Application of sildenafil in preparation of product for inhibiting adipogenic differentiation of mesenchymal stem cells

Abstract

The invention relates to the technical field of biological medicine. The invention provides an application of sildenafil in preparation of a product for inhibiting adipogenic differentiation of mesenchymal stem cells. The use concentration of the sildenafil is 5-20 mg / L. The sildenafil with the concentration can remarkably inhibit the adipogenic differentiation effect of mesenchymal stem cells, and possibly inhibits the expression of adipogenic related genes by regulating a PI3K-AKT signal channel, so that bone marrow adipogenesis is reduced, the bone microstructure is improved, and osteoporosis and other diseases related to excessive adipogenic differentiation are improved. A new thought and application value are provided for reutilization of sildenafil in bone metabolism regulation and stem cell differentiation directions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of sildenafil in preparation of products for inhibiting adipogenic differentiation of mesenchymal stem cells. BACKGROUND

[0002] Tissue engineering has developed rapidly in recent years, and mesenchymal stem cells (MSCs) are widely used due to their multi-directional differentiation potential. Adipogenic differentiation, as an important differentiation direction of MSCs, is not only of physiological significance, but also closely related to various pathological conditions. Studies have shown that excessive adipogenic differentiation of MSCs is closely related to diseases such as osteoporosis and obesity. Osteoporosis is a common metabolic bone disease. Its characteristics are bone mass loss, bone tissue microstructure degradation, and excessive accumulation of bone marrow fat. In the bone marrow microenvironment, the balance between adipogenic and osteogenic differentiation of MSCs is crucial for maintaining bone homeostasis. The bone marrow mesenchymal stem cells (BMSCs) of osteoporosis patients tend to differentiate into adipocytes, leading to increased bone marrow fat, and ultimately increasing bone fragility. Therefore, reducing bone marrow adiposity and inhibiting the adipogenic differentiation of MSCs may be an important entry point for the treatment of osteoporosis and other diseases related to excessive adipogenic differentiation of MSCs.

[0003] Diseases related to excessive adipogenic differentiation of stem cells (including osteoporosis, bone marrow adiposity, bone marrow aging, diabetic bone metabolism disorder, etc.) are prevalent in clinical practice. Their common feature is that mesenchymal stem cells differentiate into adipocytes excessively, accompanied by decreased osteogenic differentiation ability, leading to decreased bone mass, fragile bone, and weakened bone remodeling ability. At present, the intervention for excessive adipogenic differentiation of stem cells mainly focuses on the following categories: 1. Hormone drugs (such as glucocorticoid antagonists), which can inhibit adipogenesis to some extent, but have obvious side effects when used for a long time, such as metabolic disorders, immunosuppression, and osteonecrosis; 2. Anti-osteoporosis drugs (such as bisphosphonates, SERMs, calcitonin, etc.), which mainly inhibit bone resorption or promote osteogenesis to achieve the effect, and the regulation of adipogenesis is relatively indirect, and cannot inhibit the abnormal adipogenic differentiation of MSCs from the source; 3. PPAR-gamma inhibitors or small molecule modulators: which can directly inhibit the expression of stem cell adipogenic genes, but have poor specificity, often accompanied by hepatotoxicity or affecting other metabolic pathways, limiting their clinical application; 4. Emerging signal pathway targeting drugs (such as AKT, Wnt modulators): most of which are currently in the basic research stage, and there is a lack of safe and effective clinically available drugs. In summary, the existing technologies generally have the following defects: a lack of mature drugs for targeted regulation of MSCs adipogenic differentiation; the existing drugs have a single mechanism of action, and are mostly one-way regulation of “lipid inhibition or bone promotion”; there is a lack of safe and clinically convertible compounds for improving bone marrow adiposity and related pathological conditions; and the potential of PI3K-AKT signaling pathway in adipogenic inhibition is not well studied.

