Application of polyene phosphatidylcholine in preparation of medicine for treating bone fracture

Abstract

The invention relates to application of polyene phosphatidylcholine in preparation of a medicine for treating bone fracture. Through a rat tibia fracture model and a vascular endothelial cell model, polyene phosphatidylcholine can remarkably accelerate callus formation and promote bone regeneration and angiogenesis, so that fracture healing can be promoted, and the polyene phosphatidylcholine has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to new uses of polyene phosphatidylcholine. Background Technology

[0002] Approximately 5%-10% of fracture patients experience serious complications such as delayed healing or nonunion due to various reasons, becoming an increasingly prominent public health issue. Long bone fractures have a relatively high likelihood of nonunion, with some injuries exhibiting an incidence rate as high as 20%. The management of these long bone fractures is complex; poor healing can lead to disability, decreased quality of life, and high treatment costs. Currently, the main treatment methods for fractures are conservative treatment following fixation and repair, surgical fixation combined with autologous or allogeneic bone grafting, or the application of exogenous cytokines that promote bone cell growth. Surgical treatment carries the risk of serious complications, and current drug treatments remain unsatisfactory. For example, nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce pain during the fracture process but may delay fracture healing. Calcium supplements, such as calcium carbonate or bisphosphonates, can help improve fracture healing. However, long-term use of bisphosphonates may cause adverse reactions such as gastrointestinal symptoms, osteonecrosis, or atypical fractures.

[0003] Polyunsaturated phosphatidylcholine (PPC) is a polyunsaturated phosphatidylcholine extracted from soybeans. The role of PPC in liver repair has been well-established, and it is frequently used in clinical practice to treat various types of liver disease, fatty liver, and bile duct obstruction. It can protect hepatocyte structure and phospholipid-dependent enzyme systems, and can also bind to and enter biological membrane components, increasing membrane fluidity and maintaining or promoting membrane function in different organs and tissues.

[0004] It is unknown whether PPC affects fracture healing. Summary of the Invention

[0005] The technical problem to be solved by this invention is the novel use of polyene phosphatidylcholine.

[0006] The novel use of polyene phosphatidylcholine provided by this invention is: its application in the preparation of fracture treatment drugs.

[0007] The function of the fracture treatment drug is to promote angiogenesis at the fracture site.

[0008] The fracture treatment drug can promote the growth of cartilage at the fracture site and accelerate ossification and new bone formation.

[0009] This invention provides a series of biological experiments on polyene phosphatidylcholine (PPC), providing a basis for exploring new applications of PPC in promoting fracture healing. Specific biological experimental findings include the following: A rat tibial fracture model was established using a modified Einhorn model. X-rays were used to detect the progress of fracture healing. Safranin O / Fix Green staining and CD31 immunohistochemistry were used to analyze the progress of ossification and angiogenesis at the fracture site. To investigate whether PPC has a direct angiogenic effect, HUVECs (vascular endothelial cells) were used. We performed MTT assays, wound healing assays, Transwell migration assays, and tube formation assays. Finally, RT-qPCR and Western blot analysis were used to investigate its potential mechanisms. Polyene phosphatidylcholine significantly shortened the apparent recovery time of activity in rats, significantly promoted callus formation, endochondral ossification, and angiogenesis at the fracture site. In vitro, PPC promoted the proliferation activity of HUVECs, their wound healing ability, and their ability to penetrate the membrane in the Transwell apparatus, and increased tubule formation in vascular endothelial cells. PPC significantly upregulated the transcription of VEGFA, VEGFR2, PLCγ, RAS, ERK1 / 2, and MEK1 / 2. Furthermore, protein level results showed a significant increase in the expression of VEGFA, VEGFR2, MEK1 / 2, and ERK1 / 2 proteins. PPC promotes angiogenesis by activating VEGFA / VEGFR2 and downstream signaling pathways, thereby accelerating fracture healing. Attached Figure Description

[0010] Figure 1 This indicates that PPC shortened the apparent recovery time of mobility in rats with fractures. PPC: Polyene phosphatidylcholine; LPPC: Low-concentration PPC group; MPPC: Medium-concentration PPC group; HPPC: High-concentration PPC group; NC: Negative control group. *p<0.05, **p<0.01.

