Application of farrerol in preparation of medicine for preventing osteoporotic diseases caused by inflammatory bowel diseases

Abstract

The invention provides an application of farrerol in preparation of a medicine for preventing osteoporotic diseases caused by inflammatory bowel diseases, and belongs to the technical field of inflammatory bone disease treatment.The application comprises the steps that firstly, an ulcerative colitis (UC) mouse is constructed to simulate occurrence of the osteoporosis induced by enteritis, and the occurrence of the osteoporosis induced by enteritis is simulated through microcomputer tomography (CT), hematoxylin and eosin (Hamp; e) and tartrate-resistant acid phosphatase (TRAP) staining are used for analyzing the bone destruction degree and the number of osteoclasts, and the influence of the farrerol on osteoclast generation and bone loss is detected. The influence of the farrerol on the inflammatory bowel disease is analyzed through 16S rRNA (ribosomal Ribonucleic Acid) sequencing. In addition, PI3K-AKT and MAPK signal pathways related to osteoclast differentiation of the farrerol are detected through WB and PCR experiments to clarify the basic molecular mechanism of the farrerol for preventing osteoporosis. Results show that the farrerol can improve bone loss caused by enteritis, recover intestinal flora composition and repair intestinal barriers.

Description

Technical Field

[0001] The present invention relates to the technical field of inflammatory bone disease treatment, and in particular to the use of rhododendron in the preparation of a drug for preventing osteoporosis caused by inflammatory bowel disease. Background Art

[0002] As the population ages around the world, the frequency of osteoporosis (OP) is increasing, characterized by decreased bone mineral density, increased bone fragility, and increased susceptibility to fractures. Under normal physiological conditions, the formation of osteoporosis is associated with a decrease in osteoblasts and an increase in osteoclastogenesis. Osteoclasts coordinate bone resorption by secreting acid and collagenolytic enzymes, while in various diseases with an osteoporotic phenotype, excessive osteoclasts lead to localized bone defects, i.e., bone loss. Systemic bone loss is often associated with chronic inflammation, which is manifested in various diseases such as rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus. Among them, inflammatory bowel disease (IBD) is closely related to bone diseases, but its specific mechanism of action has not been fully studied.

[0003] Inflammatory bowel diseases include ulcerative colitis and Crohn's disease. Among them, ulcerative colitis (UC) has mucus, purulent bloody stools and abdominal pain as typical clinical symptoms. Many studies have found that the antigenic particles of the COVID-19 virus will exist in the human intestine for a long time, causing abdominal pain, diarrhea and other new crown sequelae, and these viruses will also activate intestinal inflammation. A large number of inflammatory cells infiltrating the intestinal mucosal layer can cause damage to the intestinal mucosal barrier, intestinal flora disorders, and immune dysfunction, which in turn cause severe inflammatory responses. At the same time, the long-term inflammatory state of the intestine will also increase the risk of osteoporosis, and in many clinical studies, the intestinal microbiota has been shown to have a profound effect on the quantity, quality and overall strength of bones.

[0004] Clinically, the main treatments for ulcerative colitis are 5-aminosalicylic acid and glucocorticoids. Although these drugs can temporarily control symptoms, their long-term efficacy is not ideal and they have many side effects. Osteoporosis secondary to inflammatory bowel disease is mainly treated with anti-resorptive drugs, including bisphosphonates and denosumab, but safety and efficacy issues limit their application. These limitations have prompted us to further comprehensively and in-depth study the biological characteristics of osteoclasts and related inflammatory pathways in order to identify new therapeutic drugs.

[0005] Traditional Chinese medicine can reduce the incidence or prevent diseases by regulating the body's state, improving immunity, and blocking the progression of the disease. Many studies have shown that traditional Chinese medicine can inhibit the occurrence and development of inflammatory bowel disease and osteoporosis by exerting anti-inflammatory and antioxidant effects and regulating the body's immune response. Rhododendron is one of the largest genera of vascular plants, and its main functions are to treat inflammation, pain, common colds and gastrointestinal diseases. Its main active ingredient, rhododendron (rhododendron rrerol, rhododendron), is a 2,3-dihydroflavonoid compound. Modern pharmacological studies have shown that rhododendron has anti-inflammatory, antioxidant and anti-cancer effects. However, it is unclear whether rhododendron affects osteoclastogenesis and bone mass changes during ulcerative colitis.

[0006] In view of this, the present invention provides an application of rhododendron in the preparation of a drug for preventing osteoporosis caused by inflammatory bowel disease, so as to solve the above problems. Summary of the invention

[0007] The purpose of the present invention is to provide the use of rhododendrin in the preparation of a drug for preventing osteoporosis caused by inflammatory bowel disease, and to solve the technical problems mentioned in the background technology.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] Application of rhododendron in the preparation of drugs for preventing osteoporosis caused by inflammatory bowel disease.

[0010] In order to explore whether rhododendron prevents bone loss in UC mice, the present invention divides mice into a control group, a UC group and a drug-added group. First, rhododendron treatment improves the colon length of UC mice and has no obvious toxic effects on organs. Secondly, CT and staining results show that rhododendron improves bone loss caused by enteritis. At the same time, we conducted serological tests and histochemical staining experiments, and the results showed that bone resorption indicators, osteoclast numbers and specific protein expressions of UC mice decreased after rhododendron treatment. In order to detect changes in the intestinal flora of mice, we used mouse feces for 16sRNA sequencing. The results showed that rhododendron treatment improved the types and abundance of intestinal flora, increased the number of beneficial bacteria, and improved the intestinal flora disorder caused by enteritis. At the same time, we detected short-chain fatty acids and inflammatory indicators in the serum levels of mice. The results showed that rhododendron treatment reduced the level of inflammatory factors and increased the content of short-chain fatty acids. Immunohistochemical staining showed that rhododendron repaired intestinal integrity. Then we extracted BMMs cells in UC mice and mice in the drug-added group to induce osteoclasts, and then performed RNA sequencing. The results showed that differential genes were involved in the regulation of inflammatory response and osteoclast differentiation. In addition, after rhododendron treatment, multiple pathways such as PI3K-AKT and MAPK were significantly inhibited, and osteoclast-related genes and inflammation-related genes were decreased, indicating that rhododendron may be involved in inhibiting the formation of osteoclasts caused by inflammation.