[0004] Sildenafil is a small molecule drug currently approved by the FDA for the treatment of pulmonary hypertension and erectile dysfunction, and has also shown promising effects in studies of heart failure and other diseases. As a PDE5 inhibitor, sildenafil can enhance the signaling effects of the NO / sGC / cGMP pathway, thereby regulating cell growth and differentiation. Studies have shown that sildenafil can promote angiogenesis and restore DNA damage in bone marrow cells. Other studies have shown that sildenafil can accelerate fracture healing. Previous research by the inventors has also indicated that sildenafil can promote osteogenic differentiation of hMSCs and reduce bone loss. The balance between osteogenic and adipogenic differentiation of hMSCs is crucial for maintaining bone homeostasis. Previous studies have suggested that sildenafil may have a regulatory effect on adipogenic differentiation of hMSCs and bone marrow steatosis, but these effects have not yet been confirmed by research. Summary of the Invention

[0005] The purpose of this invention is to provide the application of sildenafil in inhibiting the adipogenic differentiation of mesenchymal stem cells and inhibiting bone marrow steatosis, which can effectively improve osteoporosis and other diseases related to excessive adipogenic differentiation.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of sildenafil in the preparation of products that inhibit adipogenic differentiation of mesenchymal stem cells.

[0007] Preferably, the concentration of sildenafil used is 5-20 mg / L, and the in vivo dose is 1 mg / kg.

[0008] This invention provides the use of sildenafil in the preparation of products that inhibit bone marrow steatosis.

[0009] Preferably, the concentration of sildenafil used is 5-20 mg / L, and the in vivo dose is 1 mg / kg.

[0010] This invention provides a medicament for treating diseases related to excessive adipogenic differentiation of mesenchymal stem cells, including sildenafil or a pharmaceutically acceptable salt thereof.

[0011] Preferably, the diseases associated with excessive adipogenic differentiation of mesenchymal stem cells include osteoporosis, myelostematosis, metabolic bone disease, diabetes-related bone loss, obesity, and metabolic syndrome.

[0012] Preferably, the concentration of sildenafil used is 5-20 mg / L, and the in vivo dose is 1 mg / kg.

[0013] By adopting the above technical solution, the present invention has the following beneficial effects: The technical solution of this invention demonstrates that sildenafil significantly inhibits adipogenic differentiation of stem cells under non-traditional indications. It reduces bone marrow steatosis and improves osteoporosis and other diseases related to excessive adipogenic differentiation by regulating the PI3K-AKT signaling pathway and inhibiting the expression of adipogenic genes in stem cells. In vitro, sildenafil can inhibit the adipogenic differentiation of hBMSCs (human bone marrow mesenchymal stem cells) and hASCs (human adipose mesenchymal stem cells), with a more pronounced effect at 10 mg / L concentration.

[0014] The embodiments of this invention also demonstrate that appropriate concentrations of sildenafil inhibit the adipogenic differentiation of hBMSCs in nude mice; ovariectomized mice and suspension mice experiments show that sildenafil can effectively inhibit bone marrow steatosis in osteoporosis models caused by different etiologies, and also has good biocompatibility; the PI3K-AKT pathway can promote the differentiation of stem cells into adipocytes by regulating multiple key transcription factors such as PPARγ and C / EBPα. Attached Figure Description

