Application of fluvastatin combined with tanshinone in preparation of preparation for preventing and / or treating radiation-induced lung injury

Abstract

This invention relates to the field of biomedical technology. It provides a pharmaceutical composition comprising fluvastatin and tanshinone as active ingredients, and further provides its application in the preparation of formulations for the prevention and / or treatment of radiation-induced lung injury. This invention utilizes the synergistic effect of fluvastatin and tanshinone to intervene in the pathogenesis of radiation-induced lung injury from multiple targets and pathways, significantly improving prevention and treatment efficacy. It can alleviate the inflammatory symptoms of radiation-induced pneumonia and delay the progression of radiation-induced pulmonary fibrosis, with low side effects, high safety, and wide applicability, providing a new drug option and technical solution for the clinical prevention and treatment of radiation-induced lung injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of fluvastatin in combination with tanshinone in the preparation of formulations for the prevention and / or treatment of radiation-induced lung injury. Background Technology

[0002] Radiation-induced lung injury (RILI) is one of the most common serious complications during radiotherapy for thoracic tumors (such as lung cancer, esophageal cancer, breast cancer, and malignant lymphoma). It mainly manifests as two stages: radiation pneumonitis and radiation pulmonary fibrosis. These two stages are interconnected and progress gradually, seriously affecting the patient's treatment outcomes and quality of life. It may even lead to respiratory failure and become a key factor limiting the increase of thoracic radiotherapy dose and affecting the patient's prognosis.

[0003] The pathogenesis of radiation-induced lung injury is complex, involving a multi-celled and interactive process. The core mechanisms include: radiation causing direct damage to target cells in lung tissue (vascular endothelial cells, alveolar epithelial cells, etc.), leading to vascular embolism, pulmonary interstitial edema, and inflammatory cell infiltration; radiation generating a large number of free radicals, which disrupt the structure and function of biomolecules, increase alveolar-capillary membrane permeability, stimulate fibroblast proliferation and collagen secretion, ultimately leading to pulmonary fibrosis; at the same time, abnormal activation of the transforming growth factor-β (TGF-β) / Smad signaling pathway and the NF-κB signaling pathway, and the release of large amounts of inflammatory factors such as IL-8, TNF-α, and IL-6, further exacerbating the inflammatory response and fibrotic process.

[0004] Currently, clinical methods for the prevention and treatment of radiation-induced lung injury are relatively limited. Commonly used drugs mainly include glucocorticoids, free radical scavengers, and anticoagulants. However, these drugs have problems such as limited efficacy, significant side effects, and narrow applicability. For example, although glucocorticoids can relieve inflammation in the short term, long-term use can easily lead to adverse reactions such as infection, osteoporosis, and elevated blood sugar, and cannot effectively delay the progression of pulmonary fibrosis. Free radical scavengers (such as reduced glutathione) can only specifically remove free radicals, and their intervention on the regulation of inflammatory factors and the fibrosis process is weak, resulting in poor overall efficacy.

[0005] Based on the above, it is essential to obtain a drug that can address the limitations of single-drug efficacy, the inability to fully intervene in the pathogenesis of radiation-induced lung injury, and the significant side effects. Summary of the Invention

[0006] The purpose of this invention is to provide the application of fluvastatin combined with tanshinone in the preparation of formulations for the prevention and / or treatment of radiation-induced lung injury, effectively solving the problems of limited efficacy, significant side effects, and inability of a single drug to comprehensively intervene in the disease process of existing radiation-induced lung injury prevention and treatment drugs.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The present invention provides a pharmaceutical composition in which the active ingredients include fluvastatin and tanshinone.

[0008] Preferably, the tanshinone is any one or more of tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone.

[0009] Preferably, the mass ratio of fluvastatin to tanshinone is 1:(0.5-4).

[0010] This invention provides the use of the pharmaceutical composition in the preparation of formulations for the prevention and / or treatment of radiation-induced lung injury.

[0011] Preferably, the effective dose of fluvastatin in the pharmaceutical composition is 5-30 mg / kg / day, and the effective dose of tanshinone is 10-50 mg / kg / day.

