Application of wogonoside in preparation of medicine for preventing and treating inflammatory osteolysis

Abstract

The invention provides application of wogonoside in preparation of a medicine for preventing and treating inflammatory osteolysis, and belongs to the technical field of bone disease treatment, wogonoside has an inhibiting effect on RANKL-induced osteoclast generation and bone resorption, and MAPK and NF-kB signal channels related to osteoclast differentiation are detected through WB and PCR experiments. The wogonoside has an inhibiting effect on osteoclast differentiation, bone resorption function and bone loss of LPS-induced inflammatory osteolysis mice, and the effect is possibly related to inhibition of RANKL-mediated ROS level and a downstream MAPK signal channel. Therefore, the invention possibly provides a new treatment thought for preventing inflammatory osteolysis diseases.

Description

Technical Field

[0001] This invention relates to the field of bone disease treatment technology, and in particular to the application of baicalin in the preparation of drugs for the prevention and treatment of inflammatory osteolysis. Background Technology

[0002] Inflammatory osteolytic diseases are a class of inflammatory bone degenerative diseases closely related to chronic inflammatory responses and bone resorption. The pathological characteristics of osteolytic diseases are mainly manifested in enhanced osteoclast activity and the massive release of pro-inflammatory cytokines (such as IL-1 and TNF-α), accelerating bone resorption. If left untreated, they may develop into chronic inflammation, leading to excessive bone resorption and destruction, eventually evolving into rheumatoid arthritis, osteoarthritis, infectious bone diseases, and osteoporosis. Inflammatory osteolytic diseases impose a severe burden on individuals and the socioeconomic system. Studies have shown that LPS activates nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) through the TLR4 signaling pathway, leading to increased ROS production. ROS-mediated enhancement of osteoclast activity results in severe inflammatory bone degeneration.

[0003] In inflammatory osteolytic diseases, osteoclastogenesis is primarily promoted through the synergistic effect of macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL). During inflammation, macrophages and T cells secrete large amounts of RANKL, and pro-inflammatory cytokines such as TNF-α and IL-1β can also enhance RANKL expression. After binding to RANK on the surface of osteoclast precursor cells, RANKL aggregates TNF receptor-associated factor 6 (TRAF6) and activates downstream MAPK signaling pathways and activates T cell nuclear factor 1 (NFATc1). Activated NFATc1 automatically amplifies and promotes its target genes, including the ATPase H+ transport VO subunit D2 (Atp6v0d2) and cathepsin K (Ctsk), thereby regulating osteoclast development, cell fusion, and bone degradation capacity, accelerating bone resorption.

[0004] Currently, drugs used to treat inflammatory osteolytic diseases include biologics (denosumab), bisphosphonates (alendronate sodium), and glucocorticoids (dexamethasone). While these drugs have potent anti-inflammatory and bone-destructive effects, long-term use can lead to various adverse reactions. For example, denosumab can cause bone and muscle pain, alendronate sodium can cause gastrointestinal discomfort, and dexamethasone can actually cause osteoporosis. Glycosides, a class of secondary metabolites widely found in traditional Chinese medicine, are composed of aglycones and glycosyl groups linked by glycosidic bonds. They possess various pharmacological properties such as anti-inflammatory and antioxidant effects while having fewer side effects. For instance, ginsenosides have been shown to slow down LPS-induced bone damage.

[0005] Baicalin is a natural flavonoid compound extracted from the roots of Scutellaria baicalensis, possessing pharmacological effects such as anti-inflammatory and antioxidant properties. Studies have shown that baicalin can inhibit MDA-MB-231 cells through the MAPK-mTOR pathway. Other studies have found that baicalin can inhibit LPS-induced increases in lung tissue macrophage and neutrophil infiltration and significantly reduce myeloperoxidase activity. However, the role of glutamic acid (WG) in inflammatory osteolysis has not yet been reported. Summary of the Invention

[0006] The purpose of this invention is to provide an application of baicalin in the preparation of drugs for the prevention and treatment of inflammatory osteolysis, thereby solving the technical problems mentioned in the background art. Schisandrin B has osteoclast-forming and absorption effects.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] Application of baicalin in the preparation of drugs for the prevention and treatment of inflammatory osteolysis.

[0009] Baicalin inhibits RANKL-induced osteoclastogenesis and bone resorption, and the MAPK and NF-κB signaling pathways involved in osteoclast differentiation were detected by Western blotting and PCR experiments.

[0010] Furthermore, an LPS-induced inflammatory osteolysis mouse model was constructed to simulate the occurrence of inflammatory bone loss. The degree of bone destruction and the number of osteoclasts were analyzed by microcomputed tomography and staining with hematoxylin, eosin and tartrate-resistant acid phosphatase to determine the molecular mechanism by which baicalin prevents inflammatory osteolysis.