[0015] Figure 1 The effect of different concentrations of sildenafil on the inhibition of adipogenic differentiation of hBMSCs in vitro ( Figure 1 In the diagram, A represents the OilRed O staining result, B represents the Oil Red O staining quantitative analysis, C represents the relative mRNA expression level of PPAR-γ analyzed by qRT-PCR, D represents the relative mRNA expression level of C / EBP-α analyzed by qRT-PCR, and E represents the relative expression level of PPAR-γ analyzed by immunofluorescence. P<0.05, P<0.01, (P<0.001 compared with AM group); Figure 2 The effect of different concentrations of sildenafil on the inhibition of adipogenic differentiation of hASCs in vitro ( Figure 2 In the diagram, A represents the OilRed O staining result, B represents the Oil Red O staining quantitative analysis, C represents the relative mRNA expression level of PPAR-γ analyzed by qRT-PCR, D represents the relative mRNA expression level of C / EBP-α analyzed by qRT-PCR, and E represents the relative expression level of PPAR-γ analyzed by immunofluorescence. P<0.05, P<0.01, P<0.001); Figure 3 The inhibition of adipogenic differentiation of hBMSCs in a subcutaneous ectopic osteogenic model in different treatment groups ( Figure 3In the table, A represents the H&E staining result, B represents the Oil Red O staining result, C represents the Nile red staining result, D represents the statistical analysis result of the adipocyte area / tissue area in H&E staining, E represents the statistical analysis result of the adipocyte area / tissue area in Oil Red O staining, and F represents the statistical analysis of the relative fluorescence intensity in Nile red staining. P<0.05, P<0.01, P<0.001); Figure 4 Bone marrow fat deposition in OVX mice under different treatment groups ( Figure 4 In the diagram, A represents a schematic diagram of the experimental procedure, B represents the H&E staining results of different treatment groups, C represents the statistical analysis of adipocyte area, and D represents the statistical analysis of adipocyte area / tissue area. P<0.05, P<0.01, P<0.001); Figure 5 Image showing the H&E staining results of major organs in OVX mice; Figure 6 Bone marrow fat deposition in TS mice under different treatment groups ( Figure 6 In the diagram, A represents a schematic diagram of the experimental procedure, B represents the H&E staining results of different treatment groups, C represents the statistical analysis of adipocyte area, and D represents the statistical analysis of adipocyte area / tissue area. P<0.05, P<0.01, P<0.001); Figure 7 Image showing the H&E staining results of major organs in TS mice; Figure 8 Figure showing the results of the analysis of the mechanism of sildenafil against hBMSCs adipogenic differentiation. Figure 8 In the table, A represents the relative mRNA level of PI3K analyzed by qRT-PCR, B represents the relative mRNA level of AKT analyzed by qRT-PCR, C represents the relative mRNA level of FOXO1 analyzed by qRT-PCR, D represents the relative mRNA level of FOXO3 analyzed by qRT-PCR, E represents the relative mRNA level of FOXO4 analyzed by qRT-PCR, F represents the expression level of each gene shown by Western blot, G represents the quantitative analysis of p-PI3K / PI3K expression, and H represents the quantitative analysis of p-AKT / AKT expression. P<0.05, P<0.01, P<0.001). Detailed Implementation

[0016] This invention provides the application of sildenafil in the preparation of products that inhibit adipogenic differentiation of mesenchymal stem cells.

[0017] In this invention, the preferred concentration of sildenafil used is 5-20 mg / L, more preferably 10 mg / L; the in vivo dose is 1 mg / kg.

[0018] This invention provides the use of sildenafil in the preparation of products that inhibit bone marrow steatosis.

[0019] In this invention, the preferred concentration of sildenafil used is 5-20 mg / L, more preferably 10 mg / L; the in vivo dose is 1 mg / kg.

[0020] This invention provides a medicament for treating diseases related to excessive adipogenic differentiation of mesenchymal stem cells, including sildenafil or a pharmaceutically acceptable salt thereof.

[0021] In this invention, the diseases associated with excessive adipogenic differentiation of mesenchymal stem cells include osteoporosis, myelostematosis, metabolic bone disease, diabetes-related bone loss, obesity, or metabolic syndrome.

[0022] In this invention, the preferred concentration of sildenafil used is 5-20 mg / L, more preferably 10 mg / L; the in vivo dose is 1 mg / kg.

[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1: Sildenafil inhibits adipogenic differentiation of hBMSCs and hASCs in vitro.

[0025] Sildenafil (Y0001578, Sigma-Aldrich, China) was dissolved in proliferation medium (PM) containing 0.1% DMSO (composed of α-MEM + 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin) to prepare a 100 mg / L stock solution, which was then diluted to final concentrations of 1, 5, 10, 20 and 40 mg / L.

[0026] (I) Oil Red O staining and quantification

[0027] hBMSCs and hASCs (both purchased from ScienCell, USA) were respectively 2 × 10⁻⁶ per well. 4 Cells were seeded at a density of 1000 cells per well in 12-well plates and cultured in proliferation medium (PM) at 37°C and 5% CO2 for 14 days (PM group).

[0028] hBMSCs and hASCs (both purchased from ScienCell, USA) were used at a ratio of 2 × 10⁻⁶ per well. 4 Cells were seeded at a density of 100% in 12-well plates. After the cells reached 90-100% confluence, adipogenic medium (AM) was added, and adipogenic differentiation was induced for 14 days at 37°C and 5% CO2 (as the AM group).