[0011] For in vitro verification, LPS was used to simulate the intestinal inflammation environment in vivo, and rhododendronin had an inhibitory effect on RANKL and LPS-induced osteoclastogenesis, F-actin ring formation and bone resorption; and the basic molecular mechanism was elucidated by detecting the PI3K-AKT and MAPK signaling pathways involved in osteoclast differentiation through WB and PCR experiments.

[0012] After a large number of experiments, the results showed that rhododendron improved the bone loss caused by enteritis and inhibited the activity of osteoclasts by improving the expression of inflammatory factors and restoring the composition of intestinal flora. In vitro experiments showed that rhododendron can effectively inhibit the formation of osteoclasts induced by RANKL and LPS, reduce bone resorption and the formation of F-actin rings and the expression of osteoclast marker genes; it was further discovered that rhododendron inhibited the formation of osteoclasts by mediating the PI3K-AKT and MAPK signaling pathways.

[0013] Therefore, rhododendrin is a very promising drug for preventing osteoporosis caused by inflammatory bowel disease.

[0014] The present invention found that: in UC mice, rhododendron significantly increased the volume fraction and number of femoral trabecular bone, reduced the content of CTX-1 and TRACP5b in serum and increased the content of PINP. It can be seen that rhododendron significantly inhibited the number and activity of osteoclasts to protect bone loss. In addition, rhododendron improved the inflammatory environment in mice, increased the types and number of beneficial intestinal flora, and repaired the intestinal barrier.

[0015] In addition, in terms of the biological behavior of osteoclasts, rhododendrin inhibited RANKL and LPS-induced osteoclastogenesis and showed no toxic effect on the cell activity of BMMs. Functionally, rhododendrin can inhibit the expression of osteoclast-related genes and attenuate the ability of osteoclast actin ring formation and bone resorption. In terms of the mechanism of inhibiting osteoclastogenesis, d-rhododendrin B inhibited the expression of intracellular transcription factor N-rhododendrin Tc1. At the same time, rhododendrin inhibited osteoclast formation and bone resorption-related protein expression mediated by RANKL and LPS, thereby inhibiting osteoclasts. And rhododendrin inhibited the phosphorylation of PI3K and AKT proteins in the PI3K-AKT signaling pathway; reduced the phosphorylation levels of JNK, ERK and p38 proteins in the MAPK signaling pathway. In addition, rhododendrin can reduce inflammation by inhibiting the expression of IL6, IL10, IL17 and TNF-α.

[0016] In summary, rhododendrin inhibits RANKL and LPS-mediated PI3K-AKT and MAPK signaling pathways to reduce the expression of intracellular transcription factor N-rhododendrin Tc1, thereby inhibiting osteoclastogenesis and bone resorption. Rhododendrin can effectively protect UC mice from bone loss by inhibiting the expression of inflammatory factors.

[0017] The experimental method for applying rhododendron in the preparation of drugs for preventing osteoporosis caused by inflammatory bowel disease includes: cell culture, cell viability assay, osteoclast differentiation assay and TRAP staining, bone resorption function assay, F-actin ring formation assay, RNA extraction and quantitative PCR assay, protein blot analysis, model construction and data statistical analysis.

[0018] The present invention has the following beneficial effects due to the adoption of the above technical solution:

[0019] The rhododendron of the present invention can improve bone loss caused by enteritis, restore the composition of intestinal flora, and repair the intestinal barrier. Rhododendron can effectively inhibit the formation of inflammatory osteoclasts induced by RANKL and LPS, reduce bone resorption and the formation of F-actin rings and the expression of osteoclast marker genes. Rhododendron inhibits the generation of inflammatory osteoclasts by inhibiting PI3K-AKT and MAPK signaling pathways. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a formation diagram that rhododendrin inhibits RANKL-mediated osteoclastogenesis in vitro;

[0021] Figure 2 It is rhododendrin that improves the enteritis-induced bone loss picture;

[0022] Figure 3 Schematic diagram of the regulatory effect of rhododendrin on bone turnover in UC mice.

[0023] Figure 4 This is the regulatory diagram of rhododendron tinea on the intestinal flora of UC mice.

[0024] Figure 5 It is the effect of rhododendron on intestinal flora metabolites and inflammatory factors.

[0025] Figure 6 It is a diagram of the inhibition of RANKL- and LPS-induced inflammatory osteoclastogenesis and inflammatory osteoclast resorption function by rhododendrin;

[0026] Figure 7 is the specific gene expression map of rhododendrin inhibiting inflammatory osteoclasts;

[0027] Figure 8 This is a schematic diagram showing that rhododendrin inhibits the conduction of MAPK and PI3K-AKT signaling pathways in OCs differentiation; DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is 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 only for the purpose of enabling the reader to have a thorough understanding of one or more aspects of the present invention, and these aspects of the present invention can be implemented even without these specific details.

[0029] 1. Materials and Methods

[0030] 1.1 Pharmaceuticals and Chemicals

[0031] Rhododendron was purchased from Munster (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). LPS was purchased from ThermoFisher Scientific (Shanghai, China). Specific antibodies were purchased from Cell Signaling Technology (Danvers, MA). TRAP staining kit was purchased from Sigma (St. Louis, MO, USA). 96-well hydroxyapatite plates were purchased from Corning Osteoassay. Hematoxylin and eosin staining kit was purchased from Solebol Technology (Beijing, China).

