comprises weighing the raw medicines according to the ratio, soaking them in clean water, heating and boiling them, discarding the drug residue, collecting the decoction, taking the supernatant after centrifugation, and freeze-drying the decoction to obtain the lyophilized powder of the pharmaceutical composition.

[0031] Tongbi Shenggu Tang, also known as Huoxue Huayu Tang, is a proven formula developed by Dr. Zhou Mingwang and his team based on the theory of "activating blood circulation and removing blood stasis" and clinical experience. This formula has been used clinically for over 20 years and has proven efficacy in treating NONFH. Tongbi Shenggu Tang is formulated based on the Traditional Chinese Medicine theory of tonifying the kidneys and activating blood circulation, and is derived from Taohong Siwu Tang with modifications. It boasts multiple benefits, including tonifying the kidneys and strengthening bones, replenishing blood and promoting qi, activating blood circulation and removing blood stasis, and dredging the meridians and relieving pain. It dispels blood stasis without damaging vital energy, promoting the regeneration of new blood after the stasis is eliminated. Furthermore, the formula utilizes Qi-regulating methods, as Qi circulation aids blood circulation, thereby promoting blood circulation throughout the body, nourishing tendons and veins, and strengthening bones. This research team conducted in-depth research with the help of network pharmacology and other aspects, and found that Tongbi Shenggu Decoction can exert a therapeutic effect on NONFH through multiple pathways and numerous targets. This prescription may regulate lipid metabolism, affect osteoblast differentiation, osteoblast and osteoclast activity, angiogenesis, and inflammatory immune response, alleviate patients' clinical symptoms, promote bone remodeling and angiogenesis in the necrotic area of ​​the femoral head, and ultimately achieve further repair of the necrotic femoral head.

[0032] Compared with the prior art, the present application has the following advantages: the present application provides a pharmaceutical composition for preventing and treating non-traumatic femoral head necrosis, a preparation method thereof, and an application thereof; experiments have shown that Tongbi Shenggu Decoction and β-oxanthosterone can promote angiogenesis and osteogenesis by increasing the expression of miR-365a-3p, thereby playing a positive role in the treatment of NONFH; a SONFH rabbit model was successfully constructed by combining LPS with MPS, and this model was used to determine that Tongbi Shenggu Decoction can promote bone tissue repair and inhibit fat formation in SONFH model rabbits, and can effectively regulate the imbalance of bone fat metabolism homeostasis to achieve bone fat metabolism balance, suggesting that Tongbi Shenggu Decoction can intervene in the process of SONFH by improving the imbalance of bone fat metabolism homeostasis; at the same time, it was confirmed that the medium-dose group (7.7 g / kg / d) was the optimal dose group; this further demonstrated that Tongbi Shenggu Decoction regulates the dynamic balance of bone and fat through a multi-target synergistic mechanism of "regulating lipid metabolism-promoting osteogenesis-improving microenvironment", and its target is highly consistent with the TCM theory of "removing blood stasis and unblocking meridians-tonifying the kidney and filling the marrow", providing an experimental basis for the treatment of SONFH with TCM. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Shown is a technical roadmap for validating the intervention effect of Tongbi Shenggu Recipe on the angiogenic differentiation of BMECs in non-traumatic femoral head necrosis based on miR-365a-3p in one embodiment of the present application.

[0034] Figure 2 The figure shows the growth of primary bone marrow mesenchymal stem cells in culture for 4 days (40×) in one embodiment of the present application.

[0035] Figure 3The figure shows the growth of primary bone marrow mesenchymal stem cells in culture for 7 days (40×) in one embodiment of the present application.

[0036] Figure 4 The figure shows the growth of bone marrow mesenchymal stem cells at passage 3 in one embodiment of the present application (40×).

[0037] Figure 5 The results of flow cytometric identification of human bone marrow mesenchymal stem cells in one embodiment of the present application are shown; wherein, Figure 5 A is CD34, Figure 5 B is CD44, Figure 5 C stands for CD90.

[0038] Figure 6 Shown is the microscopic growth of third-generation human bone microvascular endothelial cells (40×) in one embodiment of the present application.

[0039] Figure 7 Shown is the result of immunocytochemical staining of human bone microvascular endothelial cells in one embodiment of the present application.

[0040] Figure 8 In one embodiment of the present application, the cell transfection status was observed under an inverted fluorescence microscope.

[0041] Figure 9 Shown are the RT-qPCR verification results after miR-365a-3p transfection in one embodiment of the present application; wherein, CK: blank group, M: miR-365a-3p mimic group, M NC: miR-365a-3p mimic NC, I: miR-365a-3p inhibitor group, I NC: miR-365a-3p inhibitor NC group; vs. blank group, * P < 0.05, ** P < 0.01.

[0042] Figure 10 Shown are the experimental results of Tongbi Shenggu Recipe intervening in bone marrow mesenchymal stem cells CCK8 in one embodiment of the present application.

[0043] Figure 11 The results are shown in an embodiment of the present application, in which β-hypocrea sterol intervenes in the CCK8 of bone marrow mesenchymal stem cells.

[0044] Figure 12 Shown is the observation result of exosomes under electron microscope in one embodiment of the present application; wherein, Figure 12 A is the blank group; Figure 12 B, miR-365a-3p inhibitor group; Figure 12C is the miR-365a-3p mimic group; Figure 12 D is the Tongbi Shenggu prescription group; Figure 12 E is the β-oxadosterone group.

[0045] Figure 13 Shown is the protein expression of CD63 and TSG101 in one embodiment of the present application.

[0046] Figure 14 The expression of miR-365a-3p, BTG2, PTEN and VEGF genes in human bone microvascular endothelial cells in each group is shown in one embodiment of the present application; wherein, Figure 14 A: miR-365a-3p gene expression statistics, Figure 14 B: BTG2 gene expression statistics, Figure 14 C: PTEN gene expression statistics, Figure 14 D: Statistical graph of VEGF gene expression; CK: blank group, I: miR-365a-3p inhibitor group, M: miR-365a-3p mimetic group, ZY: Tongbi Shenggu prescription group, DT: β-oxadiasterone group; * P < 0.05, ** P < 0.01.

[0047] Figure 15 Shown is the expression of BTG2, PTEN and VEGF proteins in each group of human bone microvascular endothelial cells in one embodiment of the present application.

[0048] Figure 16 As shown in one embodiment of the present application, Figure 15 Statistical data of BTG2, PTEN and VEGF protein expression in human bone microvascular endothelial cells of each group; Figure 16 A: BTG2 protein relative expression statistics, Figure 16 B: Statistical graph of relative expression of PTEN protein, Figure 16 C: Statistical graph of relative expression of VEGF protein; CK: blank group, I: miR-365a-3p inhibitor group, M: miR-365a-3p mimetic group, ZY: Tongbi Shenggu prescription group, DT: β-oxadiasterone group, * P < 0.05, ** P < 0.01.

[0049] Figure 17 Shown is a technical roadmap for verifying the intervention effect of Tongbi Shenggu Recipe on the osteogenic differentiation of BMSCs in non-traumatic femoral head necrosis based on miR-365a-3p in one embodiment of the present application.

[0050] Figure 18 Shown are the results of alizarin red staining of each group in one embodiment of the present application; wherein, Figure 18 A: blank group, Figure 18B: miR-365a-3p inhibitor group, Figure 18 C: miR-365a-3pmimic (mimic) group, Figure 18 D: Tongbi Shenggu prescription group, Figure 18 E: β-hysterone group.

[0051] Figure 19 The expression of miR-365a-3p, OCN, OPN and Runx2 genes in human bone microvascular endothelial cells in each group is shown in one embodiment of the present application; wherein, Figure 19 A: miR-365a-3p gene expression statistics, Figure 19 B: OPN gene expression statistics, Figure 19 C: OCN gene expression statistics, Figure 19 D: Runx2 gene expression statistics; CK: blank group, I: miR-365a-3p inhibitor group, M: miR-365a-3p mimetic group, ZY: Tongbi Shenggu prescription group, DT: β-oxadiasterone group; * P < 0.05, ** P < 0.01.

[0052] Figure 20 The expression of OPN, OCN and Runx2 proteins in human bone marrow mesenchymal stem cells in each group in one embodiment of the present application is shown; among them, CK: blank group, I: miR-365a-3p inhibitor group, M: miR-365a-3p mimetic group, ZY: Tongbi Shenggu prescription group, DT: β-oxanthellone group.

[0053] Figure 21 As shown in one embodiment of the present application, Figure 20 The protein expression data statistics of OPN, OCN and Runx2 protein expression in each group of human bone marrow mesenchymal stem cells are shown; Figure 21 A: Statistical graph of relative expression of OPN protein, Figure 21 B: OCN protein relative expression statistics, Figure 21 C: Statistical graph of relative expression of Runx2 protein; * P < 0.05, ** P < 0.01.

[0054] Figure 22 Shown is an experimental flow chart of an animal experiment in one embodiment of the present application.

[0055] Figure 23 Shown is the weight change of model animals in one embodiment of the present application; ** indicates P < 0.01; * indicates P < 0.05; colors are shown in the figure: black indicates the blank group, and red indicates the model group.

[0056] Figure 24Shown is the change in body temperature of model animals in one embodiment of the present application; ** indicates P < 0.01; colors are as shown in the figure: black indicates the blank group, and red indicates the model group.

[0057] Figure 25 The figure shows the weekly weight changes of animals in each group during the oral gavage process in one embodiment of the present application.

[0058] Figure 26 Shown is a general view of different groups of femoral heads in one embodiment of the present application; wherein, Figure 26 A-F represent the control group, model group, rosuvastatin group, and Tongbi Shenggu prescription low-dose, medium-dose, and high-dose groups, respectively.

[0059] Figure 27 Shown are MRI examination images of rabbits in different groups in one embodiment of the present application; wherein KG ( Figure 27 A, Figure 27 B), MG ( Figure 27 C, Figure 27 D), RG ( Figure 27 E, Figure 27 F), L-TSFG ( Figure 27 G. Figure 27 H), M-TSFG ( Figure 27 I. Figure 27 J) and H-TSFG ( Figure 27 K. Figure 27 L) Representative images of MRI scans of bilateral femoral heads in rabbits; the red circle represents the location of the femoral head, and the yellow arrow indicates slight collapse and flattening of the femoral head with abnormal signals.

[0060] Figure 28 Shown are ultrasound quantitative examination images of liver fat in different groups of rabbits in one embodiment of the present application; Figure 28 A is the blank group; Figure 28 B is the model group; Figure 28 C, rosuvastatin group; Figure 28 D is the low-dose group of Tongbi Shenggu prescription; Figure 28 E is the medium dose group of Tongbi Shenggu prescription; Figure 28 F is the high-dose group of Tongbi Shenggu prescription.

[0061] Figure 29 Shown are the liver fat CAP values ​​of rabbits in the same group in one embodiment of the present application; where * represents P < 0.05; **** represents P < 0.0001.

[0062] Figure 30 Shown are the CT and 3D reconstruction images of the femoral heads of rabbits in different groups in one embodiment of the present application; Figure 30 A is the blank group; Figure 30 B is the model group; Figure 30C, rosuvastatin group; Figure 30 D is the low-dose group of Tongbi Shenggu prescription; Figure 30 E is the medium dose group of Tongbi Shenggu prescription; Figure 30 F is the high-dose group of Tongbi Shenggu prescription.

[0063] Figure 31 Shown are the bone tissue parameters of each group in one embodiment of the present application.

[0064] Figure 32 The figure shows the pathological changes of the femoral head of SONFH rabbits treated with Tongbi Shenggu Recipe in one embodiment of the present application (×40, ×200); wherein, Figure 32 A is the blank group; Figure 32 B is the model group; Figure 32 C, rosuvastatin group; Figure 32 D is the low-dose group of Tongbi Shenggu prescription; Figure 32 E is the medium dose group of Tongbi Shenggu prescription; Figure 32 F is the high-dose group of Tongbi Shenggu Fang; black arrows indicate trabeculae; green arrows indicate normal osteocytes; red arrows indicate empty bone lacunas; and yellow arrows indicate adipocytes.

[0065] Figure 33 The figure shows the empty bone pit rate of each group in one embodiment of the present application; where * represents P < 0.05; *** represents P < 0.001.

[0066] Figure 34 The figure shows the ELISA analysis of osteogenesis-related factors in the serum of rabbits of different groups in one embodiment of the present application; wherein, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001.