[0029] hBMSCs and hASCs (ScienCell, USA) were used at 2 × 10⁻⁶ per well. 4 Cells were seeded at densities of 1,000 mg / L in 12-well plates. After reaching 90-100% confluence, adipogenic differentiation induction medium (AM) containing final concentrations of 1, 5, 10, 20, and 40 mg / L sildenafil was added, and adipogenic differentiation was induced for 14 days at 37°C and 5% CO2 (referred to as AM+1 mg / L, AM+5 mg / L, AM+10 mg / L, AM+20 mg / L, and AM+40 mg / L groups, respectively). (Once the desired density was reached, the medium was replaced and cultured again; reseeding was not required.)

[0030] The culture time was the same for all groups.

[0031] The proliferation medium (PM) for hBMSCs consisted of α-minimum essential medium (α-MEM) + 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin; the proliferation medium (PM) for hASCs consisted of Dulbecco modified Eagle medium (DMEM) + 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. All components of the proliferation media were purchased from Gibco (Grand Island, USA).

[0032] The lipid induction medium for hBMSCs consisted of α-MEM, 10% FBS, 1% penicillin and streptomycin, and a lipid inducer; the lipid induction medium for hASCs consisted of DMEM, 10% FBS, 1% penicillin and streptomycin, and a lipid inducer. The lipid inducer consisted of 500 μM 3-isobutyl-1-methylxanthine (IBMX), 200 μM indomethacin, 10 μM insulin, and 100 nM dexamethasone.

[0033] Cells from each group were washed with PBS, fixed in 10% neutral formalin for 70 min, washed again with PBS, treated with 60% isopropanol, and stained with Oil Red O. After 5 min, cells were observed under a microscope, with successful staining of lipid droplets as the standard. The staining solution was then removed and replaced with PBS. Lipid droplets were observed and imaged using an inverted optical microscope (TE2000-U, Nikon, Japan). For quantification, cells were washed and incubated with 100% isopropanol for 10 min, and absorbance was measured at 500 nm using a microplate reader (ELx800, Biotec, USA).

[0034] Oil Red O staining showed that AM-treated cells accumulated a large number of lipid droplets, indicating successful adipogenic differentiation. Sildenafil reduced lipid droplet formation at concentrations of 1, 5, 10, and 20 mg / L, with the strongest inhibitory effect observed in the AM+10 mg / L group, while no significant difference was observed in the AM+40 mg / L group (e.g., ...). Figure 1 A and Figure 2 (A) Quantitative results are as follows: Figure 1 B and Figure 2 In group B, the number of lipid droplets decreased in all groups treated with sildenafil, with 10 mg / L sildenafil showing the strongest inhibitory effect.

[0035] (ii) Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR)

[0036] Total RNA was extracted from hBMSCs and hASCs from the cells in the above groups using TRIzol. The specific procedures for RNA quantification, cDNA synthesis, and qRT-PCR were performed according to the published literature (Liu X, Li Z, Liu H, Zhu Y, Xia D, Wang S, et al. Low concentration flufenamic acid enhances osteogenic differentiation of mesenchymal stem cells and suppresses bone loss by inhibition of the NF-κB signaling pathway. Stem cell research & therapy. 2019; 10: 213).

[0037] Table 1 Primer sequence information

[0038] qRT-PCR results showed that the expression of PPAR-γ and C / EBP-α was significantly decreased, with the most significant decrease observed in the AM+10 mg / L group (e.g., ...). Figure 1 CD and Figure 2 (CD in the middle).

[0039] (III) Immunofluorescence (IF) staining

[0040] Cells from each group were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with 0.8% bovine serum albumin (BSA) for 1 h. They were then incubated overnight at 4°C with anti-PPAR-γ antibody [EPR23297-111] (ab272718, Abcam, UK), followed by incubation with goat anti-rabbit IgG secondary antibody (ab150077, Abcam, UK) for 1-2 h. Nuclei were stained with DAPI.

[0041] Immunofluorescence results showed that PPAR-γ expression was downregulated in sildenafil-treated groups, with significant downregulation in the AM+10 mg / L group (e.g., ...). Figure 1 E and Figure 2 (E in the text).