[0032] 1.2 Cell culture

[0033] Extraction and culture of primary bone marrow macrophages (BMMs): C57BL / 6 mice of about six weeks old that grew well were killed by cervical dislocation in the sampling room. Ethanol disinfectant was sprayed on the mouse body to achieve the purpose of sterilization. Ophthalmic scissors that were sterilized in advance were used to quickly cut the Achilles tendon of the lower limb, peel off the muscle from the Achilles tendon to the femoral head, cut the lower limb at the hip joint and cut off the claws (avoid exposing the bone marrow cavity), put them in a culture dish (the culture dish was placed on ice), and transferred to a biosafety cabinet to further remove the remaining muscle tissue of the tibia and femur. Add 5mL PBS to wash once, then add 3mL MEMAlpha complete medium (M-CSF final concentration is 25ng / mL), clamp the lower limb with forceps and cut the epiphysis, then use a 1mL syringe to absorb the complete medium and repeatedly rinse the medullary cavity until the bone head turns white. Transfer the mixture of bone marrow and culture medium to a 50mL centrifuge tube and blow it away with a Pasteur pipette. Centrifuge at 1200rpm for 5min and discard the supernatant. Resuspend the cells with 3mL MEMAlpha complete medium, filter the cells with a 100μm filter and drop into a T75 culture flask. Make up the medium to 15mL and let it stand in a cell culture incubator for 2 days. After that, remove the old medium in the flask, rinse the cells twice with PBS to remove dead cells and impurities, replace with new MEMAlpha complete medium and continue to culture for two days. The adherent cells are considered to be bone marrow-derived macrophages and can be used for subsequent experiments.

[0034] 1.3 Cell viability assay

[0035] Effects of the main components of rhododendron on the activity of BMMs cells: The counted BMMs cells were adjusted to a density of 6×10 3 / mL, 100μL per well was inoculated in a 96-well plate, and an equal amount of PBS solution was added around the wells where the cells were inoculated to reduce the evaporation of the cell culture medium, and then placed in a cell culture incubator for overnight culture. The next day, after the cells were fully attached, the original culture medium was discarded and replaced with MEMAlpha culture medium containing different monomers (concentrations of 10μM and 20μM), and a control group without drugs was set up, i.e., containing the same volume of DMSO. After the cells were cultured in the incubator for 48 hours, CCK-8 solution (10μL) was added to the cells in each well, and care was taken to avoid bubbles. After the culture plate was placed in the incubator for another 2 hours, the absorbance at 450nm was detected by an enzyme reader and the experimental results were derived.

[0036] 1.4 Osteoclast differentiation assay and TRAP staining

[0037] Different concentrations of rhododendronine were used to differentiate osteoclasts: 6×10 3BMMs cells per well were cultured overnight. The cells were observed under a microscope the next day to confirm whether the cells grew well. Grouping was set up: negative control group (culture medium was MEMAlpha complete culture medium), positive control group (culture medium was MEM Alpha complete culture medium + 50ng / mL RANKL + 100ng / mL LPS) drug treatment group (MEMAlpha complete culture medium + 50ng / mL RANKL + 100ng / mL LPS + 30μM rhododendron). Unless otherwise specified, the RANKL working concentration used in this embodiment was 50ng / mL. Remove the original culture medium, replace the above culture medium and change the medium every other day until the cells in the positive control group fuse to form giant mature osteoclasts containing multiple nuclei. Take the culture plate out of the incubator, gently aspirate the culture medium, fix it with 4% paraformaldehyde for 1h, rinse the bottom cells 3 times with 1×PBS, add TRAP staining solution to each well, incubate in the dark for 30min, check the degree of cell staining under the microscope, and remove the staining solution when the overall color is pink. Each well was then rinsed three times with double distilled water to remove excess staining solution, dried in a 37°C oven, and scanned with a cell imaging instrument, Cytation5.

[0038] 1.5 Bone resorption function experiment

[0039] Bovine cortical bone slices were placed in a 10 cm cell culture dish and marked with pencil on the back to divide them into negative control group and drug treatment group (30 μM rhododendron). Ethanol disinfectant was added to cover the bone slices, and then the culture dish was sealed with sealing film and placed in a 4°C decolorizing shaker for 24 hours. After 24 hours, the medium was replaced with MEMAlpha medium and continued to shake for 24 hours to replace the remaining ethanol disinfectant. The bone slices were transferred to a 96-well cell culture plate with ophthalmic forceps, and the BMMs cells counted were removed and 10 4 The number of cells / well was inoculated on the bone slices, and 6×10 3 The number of cells / well was inoculated in a 96-well plate for TRAP staining control and placed in a cell culture incubator overnight. RANKL and LPS were added for stimulation on the second day, and the culture medium was changed every two days until mature osteoclasts appeared in the 96-well plate used for control. The 96-well plate was removed, and the culture medium was aspirated and 4% paraformaldehyde was added to fix the cells for 1 hour. The 96-well cell culture plate containing bone slices was changed once, and the culture was continued for 48 hours before being fixed with electron microscopy fixative. 50 μL of TRAP staining solution was added to each well for staining for 20 minutes, and the excess staining solution was washed off with double distilled water. After drying, the cells were photographed. Cells with positive staining and ≥3 nuclei were considered mature osteoclasts. The bone slices were taken out with ophthalmic forceps, and the cells were brushed off with a brush and dried. After gold spraying, electron microscopy scanning and photography were performed.

[0040] 1.6 Determination of F-actin ring formation

[0041] 6×103 BMMs cells were placed in each well and grouped as control group (DMSO in equal volume) and rhododendronine treatment group (30 μM) for overnight culture. The cells were replaced every two days until most cells in the control group were fused and mature osteoclasts appeared. The culture medium was then removed and the cells were fixed with 4% paraformaldehyde for 1 hour. The cells were rinsed twice with 1×PBS buffer and 100 μL of Triton X-100 was added to each well to permeabilize the cell membrane for 10 minutes. The original liquid was removed and 100 μL of 3% BSA-PBS was added to each well to block the cells for 1 hour. After calculating the required volume according to the number of wells, tetramethylrhodamine isothiocyanate-labeled phalloidin was diluted in 0.2% BSA-PBS at a ratio of 1:200. After blocking, the cells were washed 3 times with 0.2% BSA-PBS and 100 μL of diluted TRITC-labeled phalloidin was added to each well for staining in the dark for 30 minutes. DAPI was diluted in 0.2% BSA-PBS (1:500 ratio), 100 μL was added to each well for 5 min in the dark, and the cell nucleus was counterstained. The excess DAPI stain was washed away with double distilled water, and the cells were photographed using the fluorescence channel of the cell imaging instrument Cytation5, and the pictures were saved.

