Application of brucea javanica oil in preparation of medicine for inhibiting radiation pneumonitis or idiopathic pulmonary fibrosis

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses application of brucea javanica oil in preparation of a medicine for inhibiting radiation pneumonitis or idiopathic pulmonary fibrosis. When the brucea javanica oil is used for preparing the medicine for inhibiting radiation pneumonitis, the symptom of radiation pneumonitis can be remarkably relieved, the curative effect of the medicine is equivalent to that of dexamethasone, release of proinflammatory factors can be effectively inhibited, and inflammatory cascade reaction is blocked and lung tissue injury is relieved by down-regulating mRNA and protein expression of an HMGB1 / TLR4 / NF-kappa BP65 axis. The brucea javanica oil is applied to preparation of the medicine for treating idiopathic pulmonary fibrosis, lung tissue inflammatory cell infiltration can be effectively inhibited, abnormal collagen deposition can be reduced, expression of fibrosis promoting proteins COL1A1, VIM and ACTA2 can be down-regulated, and the pulmonary fibrosis process can be relieved by inhibiting fibroblast activation and extracellular matrix excessive deposition.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of Brucea javanica oil in the preparation of drugs for inhibiting radiation-induced pneumonia or idiopathic pulmonary fibrosis. Background Technology

[0002] Radiation pneumonitis (RP) is a common complication of radiotherapy for thoracic malignancies. Clinically, RP patients may present with symptoms such as low-grade fever, cough, shortness of breath, and chest pain. In severe cases, it can lead to respiratory distress, respiratory failure, and even death. The treatment effect of RP significantly impacts patient prognosis and can substantially reduce patients' quality of life and clinical outcomes. The pathogenesis of radiation pneumonitis is not yet fully understood, and there are no specific guidelines for its treatment. Clinically, after ruling out pulmonary infection, systemic glucocorticoids are usually used to treat RP patients with obvious symptoms. However, glucocorticoid therapy is dose-dependent, and symptom relapse is common after dose reduction or discontinuation. Long-term use can also lead to decreased immunity and adverse consequences such as pulmonary infection and hormone-induced osteodystrophy. Amifostine (amifotin) was the first approved radiation protection drug, which prevents and alleviates radiation-induced lung injury by scavenging free radicals and protecting and accelerating DNA repair. However, its clinical use is greatly limited due to side effects such as hypotension, severe nausea, and poor tolerability. Besides the drugs mentioned above, most treatments for radiation-induced pneumonia (RP), such as angiotensin-converting enzyme inhibitors, colchicine, and interferon, are still in the experimental stage, and their clinical efficacy and adverse reactions remain unclear. Furthermore, some existing studies suggest that the occurrence of radiation-induced pneumonia may be related to multiple factors, including oxidative stress, inflammatory factors, and immune cell imbalance, and involves a network pathological process involving various cells and inflammatory factors. Therefore, researching and developing more drugs with proven efficacy against radiation-induced pneumonia is of significant practical importance, especially in-depth research into the molecular mechanisms of radiation-induced pneumonia and the development of targeted drugs, which are crucial for improving the clinical treatment outcomes of radiation-induced pneumonia.

[0003] Furthermore, idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease characterized by excessive extracellular matrix deposition, inflammatory damage, and irreversible structural remodeling in lung tissue. In recent years, the prevalence and mortality rates of IPF have continued to rise. Currently, drug treatment for IPF mainly relies on antifibrotic drugs, among which nintedanib and pirfenidone are standard treatments recommended by international guidelines. Nintedanib, as an intracellular multi-target tyrosine kinase inhibitor, primarily slows the fibrotic process by precisely blocking key signaling pathways such as PDGFR, FGFR, and VEGFR, directly inhibiting the proliferation, migration, and transformation of fibroblasts. Pirfenidone, on the other hand, can antagonize disease progression through multiple pathways by downregulating the expression of the core pro-fibrotic factor TGF-β, inhibiting collagen synthesis, and exerting anti-inflammatory and antioxidant effects. Although both can slow the annual decline in forced vital capacity in IPF patients to some extent, they cannot reverse the disease progression and have limitations such as significant adverse reactions, large differences in patient response, and high treatment costs. While lung transplantation is an effective treatment option for end-stage interstitial lung disease (IPF), its effectiveness is limited by factors such as donor scarcity, high surgical risks, and complex postoperative management. Therefore, given the high mortality rate, lack of effective drugs, and heavy medical burden associated with IPF, in-depth research into the pathogenesis of IPF, overcoming treatment bottlenecks, and developing highly effective drugs that can reverse fibrosis are key scientific challenges and urgent clinical needs in the field of IPF research.