[0042] Example 2: Sildenafil inhibits adipogenesis of hBMSCs in vivo

[0043] Based on in vitro results, the AM+10 mg / L sildenafil group was selected for in vivo validation.

[0044] Twelve 6-week-old female BALB / c nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., housed under SPF conditions, humidity 40-70%, temperature 20-26℃, 12-hour light-dark cycle) were randomly divided into three groups (n=6): PM, AM, and AM+Sildenafil groups. hBMSCs were cultured for 7 days in PM, AM, or AM containing 10 mg / L sildenafil, and then mixed with collagen membranes (Wuxi Bote Biotechnology Research Institute, Wuxi, China) (one collagen membrane could be cut into 6 pieces, approximately 5...). 5 mm, no special requirements. Cells and collagen membranes are co-cultured in tubes, one tube per site, with a seeding density of approximately 2 × 10⁶ cells per site. 6 (Each implant) was incubated at 37°C for 2 hours and then subcutaneously implanted into the back. Four weeks later, the implants were removed and stained with H&E, Oil Red O, and Nile Red. The specific staining steps are as follows: 1. H&E staining (1) Dewaxing and hydration: The sections were placed in xylene (twice, 5 minutes each time) for dewaxing. Then the sections were placed in anhydrous ethanol (twice, 2 minutes each time), 95% ethanol (2 minutes), 75% ethanol (2 minutes), and distilled water (2 minutes) for hydration step by step.

[0045] (2) Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 5-8 minutes. Hematoxylin can stain the cell nuclei blue. Rinse the sections with tap water for 3 minutes to remove unbound dye. Use 1% hydrochloric acid alcohol (1% hydrochloric acid dissolved in 70% ethanol) for rapid differentiation for a few seconds, then immediately rinse with tap water for about 20 minutes, and then use 1% ammonia water for 2 seconds to "return to blue".

[0046] (3) Eosin staining: Rinse with tap water, immerse the slide in 0.5%~1% eosin staining solution for 1-3 minutes to stain the cytoplasm and extracellular matrix pink. Rinse with tap water for a few seconds to remove excess staining solution.

[0047] (4) Dehydration and mounting: The sections were dehydrated by treating them with 75% ethanol, 95% ethanol and anhydrous ethanol for 2 minutes each. They were then cleared by placing them in xylene (twice, 5 minutes each time). Neutral resin was added to mount the sections, and a coverslip was placed on top and pressed down gently.

[0048] (5) Microscopic observation: assess tissue structure and cell morphology, take pictures with a microscope and save them for subsequent data analysis.

[0049] (6) Precautions: The concentration of the staining solution and the staining time need to be adjusted appropriately according to the thickness of the section and the characteristics of the tissue. Before mounting, ensure that the sections are free of air bubbles and dust.

[0050] 2. Oil Red O staining

[0051] (1) Preparation of frozen sections: The fixed adipose tissue was prepared into frozen sections of 8-10 μm and attached to a clean glass slide. The sections were then air-dried at room temperature for later use.

[0052] (2) Slice fixation: Place the slices in 4% paraformaldehyde for 10 minutes, then rinse with distilled water 3 times for 2 minutes each time.

[0053] (3) Staining: Use freshly prepared 0.3% Oil Red O staining solution (diluted with 60% isopropanol), immerse the sections in the staining solution and stain at room temperature for 10 minutes.

[0054] (4) Differentiation: Rinse gently with 60% isopropanol for 10-15 seconds to remove excess dye and ensure that the adipose tissue appears red.

[0055] (5) Rinse thoroughly with distilled water to remove background staining.

[0056] (6) Counterstaining: Counterstain cell nuclei with hematoxylin solution for 1 minute, then rinse to make the background clear.

[0057] (7) Mounting: Use water-based mounting medium to mount the slide and cover it with a coverslip to prevent air bubbles from forming.

[0058] (8) Microscopic observation: lipid droplets are red and cell nuclei are blue.

[0059] (9) Precautions: Ensure that the Oil Red O staining solution is freshly prepared and avoid exposure to light for extended periods. Avoid over-differentiation during staining to prevent affecting the clear development of lipid droplets. Use an aqueous mounting medium to maintain the integrity of the lipid droplets and prevent lipid droplet loss due to mounting with organic solvents.