[0011] Furthermore, baicalin inhibited RANKL-induced osteoclastogenesis in a concentration- and time-dependent manner and showed no toxicity to the cellular activity of bone marrow mesenchymal stem cells (BMMs). Baicalin inhibited the expression of osteoclast-related genes and weakened the ability of osteoclasts to resorb bone. In terms of the mechanism of inhibiting osteoclastogenesis, baicalin inhibited the expression of the intracellular transcription factor NFATc1. Baicalin inhibited osteoclasts by inhibiting the expression of RANKL-mediated TRAF6 and related downstream osteoclast formation and bone resorption-related proteins. For the classical signaling pathway mediated by RANKL, baicalin inhibited the phosphorylation levels of JNK, ERK, and p38 proteins in the MAPK signaling pathway.

[0012] Baicalin reduces ROS production by inhibiting NOX1 expression, while promoting the expression of antioxidant enzymes Nrf2, HO-1, and Cat to clear ROS from intracellular and mitochondrial spaces. In mice with inflammatory osteolysis, baicalin significantly increased the volume fraction and number of trabeculae in the femur, and reduced the levels of CTX-1, IL-1β, IL-6, and TNF-α in serum. Baicalin also significantly reduced the number of osteoclasts, the expression of NFATc1 transcription factor, and NOX1 protein in the mouse femur. In vivo reverse transcription quantitative PCR and Western blotting results showed that baicalin inhibited the expression of NFATc1 and CTSK, while increasing the expression of antioxidant enzymes to clear ROS, and inhibiting the number and activity of osteoclasts to protect against bone loss.

[0013] Furthermore, baicalin reduces intracellular transcription factor NFATc1 expression by inhibiting the RANKL-mediated TRAF6-MAPK signaling pathway, enhances antioxidant enzyme expression to clear intracellular ROS, thereby inhibiting osteoclast formation and bone resorption. By enhancing antioxidant enzyme expression and reducing ROS, it effectively protects against bone loss in mice with inflammatory osteolysis.

[0014] The present invention, by adopting the above-described technical solution, has the following beneficial effects:

[0015] (1) In this invention, baicalin reduces intracellular ROS by inhibiting the RANKL-mediated TRAF6-MAPK signaling pathway, thereby reducing the expression of the transcription factor NFATc1 and enhancing the expression of antioxidant enzymes, thus inhibiting osteoclast formation and bone resorption. Baicalin can effectively protect against bone loss in mice with inflammatory osteolysis by enhancing the expression of antioxidant enzymes and reducing ROS.

[0016] (2) Baicalin inhibits osteoclast differentiation, bone resorption, and bone loss in LPS-induced inflammatory osteolysis mice. This effect may be related to the inhibition of RANKL-mediated ROS levels and its downstream MAPK signaling pathway. Therefore, this invention may provide a new therapeutic approach for the prevention of inflammatory osteolysis. Attached Figure Description

[0017] Figure 1 This is a diagram illustrating the in vitro osteoclast differentiation induced by baicalin of the present invention, which inhibits RANKL and LPS-induced osteoclast differentiation.

[0018] Figure 2 This is a diagram illustrating how baicalin inhibits RANKL and LPS-induced ROS generation during osteoclast formation, as described in this invention.

[0019] Figure 3This is a diagram showing the expression of the MAPK signaling pathway and its downstream proteins during the inhibition of osteoclast differentiation by baicalin in this invention.

[0020] Figure 4 This is a diagram illustrating the inhibition of NFATc1 signaling pathway-related protein expression by baicalin according to the present invention.

[0021] Figure 5 This invention demonstrates that baicalin can prevent bone loss caused by lipopolysaccharide in vivo.

[0022] Figure 6 The diagram shows that the baicalin of this invention has no significant toxic effects on the kidneys, liver, and heart. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, it should be noted that many details listed in the specification are merely to provide the reader with a thorough understanding of one or more aspects of the present invention, and these aspects of the invention can be implemented even without these specific details.

[0024] I. Materials and Methods

[0025] 1.1 Pharmaceuticals and Chemicals

[0026] Baicalin was purchased from Chengdu PUREF Technology Development Co., Ltd. (Chengdu, Sichuan, China). α-MEM and FBS were purchased from Gibco (USA). RANKL and M-CSF were purchased from R&D Systems (Minneapolis, MN, USA). Specific antibodies were purchased from Cell Signaling Technology (Danvers, MA). TRAP staining kits were purchased from Sigma (St. Louis, MO, USA). 96-well hydroxyapatite plates were purchased from Corning Osteoassay. Hematoxylin and eosin staining kits were purchased from Solarbio Science & Technology (Beijing, China).