[0004] Brucea javanica is a traditional Chinese medicine, often used to treat warts and corns with significant effects and low price. It also has other uses such as stopping diarrhea, killing parasites, treating malaria, and clearing heat and detoxifying. Brucea javanica oil is a fatty oil obtained by petroleum ether extraction from mature fruits. Its main components can be divided into unsaturated fatty acids, oleic acid, and linoleic acid. However, there are currently no reports on the use of Brucea javanica oil for the treatment of radiation-induced pneumonia or idiopathic pulmonary fibrosis, or in drug preparation. Summary of the Invention

[0005] One of the technical problems solved by this invention is to provide the application of Brucea javanica oil in the preparation of drugs to inhibit radiation pneumonia, thus providing more effective treatment options for radiation pneumonia.

[0006] The second technical problem solved by this invention is to provide the application of Brucea javanica oil in the preparation of drugs to inhibit idiopathic pulmonary fibrosis, thus providing more effective treatment options for the treatment of idiopathic pulmonary fibrosis.

[0007] The technical problem solved by this invention is to study the mechanism of action of Brucea javanica oil on radiation-induced pneumonia, so as to provide direction for further development and preparation of drugs for the treatment of radiation-induced pneumonia.

[0008] The technical problem solved by this invention is achieved by the following technical solution: Application of Brucea javanica oil in the preparation of drugs to inhibit radiation-induced pneumonia or idiopathic pulmonary fibrosis.

[0009] Furthermore, the drug contains a therapeutically effective amount of Brucea javanica oil and a pharmaceutically acceptable carrier.

[0010] Furthermore, the drug is formulated into a pharmaceutically permissible dosage form.

[0011] Furthermore, the drug is formulated into an injectable form.

[0012] Furthermore, the drug that inhibits radiation pneumonitis downregulates the levels of inflammatory factors in patients with radiation pneumonitis.

[0013] Furthermore, the inflammatory factors include IL-1β, IL-6, TNF-α, and NF-κB.

[0014] Furthermore, the drug that inhibits radiation-induced pneumonia reduces radiation-induced lung injury by targeting and regulating the HMGB1 / TLR4 / NF-κBP65 axis.

[0015] Furthermore, the drug for inhibiting idiopathic pulmonary fibrosis alleviates the progression of pulmonary fibrosis by inhibiting fibroblast activation and excessive deposition of extracellular matrix.

[0016] Furthermore, the drug for inhibiting idiopathic pulmonary fibrosis downregulates the expression of profibrotic proteins COL1A1, VIM, and ACTA2.

[0017] Beneficial Effects: The Brucea javanica oil described in this invention, when used in the preparation of drugs to inhibit radiation-induced pneumonia, can significantly alleviate the symptoms of radiation-induced pneumonia, with efficacy comparable to dexamethasone. It can effectively inhibit the release of pro-inflammatory factors IL-1β, IL-6, TNF-α, and NF-κB. The mechanism of action of Brucea javanica oil is related to the targeted regulation of the HMGB1 / TLR4 / NF-κB signaling axis. By downregulating the mRNA and protein expression of the HMGB1 / TLR4 / NF-κBP65 axis, it blocks the inflammatory cascade response and reduces lung tissue damage.

[0018] The Brucea javanica oil described in this invention is used in the preparation of a drug for idiopathic pulmonary fibrosis. This drug can effectively inhibit the infiltration of inflammatory cells in lung tissue and reduce abnormal collagen deposition, downregulate the expression of profibrotic proteins COL1A1, VIM, and ACTA2, and alleviate the process of pulmonary fibrosis by inhibiting fibroblast activation and excessive deposition of extracellular matrix. Attached Figure Description

[0019] Detailed Implementation

[0020] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments.

[0021] Example 1 This embodiment is used to verify the application of Brucea javanica oil in the preparation of drugs to inhibit radiation pneumonia.

[0022] (I) Establishment of the RP mouse model: Mice were anesthetized by intraperitoneal injection of 2% sodium phenobarbital (dose 50 mg / kg) and subjected to whole-chest irradiation with 6 MV X-rays from a linear accelerator. The irradiation dose was 20 Gy per irradiation. The irradiation range was determined using a conventional X-ray simulation system, with the upper boundary extending to the top of the axilla, the lower boundary to the end of the xiphoid process, the medial boundary along the midline of the sternum, and the lateral boundary to the lateral edge of the axilla. Non-target areas were protected with 10 mm lead shields. The irradiation field was the entire chest, the irradiation rate was 3 Gy / min, and the source-skin distance was 100 cm. A single irradiation RP model was established.

[0023] (II) Experimental grouping and drug administration: Forty SPF-grade C57BL / 66-8-week-old female mice (weighing 20g±2g) were observed at the medical school quarantine center for one week. After ensuring no abnormalities, they were randomly divided into four groups of 10 mice each, and all mice were sacrificed at the end of the fourth week.