[0011] Figure 2 Radiometric analysis of the healing process of rat tibial fractures. A. X-ray images after MPPC treatment on days 1, 3, 7, 14, and 21 post-surgery. White scale indicates 1 cm. Fracture sites are circled in yellow. B: Fracture grading results at different healing stages. The value on day 0 is consistently 1. N=6 for each group. Values ​​given are the mean standard error. *** indicates significant differences between the treatment group and the negative control (NC) (p<0.05 and p<0.01, respectively). MPPC: Medium-dose polyene phosphatidylcholine.

[0012] Figure 3The results of Safranin O / Fix Green staining at the tibial fracture site are shown. A. Bone tissue sections stained with Safranin O / Fix Green were obtained from the control group or PPC treatment at 1, 3, 7, 14, and 21 days later. The top image shows the control group, and the bottom image shows the PPC treatment group. B. Quantitative analysis of cartilage tissue was performed using ImageJ software. (Red areas represent stained cartilage tissue, and blue areas represent bone tissue). Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01; determined by Student's t-test.

[0013] Figure 4 The images show that CD31 immunohistochemical staining indicates that PPC promotes angiogenesis at the fracture site. A: Bone tissue sections were stained with CD31 antibody via immunohistochemical staining after 1, 3, 7, 14, and 21 days of treatment with saline (control) or MPPC. The top image shows the control group, and the bottom image shows the MPPC treatment group. The scales in the top and bottom images represent 200 μm and 40 μm, respectively. B (CD31 staining area ratio) and C (vascular cross-sectional area) are quantitative analyses of the images in A using ImageJ software. MPPC: Medium-dose polyvinylphosphatidylcholine.

[0014] Figure 5 PPC promotes HUVEC proliferation. A: Photographs of cell growth status after 12 and 24 hours of treatment with different concentrations of PPC. Scale bar indicates 100 μm. B shows the changes in cell proliferation activity after 24 hours of treatment with different concentrations of PC. Compared with the control group, *p<0.05, **p<0.01.

[0015] Figure 6 This shows that PPC promotes wound healing in HUVECs. A: Photographs of scraped cells before and after 24 hours of treatment with different concentrations (0, 2, 4, 8 μM) of PPC. Scale bar indicates 100 μM. B: Wound closure rates of the treatment group and control group at different concentrations of PPC were calculated based on A.**p<0.01, ***p<0.001, ***p<0.0001.

[0016] Figure 7 PPC promotes the transwell migration of HUVECs. A: Photographs of migrating cells before and after 24 hours of treatment with 0, 2, 4, and 8 μM PPC. Scale bar indicates 100 μM. B: Relative number of migrating cells compared to the control group (**p < 0.01, ***p < 0.001).

[0017] Figure 8PPC promotes tubule formation in HUVECs. A: Photographs of tubule formation in HUVECs after treatment with PPC at concentrations of 0, 2, 4, and 8 μM for 12 hours. Scale bar indicates 100 μM. B: Relative number of tubules calculated from A compared to the control group. C: Number of branching points calculated from A compared to the control group. (**p < 0.01, ***p < 0.001, ***p < 0.0001)

[0018] Figure 9 The relative mRNA levels of VEGFA, VEGFR2, PLCγ, RAS, MEK1 / 2, ERK1 / 2, eNOs, PI3K, and bFGF in HUVECs after 24 hours of treatment with 8 μM PPC are shown. (*p < 0.1, **p < 0.01, ***p < 0.0001)

[0019] Figure 10 The immunoblotting results of HUVECs on the VEGF / VEGFR2 signaling pathway are shown. A: Immunoblotting images of VEGF, VEGFR2, p-ERK1 / 2, p-MEK1 / 2, and eN0 in HUVECs treated with different concentrations of PPC (0, 2, 4, 8 μM) for 24 hours. B: Quantitative analysis of the results in A was performed using ImageJ software (*p < 0.1, ***p < 0.001). Detailed Implementation

[0020] The present invention will be described in detail below with reference to the embodiments, but these are not intended to further limit the present invention.

[0021] Example 1

[0022] 1. Materials and Methods

[0023] 1.1. Drugs and Antibodies

[0024] PPC is a product of Sanofi Pharmaceuticals. VEGF-specific antibody (A12303) and β-actin-specific antibody (AC038), as well as the secondary antibody HRP goat anti-rabbit IgG (H+L) (AS014), were purchased from ABclone Technology (Wuhan, China). Specific antibodies for pERK (GB11510-100), VEGFR2 (GB11190), pMEK (GB115603-100), eNOs (GB115277-100), and CD31 (GB12064-100) were obtained from Servicebio (Wuhan, China).