[0060] 3. Nile Red staining

[0061] (1) Preparation of Nile Red staining solution: Prepare a 0.1 mg / ml stock solution with ethanol, and dilute to the working concentration before use (usually 1-10 μg / ml, diluted with PBS or distilled water). Nile Red is easily degraded, so the staining solution should be prepared and used immediately to avoid long-term storage.

[0062] (2) Preparation of frozen sections: Adipose tissue was prepared into frozen sections with a thickness of 8-10 μm, attached to a clean glass slide, and air-dried for later use.

[0063] (3) Section fixation: Immerse the sections in 4% paraformaldehyde fixative for 10 minutes to maintain tissue stability. Rinse with distilled water 3 times, 2 minutes each time.

[0064] (4) Staining: Cover the sections with Nile Red staining solution of working concentration and stain at room temperature in the dark for 5-10 minutes.

[0065] (5) Rinsing: Rinse the slides gently with PBS or distilled water three times for one minute each time to remove non-specific dyes.

[0066] (6) Mounting: Use water-based mounting medium to mount the slide, cover it with a coverslip, and ensure there are no air bubbles.

[0067] (7) Microscopic observation: Under a fluorescence microscope, neutral lipids (such as triglycerides) in adipose tissue will emit yellow fluorescence, while polar lipids (such as phospholipids) will emit red fluorescence. Nile red is photosensitive, and the staining and observation process should be carried out under light-protected conditions.

[0068] H&E staining results showed that the AM group had abundant fat vacuoles, which were significantly increased compared to the PM group; while the AM+sildenafil group had significantly reduced lipid droplets and a significantly decreased adipocyte area. Figure 3 A and D in the text). Oil Red O ( Figure 3 (B and E) and Nile Red ( Figure 3The staining results for C and F in the samples were consistent, with abundant lipid accumulation in the AM group and a significant reduction in lipid accumulation in the AM+sildenafil group. This indicates that 10 mg / L sildenafil effectively inhibits adipogenic differentiation of hBMSCs in a nude mouse subcutaneous model.

[0069] Example 3: Sildenafil reduces bone marrow fat accumulation in OVX mice

[0070] An OVX model was established to simulate estrogen-deficiency osteoporosis. Bilateral ovariectomy was performed under 0.25% avertin anesthesia (150 mg / kg, MA04781, Meilun Biotechnology, China), while the control group underwent sham surgery (exposed but not resected). Twenty 8-week-old female C57BL / 6N mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., housed under SPF conditions, humidity 40-70%, temperature 20-26℃, 12-hour light-dark cycle) were randomly divided into four groups (n=5): Sham+PBS, Sham+Sildenafil, OVX+PBS, and OVX+Sildenafil. Starting 3 months post-surgery, mice received daily intraperitoneal injections of sildenafil (1 mg / kg) or PBS for 4 weeks. Femurs were harvested for H&E staining to quantify adipocyte area and bone marrow fat percentage. Major organs were also collected for histological safety assessment.

[0071] The above experimental design process is as follows: Figure 4 A in the middle.

[0072] H&E staining results showed that the adipocyte area was similar in the Sham+PBS group and the Sham+sildenafil group, while the adipocyte count was significantly increased in the OVX+PBS group. In contrast, adipogenesis was significantly reduced in the OVX+sildenafil group. Figure 4 The results of quantitative analysis (B in the text) are consistent. Figure 4 (C in the text). Furthermore, H&E staining of the heart, liver, spleen, lungs, and kidneys showed no significant inflammatory response (C in the text). Figure 5 The results indicate good biocompatibility. This experimental result demonstrates that sildenafil can alleviate OVX-induced myeloma steatosis.

[0073] Example 4: Sildenafil reduces bone marrow fat accumulation in TS mice

[0074] To simulate mechanical loss of load, mice were suspended by their tails at a 30° angle for 14 days, while their forelimbs were free to move and they could eat and drink. The control group was not suspended.