[0042] 1.7 RNA extraction and quantitative PCR assay

[0043] According to the experimental protocol of the kit manual, total RNA was isolated and extracted from osteoclasts using Trizol reagent. To synthesize cDNA, 1 μg RNA was reverse transcribed with oligo-dT primer (Thermo Fisher kit, USA). Real-time PCR was performed using LightCycler 96 (Switzerland, Basel), and the amplification process was: 94°C pre-denaturation for 10 minutes, followed by 35 cycles, each cycle of 94°C (10 seconds), 60°C (15 seconds), and finally at 72°C (10 seconds), and then the PCR data was read after the reverse transcription was terminated.

[0044] Table 1 below lists the specific primers for osteoclast-related genes used in this experiment:

[0045] Table 1

[0046]

[0047] 1.8 Western blot analysis

[0048] BMMs were treated with rhododendron in advance for 3 hours, and then stimulated with 50ng / mL of RANKL and 100ng / mL of LPS for 0 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes and 60 minutes respectively. Subsequently, cells were washed with 1x PBS and lysed with ice-cold RIPA buffer containing a mixture of phosphatase and protease inhibitors. Protein concentration was then assessed using BCA protein assay reagent (Beyotime, China). Whole cell lysate (15 μL) was subjected to 10% SDS-PAGE and then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA).

[0049] 1.9 Construction of UC mice

[0050] 6-week-old male C57BL / 6 mice were purchased from GemPharmatech (Guangdong, China). These mice were maintained at a density of 5 mice per cage in a standard SPF facility at Guangxi Medical University. The environmental conditions of temperature, humidity and light-dark cycle were 20-24°C, 40-50% and 12h, respectively. For the UC model induced by M.Wt36,000-50,000 dextran sulfate sodium (DSS) (MP Biomedicals, 9011-18-1), 3% DSS was solvated in drinking water and replaced every two days. After one week, the concentration was adjusted to 0-1.5% every day to maintain mild to moderate inflammation according to DAI, which was determined by blood in the stool, weight loss and stool properties. The score range for each item was 0-4, which was positively correlated with severity. The grade of intestinal inflammation was then defined as severe (8-12), moderate (4-8) and mild (0-4) according to the total DAI score. DAI was assessed at the same time point every day since the start of the experiment. This dynamic adjustment continued until the end of the experiment, and the control group was fed with fresh water. The chronic UC model was established by the above-mentioned DSS, and the concentration changed dynamically from the 8th day to the 28th day.

[0051] 1.10 Statistical analysis

[0052] The experiments involved were completed at least three times. The experimental results were statistically analyzed using GraphPad Prism (9.0), and the data results were presented as mean ± standard deviation. Statistical analysis was performed using one-way / two-way analysis of variance. When the p value was less than 0.05, the difference was considered statistically significant.

[0053] 2. Results

[0054] 2.1 Rhododendrin inhibits RANKL-mediated osteoclastogenesis in vitro

[0055] In order to more accurately detect the effects of more different concentrations of rhododendron (0, 5, 10, 20, 30 and 40 μM) on BMMs cell proliferation, the present invention uses real-time cell analysis technology to continuously monitor the cells. First, the toxicity of rhododendron to BMMs was examined after 96 hours. CCK-8 assay showed that within the concentration range of 0-40 μM, rhododendron treatment did not affect cell proliferation or apoptosis in BMMs ( Figure 1 B). To explore whether rhododendrin can inhibit osteoclastogenesis, we cultured BMMs with different doses of rhododendrin (0, 5, 10, 20, 30, and 40 μM) and stimulated with RANKL. It was found that rhododendrin reduced osteoclast formation in a dose-dependent manner within a safe range ( Figure 1 CD). According to the above results, rhododendrin inhibited RANKL-induced osteoclast development in vitro and had no cytotoxic effect on osteoclasts. (A) is the structural formula of rhododendrin; (B) is the toxicity test of rhododendrin on BMMs cells at 96 hours. (C) is the TRAP staining image of BMMs cultured with or without RANKL in the presence of indicated concentrations of rhododendrin from 0 to 40 μM, n=4. (D) Quantification of TRAP in each well + Osteoclasts (nuclei ≥ 3). Data are expressed as mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.001).

[0056] 2.2 Rhododendronine improves bone microstructure in ulcerative colitis mice

[0057] In order to explore whether rhododendron can prevent bone loss in UC mice, the mice were divided into a control group, a DSS group and a rhododendron treatment group ( Figure 2 A). UC mice showed significant weight loss and increased disease activity index (DAI) ( Figure 2 To investigate the effect of rhododendron on IBD-induced bone loss, the bone microarchitecture of the distal femur was evaluated using CT. Three-dimensional (3D) images of the distal femur were used to visualize the microstructure of trabecular bone ( Figure 2 D). The distal femoral trabeculae in the DSS group were thinner, fewer, less dense, sparsely arranged, and had an enlarged bone marrow cavity. Rhododendron treatment improved bone loss and the microstructure of trabecular bone. Bone volume fraction (BV / TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and bone surface area density (BS / TV) were also increased, as well as lower trabecular separation (Tb.Sp) and structural model index (SMI) ( Figure 2 E). HE staining also showed that the trabecular area of ​​mice treated with rhododendrin increased ( Figure 2FG). Overall, rhododendrin treatment improved bone loss in UC mice. (A) Schematic diagram of UC mice treated with rhododendrin. Femurs were finally collected on day 28. (B) Percentage of body weight change, followed by statistical analysis on day 14. n=6. (C) Changes in disease activity index (DAI). n=6. (D) Representative micro-CT reconstruction images of femurs. Overall view (top), cortical bone (bottom). n=6. (E) Quantitative analysis of trabecular bone, including BV / TV, Tb.Th, Tb.N, BS / TV, Tb.Sp and SMI. n=6. (F) Representative HE staining images of mouse femoral tissue sections. (G) Quantification of BV / TV volume in HE stained sections. n=6. Data are expressed as mean ± SD (*p<0.05; **p<0.01; ***p<0.001).