[0027] 1.2 Cell Culture

[0028] Primary bone marrow-derived macrophages (BMMs) extraction and culture: Six-week-old C57BL / 6 mice with good growth were euthanized by cervical dislocation in the sampling room. The mice were sterilized by spraying their bodies with ethanol disinfectant. Using pre-autoclaved ophthalmic scissors, the Achilles tendon of the lower limb was quickly cut open, and the muscles from the Achilles tendon to the femoral head were dissected. The lower limb was severed at the hip joint, and the claws were removed (avoiding exposure of the medullary cavity). The limbs were placed in a culture dish (placed on ice) and transferred to a biosafety cabinet for further removal of remaining tibia and femur muscle tissue. The limbs were washed once with 5 mL of PBS, followed by 3 mL of MEMAlpha complete medium (M-CSF final concentration 25 ng / mL). The lower limb was held with forceps, and the epiphysis was cut open. The medullary cavity was then repeatedly rinsed with 1 mL of complete medium using a syringe until the bone turned white. The bone marrow and medium mixture was transferred to a 50 mL centrifuge tube and dispersed using a Pasteur pipette. Centrifuge at 1200 rpm for 5 min and discard the supernatant. Resuspend the cells in 3 mL of MEMAlpha complete culture medium, filter the cells through a 100 μm filter, and add the cells dropwise into a T75 culture flask.

[0029] Add culture medium to the appropriate level.

[0030] 15 mL of culture medium was placed in a cell culture incubator for 2 days. Afterward, the old culture medium was removed from the bottle, and the cells were rinsed twice with PBS to remove dead cells and impurities. The culture was then replaced with fresh MEMAlpha complete medium and cultured for another two days. The adherent cells were considered to be macrophages derived from bone marrow cells and could be used for subsequent experiments.

[0031] 1.3 Cell viability assay

[0032] Effects of different concentrations of WG on the viability of BMMs cells: Counted BMMs cells were seeded at a rate of 6 × 10⁴ cells / mL, 100 μL per well, with at least three replicates per experimental group. An equal volume of PBS was added around each well to reduce evaporation of the cell culture medium. The wells were then incubated overnight at 37°C with 5% CO₂. The next day, after complete cell attachment, the original culture medium was aspirated and replaced with MEMAlpha medium containing different concentrations of WG (1 μM, 5 μM, 10 μM, 15 μM, and 20 μM). A control group without WG and an LPS group were also included. Unless otherwise specified, LPS (100 ng / mL) was added to the LPS group 24 hours before plate collection. After 48 hours of incubation, 10 μL of CCK-8 solution was added to each well, taking care to avoid generating air bubbles. After the culture plates were placed in an incubator and incubated for another 2 hours, the absorbance value at a wavelength of 450 nm was detected using a microplate reader to determine whether each monomer had an inhibitory effect on the activity of BMMs. GraphPadPrism (9.0) was used for statistical analysis and plotting.

[0033] 1.4 Osteoclast differentiation assay and TRAP staining

[0034] Osteoclast differentiation under different concentrations of WG: Counted BMM cells were seeded at 6 × 10⁴ cells / mL, 100 μL per well, and cultured overnight at 37°C in a 5% CO₂ incubator. Cells were observed under a microscope the following day. If the cells were in good condition, the medium was changed according to different experimental groups. Experimental groups included: negative control (MEM Alpha complete medium), positive control (MEM Alpha complete medium + 50 ng / mL RANKL + 100 ng / mL LPS), and drug treatment groups (MEM Alpha complete medium + 50 ng / mL RANKL + 100 ng / mL LPS + 0 / 1 / 5 / 10 / 15 / 20 μM).

[0035] (WG). Remove the original culture medium and replace it every two days. In the positive control group, cells fused and giant mature osteoclasts with multiple nuclei appeared. Collect the plate. Remove the 96-well plate containing cells from the incubator, gently aspirate the culture medium with a pipette, fix with 4% paraformaldehyde for 1 h, wash the bottom cells three times with 1×PBS, add 50 μL of TRAP staining solution to each well, and incubate at 37°C in the dark for 15-20 min. Observe under a microscope every 5 min. When the overall color of osteoclasts is pink, aspirate the TRAP staining solution. Then, to remove excess TRAP staining solution, wash each well three times with double-distilled water, dry in a 37°C oven, scan each well with Cytation5 cell imaging instrument, and count the number of mature osteoclasts (3 or more nuclei) in each well using Image J (1.8) software. Plot the graph using GraphPadPrism (9.0).