[0024] Grouped administration: Control group: After sham irradiation, 0.2 ml of 0.9% saline was injected intraperitoneally every other day until the end of the experiment; Model group (IR): After a single irradiation of 20 Gy, 0.2 ml of saline was injected intraperitoneally every other day; Dexamethasone group (IR+DM): After a single irradiation of 20 Gy, 0.1 mg / 20 g of dexamethasone was injected intraperitoneally every other day; Brucea javanica oil group (IR+YDZ): After a single irradiation of 20 Gy, 0.2 ml of Brucea javanica oil emulsion was injected intraperitoneally every other day. The Brucea javanica oil was purchased from Shenyang Pharmaceutical University Lei Yunshang Pharmaceutical Co., Ltd., and the concentration of Brucea javanica oil was 1 g / 10 ml.

[0025] (III) Testing and Results 1. Observation of the external condition of mice Following radiation, mice in each group were observed every two days for changes in body weight, food intake, water consumption, fur, movement, mental state, and survival rate. Mice in the IR, IR+DM, and IR+YDZ groups began to show reduced food and water intake, decreased activity, and lethargy on the second day. Body weight decreased on the second day, reaching its lowest point on the sixth day, with significantly reduced food and water intake, dry fur, sluggish movement, and lethargy. Some mice also showed eye discharge. These symptoms gradually improved one week after radiation exposure, and body weight gradually increased. On day 21 post-radiation, hair loss was observed on the chest of mice in the IR, IR+DM, and IR+YDZ groups. Over time, the irradiated area in the IR group mice completely lost its hair, exposing bare skin with some scar tissue regeneration and minor skin traction. The degree of hair loss in the IR+DM and IR+YDZ groups was less severe than in the IR group. Figure 1 As shown in the figure. No mice died during the experiment.

[0026] 2. Pathological manifestations of lung tissue Lung tissue pathological sections: Lung tissue was collected from mice in each group after dissection at the end of the 4th week after irradiation. The lung tissue was fixed with 4% paraformaldehyde, dehydrated by soaking in ethanol solution, embedded in liquid paraffin, and then sectioned. The section thickness was adjusted to 4-5 μm, and the sections were cut evenly to ensure that the sections were intact and without wrinkles.

[0027] HE staining: Dewax and rehydrate lung tissue sections, stain the nuclei with hematoxylin for 8 minutes, rinse with water for 10 seconds; continue to use 1% hydrochloric acid ethanol for differentiation for 5 seconds, then rinse with water, blue cell nuclei are visible, then perform blue staining again, rinse with running water; Eosin counterstaining: treat with eosin staining solution for 1 minute, rinse with running water for 5 seconds; dehydrate after staining, mount with neutral resin and cover with coverslip.

[0028] Panoramic scanning of stained sections was performed using an optical microscope (40× for initial screening, 200× for high-power observation), and images were taken of typical lesion areas. Three different fields of view were randomly selected from each section, and pathological scoring was performed with reference to the Szapiel score. The Szapiel scoring table is shown in Table 1.

[0029] Table 1 Szapiel rating table level level score - The morphology and structure of the bronchi and alveoli were clear and intact, and no infiltration of inflammatory cells was observed. 0 + Mild inflammation in the alveolar region, localized mononuclear cell infiltration, and partial thickening of the alveolar septa. The lesions are limited to the local pleural region, accounting for no more than 20% of the total lung area, and the alveolar structure is well preserved. 1 ++ Moderate alveolar inflammation, affecting 20% ​​to 50% of the entire lung, with lesions primarily in the pleural region. 2 +++ Extensive and severe alveolar inflammation, with inflammation covering more than 50% of the total lung area. 3 HE staining of mouse lung tissue as shown Figure 2 As shown, the inflammation score of mouse lung tissue sections is as follows: Figure 3As shown in the figure, the alveolar structure of mice in the Control group was normal, with no thickening, collapse, or rupture of the alveolar walls, and no obvious inflammatory cell exudation. The inflammation scores of three samples from the same group under a microscope were 0 / 1 / 0, respectively. Compared with the Control group, the IR group showed more severe alveolar congestion and exudation, less alveolar cavity collapse, and thickened alveolar walls. The inflammation scores of three samples from the same group under a microscope were 3 / 2 / 3, respectively. Compared with the IR group, it was clearly observed that the alveolar congestion, exudation, and inflammatory cell infiltration in the IR+YDZ group were alleviated. The inflammation scores of three samples from the same group under a microscope were 1 / 1 / 1, respectively. The inflammation level in the IR+DM group was also significantly reduced compared to the IR group. The inflammation scores of three samples from the same group under a microscope were 1 / 1 / 1, respectively. These results indicate that Brucea javanica oil can significantly reduce lung tissue inflammation in mice after radiation (P<0.01). 3. Serum inflammatory factor level detection Serum separation: Before euthanasia of mice, orbital blood was collected and allowed to stand at room temperature for 60 minutes to allow complete coagulation. Then, the blood was centrifuged at 2000×g for 15 minutes at 4°C, and the supernatant serum was collected and stored at -80°C for analysis.