[0075] Twenty 8-week-old female C57BL / 6N mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., housed under SPF conditions, humidity 40-70%, temperature 20-26℃, 12h light-dark cycle) were randomly divided into four groups (n=5): Sham + PBS, Sham + sildenafil, suspension + PBS, and suspension + sildenafil. From day 14, mice were intraperitoneally injected with sildenafil (1 mg / kg) or PBS daily for 14 days. After the experiment, femurs and major organs (heart, liver, spleen, lungs, and kidneys) were collected for H&E staining.

[0076] The above experimental design process is as follows: Figure 6 A in the middle.

[0077] H&E staining results showed that, consistent with the OVX model, sildenafil treatment significantly reduced bone marrow fat accumulation in TS mice, with both adipocyte area and adipocyte area / tissue area ratio being significantly reduced. Figure 6 (B and C in the text). No inflammatory reaction was observed in the H&E staining of major organs. Figure 7 This indicates that sildenafil has good biocompatibility. The experimental results show that sildenafil can inhibit bone marrow lipogenesis in the TS model.

[0078] Example 5: Sildenafil inhibits adipogenic differentiation of hBMSCs by inhibiting the PI3K-AKT pathway.

[0079] To investigate the anti-adipogenic differentiation mechanism of sildenafil, RNA-Seq analysis was performed on hBMSCs treated with AM and AM+sildenafil. After 14 days of induction culture of hBMSCs under AM or AM+10 mg / L sildenafil conditions, total RNA was extracted and sequenced (RNA sequencing was performed by Beijing Novogene Bioinformatics Co., Ltd.), followed by qRT-PCR analysis. The qRT-PCR procedure was the same as in Example 1, and the primers are shown in Table 1. The qRT-PCR results showed that the expression of PI3K, AKT, FOXO1, FOXO3, and FOXO4 mRNA was significantly downregulated in the sildenafil group (…). Figure 8 (AE in the text).

[0080] Proteins were extracted from hBMSCs cultured for 14 days after induction with AM or AM + 10 mg / L sildenafil. The protein levels of PI3K, p-PI3K, AKT, and p-AKT were detected using an automated Western blot system (Wes, ProteinSimple, USA), and the data were analyzed using Compass software. Antibodies used included: PI3K p85α monoclonal antibody (60225-1-Ig, Proteintech, China), phosphorylated PI3K P85α / P55γ / P85β-Y467 / Y199 / Y464 antibody (AP0854, Abclonal, China), AKT polyclonal antibody (10176-2-AP, Proteintech, China), and phosphorylated AKT (Ser473) antibody (T40067F, Abmart, China).

[0081] Western blot results showed that the ratios of p-PI3K / PI3K and p-AKT / AKT decreased, such as Figure 8 The results suggest that sildenafil may inhibit adipogenic differentiation of hBMSCs by suppressing the PI3K-AKT signaling pathway.

[0082] In summary, the technical solution of this invention reveals the inhibitory effect of sildenafil on adipogenic differentiation and bone marrow steatosis of hMSCs, by affecting the PI3K-AKT signaling pathway to inhibit adipogenic differentiation of human mesenchymal stem cells and improve bone marrow steatosis. Appropriate concentrations of sildenafil can inhibit adipogenic differentiation of hMSCs both in vitro and in vivo. In animal experiments, 10 mg / L sildenafil can simultaneously improve bone marrow steatosis in OVX mice and TS mice.

[0083] 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 sildenafil in the preparation of products that inhibit adipogenic differentiation of mesenchymal stem cells.

2. The application according to claim 1, characterized in that, The concentration of sildenafil used is 5-20 mg / L, and the in vivo dose is 1 mg / kg.

3. Application of sildenafil in the preparation of products that inhibit bone marrow steatosis.

4. The application according to claim 3, characterized in that, The concentration of sildenafil used is 5-20 mg / L, and the in vivo dose is 1 mg / kg.

5. A drug for treating diseases related to excessive adipogenic differentiation of mesenchymal stem cells, characterized in that, This includes sildenafil or its pharmaceutically acceptable salts.

6. The drug according to claim 5, characterized in that, The diseases associated with excessive adipogenic differentiation of mesenchymal stem cells include osteoporosis, myelostematosis, metabolic bone disease, diabetes-related bone loss, obesity, or metabolic syndrome.

7. The drug according to claim 5, characterized in that, The concentration of sildenafil used is 5-20 mg / L, and the in vivo dose is 1 mg / kg.

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