[0067] Figure 35 The figure shows the ELISA analysis of adipogenesis-related factors in the serum of rabbits from different groups in one embodiment of the present application; *** represents P < 0.001.

[0068] Figure 36 Shown are tetracycline-calcein dual fluorescence labels (×100) for each group in one embodiment of the present application; namely, blank group, model group, rosuvastatin group, Tongbi Shenggu Fang low-dose group, Tongbi Shenggu Fang medium-dose group, and Tongbi Shenggu Fang high-dose group; the red arrows indicate the intervals between the dual fluorescences.

[0069] Figure 37 Shown are the tetracycline-calcein dual fluorescence labeling bone dynamics-related parameters of each group in one embodiment of the present application; wherein, ** indicates P < 0.01; *** indicates P < 0.001. DETAILED DESCRIPTION

[0070] To make the purpose, technical solutions and advantages of this application more clear, this application is further described in detail below. However, it should be understood that the description herein is only used to explain this application and is not intended to limit the scope of this application.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are intended solely for the purpose of describing specific embodiments and are not intended to limit this application. The reagents and instruments used herein are commercially available, and the characterization methods involved can be found in the relevant descriptions in the prior art and will not be further elaborated herein.

[0072] In order to further understand the present application, the present application is further described in detail below in conjunction with the best embodiment.

[0073] Example 1

[0074] The intervention effect of Tongbi Shenggu Recipe on the angiogenic differentiation of BMECs in non-traumatic femoral head necrosis was verified based on miR-365a-3p:

[0075] 1. Materials

[0076] 1.1 Cell sources and characteristics

[0077] 1.1.1 Human bone marrow mesenchymal stem cells, obtained by isolation and extraction from the femoral heads of selected patients undergoing surgical treatment for osteonecrosis of the femoral head;

[0078] 1.1.2 Human bone microvascular endothelial cells, purchased from Laibaiha Biotechnology; catalog number YH-Y122;

[0079] 1.2 Reagents and consumables

[0080] Table 1 shows the experimental reagents and consumables.

[0081] Table 1

[0082]

[0083] 2 Methods

[0084] 2.1 Cell culture

[0085] 2.1.1 BMSCs extraction

[0086] 1) Bone Block Processing: Store clinically obtained bone blocks in 20% FBS MEM medium and transfer to the laboratory as soon as possible. Rinse the blood from the bone block with PBS until it is completely washed away. Cut the bone block into small pieces and rinse again with PBS.

[0087] 2) Adding culture medium: Add 1 ml of culture medium to a culture dish and transfer the treated bone to it. Place the dish in an incubator and let it sit for 1 hour to allow the bone to slightly adhere to the wall. Then gently add 4-5 ml of culture medium.

[0088] 2.1.2 BMSCs identification—Flow cytometry detection of cluster of differentiation antigens 34, 44, and 90 (CD34, CD44, and CD90).

[0089] 2.1.3 After culture, BMSCs and BMECs are passaged, cryopreserved, revived, counted, and plated;

[0090] 2.1.4 Cellular intervention

[0091] Gansu Provincial Hospital of Traditional Chinese Medicine purchased Tongbi Shenggu Fang slices, placed them in a container according to the original recipe ratio, and added an appropriate amount of clean water to soak them, ensuring that the medicinal materials were fully exposed to water. After the slices were soaked, the soaked medicinal materials were placed in a heating device and boiled, and the boiling state was maintained for 30 minutes to allow the active ingredients of the medicinal materials to be fully dissolved. After boiling, the remaining medicinal residues were discarded and the resulting decoction was collected. Subsequently, the collected decoction was centrifuged at a speed of 2000r / min, the centrifugal radius was set to 125mm, the centrifugal duration was 10 minutes, and the supernatant was taken after centrifugation. Then, the obtained supernatant was transferred to a freeze dryer and operated according to the predetermined freeze-drying procedure. After a series of processes such as freezing, vacuuming, and sublimation drying, freeze-dried powder was finally obtained. After the experiment, the prepared freeze-dried powder was properly stored in a refrigerator at -20°C to ensure the stability and activity of its active ingredients, providing a reliable material basis for subsequent experimental research or clinical application.

[0092] 2.1.4.2 Preparation of liquid medicine

[0093] Take an appropriate amount of the Tongbi Shenggu Fang freeze-dried powder prepared in advance, add it to the culture medium to fully dissolve it, then filter and sterilize it using a bacterial filter to make a Tongbi Shenggu Fang liquid with a concentration of 500 μg / mL, and then dilute the prepared Tongbi Shenggu Fang liquid according to different concentrations.

[0094] 2.1.4.3 Cell proliferation and toxicity assay (CCK8)

[0095] BMSCs were seeded in a 96-well plate at a density of 3,000 cells per well in advance. Drug intervention was performed after the cell density reached more than 50%, and the OD values ​​at a wavelength of 450 nm were detected at 24 h, 48 h, and 72 h. 100 μL of PBS solution was added to the edge of each plate. The second column was specially set up as a blank control group, which included six test wells containing only Opti-MEM culture medium. Ten different concentration gradients were set. The concentrations of the Tongbi Shenggu Fang group were 0 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 125 μg / mL, 150 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL and 500 μg / mL, respectively; the concentrations of the β-oxanthone group were 0 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 400 μg / mL and 500 μg / mL from the third column, respectively. Each group had 6 replicate wells.

[0096] 2.2 Cell transfection

[0097] 2.2.1 After cell transfection, the efficiency was verified by RT-qPCR

[0098] 2.2.1.1 RNA extraction

[0099] 2.2.1.2 RT-qPCR

[0100] 1) RNA extracted from the treated cells was tested for concentration and purity in each group and the loading amount was calculated based on the experimental requirements;

[0101] 2) Experimental design: blank control group, miR-365a-3p overexpression (mimic) group, miRNA mimic NC group, miR-365a-3p inhibition (inhibitor) group, and miRNA inhibitor NC group;

[0102] 3) The Poly(A) Tailing reaction system should be prepared on ice. The Poly(A) Tailing reaction system is shown in Table 2.

[0103] Table 2

[0104]

[0105] 4) Mix thoroughly and centrifuge, then place in a TOUCH gene amplifier and incubate at 37°C for 1 hour;

[0106] 5) Next, prepare the reverse transcription reaction system on ice. See Table 3 for the reverse transcription reaction system.

[0107] Table 3

[0108]

[0109] 6) Mix well, centrifuge, and incubate at 42°C for 1 hour;

[0110] 7) Store the resulting 20 μl cDNA on ice;

[0111] 8) The RT-qPCR reaction system should also be prepared on ice. The RT-qPCR reaction system is shown in Table 4.

[0112] Table 4

[0113]

[0114] 9) When setting up RT-qPCR reactions, add miDETECTA Track™ U6 Forward Primer (10 μM) to the internal control group and miDETECTA Track™ miR-365a-3p Forward Primer (also 10 μM) to the miRNA group.

[0115] 10) Three replicate wells were set up for each experiment to increase data reliability. Finally, a three-step RT-qPCR reaction was performed. The RT-qPCR reaction procedure is shown in Table 5.

[0116] Table 5

[0117]

[0118] 11) Analyze the data and calculate the expression level.

[0119] 2.3 Exosomes

[0120] Exos were extracted and identified.

[0121] 2.4 Co-culture of BMSCs-Exos with BMECs

[0122] A cell culture tank with a semipermeable membrane was used in combination with a 6-well plate for non-contact co-culture induction. The third generation vascular endothelial cells were seeded in a 6-well plate, and the extracted BMSCs-derived Exos were added at a concentration of 1×10 5 Each well was inoculated into the culture tank, with the initial ratio of the two cells being 1:5. Low-glucose DMEM culture medium containing 10% fetal bovine serum was added, and the medium was changed every three days for a total of 14 days.

[0123] 2.5 RT-qPCR detection of gene expression levels in each group

[0124] 2.5.1 Cell RNA extraction

[0125] 2.5.2 After co-culture, the gene expressions of miR-365a-3p, BTG2, PTEN, and VEGF were detected in each group. The primer sequence information is shown in Table 6;

[0126] Table 6

[0127]

[0128] Calculate the loading amount and dilute the primers based on RNA purity and concentration, see Table 7;

[0129] Table 7

[0130]

[0131] 2) Set up blank group, miR-365a-3p inhibitor group, miR-365a-3p mimic group, Tongbi Shenggu prescription group, and β-oxanthellone group;

[0132] 3) Then proceed to the cDNA synthesis step;

[0133] Table 8 Genomic DNA reaction

[0134]

[0135] 4) Thoroughly mix the above reaction system. For specific components, see Table 1.11.

[0136] 5) Centrifugation;

[0137] 6) Place in a TOUCH gene amplification instrument and perform the reaction at 42°C for 5 minutes;

[0138] 7) Ice configuration, see Table 9;

[0139] Table 9 Reverse transcription reaction system

[0140]

[0141] 8) Mix thoroughly, centrifuge, and incubate at 42°C for 1 hour.

[0142] After the reaction, the system was heated to 72°C and incubated for 10 minutes;

[0143] 10) After completion, store 20 μL of cDNA on ice for later use.

[0144] 11) Prepare the RT-qPCR reaction system on ice, see Table 10;

[0145] Table 10 RT-qPCR reaction system (20 μL system)

[0146]

[0147] 12) Thoroughly mix the RT-qPCR reaction system and set up three replicate wells for each experiment;

[0148] 13) Slightly shake to eliminate air bubbles and then centrifuge;

[0149] 14) RT-qPCR reaction of BTG2, PTEN, and VEGF genes was performed using a two-step method. Specific conditions are shown in Table 11.

[0150] 15) For miR-365a-3p, a three-step RT-qPCR reaction was performed. Specific conditions are shown in Table 12.

[0151] Table 11 Runx2, OCN, OPN - RT-qPCR cycling program

[0152]

[0153] Table 12 RT-qPCR cycling program for miR-365a-3p

[0154]

[0155] Repeat the experiment, analyze the data, and calculate the expression level.

[0156] 2.6 Western blotting to detect protein expression levels in each group

[0157] 2.6.1 Protein extraction

[0158] 2.6.2 Protein concentration detection (BCA method)

[0159] 2.6.3 Denatured proteins

[0160] 2.6.4 WB experiment

[0161] 2.7 Statistical methods

[0162] All experimental data were statistically analyzed using SPSS 27.0 software. The measured data were expressed as mean ± standard deviation (±s). Ordinary one-way ANOVA was used to compare the means among multiple groups. P < 0.05 indicated statistical significance.

[0163] 3 Technical routes such as Figure 1 shown.

[0164] 4 Results

[0165] 4.1 Identification of BMSCs and BMECs

[0166] 4.1.1 Identification of bone marrow mesenchymal stem cells (BMSCs)

[0167] 1) Morphological identification

[0168] On the fourth day of primary cell culture, spindle-shaped cells of varying sizes adhering to the wall were occasionally observed under the microscope. Figure 2 On the 7th day, the cell density increased significantly, about 50%, and the cells were arranged in a fibroblast-like manner with a long spindle-shaped morphology. Figure 3 ; The cells were subcultured to the third generation and showed a whirlpool-like growth with large, clear, spindle-shaped nuclei. The cell morphology tended to be uniform. Figure 4 .

[0169] 2) Surface marker identification

[0170] Flow cytometry was used to identify surface markers. The results showed that the positive rates of CD34 and CD44 were 0.10% and 0.30%, respectively, indicating low expression; the positive rate of CD90 was 95.1%, indicating high expression. The cultured cells were BMSCs. Figure 5 .

[0171] 4.1.2 Identification of bone microvascular endothelial cells (BMECs)

[0172] 1) Morphological identification

[0173] The cells are cobblestone-like, polygonal, and spindle-shaped, with large and clear nuclei. Figure 6 ;

[0174] 2) Identification of specific markers

[0175] The expression of CD34 was detected by immunocytochemical staining, and the positive cell rate exceeded 99%. After adding hematoxylin staining solution, the cytoplasm remained brown-red and the nucleus was blue, which was identified as BMECs. Figure 7 .

[0176] 4.2 Cell transfection efficiency assessment

[0177] The third generation BMSCs were seeded into cell culture plates and the transfection efficiency was determined at a cell density of 50%. The transfection efficiency was observed under an inverted fluorescence microscope: the general morphological characteristics and growth status of the transfected BMSCs were observed under white light; the transfection efficiency of the cells was evaluated based on the fluorescence intensity and the proportion of positive cells under green fluorescence; the morphology, number and distribution of cell nuclei were observed under blue fluorescence; and the number and area of ​​apoptotic or necrotic cells were monitored under red fluorescence. At 48 hours, the transfection efficiency of miRNA was the highest, as shown in Figure 2. Figure 8 .