[0030] The inflammatory mediators in mouse serum samples were measured using an ELISA kit, and the results were as follows: Figure 4 As shown, compared with the Control group, the expression levels of IL-1β, IL-6, TNF-α, and NF-κB in the IR group samples were significantly increased. The IR+YDZ group effectively inhibited the levels of inflammatory factors in the serum of radiation-induced pneumonia mice, indicating that Brucea javanica oil can effectively reduce the levels of IL-1β, IL-6, TNF-α, and NF-κB inflammatory factors in RP mice.

[0031] 4. mRNA expression level statistics Four weeks post-irradiation, Trizol extracted RNA from tissues and used real-time quantitative PCR to detect the expression of key genes in the HMGB1 / TLR4 / NF-κB signaling pathway, specifically targeting HMGB1, TLR4, MyD88, and NF-κBP65, as detailed below: Trizol RNA extraction from tissue: Weigh 50-100 mg of lung tissue sample, add 1 mL of Trizol lysis reagent, homogenize the tissue under ice bath conditions until no obvious lumps remain, and incubate at room temperature for 5 minutes for complete lysis; add 200 μL of chloroform, shake vigorously for 15 seconds, and incubate at room temperature for 3 minutes; centrifuge at 12000 × g for 15 minutes at 4°C, then carefully transfer the upper aqueous phase to a new centrifuge tube; add an equal volume of isopropanol solution to the upper aqueous phase, mix thoroughly, and incubate at room temperature for 10 minutes; incubate at 4°C... Centrifuge at 12000×g for 10 minutes at low temperature, remove the supernatant, and wash the precipitate with 1 mL of 75% ethanol solution prepared with DEPC water. Centrifuge at 12000×g for 3 minutes at 4℃, remove the supernatant, and dry at room temperature for 5-10 minutes. Dissolve the ribonucleic acid precipitate in 20-30 μL of nuclease-free water. Ribonucleic acid quality testing: The concentration and purity of ribonucleic acid were determined by ultraviolet spectrophotometry (the A260 / A280 ratio should be controlled within the range of 1.8-2.1), and the integrity of ribonucleic acid was assessed by agarose gel electrophoresis.

[0032] RNA reverse transcription: Reaction system preparation: Prepare 20 μL of reverse transcription reaction solution according to Table 2; vortex mix and then briefly centrifuge to ensure that the reaction solution is concentrated at the bottom of the tube; incubate at 50℃ for 50 minutes and at 85℃ for 5 minutes; the reaction product can be used directly for subsequent qPCR experiments or stored at -20℃ for a long time.

[0033] Table 2 Reverse Transcription Reaction Solution

[0034] RT-qPCR experiment Nucleotide sequences of the HMGB1, TLR4, MyD88, and NF-κBP65 genes were retrieved from the National Center for Biotechnology Information (NCBI) database. Primers were then designed using the Primer5 tool. Finally, the designed primer sequences were synthesized by Beijing Qingke Company. The primer sequences and related information are shown in Table 3.

[0035] Table 3 Primer sequences and related information

[0036] Prepare the reaction solution according to Table 4 (3 wells per sample per indicator, 30 μL total, 10 μL per well), and then perform PCR amplification: Pre-denaturation: maintain at 95℃ for 10 minutes to fully activate DNA polymerase; Cyclic amplification (40 cycles in total): denature at 95℃ for 15 seconds, anneal and extend at 60℃ for 30 seconds; Melting curve analysis: continuously increase the temperature from 60℃ to 95℃ (0.5℃ / 5 seconds) to verify the specificity of the amplified product.

[0037] Table 4 RT-qPCR reaction solution

[0038] The relative expression level of the target gene was calculated using the 2−△△Ct method, with the internal reference gene (GAPDH) as the standardization reference. The results are as follows: Figure 5 As shown, the results indicated that, compared to the Control group, the levels of HMGB1, TLR4, MyD88, and NF-κB65 in the lung tissue of mice in the IR group were significantly increased, while the IR+YDZ group effectively inhibited the expression levels of related mRNAs in the lung tissue of mice with radiation pneumonitis (P<0.05). Therefore, Brucea javanica oil alleviates radiation pneumonitis by affecting the HMGB1 / TLR4 / NF-κB pathway.