[0058] 2.3 Rhododendron regulated the expression of bone turnover markers in ulcerative colitis mice.

[0059] In order to evaluate the effect of rhododendrin in treating osteoporosis, the present invention examined the serum levels of bone biochemical markers. The level of TRACP-5b in UC mice was significantly increased ( Figure 3 A), indicating increased bone resorption, while rhododendrin significantly reduced this expression. The level of CTX-1 in the serum of UC mice was also significantly increased ( Figure 3 B), also showed that its bone resorption activity was significantly increased, and rhododendron treatment reduced bone resorption activity. In addition, compared with the control mice, the serum PINP level of UC mice was significantly reduced ( Figure 3 C), indicating that its bone formation activity was reduced, while rhododendrin significantly increased serum PINP levels. The ratio of bone turnover markers ( Figure 3 D) shows that CTX-1 / PINP in UC mice increased significantly, and returned to normal levels after rhododendrin treatment. To explore the effect of rhododendrin on osteoporosis in mice with inflammatory bowel disease, TRAP staining was used to evaluate the histomorphology of the femur, and immunohistochemical staining of the osteoclast-specific protein CTSK was performed. Positive cells with more than three nuclei and peripheral staining in red were selected as representative images of osteoclasts ( Figure 3 E), showing that inflammatory bowel disease leads to a significant increase in osteoclasts, which can be counteracted by rhododendrin treatment ( Figure 3 F). Immunohistochemical staining results showed that compared with the control group, the number of osteoclasts expressing CTSK in UC mice increased, and the number of osteoclasts expressing CTSK was reduced after rhododendrin treatment ( Figure 3GH). The number of OCN-positive cells expressing UC mice was reduced, while rhododendrin restored the expression of OCN in mice. This indicates that rhododendrin inhibited the number and specific protein expression of osteoclasts in UC mice, and promoted the expression of osteoblast bone turnover markers. (AD) The changes in serum TRACP-5b, CTX-1, PINP and CTX-1 / PINP in each group were observed. n=6. (E) Representative images of TRAP staining of decalcified bone sections. n=6. Red arrows, osteoclasts. (F) Quantitative analysis of N.Oc / B.Pm. n=6. (G) Representative images of CTSK immunohistochemical staining in femoral tissues of the three groups. (H) N.CTSK + Quantitative analysis of Oc / B.Pm. n = 6. Data are expressed as mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.001).

[0060] 2.4 Rhododendron tinea normalizes the intestinal microbiota of ulcerative colitis mice and changes the intestinal microbial diversity.

[0061] In order to clarify the effect of rhododendron on osteoporosis through the gut / bone axis, the present invention evaluated the changes in the intestinal microbiota by detecting the feces of all groups of mice and performing 16S rRNA sequencing analysis. The results of principal component analysis (PCA) showed that at the operational classification unit (OUT) level ( Figure 4 A), the DSS group and the CTRL group can be better separated, and the rhododendron group is close to and overlaps with the CTRL. This indicates that rhododendron regulates and restores the intestinal flora of UC mice. In the α diversity index, the ace and Chao indices are used to characterize the species richness of the community, and the Shannon and Simpson indices characterize the diversity of the community. Rhododendron increases the community richness index ( Figure 4 B). This indicates that the abundance and diversity of intestinal flora increased after treatment with rhododendron, and the community richness improved. At the species level, harmful bacteria such as Enterobacter were significantly distributed in the DSS group, while beneficial bacteria such as Bacteroides were significantly dominant after rhododendron treatment ( Figure 4 C). To investigate the specific changes in the bacterial community, we plotted cluster histograms to show the changes in the intestinal flora at the phylum, family, and genus levels in each sample and in the subgroups. The relative abundance of the dominant bacterial phyla, families, and genera between the groups was also compared. As shown in the figure, Firmicutes and Bacteroidetes were dominant in each group at the phylum level, and their percentage contents were analyzed separately ( Figure 4DE), the results showed that the relative abundance of Firmicutes and Desulfobacteria increased in UC mice, and the relative abundance of Bacteroidetes decreased; after treatment with rhododendrin, the relative abundance of Firmicutes and Desulfobacteria decreased, and the relative abundance of Bacteroidetes increased. In addition, the relative abundance ratio of Firmicutes / Bacteroidetes in UC mice increased, and rhododendrin reduced it and restored it to the same level as the normal control group ( Figure 4 F). Rhododendronine treatment reorganized the bacterial flora. Compared with UC mice, rhododendronine increased the relative abundance of Bacteroidetes and Lactobacillaceae, making the overall flora structure similar to that of the CTRL group at the family level ( Figure 4 GI). At the genus level, we found that the level of Desulfovibrio increased and the levels of Bacteroides and decreased in the DSS group compared with the CTRL group, while rhodopsin significantly increased the level of Bacteroides ( Figure 4 JL). This indicates that after treatment with rhododendron, the types and numbers of beneficial intestinal bacteria increased significantly and returned to the type of intestinal flora in normal mice, improving the intestinal flora disorder caused by enteritis.

[0062] (A) PCA plot of intestinal microbiota at the OUT level. (B) Reflects the changes in alpha diversity, ace, Shannon, Chao, Simpson and coverage index of intestinal microorganisms in feces. (C) Species with significant differences in abundance among the three groups, and the length of the histogram represents the significance of different species. (DE) Relative abundance of intestinal microbiota at the phylum level. (F) Ratio of Firmicutes / Bacteroidetes at the phylum level. (GH) Relative abundance of intestinal microbiota at the family level. (I) The four most abundant families were analyzed separately. (JK) Relative abundance of intestinal microbiota at the genus level. (L) The four most abundant genera were analyzed separately. Each column represents a sample, and each column represents a group. Data are expressed as mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.001).