[0025] 1.2. Animal Modeling and Processing

[0026] In accordance with the International Guidelines for Animal Biomedical Research, all procedures were reviewed and approved by the Animal Resources Committee of Beijing University of Chinese Medicine. All experiments were conducted in the Laboratory Animal Room of Beijing University of Chinese Medicine. Seventy-four male Sprague-Dawley rats (6 weeks old) were purchased from Sibeifu Biotechnology Co., Ltd. (Beijing, China) and placed in standard plastic microisolation cages, kept in an animal facility with controlled temperature (25±2 degrees Celsius) and light (12-hour light / dark cycle). The rats had unlimited access to standard food and water. After one week of acclimatization, a rat tibial fracture model was established using a modified Einhorn method. The fractured tibia was externally fixed with a small splint. The fracture model rats were then divided into two groups. The first group of 24 animals was divided into four subgroups (n=6): negative control group (NC), low-dose PPC group (LPPC), medium-dose PPC group (MPPC), and high-dose PPC group (HPPC). PPC (LPPC 50 mg / kg, MPPC 100 mg / kg, HPPC 150 mg / kg) or saline (NC) was administered daily by gavage. The rats' diet, water intake, body weight, and mental status were recorded daily, as well as the fracture healing time (when the rats could move normally). A second group of 50 fracture model rats was divided into two subgroups: a PPC (100 mg / kg) gavage group and a saline control group. On days 1, 3, 7, 14, and 21, rats (n=5) were sacrificed to obtain bone tissue for sectioning and immunohistochemical analysis.

[0027] 1.3. Radiometric Analysis

[0028] Rats in the MPPC group underwent radiography at 1, 3, 7, 14, and 21 days post-treatment. Fracture healing was measured using a BLX-5 portable X-ray machine from [Company Name]. The parameters for X-ray examination of the fracture site were an exposure time of 1.5 seconds and a voltage of 60 kV. All X-ray examinations were read by a radiologist who was unaware of all treatments. The Rat Fracture Radiotherapy Scale was used to score fracture healing

[20] . The scoring criteria were as follows: 1: No calcification; 2: Patchy calcification; 3: Calcification presenting as callus; 4: Callus bridging the fracture gap; 5: Continuity of trabecular bone; 6: Remodeling into normal bone.

[0029] 1.4 Safranin 0 / Fixed Green Staining

[0030] Tissue sections from rat tibial fractures were dewaxed and immersed in Fast Green staining solution for 3–4 minutes. They were then washed with water and treated with Safranin O solution (Servicebio) for 2 minutes, followed by sealing with neutral resin. Images were acquired and recorded using a super-resolution microscopy imaging system (E3CM0S, Leica Aperio VERSA, Weztlar, Germany).

[0031] 1.5. Immunohistochemical staining

[0032] Rat tibias were fixed in 10% formalin for 24 hours and decalcified (10% EDTA, pH 7.4) for 21 days. They were then embedded in paraffin. 6 μm longitudinal bone sections were prepared and incubated with a primary CD31 antibody after affinity removal and dehydration. These sections were then incubated with a secondary antibody (horseradish peroxidase conjugate, Thermo Fisher, Cambridge, MA, USA) at 37°C for 60 minutes. They were subsequently stained with DAB (Solarbio, Beijing, China) and hematoxylin (Solarbio, Beijing, China). The number of vessels in the perisceleural area was counted based on CD31 staining. The cross-sectional area of ​​the vessels was measured using ImageJ software.

[0033] 1.6. Cell viability assay

[0034] Human vascular endothelial cell lines were obtained from the Shanghai Institute of Cell Science, Chinese Academy of Sciences (Shanghai, China). Cell viability was measured using the MTT (3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazoline bromide) method. Logarithmically growing HUVECs were seeded in 96-well plates (3000 cells per well) and cultured overnight until cell adhesion. After incubation for 24 hours in complete medium containing different concentrations of PPC (0, 2, 4, 6, 8, 12, 16, 24 μM), 100 μL of 0.5 mg / mL MTT was added to each well and incubated for 4 hours. The formamide in the wells was dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using a microplate reader (model: EP0CH, serial number: #130315A, BioTek, USA). MTT and DMSO were purchased from Sigma (USA). The formula for calculating cell viability is as follows: Relative inhibition % = (1 - A treatment / A control) × 100%.