[0039] 5. Protein expression level At the fourth week post-irradiation, the expression levels of proteins related to the HMGB1 / TLR4 / NF-κB pathway were detected by Western blotting, as follows: Protein extraction: Mouse lung tissue from each group was placed on ice, and 0.025g of tissue was quickly cut and rinsed in pre-chilled PBS. The tissue was then placed in a new EP tube, 300µl of RIPA lysis buffer was added, along with 2-3 sterile magnetic beads. The tube was sealed tightly with sealing film, and the tissue was thoroughly homogenized using a bio-homogenizer until a complete homogenate was formed. The sample was then placed on ice for 30 minutes for further lysis. Simultaneously, the centrifuge was pre-chilled at 4°C, 12000 rpm, for 15 minutes. After lysis, the sample was rapidly centrifuged. The supernatant was carefully transferred to a new 1.5ml EP tube. The tube was vortexed, and 20µl was transferred to another EP tube for BCA detection. Unused samples could be stored at -80°C for later use.

[0040] Protein concentration detection: The BCA protein quantification kit was used to measure the protein concentration of the sample according to the instructions, and then the protein concentration of the sample was calculated based on the standard curve of the sample concentration obtained from the standard.

[0041] westernblot: Gel preparation: Use 10% separating gel and 4.8% concentrating gel to prepare the gel, and let it solidify for later use; Sample preparation: Take out the remaining protein sample and place it on ice. Add 5*loading buffer at a ratio of 4:1 and place it in a 100℃ water bath for denaturation for 10-15 minutes.

[0042] Electrophoresis: Add an appropriate volume of sample according to the BCA quantification results. Electrophoresis conditions are 75V and 130 minutes.

[0043] Primary antibody incubation: After NC transfer, dissolve 5% skim milk powder in PBST, soak the NC membrane in it, and let it stand at room temperature for 90 minutes; dilute the primary antibody with PBST, bring the NC membrane and primary antibody into close contact, incubate overnight at 4°C, and then continue incubation at room temperature for 30 minutes the next day; after completion, wash three times with PBST, 15 minutes each time.

[0044] Secondary antibody incubation: Dilute the HRP-labeled secondary antibody, incubate the secondary antibody and NC membrane together at room temperature for 90 minutes. After the reaction is complete, wash three times with PBST for 10 minutes each time.

[0045] Color development / exposure: Add ECL luminescent solution to the NC membrane and incubate in the dark for one minute. After absorbing the excess liquid, wrap it with plastic film and place it in the gel imaging system for imaging.

[0046] Data Analysis: The study data were confirmed to follow a normal distribution after a normality test, and the results are presented as mean ± standard deviation. Differences between groups were analyzed using independent samples t-tests or one-way ANOVA, with a significance threshold set at P < 0.05. All statistical calculations were performed using GraphPadPrism9 software.

[0047] The results are as follows Figure 6 As shown, compared with the Control group, the levels of HMGB1, TLR4, MyD88, and NF-κBP65 proteins in the lung tissue of mice in the IR group were significantly increased. The IR+YDZ group effectively inhibited the expression levels of related proteins in the lung tissue of mice with radiation pneumonia. This indicates that Brucea javanica oil alleviates radiation pneumonia by affecting the HMGB1 / TLR4 / NF-κB pathway. The mechanism of action of Brucea javanica oil is related to the targeted regulation of the HMGB1 / TLR4 / NF-κB signaling axis. By downregulating the mRNA and protein expression of HMGB1, MyD88, TLR4, and NF-κBP65, it blocks the inflammatory cascade response and reduces lung tissue damage.

[0048] Example 2 In this embodiment, the establishment and detection of the RP mouse model are the same as in Example 1, except for the experimental grouping and drug administration steps: Fifty SPF-grade C57BL / 66-8-week-old female mice (weighing 20g±2g) were observed at the medical school quarantine center for one week. After ensuring no abnormalities, they were randomly divided into 5 groups of 10 mice each, and all mice were sacrificed at the end of the 4th week.

[0049] Grouped drug administration: Control group: After sham irradiation, 0.2 ml of 0.9% saline was injected intraperitoneally every other day until the end of the experiment; Model group (IR): After a single 20 Gy irradiation, 0.2 ml of saline was injected intraperitoneally every other day; High-dose Brucea javanica oil group (IR + high-dose group): After a single 20 Gy irradiation, 0.2 ml of Brucea javanica oil emulsion was injected intraperitoneally every other day. The Brucea javanica oil was purchased from Shenyang Pharmaceutical University Lei Yunshang Pharmaceutical Co., Ltd., and the concentration of Brucea javanica oil was 1 g / 10 ml. The medium-dose Brucea javanica oil group (IR + medium-dose group): after a single 20 Gy irradiation, 0.1 ml of Brucea javanica oil emulsion was injected intraperitoneally every other day. The Brucea javanica oil was purchased from Shenyang Pharmaceutical University Lei Yunshang Pharmaceutical Co., Ltd., and the concentration of Brucea javanica oil was 1 g / 10 ml. The low-dose Brucea javanica oil group (IR + low-dose group): after a single 20 Gy irradiation, 0.05 ml of Brucea javanica oil emulsion was injected intraperitoneally every other day. The Brucea javanica oil was purchased from Shenyang Pharmaceutical University Lei Yunshang Pharmaceutical Co., Ltd., and the concentration of Brucea javanica oil was 1 g / 10 ml.