[0012] Preferably, the radiation-induced lung injury includes radiation-induced pneumonia or radiation-induced pulmonary fibrosis.

[0013] The present invention also provides an formulation for the prevention and / or treatment of radiation-induced lung injury, comprising the pharmaceutical composition and pharmaceutically acceptable excipients.

[0014] Preferably, the dosage form of the preparation includes tablets, capsules, granules, suspensions, injections, or lyophilized powder for injection.

[0015] Preferably, the excipients are selected from any one or more of diluents, disintegrants, binders, lubricants, solubilizers, preservatives, suspending agents, and flavoring agents.

[0016] By adopting the above technical solution, the present invention has the following beneficial effects: This invention is the first to combine fluvastatin and tanshinone as active ingredients, which can produce a significant synergistic effect, intervening in the pathogenesis of radiation-induced lung injury from multiple targets and pathways. Fluvastatin mainly exerts its anti-inflammatory and anti-fibrotic effects by inhibiting the expression of inflammatory factors such as IL-8, protecting vascular endothelial cells, and reducing collagen fiber proliferation. Tanshinone mainly exerts its anti-inflammatory and anti-fibrotic effects by inhibiting TGF-β1 expression, regulating the NF-κB signaling pathway, scavenging free radicals, and promoting matrix metalloproteinase expression. The two complement each other and work synergistically to significantly improve the prevention and treatment effects, not only effectively relieving the inflammatory symptoms of radiation pneumonitis (such as low-grade fever, cough, and dyspnea), but also significantly delaying the progression of radiation-induced pulmonary fibrosis and improving patients' lung function.

[0017] The pharmaceutical composition of the present invention uses fluvastatin and tanshinone as active ingredients. When used in combination, the efficacy can be guaranteed while reducing the dosage of a single drug, thereby reducing the side effects caused by high-dose use of a single drug. The preparation made from this pharmaceutical composition has good safety, is well tolerated by patients, and is suitable for long-term use, especially for patients with radiation-induced pulmonary fibrosis who require long-term intervention.

[0018] The pharmaceutical compositions and formulations of the present invention can be used to prevent and treat radiation-induced lung injury caused by radiotherapy for various thoracic tumors (lung cancer, esophageal cancer, breast cancer, malignant lymphoma, etc.), covering two stages: radiation pneumonitis and radiation pulmonary fibrosis. Appropriate formulation types and dosages can be selected according to the patient's condition, and the applicable population is wide. Attached Figure Description

[0019] Figure 1 The effect of different concentrations of treatment groups on the survival rate of RLE-6TN cells in Experiment Example 1; Figure 2 HE staining results for different treatment groups in Experiment Example 2; Figure 3 Masson staining results for different treatment groups in Experiment Example 2; Figure 4 The survival rates of mice in different treatment groups in Experiment 3 are shown. Detailed Implementation

[0020] The present invention provides a pharmaceutical composition in which the active ingredients include fluvastatin and tanshinone.

[0021] In this invention, the tanshinone is any one or more of tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone, and is further preferably tanshinone IIA and / or cryptotanshinone. The tanshinone monomers of this invention exhibit stronger anti-fibrotic and anti-inflammatory activities, and a more significant synergistic effect with fluvastatin, enabling more efficient intervention in the inflammatory and fibrotic processes of radiation-induced lung injury.

[0022] In this invention, the mass ratio of fluvastatin to tanshinone is preferably 1:(0.5-4), more preferably 1:(1-3), and even more preferably 1:2.

[0023] This invention provides the use of the pharmaceutical composition in the preparation of formulations for the prevention and / or treatment of radiation-induced lung injury.

[0024] In this invention, the application is in the preparation of drugs for the prevention and / or treatment of radiation-induced lung injury during radiotherapy for thoracic tumors, wherein the thoracic tumors include one or more of lung cancer, esophageal cancer, breast cancer, and malignant lymphoma.