[0036] 1.5 Bone resorption function test

[0037] Bovine cortical bone slices of similar thickness and smoothness were laid flat in 10cm cell culture dishes. The non-smooth sides were marked with a pencil to group the slices into a positive control group (RANKL group and RANKL+LPS group) and a drug treatment group (20 μMWG). The marked bovine bone slices were first sterilized by UV irradiation in a clean bench for 30 min. Then, 75% ethanol disinfectant was added to submerge the bone slices, the culture dishes were sealed with sealing film, and the dishes were placed in a 4℃ cold storage on a shaker for 24 h for decolorization. After 24 h, the medium was replaced with DMEM phenol red-free medium, and shaking was continued for another 24 h. The bovine bone slices were then turned over, replaced with fresh DMEM phenol red-free medium, and placed in a 4℃ cold storage on a shaker for another 24 h. Finally, the slices were transferred to pure MEMAlpha culture medium and shaken again at 4℃ for 24 h. The prepared bone slices were evenly placed into 96-well cell culture plates with the labeled side facing down using sterile forceps. De-counted BMMs were seeded onto the bone slices at a density of 10⁴ cells / well. Simultaneously, 6 × 10³ cells / well were seeded into 96-well plates without bone slices for TRAP staining control. The plates were incubated overnight. The next day, RANKL stimulation was added, and the culture medium was changed every two days. The plates were harvested when mature osteoclasts appeared in the control plates. The 96-well plates were removed, the culture medium was aspirated, and the cells were fixed with 4% paraformaldehyde for 1 hour. 50 μL of TRAP staining solution was added to each well for staining.

[0038] After 15-20 minutes, the cell staining was checked every 5 minutes until the cells were stained pink. Excess staining solution was washed away with double-distilled water, and the cells were air-dried and photographed. Cells with positive staining and ≥3 nuclei were considered mature osteoclasts. 96-well cell culture plates containing bone slices were cultured once with a new medium and then fixed with electron microscopy fixation solution after 48 hours. Bone slices were removed with sterile forceps and placed in culture dishes containing PBS buffer. Cells were gently brushed off with a brush, and the slices were dehydrated, dried, sputter-coated with gold, and then scanned under an electron microscope. The area of ​​bone resorption depressions on the bovine bone slices was quantitatively counted using ImageJ (1.8) software, and graphs were plotted using GraphPadPrism (9.0).

[0039] 1.6 RNA extraction and quantitative PCR assay

[0040] Total RNA was isolated and extracted from osteoclasts using Trizol reagent according to the kit instructions. To synthesize cDNA, 1 μg of RNA was reverse transcribed using oligo-dT primers (Thermo Fisherkit, USA). Real-time PCR was performed using a LightCycler96 (Switzerland, Basel) with the following amplification process: pre-denaturation at 94°C for 10 minutes, followed by 35 cycles of 94°C (10 seconds), 60°C (15 seconds), and finally 72°C (10 seconds). PCR data were read after reverse transcription termination. Table 1 below lists the specific primers used for osteoclast-related genes in this experiment.

[0041] Table 1 Primer sequence information

[0042]

[0043] 1.8 Western blot analysis

[0044] Different densities of bone marrow membranes (BMMs) were seeded. Control and WG treatment groups were both set up and allowed to remain in incubators overnight. These experiments aimed to determine how WG affects signaling pathways for osteoclast activation. After exposure to RANKL, LPS, and WG at specified concentrations and time points, cells were lysed on ice using RIPA containing a protease inhibitor (APEBIO), PMSF (Solarbio), and a phosphatase inhibitor (ComWin Biotech, Beijing, China). After heating at 100°C for 10 minutes, the protein and loading buffer mixture was stored at -20°C for later use. Finally, proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were then incubated with 5% skim milk solution at room temperature for 1 hour. After washing with Tris-buffered saline and Tween (TBST) solution, the membranes were incubated with primary antibody overnight on a shaker at 4°C. The next day, the appropriate secondary antibody was incubated with the nitrocellulose membrane for 1 hour. After TBST washing, images were captured using the ImageQuant AS-4000 system (GE Healthcare, Chicago, Illinois, USA). Statistical analysis of the images was performed using ImageJ software.

[0045] 1.9 Detection of intracellular reactive oxygen species

[0046] BMMs (8 × 10³ cells / well) were incubated for 2 days with RANKL, LPS (added on day 2), and WG (20 μM). Intracellular ROS were then detected according to the manufacturer's instructions. The H2DCFDA fluorescent probe was added to serum-free pure α-MEM medium (H2DCFDA:α-MEM medium volume ratio 1:1000) and incubated at 37°C in a 5% CO2 incubator for 30 min. Cells were washed with PBS buffer, and the fluorescence of DCF was measured using a Biotek fluorescence microscope. Image statistical analysis was performed using ImageJ software.