[0050] The inflammatory mediators in mouse serum samples were measured using an ELISA kit, and the results were as follows: Figure 7 As shown, compared with the Control group, the expression levels of IL-1β, IL-6, and TNF-α in the IR group samples were significantly increased. The experimental group supplemented with Brucea javanica oil effectively inhibited the levels of inflammatory factors in the serum of radiation-induced pneumonia mice, with the best inhibitory effect observed when the injection volume of Brucea javanica oil was greater than or equal to 0.2 ml.

[0051] This invention successfully established a mouse model of radiation-induced pneumonia. By observing and detecting the external condition, pathological changes, inflammatory factor levels, and related pathway protein expression levels of mice in each group, it was verified that *Brucea javanica* oil can target and regulate the HMGB1 / TLR4 / NF-κB axis. The *Brucea javanica* oil treatment group significantly inhibited the levels of pro-inflammatory mediators IL-1β, IL-6, TNF-α, and NF-κB. These factors are key mediators of radiation-induced tissue damage, driving neutrophil recruitment, endothelial cell dysfunction, and fibrotic remodeling. *Brucea javanica* oil significantly inhibited the production of these factors, demonstrating its significant anti-inflammatory effect. Subsequent gene and protein level analyses showed that *Brucea javanica* oil significantly downregulated the mRNA and protein expression of HMGB1, MyD88, TLR4, and NF-κBP65 in lung tissue. In existing research, HMGB1, a damage-associated molecular pattern (DAMP) released during radiation-induced cell damage, activates the TLR4 / MyD88 signaling pathway, triggering NF-κB activation and subsequent cytokine storm, leading to an inflammatory response. Brucea javanica oil, by blocking this signaling axis, can inhibit the amplification of inflammatory signals, thereby alleviating alveolar epithelial damage and maintaining normal lung tissue structure. This invention is the first to investigate the relationship between Brucea javanica oil, the HMGB1 / TLR4 / NF-κB axis, and radiation pneumonitis, suggesting that Brucea javanica oil can serve as a DAMP-driven RP therapeutic agent. This is of great significance for optimizing the prevention and treatment system of radiation pneumonitis, exploring new biological targets for the treatment and prevention of radiation pneumonitis, and developing drugs with clear efficacy and fewer adverse reactions against radiation pneumonitis. Furthermore, in terms of overall efficacy, Brucea javanica oil is comparable to dexamethasone in reducing RP inflammation levels. However, long-term use of glucocorticoids may cause side effects such as immunosuppression and metabolic disorders. Considering its natural source and traditional medical application background, Brucea javanica oil has better safety than hormone therapy and is less likely to induce drug resistance. It can act on various diseases through multiple pathways, multiple effects, and multiple targets.

[0052] Example 3 This embodiment is used to verify the application of Brucea javanica oil in the preparation of drugs for idiopathic pulmonary fibrosis.

[0053] (I) Animal experiments have verified that Brucea javanica oil can effectively alleviate the progression of IPF. 1. Establishment of IPF mouse model: Mice were anesthetized by intraperitoneal injection of 2% sodium phenobarbital at a dose of 50 mg / kg. Anesthetized mice were placed on a manipulation board in a supine position, with their heads and limbs fixed, allowing full extension of the neck. After disinfection of the anterior neck skin, a longitudinal incision of approximately 0.5 cm was made, and subcutaneous tissue and muscle were bluntly dissected to expose the trachea. Using a microsyringe, a needle was inserted between the two tracheal cartilage rings, and bleomycin solution (dissolved in sterile saline) was slowly and single-drip infused at a volume of 100 µL, at a dose of 5 mg / kg. Immediately after infusion, the mice were rotated upright for a short time to ensure even distribution of the drug in the lungs. The skin incision was then sutured, and the mice were placed on warm bedding to recover until fully awake. Control mice underwent the same method of intratracheal infusion of the same volume of saline. Twenty-eight days after administration, the mice were dissected, and histopathological examination and hydroxyproline content determination confirmed successful establishment of pulmonary fibrosis.

[0054] 2. Experimental grouping and drug administration Thirty SPF-grade C57BL / 6-8 week old female mice (weighing 20g±2g) were observed at the medical school quarantine center for one week. After ensuring no abnormalities, they were randomly divided into 3 groups of 10 mice each, and all mice were sacrificed at the end of the 4th week.