[0178] Figure 8 After the intervention of transfection reagent, Figure 8 A is the morphology of BMSCs at 12h. Figure 8 E is the morphology of BMSCs at 24 hours. Figure 8 I is the morphology of BMSCs at 48 h; Figure 8 B is the fluorescence transfection efficiency at 12 h, Figure 8 F is the fluorescence transfection efficiency at 24 h, Figure 8 J is the fluorescence transfection efficiency at 48 h; Figure 8 C is the staining of cell nuclei after 12 hours. Figure 8 G is the staining of cell nuclei after 24 hours. Figure 8 K is the staining of cell nuclei after 48 hours, Figure 8 D is the cell death of hBMSCs at 24h. Figure 8 H is the cell death of BMSCs after 24 hours. Figure 8 L is the cell death of BMSCs after 48 hours.

[0179] 4.3 RT-qPCR identification of cell transfection

[0180] 4.3.1 RNA extraction results

[0181] Table 13 RNA extraction purity and concentration

[0182]

[0183] 4.3.2 RT-qPCR test results

[0184] In the cell transfection identification experiment, the detection of transfection efficiency showed that the efficiency reached its peak at 48 hours after transfection. RT-qPCR detection was performed by extracting RNA from each group of cells 48 hours after transfection. The results showed that the expression level of miR-365a-3p was significantly upregulated in the cell group transfected with the mimic (P<0.05); in contrast, the expression level of miR-365a-3p was significantly downregulated in the cell group transfected with the inhibitor (P<0.05). The difference between the two groups of data was significant and statistically significant. Figure 9 .

[0185] 4.4 Optimal Dosage of Tongbi Shenggu Recipe Freeze-dried Powder and β-Achyranthesin for Intervention of BMSCs

[0186] 4.4.1 Optimal Dose of Tongbi Shenggu Recipe Freeze-dried Powder for BMSCs Intervention

[0187] The OD values ​​were measured 24h, 48h, and 72h after drug intervention. Figure 10 According to the CCK8 test results, the optimal drug dose of Tongbi Shenggu Fang freeze-dried powder for BMSCs intervention was finally determined to be 150 μg / mL (P<0.001), and the intervention time was 48 hours.

[0188] 4.4.2 Optimal dose of β-hypocretin for BMSCs intervention

[0189] The third generation BMSCs were plated in 96-well plates and then the cell proliferation assay was performed to observe the intervention effects of β-oxaloacetate at four time periods: 6h, 24h, 48h, and 72h. Figure 11 According to the CCK8 test results, the optimal drug dose of β-acetosterol for BMSCs intervention was finally determined to be 100 μg / mL (P<0.001), and the intervention time was 48 hours.

[0190] 4.5 Exos identification results

[0191] 1) Transmission electron microscopy

[0192] like Figure 12 As shown in the figure, under transmission electron microscopy, it has a clear membrane boundary, the center of the vesicle is slightly concave, and it presents a typical saucer-like morphological feature, which is an exosome.

[0193] 2) Western blot detection of molecular markers

[0194] There was no expression of the exosome surface marker protein CD63, but there was expression of the surface marker protein TSG101, indicating that the product obtained by ultracentrifugation was exosome. Figure 13 .

[0195] 4.6 Expression of miR-365a-3p, BTG2, PTEN, and VEGF genes after Tongbi Shenggu prescription intervention

[0196] After Tongbi Shenggu prescription intervened in human bone microvascular endothelial cells, the expression of miR-365a-3p, PTEN and VEGF in the blank group was significantly decreased compared with the miR-365a-3p inhibitor group, Tongbi Shenggu prescription group and β-oxadiasterone group (P < 0.01), and the expression of miR-365a-3p, PTEN and VEGF in the blank group was significantly increased compared with the miR-365a-3p mimic group (P < 0.05); the expression of miR-365a-3p, PTEN and VEGF in the blank group was significantly increased compared with the miR- BTG2 expression was significantly increased in the miR-365a-3p inhibitor, Tongbi Shenggu recipe, and β-oxidosterone groups (P < 0.01), while BTG2 expression was significantly decreased in the blank group compared with the miR-365a-3p mimic group (P < 0.01). The Tongbi Shenggu recipe group showed increased miR-365a-3p, PTEN, and VEGF expressions compared with the β-oxidosterone group (P < 0.01), while the Tongbi Shenggu recipe group showed decreased BTG2 expression compared with the β-oxidosterone group. This suggests that the Tongbi Shenggu recipe can upregulate the gene expression of miR-365a-3p, PTEN, and VEGF, and downregulate the expression of the BTG2 gene.

[0197] 1) RNA extraction

[0198] Table 14 RNA extraction RNA extraction purity and concentration

[0199]

[0200] 2) Gene expression

[0201] The expression of miR-365a-3p, BTG2, PTEN and VEGF genes in human bone microvascular endothelial cells in each group Figure 14 shown.

[0202] 4.7 Expression of BTG2, PTEN, and VEGF proteins after Tongbi Shenggu prescription intervention

[0203] WB test results showed that compared with the blank group, the expression of PTEN and VEGF proteins in the miR-365a-3p inhibitor group, Tongbi Shenggu Fang group, and β-oxadiasterone group were significantly increased (P<0.01). Compared with the blank group, the expression of PTEN and VEGF proteins in the miR-365a-3p mimic group was significantly decreased (P<0.01). Compared with the blank group, the expression of BTG2 protein in the miR-365a-3p inhibitor group, Tongbi Shenggu Fang group, and β-oxadiasterone group was significantly decreased (P<0.01). Compared with the blank group, the expression of BTG2 protein in the miR-365a-3p mimic group was significantly increased (P<0.01). The expression of PTEN and VEGF in the Tongbi Shenggu Fang group was increased compared with the β-oxadiasterone group (P<0.01). The expression of BTG2 in the Tongbi Shenggu Fang group was decreased compared with the β-oxadiasterone group. That is, Tongbi Shenggu Recipe can upregulate the protein expression of PTEN and VEGF, and downregulate the protein expression of BTG2.

[0204] The protein expression and protein expression statistics of BTG2, PTEN and VEGF in human bone microvascular endothelial cells of each group are as follows Figure 15 、 Figure 16 shown.

[0205] 4.8 Summary

[0206] The gene expression and protein expression trends of BTG2, PTEN, and VEGF were consistent. The above research data showed that Tongbi Shenggu Recipe can inhibit the expression of BTG2, and when BTG2 is upregulated, the function of angiogenic growth factors (VEGF and VEGFR) is inhibited. Tongbi Shenggu Recipe can promote angiogenesis by inhibiting the expression of BTG2; Tongbi Shenggu Recipe can increase the expression of PTEN and VEGF proteins in BMECs, thereby promoting angiogenesis.

[0207] Example 2

[0208] The intervention effect of Tongbi Shenggu Recipe on osteogenic differentiation of BMSCs in non-traumatic femoral head necrosis was verified based on miR-365a-3p:

[0209] 1. Materials

[0210] 1.1 Cell sources and characteristics

[0211] Human bone marrow mesenchymal stem cells, the source and characteristics are the same as those in Example 1.

[0212] 1.2 Instruments and Equipment

[0213] Same as Example 1.

[0214] 1.3 Reagents and consumables

[0215] Table 15 Experimental reagents and consumables

[0216]

[0217] 2 Methods

[0218] 2.1 Cell culture

[0219] Human BMSCs were subcultured to passage 3 for subsequent experiments.

[0220] 2.2 Cell seeding

[0221] 1) Gelatin-coated culture vessels: Cover the bottom of the culture vessel with 0.1% gelatin solution, incubate at 37°C for 30 minutes, remove excess liquid, and allow to dry before use.

[0222] 2) Inoculation of stem cells: Digest the cells according to the passaging method, rinse and allow the cells to settle to the bottom of the centrifuge tube. Add a certain amount of complete culture medium to the centrifuge tube, then add pre-chilled cell freezing solution drop by drop, gently shaking the centrifuge tube while adding. Aliquot the cell suspension into cryopreservation tubes, and label them with information such as cell name, generation number, freezing date, and name.

[0223] 2.3 Induction of BMSCs Osteogenic Differentiation

[0224] 2.3.1 Culture medium preparation

[0225] Table 16 Human bone marrow mesenchymal stem cell osteogenic differentiation medium

[0226]

[0227] 2.3.2 Induction of BMSCs Osteogenic Differentiation

[0228] The cells were cultured at 37°C and 5% CO2 for 21 days. The osteogenic differentiation medium was replaced every 2 to 3 days. The cell morphology, calcium salt crystal precipitation, and calcium nodule formation were observed. After 21 days, staining was performed for identification.

[0229] 2.4 Alizarin red staining.

[0230] 2.5 RT-qPCR detection of gene expression levels of miR-365a-3p, Runx2, OCN, and OPN in each group:

[0231] 2.5.1 Cellular RNA extraction

[0232] 2.5.2 Detection of miR-365a-3p, Runx2, OCN, and OPN gene expression in BMSCs after osteogenic differentiation

[0233] Table 17 Primer sequence information

[0234]

[0235] 1) After routine disinfection, turn on the ice machine and use an ultra-micro UV-visible spectrophotometer to check RNA purity and concentration. Calculate the sample loading amount and dilute the primers as shown in Table 18.

[0236] Table 18 Primer dilution

[0237]

[0238] 2) Set up blank group, miR-365a-3p inhibitor group, miR-365a-3p mimic group, Tongbi Shenggu prescription group, and β-oxanthellone group;

[0239] 2.6 Western blot detection of OPN, OCN, and Runx2 expression levels in each group;

[0240] The method was the same as in Example 1, wherein the dilution ratio of OPN, OCN and Runx2 was 1:1000, and the dilution ratio of GAPDH was 1:5000;

[0241] 2.7 Statistical methods

[0242] All experimental data were statistically analyzed using SPSS 27.0 software. The measured data were expressed as mean ± standard deviation (±s). Ordinary one-way ANOVA was used to compare the means among multiple groups. P < 0.05 indicated statistical significance.

[0243] 3 Technology Roadmap

[0244] like Figure 17 shown.

[0245] 4 Results

[0246] 4.1 Alizarin Red Staining

[0247] Under the intervention of different methods, BMSCs underwent osteogenic differentiation for 21 days. Microscopic observation showed the gradual formation of mineralized nodules. After staining with alizarin red dye, the mineralized nodules appeared as clear red particles under the microscope. By dissolving the mineralized nodules, semi-quantitative analysis of the mineralized nodules showed that compared with the blank group, the number of mineralized nodules formed in the miR-365a-3p inhibitor group, Tongbi Shenggu Fang group, and β-oxanthellone group after 21 days of osteogenic induction was significantly increased, while the number in the miR-365a-3p mimic group was significantly decreased. The results of alizarin red staining of each group are shown in Figure 2. Figure 18 shown.

[0248] 4.2 RT-qPCR detection of miR-365a-3p, OPN, OCN, and Runx2 gene expression levels in each group

[0249] After the Tongbi Shenggu formula was used to treat human bone marrow mesenchymal stem cells, the expression of miR-365a-3p, OPN, OCN, and Runx2 was significantly decreased in the blank group compared with the miR-365a-3p inhibitor group, the Tongbi Shenggu formula group, and the β-oxadiasterone group (P < 0.01). The expression of miR-365a-3p, OPN, OCN, and Runx2 in the blank group was significantly increased compared with the miR-365a-3p mimic group (P < 0.05). The expression of miR-365a-3p, OPN, OCN, and Runx2 in the Tongbi Shenggu formula group was increased compared with the β-oxadiasterone group (P < 0.01). In other words, the Tongbi Shenggu formula can upregulate the gene expression of miR-365a-3p, OPN, OCN, and Runx2.

[0250] 1) RNA extraction

[0251] Table 19 RNA extraction RNA extraction purity and concentration

[0252]

[0253] 2) Gene expression

[0254] The expression of miR-365a-3p, OCN, OPN and Runx2 genes in human bone microvascular endothelial cells in each group are shown in Figure 2 Figure 19 shown.