[0063] 2.5 Rhododendron oxytocin regulates inflammatory cytokine expression and improves fecal short-chain fatty acids and intestinal integrity.

[0064] The present invention found that rhododendron may affect the changes in bone mass by affecting the intestinal flora metabolites of UC mice, so we detected the content of short-chain fatty acids in the feces of all groups ( Figure 5A). Compared with CTRL, the concentrations of propionic acid, isovaleric acid, isobutyric acid, butyric acid, valeric acid, and acetic acid in UC mice were greatly decreased. Significant differences were also observed in the production of propionic acid, isovaleric acid, and isobutyric acid compared with the rhododendron group. The content of short-chain fatty acids increased after rhododendron treatment. HE staining of the colon in normal mice showed that the intestinal crypts were abundant and neatly arranged, without pathological changes such as inflammation, necrosis, and edema, and the morphological structure was normal. However, the tissues and mucosa of UC mice were locally damaged, the intestinal glands were atrophied and deformed, and there was inflammatory cell infiltration. Rhododendron treatment reduced mucosal damage and inflammatory cell infiltration, and improved the crypt structure ( Figure 5 B). In addition, the expression of tight junction proteins occludin and ZO-1, which reflect intestinal epithelial permeability, was significantly reduced in UC mice. Rhododendronine increased the expression of occludin and ZO-1 and restored intestinal integrity ( Figure 5 CD). Then, by detecting the changes of serum inflammatory factors ( Figure 5 E), showing that rhododendronine reduced the levels of IL-6, IL-17, IL-10 and TNF-α, further verifying that rhododendronine can reduce the level of inflammation in vivo. (A) Contents of propionic acid, isovaleric acid, isobutyric acid, butyric acid, valeric acid and acetic acid in mouse feces. (B) Representative HE staining of colon. n=6. (C) Representative images of Occludin immunohistochemical staining in colon tissues of the three groups. (D) Representative images of ZO-1 immunohistochemical staining in colon tissues of the three groups. (E) Changes in serum IL-6, IL-17, IL-10 and TNF-α were observed in each group. n=6. Data are expressed as mean ± SD (*p<0.05; **p<0.01; ***p<0.001).

[0065] 2.6 Rhododendron inhibits RANKL- and LPS-induced inflammatory osteoclastogenesis and inflammatory osteoclast resorption function. (A) Cell viability of BMMs treated with rhododendron was measured by CCK-8 assay, n = 4. (B) Schematic diagram of the treatments used to evaluate the osteoclastogenic activity of RANKL, LPS, and rhododendron. (C) TRAP staining images of BMMs cultured with or without RANKL and LPS in the presence of the indicated concentrations of rhododendron (30 μM), n = 4. (D) Quantification of TRAP+ osteoclasts (nuclei ≥ 3) in each well. (E) Representative images showing impaired podosome band formation in osteoclasts treated with rhododendron. (F) Quantification of relative area of ​​osteoclasts. (G) Quantification of the number of nuclei per osteoclast. (H) Representative images of bone resorption areas formed by osteoclasts treated with rhododendron obtained by scanning electron microscopy. (I) Quantification of bone resorption area.

[0066] The pathogenesis of osteoporosis is characterized by an increase in the number and activity of osteoclasts. Under inflammatory conditions, the generation of osteoclasts is further stimulated, accelerating bone loss. Therefore, in order to simulate the inflammatory environment in enteritis mice, we explored the effect of rhododendron on osteoclastogenesis under the stimulation of RANKL and LPS in vitro. First, the toxicity of rhododendron and the addition of LPS to BMMs was examined after 96 hours. CCK-8 assay showed that rhododendron (30 μM) and LPS (100 ng / ml) had no cytotoxic effect on BMMs ( Figure 7 A). This ruled out the possibility that the inhibitory effect of rhododendrin on osteoclast differentiation was caused by decreased cell proliferation or increased apoptosis in BMMs. To determine the potential role of rhododendrin in inflammatory osteoclast formation, osteoclast-related experiments were performed with or without rhododendrin treatment ( Figure 7 B), data measured by staining for tartrate-resistant acid phosphatase (TRAP), a widely used osteoclast marker, showed that when BMMs cells were pre-treated with RANKL and then treated with LPS, the number and area of ​​osteoclasts generated were greater than when RANKL was added alone. Rhododendrin inhibited the generation of osteoclasts under inflammatory conditions ( Figure 7 CD). In addition, under the intervention of rhododendrin, the formation of the F-actin-based podosome belt, which is dependent on bone resorption, was inhibited and the area was significantly reduced ( Figure 7 EG). Therefore, we further inoculated osteoclasts on sterile bovine bone slices to observe the effect of rhododendron on bone resorption in the presence of LPS. When RANKL was added, absorption pits appeared on the bone blocks, and after LPS was added to RANKL, the area of ​​absorption pits on the bone blocks increased. On the bone slices intervened by rhododendron, the area of ​​absorption pits was significantly reduced, and the bone resorption function decreased ( Figure 7HI). These results suggest that rhododendron plays a role in resisting inflammatory osteoclastogenesis and inflammatory osteoclast resorption. (AF) qPCR was used to detect the expression levels of CTSK, N-rhododendron tc1, c-Fos, ATP6V0D2, MMP9 and Traf6 relative to β-actin in BMMs stimulated with RANKL and LPS for 5 days in the presence of rhododendron (n=3). (G) Images of RANKL and LPS-induced nuclear translocation of N-rhododendron tc1 in the absence and presence of rhododendron. (H) Quantification of nuclear / cytoplasmic fluorescence ratio of N-rhododendron tc1 (n=4) (I) Representative Western blot images of the expression levels of N-rhododendron tc1, c-Fos, CTSK, ATP6V0D2 and MMP9. BMMs treated with or without rhododendron were cultured for 1, 3 and 5 days in the presence of RANKL and LPS, and the expression of related proteins was detected by Western Blot. (JN) Quantification of the band intensity ratios of N-drugine Tc1, c-FOS, CTSK, ATP6V0D2, and MMP9 relative to β-actin (n=3).