[0025] In this invention, the effective dose of fluvastatin in the pharmaceutical composition is preferably 5-30 mg / kg / d, more preferably 8-25 mg / kg / d, and even more preferably 10 mg / kg / d; the effective dose of tanshinone is preferably 10-50 mg / kg / d, more preferably 15-30 mg / kg / d, and even more preferably 20 mg / kg / d.

[0026] In this invention, the radiation-induced lung injury includes radiation-induced pneumonia or radiation-induced pulmonary fibrosis.

[0027] The present invention also provides an formulation for the prevention and / or treatment of radiation-induced lung injury, comprising the pharmaceutical composition and pharmaceutically acceptable excipients.

[0028] In this invention, the dosage form of the preparation includes tablets, capsules, granules, suspensions, injections, or lyophilized powder injections.

[0029] In this invention, the excipients are selected from any one or more of diluents, disintegrants, binders, lubricants, solubilizers, preservatives, suspending agents, and flavoring agents.

[0030] In this invention, the diluent may be starch, lactose, mannitol, or microcrystalline cellulose; the disintegrant may be sodium carboxymethyl starch, crospovidone, or low-substituted hydroxypropyl cellulose; the binder may be hydroxypropyl methylcellulose, povidone, or gum arabic; the lubricant may be magnesium stearate, talc, or sodium stearate fumarate; the solubilizer may be polyethylene glycol, propylene glycol, or Tween-80; the preservative may be sodium benzoate, potassium sorbate, or parabens; the suspending agent may be sodium carboxymethyl cellulose, sodium alginate, or xanthan gum; and the flavoring agent may be sucrose, steviol glycosides, or lemon flavoring.

[0031] 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.

[0032] In this invention, the fluvastatin was purchased from Sigma-Aldrich, USA; and the tanshinone IIA was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0033] Example 1

[0034] A pharmaceutical composition wherein the active ingredients are fluvastatin and tanshinone IIA in a mass ratio of 1:2.

[0035] Example 2

[0036] A pharmaceutical composition wherein the active ingredients are fluvastatin and cryptotanshinone in a mass ratio of 1:1.

[0037] Example 3

[0038] A pharmaceutical composition wherein the active ingredients are fluvastatin and tanshinone IIA in a mass ratio of 1:1.5.

[0039] Example 4

[0040] Fluvastatin combined with tanshinone injection (containing the pharmaceutical composition described in Example 1) consists of: 10 mg fluvastatin, 20 mg tanshinone IIA, 1 ml polyethylene glycol 400, 0.5 ml propylene glycol, and purified water to 10 ml.

[0041] The preparation method of the above-mentioned fluvastatin combined with tanshinone injection is as follows: (1) Mix polyethylene glycol 400 and propylene glycol evenly, then add fluvastatin and cryptotanshinone, and stir until completely dissolved; (2) Add an appropriate amount of purified water to the above solution, stir well, and adjust the pH value to 6.5~7.5; (3) Add 0.1% activated carbon, stir for 30 min, filter, and filter the filtrate through a microporous membrane (0.22 μm) to remove bacteria; (4) Dispense into ampoules and sterilize to obtain fluvastatin combined with tanshinone injection.

[0042] Example 5

[0043] Fluvastatin combined with tanshinone granules (containing the pharmaceutical composition described in Example 1) consists of the following components: 5 mg fluvastatin, 10 mg tanshinone IIA, 2 g lactose, 2 g mannitol, 0.3 g sodium carboxymethyl cellulose, 0.1 g steviol glycosides, and an appropriate amount of purified water.

[0044] The preparation method of the above-mentioned fluvastatin combined with tanshinone granules is as follows: (1) Take fluvastatin and tanshinone IIA, crush them, pass them through a 100-mesh sieve, and mix them evenly; (2) Add lactose, mannitol, sodium carboxymethyl cellulose and steviol glycosides to the above mixed powder, mix evenly, add purified water and then place in a granulator to granulate, dry and granulate; (3) Dispense into aluminum foil bags to obtain fluvastatin combined with tanshinone granules.

[0045] Example 6

[0046] Fluvastatin combined with tanshinone injection (containing the pharmaceutical composition described in Example 3) consists of: fluvastatin 20 mg, tanshinone IIA 30 mg, polyethylene glycol 400 1 ml, propylene glycol 0.5 ml, and purified water to 10 ml.