[0255] 4.3 Western blot detection of OPN, OCN, and Runx2 protein expression levels in each group

[0256] Western blot analysis showed that compared with the blank group, the miR-365a-3p inhibitor, Tongbi Shenggu formula, and β-oxanthellate sterol groups significantly increased the expression of OPN, OCN, and Runx2 proteins (P < 0.05). However, the miR-365a-3p mimic group significantly decreased the expression of OPN, OCN, and Runx2 proteins (P < 0.01). Furthermore, the expression of OPN, OCN, and Runx2 in the Tongbi Shenggu formula group was significantly higher than that in the β-oxanthellate sterol group (P < 0.01). This suggests that the Tongbi Shenggu formula can upregulate the protein expression of OPN, OCN, and Runx2.

[0257] The expression of OPN, OCN and Runx2 proteins in human bone marrow mesenchymal stem cells in each group is shown in Figure 2. Figure 20 The protein expression results are shown in Figure 21 shown.

[0258] 4.4 Summary

[0259] The gene expression of OPN, OCN and Runx2 was consistent with the protein expression trend. The above research data showed that Tongbi Shenggu recipe can promote the expression of OPN, OCN and Runx2 and thus promote osteogenesis.

[0260] Example 3

[0261] This application studies the mechanism of Tongbi Shenggu Decoction on bone-fat metabolism imbalance in rabbits with steroid-induced osteonecrosis of the femoral head (SONFH) model:

[0262] 1. Materials

[0263] 1.1 Animals

[0264] Forty-seven 24-week-old SPF New Zealand white rabbits were used in this experiment. They were provided by the Laboratory Animal Center of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Laboratory Animal License No. SCXK[Gan]2020-0002) and weighed (2.5±0.5) kg. They were housed at the Laboratory Animal Center of the 940th Hospital of the Joint Logistics Support Force. The rabbits were acclimated for one week before the experiment. During the formal experiment, they were housed in separate cages on a standard diet, one rabbit per cage, at room temperature of 20–28°C, relative humidity of 40%–60%, with air circulation and free access to food and water. All animal care and experimental studies adhered to the relevant in vivo animal experimentation guidelines in the International Association of Veterinary Editors' Consensus on Authors' Guidelines for Animal Ethics and Welfare and the Guidelines for Reporting Animal Research.

[0265] 1.2 Drugs and reagents

[0266] Table 20 Drugs and reagents required for the experiment

[0267]

[0268] Among them, chloral hydrate is only used to sedate experimental rabbits during imaging examinations.

[0269] 2 Experimental methods

[0270] 2.1 Modeling

[0271] A SONFH rabbit model was established using lipopolysaccharide (LPS) combined with methylprednisolone sodium succinate (MPS) for 4 weeks. The specific procedures are as follows:

[0272] Forty New Zealand white rabbits were weighed and numbered and injected with LPS (10 μg / kg) twice via the marginal ear vein, 24 hours apart. The first injection was administered on the first day. Immediately following the second LPS injection, MPS (20 mg / kg) was injected into the gluteus medius muscle once every 24 hours for a total of three injections, alternating between the gluteus medius and gluteus medius muscles. All injection doses were adjusted in real time based on the latest body weight before each administration. The model was considered complete two weeks after the last MPS injection. During the modeling period, vital signs and weight changes were monitored daily, and any deaths were promptly replaced.

[0273] A control group was set up, in which 7 New Zealand white rabbits were given equal volumes of normal saline at the same time intervals and injection routes: two injections into the marginal ear vein (10 μg / kg) and three injections into the gluteal muscles (20 mg / kg).

[0274] After the modeling was completed, one animal was randomly selected from the blank group and one animal was randomly selected from the model group for HE staining to detect the success of the modeling. At the same time, one animal was randomly selected for a preliminary experiment of double-fluorescence labeling of bone tissue.

[0275] 2.2 Preparation of drug solution

[0276] 2.2.1 Preparation of Tongbi Shenggu Recipe Liquid

[0277] 2.2.1.1 Research basis and dosage design

[0278] The Tongbi Shenggu Recipe (composed of Epimedium, Cyathula, Rehmannia root, Angelica sinensis, Peach kernel, Eucommia bark, Psoralea corylifolia, and Licorice root) is an empirical formula developed by our team. All medicinal ingredients were purchased from the Gansu Provincial Hospital of Traditional Chinese Medicine's pharmacy, processed through a standardized decoction and concentration process, and stored at 4°C. Based on the body surface area conversion principle outlined in the "Methodology of Pharmacological Research in Traditional Chinese Medicine," the following were determined:

[0279] Clinical equivalent dose: Adult (70 kg) daily crude drug dosage: 165 g;

[0280] Animal conversion factor: rabbit (1.5 kg) equivalent dose 7.7 g / kg;

[0281] Dose gradient setting:

[0282] Medium dose: 7.7 g / kg;

[0283] Low dose: 3.85 g / kg (1 / 2 of the medium dose);

[0284] High dose: 15.4 g / kg (twice the medium dose).

[0285] 2.2.1.2 Drug solution preparation process

[0286] Basic treatment: Take a certain amount of Chinese herbal medicine slices and place them in a stainless steel pot, add pure water to immerse the herbs; soak for 1 hour and then boil for 1.5 hours; filter the medicinal liquid and concentrate it using a rotary evaporator.

[0287] Repackaging and storage: After fully mixing the drug solutions of different concentrations, dispense them into measuring cups, mark the concentration and date, and store in a refrigerator at 4℃.

[0288] 2.2.1.3 Preparation parameters for each dose group (e.g. 21)

[0289] Table 21 Preparation parameters of each dosage group of Chinese herbal compound

[0290]

[0291] 2.2.1.4 Key points

[0292] (1) Standardized gavage volume: 20 ml / kg / day;

[0293] (2) Batch consistency control: All drug solutions are prepared at one time based on two weeks’ supply;

[0294] (3) Stability assurance: Concentration and packaging should be completed within 2 hours after decoction;

[0295] (4) Labeling system: clearly mark the concentration gradient (low, medium, high), preparation date and expiration date.

[0296] 2.2.2 Preparation of Rosuvastatin Suspension*

[0297] Referring to the equivalent dose formula based on the body surface area of ​​humans and rabbits, the clinical dose of rosuvastatin is 20 mg / d, and the corresponding conversion coefficient for experimental animals (rabbits 1.5 kg) is 0.07. Therefore, the equivalent dose for rabbits is 0.94 mg / kg. Based on the gavage volume of 20 ml / kg of animal body weight, once a day, the concentration of the suspension preparation should be 0.05 mg / ml.

[0298] Weigh 10 mg of rosuvastatin using an electronic balance. Measure 200 mL of normal saline using a graduated cylinder and pour it into a 250 mL beaker. Slowly add the weighed rosuvastatin to the saline, stirring with a magnetic stirrer to prevent clumping. Stir until completely dissolved, and adjust the solution to 0.05 mg / mL. Aliquot the prepared suspension into 50 mL centrifuge tubes, with each tube containing 45 mL. Ensure the suspension is homogeneous and avoid stratification. Prepare and use immediately to prevent prolonged storage that may affect efficacy.

[0299] 2.3 Animal grouping and intervention

[0300] Forty-seven 24-week-old, clean-grade, healthy New Zealand white rabbits were used in the experiment. Seven rabbits received alternating injections of 20 mg / kg of saline into the gluteal muscles as the blank group (KG). After successful model establishment, the rabbits were accurately weighed and recorded, and then randomly divided into five groups (8 rabbits each) using a random number table: model group (MG), rosuvastatin control group (RG), low-dose Tongbi Shenggu Fang group (L-TSFG), medium-dose Tongbi Shenggu Fang group (M-TSFG), and high-dose Tongbi Shenggu Fang group (H-TSFG).

[0301] All animal-related procedures in this study were performed in a clean animal laboratory. Oral gavage was initiated after successful model establishment, and drug intervention was continued for 4 weeks until the animals were sacrificed. Two people worked together to perform oral gavage while the New Zealand white rabbits were conscious. The assistant took a seated position, clamped the rabbit's lower limbs and waist and abdomen between their legs, held the rabbit's forelimbs with one hand, and held the rabbit's ears with the other hand to tilt the head upwards, ensuring a straight line between the mouth and esophagus to maintain airway. The gavage operator inserted a mouth gag into the rabbit's mouth horizontally, holding the tongue. A gastric tube was inserted 15–20 cm into the esophagus through the small central opening of the mouth gag. The outer opening of the tube was placed in a measuring cup filled with water. No bubbles indicated that the tube was not in the trachea. The drug was then injected. The rabbit was kept still and the drug solution was slowly introduced to prevent choking.

[0302] This experiment set up a blank group (KG group), a model group (MG group), a rosuvastatin positive control group (RG group), and different dose groups of Tongbi Shenggu Fang (L-TSFG, M-TSFG, and H-TSFG groups). The KG and MG groups were given equal volumes of normal saline by gavage daily; the RG group was given rosuvastatin suspension (0.05 mg / ml) by gavage; the different dose groups of Tongbi Shenggu Fang were given low (3.85 g / kg / d), medium (7.7 g / kg / d), and high (15.4 g / kg / d) doses of the drug by gavage (see Figure 22 All gavages were administered using a standardized gastric tube method at a fixed time each day. Animals were weighed weekly during the gavage process. Animals in each group were allowed free access to food and water and were free to move around their cages. The animals' mental state, diet, and coat condition were observed and recorded promptly.

[0303] 2.4 Dual fluorescence labeling of bone tissue

[0304] On days 13 and 14 before sacrifice, the animals were subcutaneously injected with tetracycline at 30 mg / kg, 24 hours apart, for a total of two doses. On days 3 and 4 before sacrifice, the animals were subcutaneously injected with calcein at 15 mg / kg, 24 hours apart, for a total of two doses. The animals were observed after the injections to ensure that there were no abnormal reactions.

[0305] Accurately weigh 500 mg of tetracycline using an electronic balance and add it to a 100 mL beaker. Calculate the required amount based on 30 mg / kg of tetracycline. Add 50 mL of normal saline and stir with a magnetic stirrer until completely dissolved. Stir until completely dissolved. Draw up the volume to 10 mg / mL. Aliquot the prepared tetracycline solution into 50 mL sterile centrifuge tubes and store in a 4°C refrigerator protected from light. Bring to room temperature before use. Accurately weigh 100 mg of calcein using an electronic balance and add it to a 100 mL beaker. Calculate the required amount based on 15 mg / kg of calcein. Add 50 mL of normal saline and stir with a magnetic stirrer until completely dissolved. Draw up the volume to 20 mg / mL. Aliquot the prepared calcein solution into 50 mL sterile centrifuge tubes and store in a 4°C refrigerator protected from light. Bring to room temperature before use. Prepare the tetracycline hydrochloride and calcein solutions in a dark environment. Store in a dark environment and prepare immediately before use.

[0306] 2.5 Imaging examination

[0307] 2.5.1 MRI examination

[0308] At week five of drug intervention, two rabbits were randomly selected from each group and fasted for 12 hours, for a total of 12 rabbits undergoing MRI examination. Prior to MRI examination, rabbits were sedated with 10% chloral hydrate (2-3 mL / kg) injected intravenously into the ear cavity (chloral hydrate was used only for sedation during imaging studies). The chloral hydrate was injected at a depth sufficient to eliminate corneal reflexes and ensure the rabbits remained in the prone position for MRI scanning. After onset of MRI, the rabbits were placed in the prone position on the MRI table, with both femoral heads positioned in the center of the scanning field of view. Conventional T1- and T2-weighted scans of both femoral heads were performed using a Philips Achieva 3.0T TX MRI machine. Following the examination, the rabbits were caged and maintained on a standard diet as normal in the animal room.

[0309] MRI imaging of the femoral head was compared between groups. MRI scans were performed to assess the normal appearance of the femoral head, the presence of abnormal high and low signal levels within the femoral head, the presence of bone marrow edema and surrounding tissue edema, and changes in the hip joint space. MRI imaging was performed by a chief physician at the Radiology Center of Gansu Provincial Hospital of Traditional Chinese Medicine.

[0310] 2.5.2 Ultrasound examination

[0311] During the fifth week of drug intervention, three rabbits were randomly selected from each group and fasted for 12 hours, resulting in a total of 18 rabbits undergoing color Doppler ultrasound examination. Before the examination, rabbits were sedated with 10% chloral hydrate (2-3 mL / kg) injected intravenously into the ear cavity (chloral hydrate was used only for sedation during imaging studies). The injection depth was at least sufficient to prevent corneal reflex and ensure the rabbits remained supine for ultrasound examination. After the hair on the chest and abdomen was removed, the rabbits were placed supine on the ultrasound examination table. After the coupling agent took effect, ultrasound scanning was initiated and liver fat quantification was performed. Ultrasound examinations were performed by the chief physician of the Ultrasound Department of Gansu Provincial Hospital of Traditional Chinese Medicine. After the examination, the rabbits were housed in separate cages and fed a standard diet.