[0067] 2.8 Rhododendron tinctorius inhibits inflammatory osteoclast-specific gene expression.

[0068] Based on the effect of rhododendron on the number of osteoclasts and bone resorption function, the present invention further explored the effect of rhododendron on osteoclast-related genes by reverse transcription quantitative PCR. The detected osteoclast-related specific expression genes included the expression of genes mediating osteoclast fusion and resorption (Ctsk, Mmp9, N-rhododendron tc1, Fos and Atp6v0d2). RANKL and LPS increased the expression of these genes in mature osteoclasts, while rhododendron intervention weakened the previous stimulation of RANKL and LPS, thereby downregulating the expression of these genes ( Figure 8AF). N-rhododendron Tc1 is a key transcription factor for osteoclastogenesis and is mainly responsible for controlling osteoclast formation. Nuclear translocation of N-rhododendron Tc1 activates the expression of osteoclast-specific genes. We used immunofluorescence staining to evaluate the effect of rhododendron on nuclear translocation, and the results showed that during the differentiation of BMMs into osteoclasts stimulated by RANKL and LPS, the proportion of nuclear translocation of N-rhododendron Tc1 was significantly decreased after rhododendron treatment, indicating that rhododendron inhibited the nuclear translocation of N-rhododendron Tc1. To learn more about the role of rhododendron in RANKL-induced osteoclastogenesis, we detected the protein expression levels in BMMs after rhododendron intervention. After 1, 3, and 5 days of RANKL stimulation in BMMs, the protein expressions of N-rhododendron Tc1, c-FOS, CTSK, ATP6V0D2, and MMP9 were significantly inhibited on days 3 and 5 after rhododendron addition compared with the control group. The results showed that rhododendron inhibited the expression of osteoclast marker genes such as N-rhododendron Tc1, c-Fos, CTSK, ATP6V0D2 and MMP9, thereby preventing osteoclast formation and function. (A) The expression of PI3K / AKT and MAPK pathways induced by RANKL and LPS from 0 to 60 minutes was evaluated by western blot. (n=3). (BC) The ratios of p-PI3K / PI3K and p-AKT / AKT (n=3). (DF) The ratios of p-P38 / P38, p-ERK / ERK, and p-JNK / JNK (n=3).

[0069] 2.9 Rhododendron inhibited the transduction of MAPK and PI3K-AKT signaling pathways in osteoclast differentiation.

[0070] In order to further understand the mechanism by which rhododendron inhibits osteoclastogenesis, the present invention studies the changes in the osteoclast-related signaling pathway MAPK pathway and the PI3k-AKT signaling pathway enriched by previous sequencing after the addition of rhododendron. By West Blotting, p-PI3K was significantly inhibited at 5, 10, 30, and 60 minutes after RANKL and LPS stimulation, and p-AKT was inhibited at 5 minutes. It was also found that rhododendron inhibited the activation of p-P38 at 5, 10, and 20 minutes after RANKL and LPS stimulation, p-ERK was inhibited at 10 and 20 minutes, and p-JNK was also significantly reduced at 5, 10, and 20 minutes. These data indicate that rhododendron inhibits osteoclastogenesis by inhibiting the conduction of MAPK and PI3K-AKT signaling pathways.

[0071] In summary, the present invention found that rhododendrin can inhibit RANKL and LPS-induced inflammatory osteoclast formation, F-actin ring formation and bone loss in UC mice in vitro and in vivo and improve inflammatory bowel disease. Further analysis of the molecular mechanism found that rhododendrin inhibits inflammatory osteoclast formation by affecting MAPK (p38, ERK and JNK) and PI3K-AKT signaling pathways. Based on this, the present invention believes that rhododendrin may have great potential and clinical value for the prevention of osteoporosis caused by inflammatory bowel disease.

[0072] In the present invention, the effect of rhododendron, the main active ingredient from the genus Rhododendron, on osteoclast formation was preliminarily explored, and it was found that rhododendron can inhibit the formation of inflammatory osteoclasts induced by inflammatory bowel disease. The present invention found that rhododendron inhibited osteoclast formation in a dose-dependent manner and had no significant inhibition on cell viability, and found that rhododendron can interfere with the differentiation process of osteoclasts.

[0073] Normal bone is in a state of constant remodeling through osteoblast-mediated bone formation and osteoclast-mediated bone resorption, while osteoporosis is the result of an imbalance in osteoblast and osteoclast function, resulting in reduced bone formation or increased bone resorption. Systemic bone loss is often associated with chronic inflammation, which is manifested in various diseases such as rheumatoid arthritis, enteritis, and systemic lupus erythematosus. Inflammatory bowel disease is closely related to bone diseases, but its specific mechanism of action has not been fully studied. Inflammatory bowel disease includes ulcerative colitis and Crohn's disease. The incidence of ulcerative colitis in China is much higher than that of Crohn's disease. Ulcerative colitis is characterized by mucopurulent bloody stools and abdominal pain as typical clinical symptoms. Currently, drugs for the treatment of osteoporosis are mainly divided into antiresorptive drugs and anabolic drugs for reducing bone resorption or increasing bone formation. Among them, antiresorptive treatments, such as bisphosphonates, estrogens, and selective estrogen receptor modulators, denosumab, target osteoclasts, while anabolic drugs, such as parathyroid hormone and parathyroid hormone-related protein, aim to improve bone formation. However, since the pathophysiology of osteoporosis varies from person to person, treatment may have serious side effects. Mesalazine remains the first choice for the treatment of colitis in clinical practice. However, as a first-line treatment for inflammatory bowel disease, mesalazine has a series of side effects that lead to a decrease in the quality of life of patients. These limitations prompted us to further comprehensively and in-depth study the biological characteristics of osteoclasts and related inflammatory pathways in order to identify new therapeutic drugs. The inventors' results demonstrated for the first time that rhododendron can improve colon inflammation and inhibit inflammatory osteoclast differentiation and bone resorption function.