[0047] 1.10 LPS-induced mouse model of inflammatory osteolysis

[0048] Thirty 8-week-old C57BL / 6J mice were housed in an SPF-grade environment with 12-hour light / dark and a room temperature of 25°C, with free access to sterilized feed and purified water. After one week of acclimatization, these 9-week-old mice were randomly divided into 5 experimental groups (n=6): sham group, LPS group (5 mg / kg), low-concentration WG group (10 mg / kg), high-concentration WG group (20 mg / kg), and dexamethasone group (1 mg / kg). The modeling process lasted for 8 days. The sham group received intraperitoneal injections of saline throughout the process. The LPS group received intraperitoneal injections of LPS on days 1 and 4, and intraperitoneal injections of saline on the remaining days. The WG and DEX groups received intraperitoneal injections of LPS on days 1 and 4, and intraperitoneal injections of the corresponding drug concentrations on days 2, 4, 6, and 8, and intraperitoneal injections of saline on the remaining days. Eight days later, ocular blood was collected from mice after anesthesia. The collected blood was placed in 1.5 mL EP tubes and allowed to stand at room temperature for 1 hour before centrifugation at 5000 rpm for 20 minutes. The tubes were then stored at -80°C. After collecting the ocular blood, the mice were euthanized, and the hind limb bones and internal organs were completely removed. The hind limb bones were fixed with 4% paraformaldehyde and then replaced with 75% alcohol at room temperature after 48 hours. The internal organs were stored in 4% paraformaldehyde at room temperature for further analysis.

[0049] 1.11 Enzyme-linked immunosorbent assay (ELISA)

[0050] Mouse ocular blood, stored in the refrigerator, was used for enzyme-linked immunosorbent assay (ELISA). The steps were as follows, following the kit instructions: Wash buffer (50×) was diluted with double-distilled water to prepare 1× wash buffer, and all reagents in the kit were thoroughly mixed. Three replicates were set up for each standard and blank well. 50 μL of diluted standard and 50 μL of mouse serum sample diluted 1:1 with standard diluent were added to each well. Immediately afterwards, 50 μL of biotin-labeled antibody was added to each well. The wells were sealed with sealing film and incubated at 37°C in the dark for 1 hour. After incubation, the liquid in the wells was discarded and the plate was thoroughly dried. Then, each well was filled with 1× wash buffer, shaken for 30 seconds, and the liquid was discarded. The plate was blotted clean on absorbent paper. This washing process was repeated three times. Finally, 80 μL of HRP-conjugated streptavidin was added to each well, and the plate was sealed with sealing film and incubated at 37°C in the dark for 30 minutes. After incubation, discard the liquid in the wells and repeat the washing procedure three times. Then, add 50 μL each of reaction substrates A and B to each well, incubate at 37°C in the dark for 10 minutes, then remove the plate and quickly add [the solution to the wells].

[0051] 50 μL of stop solution was placed in an ELISA reader, and the absorbance of each well was measured at 450 nm. A standard curve was plotted with the absorbance (OD) value on the ordinate (Y) and the corresponding concentration of the analyte standard on the abscissa (X), according to the operating instructions. The concentrations of CTX-1, IL-1β, IL-6, and TNF-α in the corresponding mouse serum samples were then calculated based on the standard curve.

[0052] 1.12 Statistical Analysis

[0053] The experiments involved were conducted at least three times. The results were statistically analyzed using GraphPadPrism (9.0) and presented as mean ± standard deviation. All statistical analyses were performed using one-way / two-way ANOVA. A p-value less than 0.05 was considered statistically significant.

[0054] II. Results

[0055] 2.1 Baicalin inhibits RANKL and LPS-induced osteoclast differentiation and bone resorption in vitro.

[0056] We cultured the cells normally for 72 hours under baicalin treatment, and then stimulated them with LPS for 24 hours to observe the activity of BMMs. The results showed that there was no effect. Figure 1 A). Next, to investigate the effects of baicalin on RANKL and LPS-induced osteoclast differentiation, we pre-stimulated BMMs with RANKL for 5 days, followed by LPS stimulation for 24 hours. The results showed that the addition of LPS stimulation promoted osteoclast differentiation, while baicalin still inhibited osteoclast differentiation. Figure 1 B, C). Subsequently, we investigated the effect of baicalin on osteoclast bone resorption function through bone resorption experiments. The results showed that, in addition to RANKL stimulation (RANKL group), LPS stimulation (LPS group) resulted in larger bone resorption pit areas, indicating that baicalin could inhibit bone resorption function. Figure 1 DF).