[0312] 2.6 Serological testing

[0313] 2.6.1 Blood samples

[0314] At week 5 of drug intervention, after imaging examinations, rabbits were fasted for 12 hours for cardiac blood sampling. The rabbits were placed in a supine position with their limbs secured to an operating table. One hand held the head. Hair was removed from the left chest over the heart to improve visibility. Sterile gauze soaked in alcohol was used to disinfect the skin. The left hand palpated the third and fourth intercostal spaces on the left side of the rabbit. A blood collection needle was inserted 3 mm to the left of the sternum, at the site where the most pronounced cardiac pulse was located. The needle was tilted toward the sternum at a 45° angle to the horizontal plane. Five milliliters of blood was collected from the rabbit heart and placed in a vacuum tube containing EDTA-K2 anticoagulant. The tubes were allowed to stand at room temperature for 30 minutes and then centrifuged at 3000 rpm for 10 minutes in a 4°C high-speed centrifuge. The pale yellow supernatant, representing plasma, was obtained after centrifugation. Using a 100-1000 μL pipette, 500 μL of the upper serum layer was aspirated into labeled cryovials and stored at -80°C until further use.

[0315] 2.6.2 ELISA for bone metabolism and lipid metabolism

[0316] The levels of serum bone-specific alkaline phosphatase (ALP), osteoblast-related factor Runt-related transcription factor 2 (Runx2), and type I collagen (Collagen I) were detected by enzyme-linked immunosorbent assay (ELISA). The expressions of serum total cholesterol (TC), adipogenic-related factors recombinant human CCAAT / enhancer binding protein (C / EBP) alpha (C / EBPα), and peroxisome proliferator-activated receptor gamma (PPARγ) were also detected.

[0317] 2.7 Micro-CT examination

[0318] 2.7.1 Femoral head tissue samples

[0319] All animals were fasted for 12 hours before sacrifice. Air embolization was performed by injecting 30 ml of air into the marginal auricular vein. An incision was made through the greater trochanter of the femur. The muscles and ligaments attached to the greater trochanter were dissected layer by layer. The joint capsule and round ligament were cut to expose the femoral head. The surrounding connecting tissues and ligaments were severed, and the proximal femur was freed. The incision was extended downward to the tibial plateau. The muscles, tendons, medial and lateral collateral ligaments, and anterior and posterior cruciate ligaments of the distal femur and proximal tibia were dissected layer by layer. The entire femur was separated using sterile gauze. The femoral heads were then quickly removed, rinsed with plenty of saline in a clean surgical tray, and placed in a 50 ml sterile cryotube. The tubes were labeled with the group and the date of collection and stored at -80°C for future use. All animal carcasses were then disposed of in a sterile manner.

[0320] 2.7.2 Observation of femoral head microstructure

[0321] To clarify the bone microstructure of the rabbit femoral head, each group removed the femoral head specimens from a -80°C freezer and placed them in an ice box to acclimate to the ambient temperature to prevent rapid thawing. The specimens were then placed in a tube and scanned and analyzed along the coronal plane of the femoral head using Micro-CT. The Micro-CT scanning parameters were set as follows: voltage 70 kVp, current 114 μA, power 8 W, medium resolution, exposure time 230 ms, and overall resolution 78 μm. After the scan, the rabbits were observed for radiographic morphological changes to evaluate the femoral head necrosis and repair in different groups, and representative slices of each specimen were selected. The proximal femur, including the femoral head, was used as the region of interest (ROI). ROI-related bone tissue parameters were obtained, and trabecular morphological parameters of the ROI were quantitatively analyzed, including bone volume per tissue volume (BV / TV), bone surface per bone volume (BS / BV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp).

[0322] 2.8 Pathological testing

[0323] 2.8.1 Preparation of femoral head slide specimens

[0324] After the Micro-CT scan, the femoral head samples were fixed in 10% neutral formalin fixative for 48 hours. After fixation, the following treatments were performed: (1) phosphate buffered saline (PBS) was shaken and washed three times (20 min / time) on a shaker; (2) distilled water was repeatedly washed three times (20 min / time) to remove the residual fixative. The samples were then transferred to JYBL-I decalcification solution and decalcified at room temperature for 24–36 hours. The decalcification endpoint was determined by needle puncture: a 5 mL syringe needle was used to vertically puncture the distal femur. When the bone tissue had no mechanical resistance and showed a wet paper-like penetration feeling, it was determined to be completely decalcified and the decalcification process was terminated.

[0325] 2.8.2 Paraffin sections

[0326] After decalcification, tissue processing and paraffin embedding were performed according to standard procedures:

[0327] (1) Sample cleaning and pretreatment: Rinse with phosphate buffered saline (PBS) three times (5 min / time) to fully remove the residual decalcification solution; cut the sample along the cross section of the femoral trochanter and transfer it to a perforated dehydration box for fixation.

[0328] (2) Gradient dehydration and clearing: Dehydration using a fully automatic tissue dehydrator: 75% anhydrous ethanol (1 h) → 80% anhydrous ethanol (1 h) → 90% anhydrous ethanol (12 h); soak in TO biological clearing agent twice (1 h / time) to replace the residual water and dehydrating agent in the tissue.

[0329] (3) Paraffin immersion and embedding: Paraffin immersion: Immerse in 60℃ molten paraffin for 3 hours to ensure that the paraffin fully penetrates the tissue; Embedding operation:

[0330] a. Place the wax-impregnated specimen at the bottom of a pre-cooled embedding cassette with the coronal surface facing downward.

[0331] b. Inject molten pure wax to 1 / 2 of the box body and pre-solidify the base on the ice table;

[0332] c. Use tweezers to adjust the sample position and fill it with paraffin, then cover the marked plastic mesh box;

[0333] d. Cool on ice until the wax block is completely solidified, then seal and store at 4°C until ready for use.

[0334] (4) Slicing and preparation

[0335] a. Sectioning parameters: Remove the wax block and use a rotary microtome with the blade tilted at 15°. Adjust the section thickness to approximately 5 μm by rotating the rotary propeller.

[0336] b. Spreading and attaching: Flatten the tissue in a 45°C warm water spreading box, attach the slides, and then mark them;

[0337] c. Baking and storage: Dry in a 65°C biological tissue drying machine for 30 min, and store in a sealed container at -20°C refrigerator until ready for use.

[0338] 2.8.3 HE staining

[0339] Paraffin sections were obtained from different groups to observe histomorphological changes within the femoral head. Automated staining was performed: Paraffin sections were dewaxed in xylene I and II for 10 minutes each and rehydrated in graded ethanol (100% → 95% → 90% → 80% → 70%), each step for 2 minutes. Sections were then immersed in hematoxylin solution for 10 minutes, and any excess stain was rinsed with running water. Sections were then differentiated in 0.5% hydrochloric acid ethanol (prepared with 70% ethanol) for 1 minute to remove nonspecific staining. The sections were then bluished in distilled water for 15 minutes. Sections were then immersed in 0.5% eosin solution for 1 minute, and excess stain was rinsed with running water. The staining reaction was terminated by rinsing with distilled water for 5 minutes. Sections were then dehydrated in graded ethanol (70% → 80% → 90% → 95% → anhydrous ethanol I / II, each step for 1 minute). Sections were then cleared in xylene twice (10 minutes each time). Excess xylene was removed with filter paper, and the sections were mounted in neutral gum and cured at room temperature in the dark.

[0340] The femoral head trabecular structure, osteocyte morphology, and bone marrow adipocyte distribution were observed under an optical microscope. High-resolution images were acquired from randomly selected non-overlapping fields of view at 10×20 magnification for each section. The number of bone lacunae in this area was determined, and the number of empty lacunae was calculated (empty lacunae rate = empty lacunae / bone lacunae × 100%). The mean values ​​of the three groups were used for statistical analysis (SPSS 26.0, one-way analysis of variance).

[0341] 2.9 Dual Fluorescence Labeling of Hard Tissue Sections

[0342] 2.9.1 Resin preparation for hard tissue sections

[0343] Each set of double-fluorescent-labeled specimens was removed from a -80°C freezer and placed on crushed ice for temperature equilibration. The femoral head was cut open along the trochanteric region for later use. Then, 40 ml of methyl methacrylate, 10 ml of dibutyl phthalate, and 3 g of benzoyl peroxide were added sequentially to a 100 ml beaker. The mixture was then placed on a magnetic stirrer with a magnetic rotor and stirred thoroughly for 2 hours. After stirring, each set of double-fluorescent-labeled specimens was fixed in a plastic bottle. The stirred liquid was poured into the plastic bottle, which was then placed in a CNC hard tissue embedding machine to vacuum extract air for 1.5 hours. After extraction, the plastic bottle was sealed and placed in a 37°C water bath. After 48 hours, the specimens were removed from the water bath and stored in a cool, dry place for 2–3 days. After complete hardening, the fixed specimens were removed.

[0344] 2.9.2 Preparation of hard tissue resin sections

[0345] The hardened specimen was fixed on the base of a CNC hard tissue slicer and sliced ​​continuously along the transverse axis of the specimen using the CNC hard tissue slicer. The slice thickness was 150-200 µm. Water was added to cool the specimen during the slicing process to avoid overheating. After slicing, the slices were fixed on a glass slide for easy observation.

[0346] 2.9.3 Dual fluorescence observation and analysis

[0347] A Nikon AXR MP 20X two-photon confocal microscope was used to observe the fluorescence spacing between groups and the new bone formation in hard tissue sections, and the distance between the two marked lines was measured using Image Pro Plus 6.0 software.

[0348] 2.10 Statistical Analysis

[0349] All data were statistically analyzed using SPSS 26.0 software and graphed using GraphPad Prism 10. ±s). The two groups were compared using the independent sample t test, multiple groups were compared using one-way analysis of variance, and further pairwise comparisons were performed using the LSD-t test. P < 0.05 indicated that the differences were statistically significant.

[0350] 3 Experimental Results

[0351] 3.1 Animal conditions

[0352] 3.1.1 General

[0353] A total of 47 animals were enrolled. One died during adaptive feeding and was promptly replaced. Five died during modeling and were promptly replaced. Two animals were sacrificed to verify modeling success with HE staining, one was sacrificed during pre-experiments with double-fluorescence labeling of bone tissue, one died due to anesthesia overdose during MRI, and one died on the second day of gavage. A total of 36 animals were included in the analysis. Animals that died during modeling showed gastric mucosal erosion, intestinal mucosal bleeding, and intestinal distension. Animals that died during drug gavage showed significant intestinal distension. This is suspected to be due to the combination of LPS and high-dose MPS, which may induce systemic inflammatory response syndrome and oxidative damage to digestive tract tissues, leading to multiple organ failure, mucus barrier disruption, and gastric mucosal erosion. Intestinal mucosal bleeding may be caused by increased vascular permeability and coagulopathy caused by inflammatory cell infiltration. Intestinal distension may be caused by intestinal motility disorders and overgrowth of pathogens such as Clostridium perfringens. Approximately 11% of the rabbits in this study died within 2 weeks of modeling, possibly related to endotoxin dose, frequency of hormone injection, or individual animal differences in stress tolerance.

[0354] 3.1.2 Weight and body temperature

[0355] During the experiment, most rabbits in the blank group were in good spirits, had normal activity and appetite, had normal fur luster, and showed no obvious abnormal behavior. Their weight increased over time. However, after LPS+MPS treatment, rabbits in the model group showed poor spirits, decreased activity, sluggish reactions, decreased appetite, sparse and dull fur, and decreased weight. These conditions improved two weeks after the drug injection, and their appetite and weight gradually increased.

[0356] During the animal modeling period, the weight of the rabbits in the model group decreased rapidly after 1 week of modeling, and gradually increased and stabilized after 2 weeks; while the weight of the animals in the blank group showed an increasing trend (see Table 22, Figure 23 Compared with the blank group, the body weight of the rabbits in the model group decreased significantly at week 1 and 2 of modeling, and the difference was statistically significant (P < 0.01). The body weight of the model group increased at week 3, but was still lower than that of the blank group, and the difference was statistically significant (P < 0.01).