[0035] 1.7. Wound Healing Assay

[0036] 3×10 6 HUVECs were seeded at 10 cells / well in 6-well plates and cultured in DMEM containing 10% fetal bovine serum. After cell attachment, the cells were scratched at the bottom of the culture wells using the tip of a 10 μL pipette. The HUVECs were then cultured in complete medium containing different concentrations of PPC (0, 2, 4, 8 μM) for 24 hours. The cell scratches were then photographed at 10x magnification using a microscope (Zeiss, Germany). The scratch area was calculated using image J, and the scratch healing rate was calculated as: Wound healing rate (%) = (width 0h - width 24h) / width 0h.

[0037] 1.8. Transwell migration assay

[0038] Add 500 μL of DMEM medium containing 20% ​​fetal bovine serum to the lower chamber of a Transwell plate (24-well plate), and add 3 × 10⁻⁶ DMEM medium containing 20% ​​fetal bovine serum. 4 DMEM containing HUVECs was added to the upper chamber of a Transwell containing different concentrations of PPC (0, 2, 4, 8 μM). The chambers were then incubated in a cell culture incubator (24°C, 5% CO2) for 24 hours. Afterward, unmigrated HUVECs were removed from the top of the membrane using a cotton swab. The Transwell apparatus was then transferred to anhydrous ethanol and immersed for 30 minutes to fix the cells. The cells were then stained using Giemsa staining. Cell images of the stained Transwell chamber membrane were taken under a microscope (4×). Four fields of view were randomly captured in each Transwell insert, and the cells were counted.

[0039] 1.9. Determination of tubule formation in HUVECs

[0040] Approximately 2×10 4 Cells / well were seeded in 96-well plates coated with growth factor reducing matrix gel (Corning). PPC was added to the cell culture medium at seeding concentrations of 0, 2, 4, and 8 μM. The plates were then incubated. After 16 hours of culture, images were acquired using a microscope (Zeiss, Germany) at 4x magnification. The number of luminal nodes and the number of luminal junction intersections were analyzed using the angiography analyzer plugin in ImageJ software. The data were analyzed using Graph Prism.

[0041] 1.10. RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0042] Total RNA was extracted from treated cells using the RNApure kit (Aidlab Biology, RN03, Beijing, China) and quantified using a microplate reader (Thermo, USA). cDNA was created using a reverse transcription kit (Novoprotein, E047, Shanghai, China) and amplified using SYBR Green Real-Time PCR Master Mix (NovopProtein, E096, Shanghai, China). Primer sequences (Sangon Biotech Company, Shanghai, China) are listed in Table 1. Table 1 Primer sequences

[0043] 1.11. Western blot analysis

[0044] HUVECs (7×10) 4 Cells (cells / mL) were treated with 0, 2, 4, and 8 μM PPC for 24 hours, followed by lysis with RIPA buffer (Solarbio Life Sciences, Beijing, China). Protein concentration of the lysates was determined using the BCA assay. Proteins were separated by 10% SDS-PAGE and electrotransferred to PVDF membranes (Millipore, USA). After blocking with 5% skim milk in 0.1% Tween 20-Tris buffer (TBST) for 1 hour at room temperature, the membranes were incubated overnight at 4°C with the corresponding primary antibody (1:1000). The membranes were then incubated with HRP-linked IgG secondary antibody for 2 hours at room temperature. Color development was performed using an ECL luminescence kit (Lambolide, Beijing, China). (ChemiDoc) TM Positive bands were exposed on the MP imaging system (Bio-Rad, California), and the intensity of the bands was analyzed using ImageJ software.

[0045] 1.12. Statistical Analysis

[0046] GraphPad Prism software was used for statistical processing of the data. Measurement data are expressed as mean ± standard error (X ± S). Significant differences between groups were assessed using one-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0047] 2. Results

[0048] 2.1. PPC promotes the recovery of activity in rats.

[0049] Postoperatively, rats with severed limbs were unable to move normally. To determine the apparent recovery time of mobility, the apparent recovery time of mobility was recorded when rats showed no tenderness or longitudinal impingement pain at the fracture site, no abnormal movement at the fracture site, and were able to walk normally and stably with stable toes, coordinated gait, and normal walking posture. As healing progressed, untreated control rats recovered normal activity by day 29; this was defined as the apparent recovery time of mobility. All concentrations of PPC treatment shortened the apparent recovery time of mobility in rats (Figure 1). The apparent recovery time of mobility in the high-concentration group was 25 days, 4 days shorter than the control group. There was no significant difference in the apparent recovery time of mobility between the high-dose and medium-dose groups; therefore, the medium-dose group was selected for subsequent experiments.