[0057] 2.2 Baicalin reduces ROS levels in LPS- and RANKL-induced BMMs

[0058] To investigate whether baicalin affects the ROS levels within BMMs induced by LPS and RANKL, we first stimulated BMMs with RANKL for two days, followed by stimulation with LPS for 24 hours. We used the oxidation-sensitive fluorescent probe H2DCFDA to detect the ROS levels within BMMs. The results showed that the fluorescence intensity in the LPS group was stronger than that in the RANKL group, indicating that baicalin treatment downregulated the ROS levels. Figure 2 (A, B). Next, we detected genes and proteins related to oxidative stress using real-time quantitative PCR.

[0059] PCR results showed that the levels of Nfe2l2, Homx-1, and Cat were downregulated in both the RANKL and LPS groups, and recovered or improved under baicalin treatment; the level of Keap1 was upregulated in both the RANKL and LPS groups, and downregulated under baicalin treatment. Figure 2 CF). Western blot results also confirmed the effects of baicalin treatment on Nrf2, Keap1, HO-1, and CAT proteins. Figure 2 GL). The Nrf2 / Keap1 ratio was decreased in both the RANKL and LPS groups, and increased after treatment with baicalin, but the difference was not significant. Figure 2 J).

[0060] 2.3 Baicalin inhibits the MAPK signaling pathway during osteoclast differentiation

[0061] The NF-κB signaling pathway, including members such as p65 and IκBα, plays an indispensable role in RANKL-induced osteoclast differentiation. The MAPK (including JNK, ERK, and p38) signaling pathway is activated during RANKL-induced osteoclast differentiation, inhibiting the activation of the NF-κB / MAPK signaling pathway to suppress osteoclast differentiation and generation. Therefore, this invention first investigated the effect of WG treatment on the MAPK signaling pathway after RANKL and LPS activation. Western blotting experiments showed that for the MAPK signaling pathway, 20 μM WG treatment of the control group reduced ERK and JNK phosphorylation levels after 10 minutes of RANKL and LPS stimulation, and reduced p38 phosphorylation levels after 10-20 minutes of RANKL and LPS stimulation. For the NF-κB pathway, WG had no effect on the degradation of IκB-α protein or the phosphorylation level of p65 at any time. Figure 3 AF).

[0062] 2.4 Baicalin inhibits the expression of osteoclast differentiation-specific genes and proteins.

[0063] Previous research revealed that WG can inhibit the MAPK signaling pathway during osteoclast differentiation. The MAPK pathway plays a crucial role in the differentiation and functional regulation of osteoclasts. This pathway, through signaling molecules such as ERK, JNK, and p38, responds to external stimuli (e.g., RANKL), thereby activating downstream transcription factors (e.g., c-Fos and NFATc1) and promoting the expression of osteoclast-related genes and proteins. Therefore, we further investigated whether baicalin affects the expression of osteoclast-specific genes and proteins. Real-time quantitative PCR was used.

[0064] PCR results showed that, compared with the RANKL group, the expression of osteoclast-related genes such as Nfatc1, Fos, Ctsk, and Atp6v0d2 was increased in the LPS group, and WG treatment significantly inhibited the expression of these genes. Figure 4 (AD). We induced osteoclast differentiation with RANKL and LPS on days 0, 1, 3, and 5, respectively, while another group received 20 μM WG. Immunoblot assays showed that the expression levels of NFATC1, c-Fos, CTSK, and ATP6VOD2 increased with increasing stimulation time. However, in the WG treatment group, the expression levels of these proteins showed a significant decreasing trend. Specifically, the inhibitory effects of WG on NFATC1 and c-Fos mainly occurred on days 1 and 3 of osteoclast differentiation, while the inhibitory effects on ATP6VOD2 and CTSK mainly occurred on days 3 and 5 of osteoclast differentiation. Figure 4 EI).

[0065] 2.5 Baicalin can inhibit trabecular bone loss in LPS-induced inflammatory osteolysis mice.

[0066] In vitro cell experiments showed that WG could inhibit osteoclastogenesis and the secretion of inflammatory factors; however, its role in inflammatory bone loss remains unclear. We evaluated its in vivo efficacy by establishing an LPS-induced mouse model of inflammatory bone loss, using WG at concentrations of 10 mg / kg and 20 mg / kg as treatment concentrations. The control group used 1 mg / kg dexamethasone. First, we performed ELISA experiments on serum from the ocular blood of mice. The marker of bone resorption, type I collagen C-terminal peptide (CTX-1), was elevated in the LPS group and decreased in the 10 mg / kg and 20 mg / kg WG treatment groups as well as the DEX group. The expression of inflammatory factors IL-1β, IL-6, and TNF-α showed similar trends. Figure 5 AD). Micro-CT analysis showed that bone mass was significantly reduced in the LPS group compared to the Sham group, while the DEX group and WG treatment group significantly prevented LPS-induced femoral bone loss. Figure 5 E). Quantitative analysis showed that, compared with the LPS group, bone tissue parameters BV / TV and Tb.N were significantly increased in the DEX group and the 20 mg / kg WG group, and also increased in the 10 mg / kg WG group, but to a lesser extent than the previous two groups. Figure 5 F,G); Tp.Sp was significantly reduced in the DEX group and the 20 mg / kg WG group, and also decreased in the 10 mg / kg WG group, but the magnitude was smaller than that in the above two groups. Figure 5 H); while the cortical bone parameter Ct.Th increased in the DEX group and the 20 mg / kg WG group, but showed no significant difference in the 10 mg / kg WG group. Figure 5 I).