[0357] LPS combined with MPS modeling also causes changes in animal body temperature. After drug injection, the body temperature of the model group rose rapidly and was higher than that of the blank group. The body temperature stabilized only after 5 days of injection (see Table 23, Figure 24 Compared with the blank group, the model group showed a significant increase in body temperature during the first four days after modeling, reaching its highest temperature on the first day (P < 0.01). This difference may be due to the drug intervention-induced inflammatory state in the body, resulting in a rapid increase in body temperature, low mental state, poor diet, and weight loss. After drug metabolism is completed, the body returns to normal weight and temperature. Therefore, when LPS combined with MPS is used for intervention, the vital status of the animal should be closely monitored to ensure the smooth progress of the experiment.

[0358] Table 22 Changes in animal body weight during modeling ( ±s, / kg)

[0359]

[0360] in: indicates P < 0.01 compared with KG; Indicates P < 0.05 compared with KG.

[0361] Table 23 Changes in animal body temperature during modeling ( ±s, / ℃)

[0362]

[0363] in, Indicates P < 0.01 compared with KG.

[0364] During the intervention of animal drugs, the mental state of the rabbits in the rosuvastatin group, the Tongbi Shenggu prescription low, medium and high dose groups was decreased, the activity was reduced, the hair was thick and smooth, the luster was slightly dull, the body weight was decreased in the first week of gavage, and then tended to be stable (see Table 24, Figure 25 ). The body weight of the rabbits in the blank group was always in a growth trend; the body weight of the rabbits in the model group recovered and grew after 3 weeks of modeling, and the growth trend was lower than that of the blank group in the third and fourth weeks of gavage, and the difference was statistically significant (P<0.05). Compared with the model group, the body weight of the rabbits in each treatment group increased, and the body weight of the rabbits in the medium and high dose groups of the Tongbi Shenggu prescription in the first week of gavage had no statistically significant difference (P>0.05) compared with the model group; the body weight of the rabbits in the high dose group of the Tongbi Shenggu prescription in the second week of gavage had no statistically significant difference (P>0.05) compared with the model group; the body weight of the rabbits in the rest groups was higher than that of the model group, and the difference was statistically significant (P<0.05).

[0365] During the gavage, the rabbits in the high dose group of each treatment group had decreased diet, sparse hair, dull luster, reduced activity, poor mental state, and lower body weight than other treatment groups. The reason may be that the drug concentration of the high dose group of the Tongbi Shenggu prescription is too high, the animal absorption is slow, and the gastrointestinal pressure is increased.

[0366] Table 24 Body weight change of animals in each group per week during gavage ±s, / kg)

[0367]

[0368] Among them, P<0.05 compared with the blank group; #P<0.05 compared with the model group.

[0369] According to the gross anatomy observation, the surface morphology of the femoral head of the New Zealand white rabbits in all groups remained normal physiological curvature, and there was no difference in the smoothness and flatness of the cartilage surface; the color of the articular cartilage was uniform; the joint surface was uniformly covered and complete, and no signs such as osteophyte formation and cartilage detachment were observed, as shown in Figure 26 .

[0370] 3.2 Imaging examination

[0371] 3.2.1 MRI examination

[0372] MRI examination is the most sensitive way and gold standard for diagnosing and evaluating early SONFH, especially for X-ray negative cases (I-II period), and long-term use of hormones may cause hip pain even if the X-ray is normal. In this study, 3 rabbits were randomly selected from each group, a total of 18 rabbits were subjected to MRI examination, and MRI was used to examine the early pathological changes of the SONFH rabbit model, and the MRI images of each group are shown in Figure 27 .

[0373] The femoral heads of rabbits in the blank group had normal morphology and signal intensity. On T1-weighted images (T1WI), the femoral head bone showed uniform isointense signals. On T2-weighted images (T2WI), the bone signal was also uniform, with no abnormal high- or low-signal areas. The femoral heads on both sides had regular shapes and smooth surfaces. The hip joint spaces on both sides were normal, and no swelling or abnormal signals were observed in the joint capsule or surrounding soft tissue. In the model group, the femoral heads showed obvious abnormal signals. On T1WI, punctate and flaky low-signal intensity areas were visible within the femoral heads, indicating bone damage. On T2WI, these areas showed high signal intensity, suggesting edema or necrosis. In addition, the femoral head epiphyses on both sides were blurred, the femoral head surface was uneven and irregular, the femoral head morphology was slightly collapsed, and the joint spaces on both sides were uneven.

[0374] Compared with the model group, the abnormal signals of the femoral heads of rabbits in the rosuvastatin group and the Tongbi Shenggu recipe groups at different doses were alleviated. The femoral epiphyseal lines on both sides were slightly blurred, the surface of the femoral heads was uneven, and the area and intensity of the low-signal and high-signal areas were reduced on T1WI and T2WI. The joint space was relatively wider than that in the model group, and the fluid accumulation in the joint cavity and surrounding soft tissue was less than that in the model group. This shows that rosuvastatin and Tongbi Shenggu recipe at different doses showed definite therapeutic effects on the treatment of SONFH, among which the efficacy of the medium-dose Tongbi Shenggu recipe was more obvious than that of the other treatment groups, suggesting that Tongbi Shenggu recipe plays a positive role in promoting bone repair in SONFH model rabbits, but the specific effects and mechanisms need further research and verification.

[0375] 3.2.2 Ultrasound liver fat quantitative examination

[0376] To further evaluate the changes in liver fat density and structure in SONFH model rabbits and clarify the degree of lipid metabolism disorder in SONFH model rabbits, this study used the controlled attenuation parameter (CAP) to reflect the degree of fatty degeneration in the rabbit liver. CAP can provide accurate quantitative data through non-invasive imaging methods. The unit is dB / cm. The higher the CAP value, the more severe the fatty degeneration of the liver; it leads to lipid metabolism disorder, resulting in increased lipid levels in the blood. Long-term lipid deposition in the small blood vessels of the femoral head causes vascular blockage, affecting the blood supply to the femoral head and the differentiation ability of bone marrow mesenchymal stem cells, which hinders new bone formation and further aggravates ischemic necrosis of the femoral head.

[0377] In this study, 3 rabbits were randomly selected from each group, a total of 18 rabbits, for ultrasound examination. The liver fat of each group of rabbits was quantitatively analyzed. The ultrasound examination results are shown in Figure 28The results of CAP value of each group showed that the CAP of model group rabbits (1.1 ± 0.16 dB / cm) was significantly higher than that of blank group (0.62 ± 0.01 dB / cm), and the difference was statistically significant (P < 0.05); the CAP of rosuvastatin group (0.68 ± 0.01 dB / cm), Tongbijingugufang low-dose group (0.93 ± 0.05 dB / cm), Tongbijingugufang middle-dose group (0.74 ± 0.01 dB / cm), and Tongbijingugufang high-dose group (0.82 ± 0.04 dB / cm) were significantly lower than that of model group rabbits, and the difference was statistically significant (P < 0.05); the CAP of Tongbijingugufang middle-dose group was compared with that of Tongbijingugufang low-dose group and Tongbijingugufang high-dose group, and the difference was statistically significant (P < 0.05); but there was no significant difference between Tongbijingugufang middle-dose group and rosuvastatin group (P > 0.05). See Table 25, Figure 29 .

[0378] According to the above ultrasound examination results and liver fat CAP value analysis, it is confirmed that the liver fat density and structure of SONFH model rabbits have specific changes, which aggravate the lipid metabolism disorder of SONFH; in addition, rosuvastatin and different doses of Tongbijingugufang play an active role in improving the degree of fatty degeneration and lipid metabolism disorder of SONFH model rabbits, among which the curative effect of rosuvastatin and Tongbijingugufang middle dose is more significant.

[0379] Table 25 Ultrasonic attenuation coefficient of each group of animals ± s, %

[0380]

[0381] Among them, P < 0.05 compared with KG; #P < 0.05 compared with MG; *P < 0.05 compared with M-TSFG.

[0382] 3.3 Micro-CT examination analysis

[0383] In order to further determine the improvement of bone microstructure of femoral head of SONFH model rabbits by Tongbijingugufang, Micro-CT analysis (such as Figure 30 ) was used, and the bone tissue parameters were quantitatively analyzed. The results showed that the bone microstructure of femoral head of model group and each treatment group changed significantly compared with blank group, the bone mass lost to different degrees, and the bone trabecula morphology was damaged to different degrees, and the bone microstructure of femoral head of rabbits in Tongbijingugufang middle-dose group after intervention was less damaged.

[0384] Quantitative evaluation of bone microstructure and bone tissue parameters of femoral head of each group showed (see Table 26, Figure 31), the BV / TV, Tb.N and Tb.Th of the rabbits in the model group were significantly lower than those in the control group, and the differences were statistically significant (P<0.05), while the BV / TV and Tb.N of the rabbits in the rosuvastatin group were higher than those in the model group, but the differences were not statistically significant (P>0.05). The BV / TV, Tb.N and Tb.Th of the rabbits in the other treatment groups were significantly higher than those in the model group, and the differences were statistically significant (P<0.05). The BV / TV, Tb.N and Tb.Th of the medium-dose Tongbi Shenggu Fang group were higher than those in the other treatment groups, among which the BV / TV and Tb.N of the rosuvastatin group and the high-dose Tongbi Shenggu Fang group were lower than those in the medium-dose Tongbi Shenggu Fang group, and the differences were statistically significant (P<0.05). The Tb.Th of the rosuvastatin group, the low-dose Tongbi Shenggu Fang group and the high-dose Tongbi Shenggu Fang group was lower than that in the medium-dose Tongbi Shenggu Fang group, and the differences were statistically significant (P<0.05). The Tb.Sp and BS / BV of rabbits in the model group were significantly higher than those in the control group (P<0.05), while those in the other treatment groups were significantly lower than those in the model group (P<0.05). The BS / BV in the medium-dose Tongbi Shenggu Recipe group was significantly lower than that in the other treatment groups (P<0.05), and the Tb.Sp in the medium-dose Tongbi Shenggu Recipe group was significantly lower than that in the rosuvastatin group (P<0.05). After intervention, BV / TV, Tb.N, and Tb.Th in all treatment groups were significantly increased, while Tb.Sp and BS / BV were decreased compared with the model group.

[0385] The above micro-CT images and quantitative analysis of bone tissue parameters confirmed that rosuvastatin and Tongbi Shenggu recipe had a bone repair effect on SONFH rabbits and a positive promoting effect on bone microstructure, among which the medium-dose group of Tongbi Shenggu recipe had a better therapeutic effect.

[0386] Table 26 Comparison of bone tissue parameters in different groups ( ±s, n=6)

[0387]

[0388] in, Indicates P < 0.05 compared with KG; # indicates P < 0.05 compared with MG; Indicates P < 0.05 compared with M-TSFG.

[0389] 3.4 HE staining analysis

[0390] Consistent with the micro-CT results, HE staining showed that the model group had obvious bone necrosis, with thinning, sparse and disordered trabeculae in the femoral head; osteocytes decreased, with a large number of empty bone pits and an increase in fat cells; the pathological changes in the trabecular tissue structure of rabbits in the low-dose, medium-dose and high-dose rosuvastatin and Tongbi Shenggu Fang groups were significantly reduced, significantly alleviating the bone necrosis and repair process of SONFH model rabbits; while no obvious pathological changes were observed in the normal group. Figure 32 .

[0391] In the control group, trabeculae were dense and regularly arranged, with distinct and evenly distributed osteocytes within the trabeculae and abundant lacunae. Adipocyte morphology and size were normal, with normal osteocyte morphology and nuclei located centrally within the lacunae. In the model group, trabeculae were sparsely arranged and disorganized, with thinner and some even broken trabeculae. The number of osteocytes within the trabeculae was significantly reduced, with numerous empty lacunae. Adipocytes increased and were abundantly distributed within the medullary cavity, and bone marrow cellularity decreased. In the rosuvastatin group, trabeculae were sparsely arranged and disorganized, with thinner and some broken trabeculae. The number of osteocytes was significantly reduced, with fewer empty lacunae than in the model group. Adipocyte morphology and size were similar to those in the control group. In the low-, medium-, and high-dose Tongbi Shenggu Fang groups, trabeculae were sparsely arranged, with thinner trabeculae, fewer osteocytes, and fewer empty lacunae than in the model group. Adipocyte morphology and size were similar to those in the control group. The empty bone lacuna rates of the six groups of rabbits were significantly different (P < 0.05). The empty bone lacuna rate of the model group was higher than that of the blank group (P < 0.05). The empty bone lacuna rate of the rosuvastatin group, the low-dose, medium-dose, and high-dose Tongbi Shenggu Fang groups was lower than that of the model group (P < 0.05). The empty bone lacuna rate of the medium-dose Tongbi Shenggu Fang group was lower than that of the rosuvastatin group and the low-dose Tongbi Shenggu Fang group (P < 0.05). There was no statistically significant difference between the medium-dose and high-dose Tongbi Shenggu Fang groups (P > 0.05). See Table 27. The results showed that after intervention with four different treatment methods, the medium-dose group of Tongbi Shenggu Recipe had the best effect in improving the empty bone pit rate in rabbits, further confirming that Tongbi Shenggu Recipe can restore bone loss in SONFH model rabbits and promote bone repair to a certain extent.