[0050] 2.2. X-ray analysis results

[0051] X-rays are used to observe fracture healing. Figure 2A). All rats showed obvious tibial fractures on the day of modeling. Fracture healing was examined on days 1, 3, 7, 14, and 21 post-surgery. On days 1 and 3 of treatment, there was no significant change in the fracture. On day 7 of treatment, a cloud-like shadow indicating early callus formation appeared around the fracture in the PPC treatment group, while the fracture in the Nc group was slightly blurred. On day 14 of treatment, a small amount of callus formed at the fracture site in the PPC treatment group rats. On day 21 of treatment, the fracture defect area in the PPC group was filled with a relatively complete callus, and a large amount of external callus formed, significantly better than the control group, and tending towards basic healing. At this time, cartilage callus filling was observed in the control group. (Comparison...) Figure 2 Radiographic scores from X-rays were obtained in group B. At postoperative days 7, 14, and 21, fracture healing in the PPC group was significantly better than that in the NC group (P < 0.05). The fracture healing score table showed that on day 7, the fracture healing degree in the PPC treatment group was significantly better than that in the control group, and the difference gradually widened thereafter. By days 14 and 21, the fracture healing scores in the PPC treatment group were 1.33 times and 1.47 times that of the control group, respectively.

[0052] 2.3. PPC promotes the progression of ossification at fracture sites in rats

[0053] Fracture healing in the bony diaphysis occurs through intrachondral and intramembranous ossification. For example... Figure 6 As shown, 3A and PPC significantly accelerated bone formation during fracture healing, as evidenced by the increase in cartilage area at the fracture site in rats. On day 7 post-fracture, chondrocytes began to hypertrophy, undergoing intramembranous and intrachondral ossification. The PPC treatment group achieved the highest cartilage area ratio. Figure 3 In group B), endochondrial ossification occurred, while cartilage formation in the control group was relatively delayed. On day 14, the hypertrophic cartilage began to calcify into woven bone. In the PPC-treated group, woven bone dominated callus formation, while in the control group, more cartilage remained in the hypertrophic phase. Furthermore, on day 21, woven bone remodeling and new bone formation were observed in the PPC-treated group. A small amount of cartilage hypertrophy was still visible in the control group, indicating that the endochondrial ossification phase had not yet ended.

[0054] 2.4. PPC promotes angiogenesis at fracture sites in rats.

[0055] To observe the temporal pattern of the effect of PPC on angiogenesis at the fracture site in rats, bone tissue was collected on days 1, 3, 7, 14, and 21 postoperatively, and the effect was observed by immunohistochemical staining for CD31. Figure 4 A). The results showed that on the first and third days after surgery, the number of CD31-positive vessels in the PPC treatment group was significantly increased ( Figure 4B). On day 1, the CD31 area ratio in the PPC treatment group was more than 10 times higher than that in the control group (P < 0.0001). After 7 days, there was no significant difference in the CD31 area ratio between the PPC group and the control group. The effect of PPC on vascular cross-sectional area differs from its effect on vascular number. Figure 4 C). PPC treatment significantly increased the number of larger vessels. From day 3 to day 14, the vascular cross-sectional area in the PPC treatment group was much larger than that in the control group, reaching the maximum difference on day 14 (P = 0.0001), at which point the vascular area in the PPC treatment group was more than 15 times larger than that in the control group. After 21 days, the difference in vascular area between the PPC treatment group and the control group decreased (P = 0.0089).

[0056] 2.5. PPC promotes the proliferation activity of HUVECs

[0057] To evaluate the effect of PPC on HUVECs cell viability, an MTT assay was performed. The results showed that 0–24 μM doses of PPC had no cytotoxic effect on HUVECs. Figure 5 Compared with the control group (0 μM), treatment with doses of 4, 6, and 8 μM significantly promoted the proliferation of HUVECs. Subsequently, HUVECs were photographed microscopically every 12 hours. Microscopic examination revealed a significant increase in cell number in the PPC-treated groups, exhibiting a dose-response relationship within the concentration range of 4–8 μM.