[0055] Grouped administration: Control group: After intratracheal infusion of the same dose of normal saline as described above, 0.2 ml of 0.9% normal saline was injected intraperitoneally every other day until the end of the experiment; Fibrosis group: After bleomycin modeling, 0.2 ml of normal saline was injected intraperitoneally every other day; Brucea javanica oil group (BJOE): After bleomycin modeling, 0.2 ml of Brucea javanica oil emulsion was injected intraperitoneally every other day. The Brucea javanica oil was purchased from Shenyang Pharmaceutical University Lei Yunshang Pharmaceutical Co., Ltd., and the concentration of Brucea javanica oil was 1 g / 10 ml.

[0056] 3. Test Results Results of HE and Masson staining of mouse lung tissue are as follows Figure 8 As shown.

[0057] HE staining results showed that the alveolar structures of mice in the Control group were clear and intact, with uniform alveolar septa and no obvious thickening, congestion, or inflammatory cell infiltration. The lung tissue structure was normal, and the inflammation scores of the three samples in the same group were 0 / 0 / 1 points, respectively. In contrast, the lung tissue structure of the Fibrosis group was disordered, with significant thickening of alveolar septa, alveolar collapse and fusion, accompanied by a large number of inflammatory cells (mainly lymphocytes and macrophages) infiltration and focal hemorrhage. The inflammation scores of the three samples in the same group were significantly increased, at 3 / 3 / 4 points, respectively. The pathological changes in the lung tissue of the BJOE group were significantly reduced compared with the Fibrosis group, with decreased alveolar structural damage, reduced inflammatory cell infiltration, and some relief of alveolar septal thickening. The inflammation scores of the three samples in the same group were 1 / 2 / 1 points, respectively.

[0058] Masson staining results showed that in the Control group, only a very small amount of blue collagen fibers were distributed around blood vessels and bronchi in the lung tissue, with almost no collagen deposition in the lung parenchyma. In the Fibrosis group, a large number of diffuse blue collagen fibers were deposited in the lung tissue, especially forming dense blue areas in the thickened alveolar septa and lung interstitium, indicating severe pulmonary fibrosis. In contrast, the area and density of blue collagen deposition in the BJOE group were significantly reduced compared to the Fibrosis group, and the collagen fibers were relatively loosely arranged, mainly limited to some thickened septa, with better preservation of the lung parenchyma structure.

[0059] It is known that bleomycin can successfully induce significant alveolitis and pulmonary fibrosis in mice. However, after intervention with injection of Brucea javanica oil emulsion (concentration 1g / 10ml), it can effectively inhibit the infiltration of inflammatory cells in lung tissue and reduce abnormal collagen deposition, thereby alleviating the pathological process of bleomycin-induced pulmonary fibrosis.

[0060] 4. Determination of hydroxyproline content Sample processing and assay methods: After euthanizing mice, approximately 50 mg of tissue from the right middle lobe of the lung was collected, rinsed with pre-cooled physiological saline, blotted dry with filter paper, and weighed. The tissue sample was placed in a hydrolysis tube, 2 mL of 6 M hydrochloric acid was added, and hydrolysis was carried out in a 110°C oven for 24 hours. After hydrolysis, an appropriate amount of the hydrolysate was neutralized to approximately pH 7.0 with sodium hydroxide solution and brought to a suitable volume. Subsequently, the processed sample and standards were added sequentially to chloramine T solution, perchloric acid solution, and p-dimethylaminobenzaldehyde solution, and the mixture was incubated in a 60°C water bath for 20 minutes for color development. After cooling, the absorbance of each well was measured at a wavelength of 560 nm using a microplate reader. The concentration of hydroxyproline in the sample was calculated based on the standard curve, and the final result was expressed as micrograms (µg / g) of hydroxyproline per gram of wet-weight lung tissue.

[0061] Test results: The results of the hydroxyproline content determination are as follows: Figure 9 As shown in the results, the hydroxyproline content in the lung tissue of the Control group was the lowest, within the normal physiological range. The hydroxyproline content in the lung tissue of the Fibrosis group was significantly increased, showing a highly significant difference compared to the Control group, indicating that bleomycin successfully induced a large amount of abnormal accumulation of collagen in the lungs. After intervention with Brucea javanica oil, the hydroxyproline content in the lung tissue of the BJOE group was still higher than that of the Control group, but significantly lower than that of the Fibrosis group. This indicates that Brucea javanica oil can effectively inhibit excessive deposition of extracellular matrix, thereby delaying or alleviating the pathological process of bleomycin-induced pulmonary fibrosis.