[0074] Today, the COVID-19 virus is still repeatedly infected and mutated, and patients show long-term persistent atypical sequelae after the acute infection is cured, which greatly affects the quality of life of patients. COVID-19 will change the intestinal flora, reduce the number of certain beneficial bacteria, lead to an increase in inflammatory cytokines, and these changes may still persist after recovery. In the state of long-term chronic intestinal inflammation, inflammatory cytokines will disrupt the balance of human bone metabolism, promote bone resorption, and increase the risk of osteoporosis. The present invention constructs UC mice to explore the preventive effect of rhododendron on this disease. The results showed that rhododendron had a significant protective effect on DSS-induced colitis, restored the weight of mice, and reduced mortality and DAI scores. In addition, CTX-1 and PINP are two references for bone resorption and bone formation, respectively, as representative markers of osteogenesis and osteoclasts to reflect the bone turnover state of mice. We found that rhododendron not only reduced the level of CTX-1 in UC mice, but also increased the level of PINP in the serum of UC mice. In addition, TRACP-5b secreted by osteoclasts is also considered to be a specific marker of bone resorption, and OCN is a marker of late osteoblasts. The level of TRACP-5b in UC mice was reduced by treatment with rhododendron. The present invention found that the UC model is accompanied by damaged intestinal barrier. ZO-1 and Occludin are important proteins for the physical defense of the intestinal barrier, and rhododendron allows ZO-1 and Occludin in the colon tissue of UC mice to recover. In the treatment of inflammatory bowel disease, the regulatory role of cytokines is very important. When the secretion of proinflammatory factors increases and the proinflammatory / anti-inflammatory factors are unbalanced, it will cause persistent inflammation of the intestinal mucosa and damage to the barrier function. Tumor necrosis factor has also been shown to play various proinflammatory functions in mucosal inflammation of inflammatory bowel disease. The present invention found through Elisa detection of mouse serum that rhododendron reduced the levels of related inflammatory factors IL6, IL10, IL17 and TNF-α in UC mice. In addition, the intestine has a large number of microbial communities, and regulating the composition of the microbiome is one of the mechanisms for treating metabolic diseases. The present invention shows the changes in the diversity and species richness of the intestinal flora in the UC model. Bacteroidetes, Firmicutes and Verrucomicrobia are beneficial flora, while Desulfobacteria are harmful flora, and the Firmicutes / Bacteroidetes ratio increases in UC mice. This suggests that rhododendron can regulate the abundance and composition of intestinal flora to exert its function. In addition, the present invention found that rhododendron treatment changed the fecal metabolism of UC mice and increased the content of short-chain fatty acids. Among them, propionic acid, a metabolite closely related to inflammation and bone, also has significant differences, which may be a key potential metabolite for rhododendron to treat osteoporosis caused by enteritis. More research is still needed to explore the relationship between metabolites and intestinal flora and osteoporosis.

[0075] In order to study the specific molecular mechanism of rhododendron inhibiting osteoclast differentiation, the inventors studied the main signaling pathways that affect osteoclast differentiation. MAPK and PI3K-AKT pathways are two classic pathways for osteoclast differentiation. MAPK signaling pathways (including p38, ERK and JNK) are mainly involved in the regulation of osteoclast growth, differentiation, inflammatory response and other important physiological processes. The combination of RANKL and RANK will lead to an increase in the phosphorylation levels of JNK, p38 and ERK, and finally start the increase in the expression of osteoclast marker genes in the nucleus, promoting the formation of osteoclasts. Among these signaling molecules, ERK is very important for the survival and differentiation of osteoclasts, P38 participates in the differentiation of osteoclasts but does not affect its bone absorption function, and blocking the activation of JNK can affect the formation of osteoclasts. Mice with JNK gene deletion will develop osteosclerosis due to incomplete bone absorption function. Therefore, inhibiting p38, ERK and JNK can effectively block the formation of osteoclasts induced by RANKL. In the present invention, it was found through experiments that rhododendron inhibited the expression of TRAF6 and the transcriptional expression of NFATc1. The PI3K-AKT signaling pathway is mainly involved in promoting the process of osteoclast proliferation and activation and inflammatory response. The results of the immunoblotting study showed that rhododendron significantly inhibited the phosphorylation of PI3K and AKT proteins in the PI3K-AKT signaling pathway; and significantly reduced the phosphorylation levels of JNK, ERK and p38 proteins in the MAPK signaling pathway. The above results prove that rhododendron can inhibit osteoclast differentiation by inhibiting the PI3K-AKT and MAPK pathways and affecting NFATc1 transcription. The present invention found that after intervention with d rhododendron, WB experiments found that c-Fos and NFATc1 were significantly inhibited in terms of protein expression levels. When PCR tests were performed, it was also found that the expression of osteoclast marker genes such as Nfatc1, c-Fos, Mmp9 and Atp6v0d2 in the drug group was reduced in a dose-dependent manner. In summary, the above analysis results indicate that rhododendrin may affect the expression levels of downstream important transcription factors c-Fos and NFATc1 proteins by blocking the MAPK and PI3K-AKT signaling pathways, thereby inhibiting the expression of osteoclast marker genes and thus inhibiting osteoclast differentiation and bone resorption.

[0076] Conclusion: Rhododendrin has an inhibitory effect on osteoclast differentiation, bone resorption function and bone loss induced by UC mice in vivo and in vitro, which may be related to the inhibition of MAPK and PI3K-AKT signaling pathways mediated by RANKL and LPS. Therefore, the present invention may provide a new therapeutic idea for preventing osteoporosis caused by inflammatory bowel disease.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The application of rhododendron in the preparation of drugs for preventing osteoporosis caused by inflammatory bowel disease.

2. An experimental method for using rhododendron in the preparation of a drug for preventing osteoporosis caused by inflammatory bowel disease, characterized in that: The specific process is: cell culture, cell viability assay, osteoclast differentiation assay and TRAP staining, bone resorption function assay, F-actin ring formation assay, RNA extraction and quantitative PCR assay, western blot analysis, model construction and data statistical analysis.

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