[0047] The preparation method of the above-mentioned fluvastatin combined with tanshinone injection is as follows: (1) Mix polyethylene glycol 400 and propylene glycol evenly, then add fluvastatin and cryptotanshinone, and stir until completely dissolved; (2) Add an appropriate amount of purified water to the above solution, stir well, and adjust the pH value to 6.5~7.5; (3) Add 0.1% activated carbon, stir for 30 min, filter, and filter the filtrate through a microporous membrane (0.22 μm) to remove bacteria; (4) Dispense into ampoules and sterilize to obtain fluvastatin combined with tanshinone injection.

[0048] Test Example 1 Safety Test

[0049] (a) In vitro cytotoxicity test

[0050] RLE-6TN cell line (purchased from Hefei Wanwu Biotechnology Co., Ltd., catalog number: Delf-16735) was seeded in DMFM / F12 medium containing 10% fetal bovine serum and cultured at 37℃ and 5% CO2 until the logarithmic growth phase. Then, it was seeded into 96-well plates and divided into a fluvastatin group, a tanshinone IIA group, and a control group. After culturing for another 24 hours, fluvastatin was added to the wells of the fluvastatin group to final concentrations of 5 μmol / L, 10 μmol / L, and 20 μmol / L, respectively. Tanshinone IIA was added to the wells of the tanshinone IIA group at concentrations of 10 μmol / L, 20 μmol / L, 40 μmol / L, and 80 μmol / L, respectively, to achieve final concentrations of 10 μmol / L, 20 μmol / L, 40 μmol / L, and 80 μmol / L, respectively. No reagent was added to the control group. After culturing for 48 h, CCK-8 reagent (purchased from Dojin Chemical Research Institute, Japan) was added to each well according to the instructions, and the wells were incubated for 2 h. The OD values ​​of each well were then measured using a microplate reader. 450nm The values ​​were then calculated to determine cell viability.

[0051] Cell viability (%) = (fluvastatin OD)450nm Or tanshinone IIA OD 450nm ) / Control group OD 450nm ×100%

[0052] The results are as follows Figure 1 As shown, fluvastatin with a final concentration ≤10 μmol / L or tanshinone IIA with a final concentration ≤20 μmol / L did not inhibit the growth and proliferation of the RLE-6TN cell line.

[0053] (ii) Long-term toxicity test

[0054] Sixty female C57BL / 6 mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged 6-8 weeks and weighing 18-22g were randomly selected and fed for 1 week. They were then randomly divided into three groups of 20 mice each: a blank control group, a fluvastatin combined with tanshinone conventional dose group (fluvastatin 10mg / kg / d + tanshinone IIA 20mg / kg / d), and a fluvastatin combined with tanshinone high dose group (fluvastatin 30mg / kg / d + tanshinone IIA 60mg / kg / d).

[0055] The blank control group was administered an equal volume of normal saline by gavage; the combined conventional dose group was given fluvastatin 10 mg / kg / d + tanshinone IIA 20 mg / kg / d by gavage; the combined high dose group was given fluvastatin 30 mg / kg / d + tanshinone IIA 60 mg / kg / d by gavage; once daily for 90 consecutive days, with an administration volume of 1 ml / 100 g body weight.

[0056] Mice were observed daily for their mental state, appetite, activity, and excretion, and weighed weekly. Results showed that no mice died in any of the three groups, their mental state was good, their appetite, activity, and excretion were normal, and their weight gain trends were consistent, with no significant differences between groups. P >0.05), indicating that long-term administration had no significant effect on the growth and development of mice.

[0057] Ninety days after drug administration, all mice were sacrificed, and organs such as the heart, liver, spleen, lungs, kidneys, stomach, and small intestine were dissected, weighed, and their coefficients calculated. After HE staining, the pathological morphology was observed under a light microscope. The results showed no significant differences in the organ coefficients among the three groups of mice. P >0.05), the organs showed no pathological changes such as edema, hemorrhage, necrosis, or hyperplasia, and the cell morphology was normal, indicating that long-term administration had no significant toxic effects on the major organs of mice.