[0392] Table 27 Empty bone pit rate ( ±s, %)

[0393]

[0394] in,: Indicates P < 0.05 compared with KG; # indicates P < 0.05 compared with MG; Indicates P < 0.05 compared with M-TSFG; P > 0.05 compared with M-TSFG.

[0395] 3.5 Serological testing

[0396] 3.5.1 ALP, Runx2, and Col1 Levels

[0397] Elisa results showed that the levels of ALP, Runx2 and Col1 in the serum of the SONFH rabbit model were significantly lower than those in the blank group, and the difference was statistically significant (P<0.05). After intervention with different doses of rosuvastatin and Tongbi Shenggu Fang, the levels of ALP, Runx2 and Col1 were higher than those in the model group, and the difference was statistically significant (P<0.05). Compared with the medium-dose group of Tongbi Shenggu Fang, the levels of Runx2 and Col1 in the rosuvastatin group were lower, and the difference was statistically significant (P<0.05). Compared with the medium-dose group of Tongbi Shenggu Fang, the expression levels of ALP, Runx2 and Col1 in the other treatment groups were lower, but the differences were not statistically significant (P>0.05). See Table 28. Figure 34 .

[0398] The results suggest that the bone metabolism of SONFH model rabbits is disordered, and both rosuvastatin and Tongbi Shenggu recipe can improve the activity levels of osteoblast-related factors in the serum of SONFH model rabbits and correct bone metabolism disorders. This confirms the positive effects of rosuvastatin and Tongbi Shenggu recipe, which can prevent bone loss and promote bone repair. At the same time, it confirms that the medium dose of Tongbi Shenggu recipe is the optimal dose.

[0399] Table 28 Expression levels of ALP, Runx2 and Col1 ( ±s, n=6)

[0400]

[0401] in, Indicates P < 0.05 compared with KG; # indicates P < 0.05 compared with MG; Indicates P < 0.05 compared with M-TSFG.

[0402] 3.5.2 TC, C / EBPα, and PPARγ Levels

[0403] Consistent with the results of quantitative ultrasound examination of liver fat, the levels of TC, C / EBPα and PPARγ in the serum of SONFH model rabbits were significantly higher than those in the blank group, and the differences were statistically significant (P<0.05). After intervention with rosuvastatin and Tongbi Shenggu Fang at different doses, the levels of TC, C / EBPα and PPARγ were lower than those in the model group, and the differences were statistically significant (P<0.05). The expression levels of TC and PPARγ in the rosuvastatin group, Tongbi Shenggu Fang low-dose group, and Tongbi Shenggu Fang high-dose group were higher than those in the Tongbi Shenggu Fang medium-dose group, but the difference in C / EBPα was not statistically significant (P>0.05). The levels of TC and PPARγ in the Tongbi Shenggu Fang low-dose group were higher than those in the Tongbi Shenggu Fang medium-dose group, and the differences were statistically significant (P<0.05). See Table 29. Figure 35 .

[0404] The results showed that the serum total cholesterol content of SONFH model rabbits was elevated, inducing hyperlipidemia and the occurrence of lipid metabolism disorders. Rosuvastatin and Tongbi Shenggu recipe could reduce the level of serum total cholesterol to a certain extent, reduce the activity level of adipogenesis-related factors in the serum to correct lipid metabolism disorders. Rosuvastatin and Tongbi Shenggu recipe could improve the lipid metabolism of SONFH model rabbits to a certain extent, and also confirmed that the medium dose of Tongbi Shenggu recipe was the optimal dose.

[0405] Table 29 Expression levels of TC, C / EBPα and PPARγ ( ±s, n=6)

[0406]

[0407] in, Indicates P < 0.05 compared with KG; # indicates P < 0.05 compared with MG; indicates P < 0.05 compared with M-TSFG; Indicates P>0.05 compared with M-TSFG.

[0408] 3.6 Analysis of Dual Fluorescence Labeled Hard Tissue Sections

[0409] Calcein-tetracycline labeling is used to assess the rate of new bone apposition, as well as bone quantity and quality, under dynamic conditions. Tetracycline and calcein are bone-binding fluorescent dyes that bind to newly formed bone tissue. Using undecalcified hard tissue sectioning techniques, the labeled bone tissue can be visualized under a fluorescence microscope. Tetracycline-labeled bone appears yellow, while calcein appears green. This study demonstrated the rate and interval between injections of calcein (green) and tetracycline (yellow) over a 10-day period.

[0410] Similar to the trend of micro-CT, the results of dual fluorescence labeling showed that the fluorescence of the cancellous bone of the femoral head in the blank group was clear, distributed, and continuous. The dual fluorescence performance was obvious, and the distance between tetracycline and calcein was wide, showing the largest distance ( Figure 36 The distance between fluorescence in blank group (22.74±1.05, μm) was significantly higher than that in model group (6.6±0.6, μm), and the difference was statistically significant (P<0.001); the mineralization area in blank group (10.11±0.4, μm) was significantly higher than that in model group (6.6±0.6, μm). 2 ) was significantly higher than that of the model group (3.02±0.27, μm 2 The difference was statistically significant (P < 0.001). In the model group, calcein was more distributed on the outside, while tetracycline was not obvious on the inside. The fluorescence intensity was weakened, the distribution was diffuse, blurred, and dim, showing the shortest distance ( Figure 36 The intensity of dual fluorescence weakened in each treatment group. The fluorescence distribution in the low-dose and high-dose Tongbi Shenggu Fang groups was less, the fluorescence spacing was narrower, and the inner fluorescence was not obvious (D, F). The mineralized area in each treatment group was significantly increased compared with the model group, and the difference was statistically significant (P < 0.001). The fluorescence spacing was wider than that in the model group and lower than that in the blank group. The low-, medium-, and high-dose Tongbi Shenggu Fang groups showed statistically significant differences compared with the model group (P < 0.001), while the rosuvastatin group showed no statistically significant difference compared with the model group (P > 0.05). There was no statistically significant difference in fluorescence brightness among the groups (P > 0.05).

[0411] The results showed that within 10 days of calcein-tetracycline labeling, the bone mass of the blank group increased, and the bone formation rate and trabecular formation increased; after 4 weeks of administration, the mineralization area and bone formation rate of the New Zealand white rabbits in the model group, rosuvastatin group and Tongbi Shenggu Fang low-, medium- and high-dose groups decreased to varying degrees compared with the blank group, but the mineralization area and bone formation rate of the rabbits after rosuvastatin and Tongbi Shenggu Fang intervention decreased more slowly than those after LPS combined with MPS intervention.

[0412] The above-mentioned quantitative analysis results of dual-fluorescence-labeled hard tissue sections and bone dynamics-related parameters further confirmed the adverse effects of the combined use of LPS and MPS on SONFH. At the same time, it also verified the positive effects of rosuvastatin and Tongbi Shenggu recipe on bone repair in SONFH model rabbits, and verified that the medium-dose group of Tongbi Shenggu recipe was the optimal dose group.

[0413] Table 30 shows the tetracycline-calcein dual fluorescence labeling bone dynamics related parameters of each group ( ±s, n=6).

[0414]

[0415] in, indicates P < 0.001 compared with the KG group; =Indicates P < 0.001 compared with the MG group; #Indicates P < 0.001 compared with the M-TSFG group; Indicates P>0.05 compared with the KG group.

[0416] Effect of Tongbi Shenggu Recipe on Bone Fat Imbalance in SONFH Rabbit Model

[0417] The specific pathological mechanism of SONFH remains to be determined, but bone-fat imbalance (an imbalance between osteogenic and adipogenic differentiation) is a major mechanism. Han et al. have shown that the C / EBPs family of transcription factors (including C / EBPα, C / EBPβ, and C / EBPδ) plays a central regulatory role in adipogenesis. By binding to target gene promoters (such as PPARγ and aP2), they directly activate the transcription of adipogenic genes, enhancing adipocyte glucose transport (e.g., upregulating GLUT4 expression), and providing a carbon source for lipid synthesis. C / EBPβ and C / EBPδ are rapidly activated during the initial stages of adipocyte differentiation, triggering the adipogenic process by phosphorylating the ERK / MAPK signaling pathway, followed by a rapid downregulation of their expression. C / EBPα is stably expressed throughout the differentiation process, forming a transcriptional complex with PPARγ to maintain the mature adipocyte phenotype and also inhibiting the Wnt / β-catenin pathway to prevent osteogenic differentiation, thereby ensuring the directionality of adipogenesis. The results of a study by Duan et al. showed that C / EBPα mediates the adipogenic differentiation of BMSCs and participates in the development of SONFH by targeting the PPARγ signaling pathway. Frequent use of GCs inhibits osteoblastogenesis and induces apoptosis of osteoblasts and osteocytes, enhancing the differentiation, maturation, and lifespan of osteoclasts. At the same time, the gradual accumulation of fat in the femoral head medullary leads to increased intraosseous pressure, impaired blood flow, and ischemia, with accumulation of apoptotic cells and a gradual increase in osteoclasts, ultimately leading to femoral head necrosis. Therefore, restoring bone-fat balance by promoting osteogenesis, inhibiting adipogenesis, and promoting bone remodeling is crucial for the treatment of SONFH.

[0418] There are some studies on the mechanism of bone lipid imbalance in the pathogenesis of SONFH. Yu et al. found that there was an imbalance between adipogenic and osteogenic differentiation in the necrotic area of SONFH, which may be related to PPARy histone acetylation causing bone lipid imbalance to affect the progression of SONFH; Fusillo et al. reported a case of bilateral femoral head avascular necrosis secondary to familial hyperlipidemia, which provided important clinical evidence for revealing the causal relationship between lipid metabolism disorder and femoral head necrosis. Subsequently, Wang et al. found that tanshinone IIA could promote the osteogenic differentiation of BMSCs by activating AKT signaling and inhibit the adipogenic differentiation of BMSCs by activating TGFβ signaling, which had a therapeutic effect on this disease. Yang et al. found that statins could reduce the risk of GCs-related bone necrosis in animal models and to some extent could effectively prevent the occurrence of SONFH in animal models; statins act on bone marrow mesenchymal stem cells and increase the expression of Runx2 to enhance osteogenesis, increase the activity of osteocalcin promoter, and inhibit adipogenesis by reducing the expression of adipocyte-specific gene PPARγ2, thereby regulating adipogenic differentiation. Sun et al. found that PLGA microspheres loaded with lovastatin could stabilize the bone-lipid homeostasis balance by up-regulating the expression of Cbfa1 and Runx2 and inhibiting the expression of PPARγ, reducing the intramedullary pressure, improving the local blood flow, and increasing the osteogenesis, thereby reducing the occurrence of SONFH.