[0062] (II) Cellular experiments verified that Brucea javanica oil significantly reduced the levels of pulmonary fibrosis marker proteins. 1. Establishment of a Cellular Fibrosis Model: Human embryonic lung fibroblasts (MRC-5) were harvested and cultured in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a 37°C, 5% CO2 incubator. When the cells reached 80%-90% confluence, they were digested with 0.25% trypsin and passaged. Cells in the logarithmic growth phase were used for subsequent experiments. To establish an in vitro lung fibrosis model, MRC-5 cells were stimulated with transforming growth factor-β1 (TGF-β1, 10 ng / mL) for 48 hours to induce activation into a myofibroblast phenotype, mimicking key fibrosis processes. Figure 10 -A indicates that the cell fibrosis model has been successfully established. Figure 10 -B indicates that when different concentrations of Brucea javanica oil were used to conduct toxicological experiments on cell models, the optimal concentration of Brucea javanica oil was 1 mg / ml.

[0063] 2. Experimental Grouping and Drug Treatment: MRC-5 cells were randomly divided into the following three groups: Control Group: Cultured in normal culture medium without TGF-β1 stimulation. Model Group (Fibrosis): Treated with culture medium containing TGF-β1 (10 ng / mL) for 48 hours. Brucea javanica Oil Group (BJOE): Two hours before TGF-β1 stimulation, pretreated with culture medium containing Brucea javanica oil emulsion (final concentration 10 µg / mL), then cultured for 48 hours with the same concentration of Brucea javanica oil and TGF-β1 (10 ng / mL). The Brucea javanica oil emulsion used in the experiment was purchased from Shenyang Pharmaceutical University Lei Yunshang Pharmaceutical Co., Ltd. Before use, it was dissolved in cell culture-grade DMSO and diluted with complete culture medium to the working concentration, ensuring that the final DMSO concentration of each treatment group was consistent (≤0.1%).

[0064] 3. The levels of type I collagen (COL1A1), vimentin (VIM), and α-smooth muscle actin (ACTA2) in the cell supernatant were detected by ELISA. The results are as follows: Figure 10 As shown in Figure -c, compared with the Control group, the secretion levels of COL1A1, VIM, and ACTA2 in the cell supernatant of the Fibrosis group were significantly increased. After intervention with Brucea javanica oil, the secretion of COL1A1, VIM, and ACTA2 was significantly inhibited.

[0065] Western blot analysis was performed on cell lysates to detect the expression levels of vimentin (VIM), α-smooth muscle actin (ACTA2), and E-cadherin. The results are as follows: Figure 10 -D and Figure 10As shown in Figure -E, compared with the Control group, the expression of VIM and ACTA2 in the Fibrosis group was significantly upregulated, while the expression of the epithelial marker E-cadherin was inhibited. After treatment with Brucea javanica oil, the expression levels of VIM and ACTA2 proteins in the BJOE group decreased in a dose-dependent manner, while the expression of E-cadherin was partially restored.

[0066] In summary, in the TGF-β1-induced MRC-5 cell fibrosis model, the treatment with Brucea javanica oil described in this invention effectively downregulated the expression of key pro-fibrotic proteins (COL1A1, VIM, ACTA2) and partially reversed the loss of the epithelial marker E-cadherin. This confirms at the molecular level that Brucea javanica oil has direct anti-pulmonary fibrosis activity, which is consistent with the results of animal experiments, jointly demonstrating that specific concentrations of Brucea javanica oil can alleviate the progression of pulmonary fibrosis by inhibiting fibroblast activation and excessive extracellular matrix deposition.

[0067] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Use of brucea javanica oil in the preparation of a medicament for inhibiting radiation pneumonitis or idiopathic pulmonary fibrosis.

2. Use according to claim 1, wherein The medicament contains a therapeutically effective amount of brucea javanica oil and a pharmaceutically acceptable carrier.

3. Use according to claim 2, wherein the compound is ###0002### The medicament is prepared into a pharmaceutically acceptable dosage form.

4. The use according to claim 3, wherein the compound is ###0002### The medicament is prepared into an injection.

5. The use according to claim 1, wherein the compound is ###0002### The medicament for inhibiting radiation pneumonitis down-regulates the level of inflammatory factors in patients with radiation pneumonitis.

6. Use according to claim 5, wherein The inflammatory factors include IL-1β, IL-6, TNF-α, NF-κB.

7. The use according to claim 1, wherein The medicament for inhibiting radiation pneumonitis alleviates radiation lung injury by targeting the regulation of the HMGB1 / TLR4 / NF-κB P65 axis.

8. The use according to claim 1, wherein The medicament for inhibiting idiopathic pulmonary fibrosis alleviates the progression of pulmonary fibrosis by inhibiting fibroblast activation and excessive deposition of extracellular matrix.

9. The use according to claim 1, wherein The medicament for inhibiting idiopathic pulmonary fibrosis down-regulates the expression of pro-fibrotic proteins COL1A1, VIM, ACTA2.

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