[0058] In summary, the fluvastatin combined with tanshinone drug composition of the present invention has high cellular safety, low in vivo toxicity, and is safe for long-term use, fully meeting the clinical drug safety requirements for long-term intervention treatment of radiation-induced lung injury.

[0059] Experimental Example 2

[0060] 180 C57BL / 6 mice were divided into 9 groups: blank control group, irradiation group, fluvastatin group, tanshinone group (tanshinone IIA group), low-dose combination group, medium-dose combination group, high-dose combination group, negative control group, and positive control group, with 20 mice in each group.

[0061] One hour before ionizing radiation, mice in the fluvastatin group were injected intraperitoneally with 10 mg / kg fluvastatin, mice in the tanshinone group were injected intraperitoneally with 20 mg / kg tanshinone IIA, mice in the low-dose combination group were injected intraperitoneally with 5 mg / kg fluvastatin + 10 mg / kg tanshinone IIA, mice in the medium-dose combination group were injected intraperitoneally with 10 mg / kg fluvastatin + 20 mg / kg tanshinone IIA, mice in the high-dose combination group were injected intraperitoneally with 30 mg / kg fluvastatin + 60 mg / kg tanshinone IIA, mice in the blank control group were injected intraperitoneally with 0.1 mL of physiological saline, mice in the irradiation-only group and the negative control group were injected intraperitoneally with 0.1 mL of dimethyl sulfoxide, and mice in the positive control group were injected intraperitoneally with 20 mg / kg amifostine.

[0062] Mice in the simple irradiation group, fluvastatin group, tanshinone group, low-dose combination group, medium-dose combination group, high-dose combination group, and positive control group were anesthetized by intraperitoneal injection of 50 mg / kg 1% sodium pentobarbital solution and fixed in a supine position. The lungs of each mouse were subjected to γ-ray ionizing radiation using 60Co as the radiation source, with a radiation dose of 30 Gy and a radiation dose rate of 1 Gy / min. Lead plates were used to shield other parts of the mice to avoid ionizing radiation damage. Mice in the blank control group and negative control group were not subjected to ionizing radiation.

[0063] (a) HE staining

[0064] After the above ionizing radiation treatment was completed, bilateral lungs of mice in each group were harvested on days 7, 14, and 20. The lungs were fixed, embedded in paraffin blocks, sectioned, and then stained with hematoxylin and eosin (HE) and Masson staining.

[0065] The results of HE staining sections show (e.g.) Figure 2In the blank control group, the lung tissue structure of mice was intact, the alveolar walls were slender and elastic, and the alveolar cavities were regular in shape and evenly distributed, without any abnormal changes such as inflammatory infiltration or hemorrhage, similar to the negative control group. Twenty days after ionizing radiation modeling, the lung tissue of the irradiation-only group showed obvious pathological damage, manifested as capillary dilation and congestion, increased vascular permeability leading to extravasation of red blood cells into the alveolar cavities, partial alveolar collapse, atelectasis, or fusion to form bullae, and extensive inflammatory cell infiltration in the lung interstitium. The alveolar walls were significantly thickened, edematous, adhered, and fused in patches. Obvious hemorrhage and fibrinous exudate were observed in some areas of the alveolar cavities. Simultaneously, macrophages that had engulfed large numbers of red blood cells formed brownish-brown refractive particles, indicating successful establishment of the radiation-induced lung injury model. The positive control (amifostine), fluvastatin monotherapy group, and tanshinone IIA monotherapy group showed similar degrees of lung tissue damage, all exhibiting varying degrees of interstitial edema, alveolar wall thickening and fusion, and alveolar congestion. However, the overall degree of inflammation and damage was significantly greater than in the irradiation-only group. The effects were reduced, but the outlines of normal alveolar structures were still discernible, indicating that the three interventions, when used alone, all had a certain protective and alleviating effect on radiation-induced lung injury, and there was no significant difference in the effect of fluvastatin and tanshinone IIA alone. In the low-dose combined treatment group (fluvastatin + tanshinone IIA), mild interstitial edema and local alveolar wall fusion were still observed, but the degree of lung tissue inflammation was lower than that of the irradiation-only group, the positive control group, and the two-drug-only group. The degree of alveolar structure destruction was significantly improved, suggesting that the combination of the two drugs has a certain synergistic effect of anti-radiation and anti-inflammation. In the medium-dose combined treatment group, most alveolar cavities of mice had clear morphology and outlines, alveolar wall thickening was not obvious, interstitial edema was mild, and there was no obvious adhesion and fusion between alveoli. Although it did not recover to the level of the normal control group, the degree of alveolar inflammation and alveolar wall thickness were significantly better than those of the single-drug group and the positive control group, and the pathological manifestations were similar to those of the high-dose combined treatment group. This indicates that the combination of medium- and high-dose fluvastatin and tanshinone IIA has a significant preventive and therapeutic effect on radiation-induced lung injury, and no obvious dose dependence was observed.