[0419] Traditional Chinese Medicine (TCM) theory posits that the pathogenesis of SONFH is the result of multiple mechanisms and factors, closely related to the liver, spleen, kidneys, and other internal organs. The Suwen (Suwen) Yinyang Yingxiang Dalun states: "The kidneys produce bone marrow." The kidneys govern the bones and store essence, which in turn produces marrow. The marrow resides within the bones, nourishing them. Strengthening the kidneys governs bone production and marrow. This clearly indicates that the kidneys, essence, bones, and marrow can influence and interact with each other, forming an inextricable connection. The Neiwai Shangbing Hualun (Neiwai Shangbing Hualun) states: "The kidneys govern the bones and are associated with cold; the liver governs the tendons and is associated with wind. Since ancient times, liver and kidney diseases have been treated with the same approach, as they mutually support each other." This demonstrates the role of the liver and kidneys in skeletal growth and development, bone metabolism, and the homeostasis of the host bone microenvironment. Zhang Chengyuan et al. believe that patients with femoral head necrosis (AFN) suffer from chronic disease affecting the meridians, which can lead to a mixture of deficiency and excess. Treatment should focus on promoting blood circulation and unblocking the meridians, as well as tonifying and replenishing deficient meridians. Previous research has demonstrated clinical success in treating AFN with the Tongbi Shenggu formula. The formula primarily tonifies the kidneys and regulates the liver, replenishes essence and nourishes the marrow, and promotes the flow of qi and blood through the meridians, improving the bone microenvironment and addressing the underlying pathogenesis. The core pathological manifestation of SONFH is "bone-fat imbalance": insufficient kidney essence leads to weak bone growth (osteogenesis impaired), while stagnation of phlegm and blood stasis leads to lipid deposition (medullary seborrheic dermatitis). The interaction between the two causes organic lesions such as trabecular fracture and medullary cavity cavitation. Long-term illness invades the meridians, disrupting the smooth flow of qi and blood, causing phlegm and blood stasis to accumulate in the bone gaps and block the meridians. Pathological products further hinder the distribution of essence, forming a vicious cycle of "essence deficiency-stasis-bone erosion" and ultimately leading to the collapse of bone structure. The imbalance of bone microenvironment homeostasis together leads to the characteristic pathological phenotype of "bone-fat homeostasis imbalance", which is specifically manifested by the triple pathological characteristics of inhibited osteoblast activity, abnormal lipid droplet deposition, and trabecular microstructure damage. Therefore, in clinical practice, attention should be paid to the mixed deficiency and excess of SONFH in the early, middle and late stages of its onset. A comprehensive assessment should be conducted based on the principles of qi, blood, meridians, and underlying deficiency and superficial excess. While diagnosing the early stage of the disease, the treatment principle of "tonifying the kidney and strengthening the bones to consolidate the foundation, and activating blood circulation and unblocking the meridians to eliminate the symptoms" should be established. Methods such as promoting qi and blood circulation, removing blood stasis and unblocking the meridians, removing phlegm and dampness, and tonifying the liver and kidney should be given to maintain blood supply to the femoral head, relieve pain, and improve the condition.

[0420] In recent years, traditional Chinese medicine has demonstrated unique advantages in regulating the imbalance of bone metabolism and lipid metabolism through multiple targets, and established the treatment principle of "tonifying the kidney and strengthening bones to consolidate the foundation, and activating blood circulation and unblocking meridians to eliminate the symptoms": First, directly replenishing the innate essence, such as drugs such as Drynaria fortunei and Epimedium, to help fill the marrow sea and nourish the bones; second, unblocking the blockage of bone meridians, such as Salvia miltiorrhiza, to break up the stagnant blood stasis, resolve the accumulated phlegm, and restore the circulation of qi and blood. Tongbi Shenggu Recipe is an empirical prescription inherited and optimized by our research group based on the concept of "combining disease and syndrome". It is composed of Chinese medicinal herbs such as epimedium, cyperus rotundus, Rehmannia root, Chinese angelica root, peach kernel, Eucommia bark, Psoralea corylifolia, and licorice. This prescription uses epimedium and cyperus rotundus as the main herbs, which can nourish the kidney and strengthen bones, promote blood circulation and dredge menstruation; Rehmannia root and Chinese angelica root are used as the auxiliary herbs, which can nourish yin and tonify the kidney, promote blood circulation and produce marrow; peach kernel is used to promote blood circulation, remove blood stasis, promote qi and relieve pain; and Eucommia bark and salt Psoralea corylifolia are used to cooperate with the main herbs to nourish kidney yang and strengthen tendons and bones; finally, licorice is used to harmonize all the herbs. The synergistic effect of the various medicines in the prescription embodies the characteristics of the combination of giving equal importance to promoting blood circulation and regulating qi, relaxing muscles and tendons and activating collaterals, and nourishing yin and tonifying yang. First, it uses blood-activating and blood-stasis-removing drugs to remove stasis in the collaterals, so that blood can circulate through the original path, new blood can be regenerated, and the meridians can be unblocked; second, it directly replenishes the innate essence, such as drugs such as Drynaria fortunei and Epimedium, which help fill the marrow sea and nourish the bones, and together achieve the effect of "essence filling - blood stasis removal - bone restoration", and construct a two-way regulatory system of "blood stasis removal and collateral dredging - kidney tonifying and marrow filling", which mainly exerts its effects by regulating the balance of bone and fat metabolism, improving the bone microenvironment, promoting bone repair, and inhibiting fat formation.

[0421] Based on the balance of bone and fat metabolism, this study used LPS combined with MPS to induce SONFH in New Zealand white rabbits. Four weeks later, MRI results of the model group showed obvious abnormal signals in the femoral head area, with uneven surface, irregular shape, and mild collapse of the femoral head. Ultrasound liver fat quantitative examination showed that specific changes had occurred in fat density and structure, and the CAP value was significantly higher than that of the blank group (P < 0.05). At the same time, serological examination showed that in the SONFH model, TC, C / EBPα, and PPARγ were highly expressed in the femoral head, while ALP, Runx2, and Col1 were lowly expressed in the femoral head. Histopathological examination revealed sparse and disordered trabeculae, thinning of the trabeculae, and even fractures in some. The number of osteocytes within the trabeculae was significantly reduced, with numerous empty bone lacunas present. Adipocytes increased and were abundantly distributed within the medullary cavity, and bone marrow cells decreased. Micro-CT scans, consistent with histopathological findings, showed marked sparse and thinning of the femoral head trabeculae. Bone microarchitectural parameters BV / TV, Tb.Th, and Tb.N were significantly decreased compared with the control group, while Tb.Sp and BS / BV were increased. Furthermore, the fluorescence area and distance between the two fluorescence elements in dual-fluorescence-labeled hard tissue sections were significantly reduced. This suggests that bone-fat homeostasis is imbalanced in SONFH rabbits, inhibiting osteogenic differentiation and promoting adipogenic differentiation, leading to decreased bone formation and increased bone destruction, decreased bone mass, and increased bone marrow adipose tissue, ultimately leading to osteonecrosis.

[0422] The results of this study also showed that after the application of rosuvastatin and different doses of Tongbi Shenggu Recipe, MRI results showed that the abnormal signals in the femoral head area were reduced; the liver ultrasound CAP value was reduced, and it could inhibit the expression of TC, C / EBPα and PPARγ, and promote the expression of ALP, Runx2 and Col1; micro-CT scanning and histopathology showed that the trabecular structure was improved, the empty bone pits increased compared with the model group, the metrological parameters of bone microstructure increased, BV / TV, Tb.Th and Tb.N increased significantly compared with the model group, Tb.Sp and BS / BV decreased compared with the model group, and the fluorescent surface of the double-fluorescence labeled hard tissue sections after different treatment interventions was significantly improved. The distance between the accumulation and double fluorescence increased compared with that in the model group, indicating that rosuvastatin and Tongbi Shenggu recipe can reduce intramedullary fat production in the femoral head, and the empty bone pit rate and the bone marrow area occupied by fat cells were significantly reduced. ELISA results showed that TC, C / EBPα and PPARγ expressions were downregulated, and ALP, Runx2 and Col1 expressions were upregulated, which improved the degree of fatty degeneration and played a positive role in lipid metabolism disorders, prevented bone loss, promoted bone repair, and corrected bone-fat imbalance. Among them, the therapeutic effects of rosuvastatin and Tongbi Shenggu recipe at medium doses were more significant. Tongbi Shenggu recipe played a positive role in promoting bone-fat balance in SONFH model rabbits, confirming that the medium dose of Tongbi Shenggu recipe was the optimal dose.

[0423] In summary, this study successfully established a SONFH rabbit model using LPS combined with MPS, revealing the mechanism of glucocorticoid-induced femoral head necrosis from the perspective of bone-fat metabolic homeostasis imbalance; confirmed that the dose of Tongbi Shenggu Fang was the optimal dose, indicating that rosuvastatin and Tongbi Shenggu Fang could promote bone repair and inhibit adipogenesis, reducing the incidence of SONFH model rabbits. Its mechanism of action may be that Tongbi Shenggu Fang corrects bone-fat imbalance by upregulating the expression of ALP, Runx2 and Col1, and downregulating the expression of TC, C / EBPα and PPARγ, promoting bone formation and accelerating bone repair; reducing bone destruction and adipogenesis, and improving the imbalance of bone formation and adipogenesis in SONFH, providing new clues for the potential role of Tongbi Shenggu Fang in the prevention or treatment of SONFH, and providing a new theoretical basis for the targeted treatment of SONFH, suggesting that regulating bone-fat metabolic balance may become an important strategy to reverse early femoral head necrosis.

[0424] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements or improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. Use of an active ingredient targeting miR-365a-3p or a preparation product containing the active ingredient in the preparation of a drug for preventing and treating non-traumatic femoral head necrosis.

2. The use according to claim 1, characterized in that The active ingredient targeting miR-365a-3p includes an agonist that promotes the expression of miR-365a-3p.

3. The use according to claim 2, characterized in that The agonist for promoting the expression of miR-365a-3p includes a Tongbi Shenggu pharmaceutical composition and / or β-oxanthellate sterol.

4. The use according to claim 3, characterized in that The anti-bi and bone-building medicinal composition is prepared from the following raw materials in parts by weight: 10-20 parts of raw rehmannia, 10-20 parts of cyathula, 8-12 parts of prepared rehmannia, 8-12 parts of angelica, 8-12 parts of millettia reticulata, 8-12 parts of astragalus, 8-12 parts of epimedium, 8-10 parts of epimedium, 8-10 parts of psoralea corylifolia, 8-10 parts of eucommia bark, 8-10 parts of dipsaccharum officinale, 8-10 parts of drynaria, 8-10 parts of chuanxiong, 8-10 parts of peach kernel, 8-10 parts of safflower, 8-10 parts of white peony root, 8-10 parts of gentiana macrophylla, and 8-10 parts of liquorice.

5. The use according to claim 4, characterized in that The tonifying and bone-forming medicinal composition is prepared from the following raw materials in parts by weight: 15 parts of raw rehmannia, 15 parts of cyathula, 10 parts of prepared rehmannia, 10 parts of angelica, 10 parts of millettia reticulata, 10 parts of astragalus, 10 parts of epimedium, 9 parts of epimedium, 9 parts of psoralea corylifolia, 9 parts of eucommia bark, 9 parts of dipsaccharin, 9 parts of drynaria, 9 parts of ligusticum, 9 parts of peach kernel, 9 parts of safflower, 9 parts of white peony root, 9 parts of gentiana macrophylla, and 9 parts of liquorice.

6. The use according to any one of claims 1 to 5, characterized in that The preparation product is selected from the following groups: external preparations, oral preparations, and injections.

7. A pharmaceutical composition for preventing and treating non-traumatic femoral head necrosis, characterized in that: The pharmaceutical composition is prepared from the following raw materials in parts by weight: 10-20 parts of raw rehmannia, 10-20 parts of cyathula, 8-12 parts of prepared rehmannia, 8-12 parts of angelica, 8-12 parts of millettia reticulata, 8-12 parts of astragalus, 8-12 parts of epimedium, 8-10 parts of epimedium, 8-10 parts of psoralea corylifolia, 8-10 parts of eucommia bark, 8-10 parts of dipsaccharum officinale, 8-10 parts of drynaria, 8-10 parts of chuanxiong, 8-10 parts of peach kernel, 8-10 parts of safflower, 8-10 parts of white peony root, 8-10 parts of gentiana macrophylla, and 8-10 parts of liquorice.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition is prepared from the following raw materials in parts by weight: 15 parts of raw rehmannia, 15 parts of cyathula, 10 parts of prepared rehmannia, 10 parts of angelica, 10 parts of millettia reticulata, 10 parts of astragalus, 10 parts of epimedium, 9 parts of epimedium, 9 parts of psoralea corylifolia, 9 parts of eucommia bark, 9 parts of dipsaccharum officinale, 9 parts of drynaria, 9 parts of ligusticum, 9 parts of peach kernel, 9 parts of safflower, 9 parts of white peony root, 9 parts of gentiana macrophylla, and 9 parts of liquorice.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that The pharmaceutical composition is prepared in the form of capsules, granules or decoctions.

10. The method for preparing the pharmaceutical composition according to any one of claims 7 to 9, characterized in that: The raw materials are weighed according to the ratio, soaked in clean water, heated and boiled, the drug residue is discarded, the decoction is collected, the decoction is centrifuged, the supernatant is collected, and the lyophilized powder of the pharmaceutical composition is obtained after freeze-drying.

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