[0058] 2.6. PPC promotes HUVEC migration

[0059] To verify whether PPC promotes HUVEC migration, we performed wound healing assays and Transwell migration assays. Scratch assay images showed that PPC treatment (2, 4, 8 μM) significantly accelerated scratch healing after 24 hours compared to the control group (0 μM) (Figure 6A). This migration-promoting effect was strongest at the 8 μM concentration, with a scratch healing rate of approximately 60%, about three times that of the control group. Figure 6 B). Subsequently, in Transwell migration assays, we found that the number of migrating cells significantly increased after PPC treatment compared to the control group (0 μM), and the number of migrating cells increased significantly with increasing concentration. Figure 7 A). This migration-promoting effect was strongest when administered at a concentration of 8 μM, with cell migration rates three times higher than the control group (0 μM). Figure 7 B). Both cell wound healing assays and Transwell migration assays showed that PPC promoted the migration of HUVECs in a concentration-dependent manner.

[0060] 2.7. PPC promotes the formation of HUVEC tubules.

[0061] The endothelial cell tube formation assay is an important experimental method for studying the ability of endothelial cells to form tubular structures. To assess the effect of polycystic vasculature (PPC) on the vascularization ability of HUVECs, we performed a tubule formation assay. When PPC was applied to HUVECs, the cells lengthened at 12 hours and developed in the direction of tube formation, although no clear tube structure was yet observed. Figure 8 A). Compared with the control group (0 μM), the treated group showed a greater density of vascular analogs. Furthermore, compared with the control group, the PPC treatment group showed significantly larger and denser vascular analogs. Figure 8 B), with a higher number of nodes ( Figure 8 C)). This ability to promote tubule formation increases with increasing PPC concentration, reaching a peak at a dose concentration of 8 μM. In summary, PPC can significantly promote the vascularization of HUVECs and exhibits a concentration-dependent effect.

[0062] 2.8. PPC regulates mRNA expression associated with the VEGF / VEGFR2 signaling pathway

[0063] Using qRT-PCR, we detected the mRNA expression of nine angiogenesis-related genes in HUVECs before and after PPC treatment. The results showed that after PPC treatment, VEGFA, VEGFR2, PLCγ, RAS, ERK1 / 2, and MEK1 / 2 were significantly upregulated in HUVECs, while eNOS, PI3K, and bFGF showed no significant difference. Figure 9 Based on these results, we hypothesize that PPC may play a role in promoting angiogenesis by modulating the VEGFA / VEGFR2 signaling pathway.

[0064] 2.9. PPC regulates the expression of proteins related to the VEGF / ERK1 / 2 signaling pathway.

[0065] We used Western blotting to detect the expression of angiogenesis-related signaling pathway proteins in HUVECs after PPC treatment. The results showed that, compared with the control group, the protein expression of VEGF, VEGFR2, MEK1 / 2, and ERK1 / 2 was significantly enhanced in the treatment group in a concentration-dependent manner. Figure 10 PPC not only promotes VEGF expression but also stimulates the expression of the VEGF receptor VEGFR2. Furthermore, PPC further promotes the expression of MEK1 / 2 and ERK1 / 2, and facilitates the proliferation, migration, and differentiation of HUVECs into the tubes.

[0066] This study revealed two new findings: first, PPC promotes fracture healing; second, PPC directly promotes angiogenesis. These two effects of PPC have not been previously reported.

[0067] Many factors influence fracture healing, among which blood supply to the fracture site has a significant impact. Angiogenesis plays a crucial role in fracture healing, especially in the early stages. In this study, we found that PPC significantly promotes angiogenesis at the onset and expands the formed vessels later, thereby accelerating fracture repair. Furthermore, we found that PPC promotes HUVEC proliferation by regulating VEGF expression. Endothelial cell migration is essential in angiogenesis. PPC can significantly promote HUVEC migration, which we hypothesize is partly due to the action of VEGFA.

[0068] 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 polyene phosphatidylcholine in the preparation of drugs for treating fractures. The fractures include linear fractures, comminuted fractures, and bone defects.

2. The application according to claim 1, characterized in that, The fractures were limb fractures.

3. The application according to claim 2, characterized in that, The fracture is a femoral fracture.

4. The application according to claim 1, characterized in that, The route of administration for the polyene phosphatidylcholine is oral.

5. The application according to claim 1, characterized in that, The route of administration for the polyene phosphatidylcholine is injection.

6. The application according to claim 5, characterized in that, The injection administration includes at least one of the following: local injection at the fracture site, intraperitoneal injection, subcutaneous injection, intramuscular injection, and intravenous injection.

7. Application of polyene phosphatidylcholine in the preparation of treatment for vascular reconstruction after fracture.

8. Application of polyene phosphatidylcholine in the preparation of drugs for vascular reconstruction after fracture.

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