[0067] 2.6 Baicalin showed no toxicity to LPS-induced inflammatory osteolysis in mice.

[0068] To confirm the safety of the LPS-induced inflammatory osteolysis model in mice and whether WG would cause damage to major organs (heart, liver, and kidneys) when it took effect in mice, we performed pathological analysis on the heart, liver, and kidneys of the model mice. HE staining results showed that the heart, liver, and kidney tissues of the model mice were all normal. Figure 6 The above results indicate that WG has no toxic effect on LPS-induced inflammatory osteolysis model mice.

[0069] In summary, this invention reveals that baicalin can inhibit RANKL-induced osteoclast formation, bone resorption, and LPS-induced inflammatory osteolysis in mice both in vitro and in vivo. Further analysis of the molecular mechanism shows that baicalin inhibits RANKL-induced osteoclast formation by reducing intracellular ROS and downstream MAPK (p38, ERK, and JNK) signaling pathways. Therefore, this invention suggests that baicalin may have significant potential and clinical value in the prevention of inflammatory osteolysis.

[0070] Inflammatory osteolysis is a significant and challenging clinical problem in orthopedics. Its main cause is the enhanced osteoclast activity due to the influence of various cytokines released in the inflammatory microenvironment. Therefore, inhibiting inflammation and osteoclast generation stimulated by the inflammatory environment can effectively reduce inflammatory osteolysis. Many plant extracts have been shown to inhibit osteoclast differentiation. For example, baicalin inhibits osteoclast generation by reducing NF-κB and MAPK signaling pathways, thereby attenuating LPS-mediated inflammatory osteolysis. In this invention, we found that baicalin, an extract of Scutellaria baicalensis, inhibits RANKL and MAPK pathways in vitro by affecting ROS-related iron metabolism abnormalities and the MAPK pathway.

[0071] LPS-induced osteoclast differentiation and its ability to alleviate LPS-induced inflammatory osteolysis in mice in vivo. These results provide strong evidence that WG is a candidate drug for anti-inflammatory osteolysis.

[0072] We first investigated the effect of WG on RANKL-induced osteoclast differentiation in vitro, and also verified in vivo that WG could inhibit the expression of serum inflammatory cytokines IL-1β, IL-6, and TNF-α. IL-1β, IL-6, and TNF-α are three important pro-inflammatory cytokines that play key roles in bone metabolism and osteoclast regulation. IL-1β can promote RANKL expression in osteoblasts, bone marrow stromal cells, and osteoclast precursor cells; RANKL is a key factor in osteoclast differentiation and activation. IL-6 and RANKL have a synergistic effect in bone resorption. TNF-α can directly activate osteoclast precursor cells, increase RANKL expression, and further promote bone resorption by binding to its receptors (TNFR1 and TNFR2). Therefore, our subsequent study focused on the effect of WG on osteoclast differentiation co-stimulated by RANKL and LPS. Our study compared osteoclast differentiation with RANKL stimulation alone (RANKL group) and with combined RANKL and LPS stimulation (LPS group). As expected, our study showed that osteoclast differentiation in the LPS group was indeed enhanced compared to the RANKL group. Bone resorption experiments also showed that the osteoclasts in the LPS group had stronger bone resorption capacity than those in the RANKL group.

[0073] ROS (Reactive Organic Compounds) are a class of highly reactive molecules produced during cellular metabolism, mainly including hydrogen peroxide, superoxide anion, chloride radicals, and hydroxyl radicals. Osteoclast precursor cells are the precursors to mature osteoclasts; they need to differentiate into mature osteoclasts to perform bone resorption. In this process, ROS participate in the proliferation, differentiation, and activation of osteoclast precursor cells by regulating multiple signaling pathways. Studies have shown that ROS can promote the differentiation of osteoclast precursor cells into mature osteoclasts by stimulating the RANKL (receptor-activated nuclear factor κB ligand) / RANK (RANK receptor) signaling pathway. Nrf2, as a major regulator of intracellular oxidative stress, usually forms a complex with Keap1 in the cytoplasm. Under normal circumstances, Keap1 acts as a negative regulator of Nrf2, promoting...

[0074] Nrf2 is maintained at low levels through ubiquitination and degradation. When cells are subjected to oxidative stress or chemical stimulation, the cysteine ​​residues of Keap1 are oxidized, disrupting the binding of Keap1 to Nrf2. Nrf2 then translocates to the nucleus and binds to the antioxidant response element ARE, initiating the transcription of a series of antioxidant-related genes. In our study, WG treatment inhibited intracellular ROS generation during osteoclast differentiation of BMMs by reducing Keap1 and activating Nrf2. Simultaneously, the antioxidant enzyme CAT and the heme oxygenase isoform HO-1 were also increased after WG treatment; these enzymes can help scavenge hydrogen peroxide and catalyze the breakdown of heme, respectively, thus enhancing the cell's antioxidant capacity.

[0075] Based on the anti-inflammatory properties of WG and its inhibitory effect on osteoclastogenesis in vitro, we established an LPS-induced mouse model of inflammatory bone loss. Micro-CT scanning and analysis of bone tissue parameters showed that WG treatment significantly reduced LPS-induced bone loss, and the high-concentration group exhibited similar therapeutic potential to DEX. Furthermore, HE staining of the mouse heart, liver, and kidneys showed no pathological changes, indicating that the WG concentration used in this experiment was safe and effective.

[0076] Conclusion: Baicalin inhibits osteoclast differentiation, bone resorption, and LPS-induced inflammatory osteolysis in mice, potentially by suppressing RANKL-mediated ROS levels and the downstream MAPK signaling pathway. Therefore, this invention may provide a novel therapeutic approach for the prevention of inflammatory osteolysis.

[0077] Matters not covered in this invention are common knowledge.

[0078] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of baicalin in the preparation of drugs for the prevention and treatment of inflammatory osteolysis.

2. The application of baicalin according to claim 1 in the preparation of drugs for the prevention and treatment of inflammatory osteolysis, characterized in that: Baicalin inhibits RANKL-induced osteoclastogenesis and bone resorption, and the MAPK and NF-κB signaling pathways involved in osteoclast differentiation were detected by Western blotting and PCR experiments.

3. The application of baicalin according to claim 1 in the preparation of drugs for the prevention and treatment of inflammatory osteolysis, characterized in that: A mouse model of LPS-induced inflammatory osteolysis was constructed to simulate the occurrence of inflammatory bone loss. The degree of bone destruction and the number of osteoclasts were analyzed by microcomputed tomography and staining with hematoxylin, eosin and tartrate-resistant acid phosphatase to determine the molecular mechanism by which baicalin prevents inflammatory osteolysis.

4. The application of baicalin according to claim 1 in the preparation of drugs for the prevention and treatment of inflammatory osteolysis, characterized in that: Baicalin inhibited RANKL-induced osteoclastogenesis in a concentration- and time-dependent manner and showed no cytotoxicity to bone marrow cytokines (BMMs). Baicalin inhibited the expression of osteoclast-related genes and reduced the ability of osteoclasts to resorb bone. In terms of the mechanism of inhibiting osteoclastogenesis, baicalin inhibited the expression of the intracellular transcription factor NFATc1. Baicalin inhibited osteoclasts by inhibiting the expression of RANKL-mediated TRAF6 and related downstream osteoclast formation and bone resorption-related proteins. For the classical signaling pathway mediated by RANKL, baicalin inhibited the phosphorylation levels of JNK, ERK, and p38 proteins in the MAPK signaling pathway. Baicalin reduces ROS production by inhibiting NOX1 expression, while promoting the expression of antioxidant enzymes Nrf2, HO-1, and Cat to clear ROS from intracellular and mitochondrial spaces. In mice with inflammatory osteolysis, baicalin significantly increased the volume fraction and number of trabeculae in the femur, and reduced the levels of CTX-1, IL-1β, IL-6, and TNF-α in serum. Baicalin also significantly reduced the number of osteoclasts, the expression of NFATc1 transcription factor, and NOX1 protein in the mouse femur. In vivo reverse transcription quantitative PCR and Western blotting results showed that baicalin inhibited the expression of NFATc1 and CTSK, while increasing the expression of antioxidant enzymes to clear ROS, and inhibiting the number and activity of osteoclasts to protect against bone loss.

5. The application of baicalin according to claim 1 in the preparation of drugs for the prevention and treatment of inflammatory osteolysis, characterized in that: Baicalin reduces intracellular ROS by inhibiting the RANKL-mediated TRAF6-MAPK signaling pathway, thereby reducing the expression of the transcription factor NFATc1 and enhancing the expression of antioxidant enzymes. This leads to the elimination of intracellular ROS, which in turn inhibits osteoclast formation and bone resorption. By enhancing the expression of antioxidant enzymes and reducing ROS, it effectively protects against bone loss in mice with inflammatory osteolysis.

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