[0066] The results of Masson staining show that (e.g.) Figure 3 In the blank control group, the lung tissue of mice showed normal structure and morphology, exhibiting a typical honeycomb structure with uniformly distributed alveolar cavities and thin alveolar septa containing numerous red-stained capillary networks. The lung interstitium contained only a small amount of blue-stained collagen fibers, which stabilized the normal lung structure. The epithelial tissue, smooth muscle layer, and connective tissue of the right bronchioles were clearly defined and structurally intact, without obvious inflammatory infiltration, fibrosis, or pathological damage, exhibiting the morphological characteristics of normal lung tissue. Furthermore, no significant difference in lung tissue staining morphology was observed between the negative control group and the blank control group.

[0067] Six months after radiation intervention, mice in the irradiation-only group showed typical pathological changes of chronic radiation-induced pulmonary fibrosis in their lung tissue. In this group, a large amount of blue-stained collagen fibers abnormally proliferated and deposited disorderly in the lung interstitium, mainly concentrated around blood vessels and alveoli, forming obvious fibrotic lesions. The proliferating interstitial tissue compressed the alveolar structure, leading to disordered alveolar morphology and structural deformation, accompanied by local inflammatory cell infiltration. Combined with staining characteristics, the cell nuclei were blue-black, while the smooth muscle of the blood vessel walls and erythrocytes were red, demonstrating significant characteristic staining. This confirms that long-term exposure to ionizing radiation can induce excessive collagen deposition in the lung interstitium, leading to persistent pulmonary fibrosis.

[0068] After intervention in the fluvastatin monotherapy group, tanshinone monotherapy group, and amifostine positive control group, lung tissue damage in mice was effectively improved. The basic alveolar morphology was preserved in all groups, the honeycomb alveolar cavity structure remained intact without significant collapse, and there was no abnormal thickening of the alveolar septa. The degree of interstitial collagen deposition was significantly reduced, with only a small amount of blue-stained collagen fibers present around the airways and blood vessels, and no widespread fibrotic lesions were observed. Tissue staining was clear, with cytoplasm and smooth muscle appearing red, cell nuclei appearing purplish-blue, and only a small amount of inflammatory cell infiltration observed locally. These results indicate that all three intervention methods can effectively inhibit the process of ionizing radiation-induced interstitial fibrosis, reduce abnormal collagen accumulation, alleviate lung tissue structural damage, and maintain the normal morphology and structural integrity of lung tissue.

[0069] Compared to the irradiation-only group and each single-drug intervention group, fluvastatin combined with tanshinone intervention achieved a superior lung protective effect. Mice in the combined-drug group exhibited well-defined and regular alveolar structures, normal honeycomb alveolar cavity morphology, and no collapse, deformation, or other abnormal changes. Collagen deposition in the pulmonary interstitium was extremely mild, with only trace amounts of blue-stained collagen fibers around blood vessels, and no diffuse fibrotic lesions were formed. Simultaneously, the alveolar septa were thin, with good blood supply and mild inflammatory cell infiltration. Furthermore, as the combined drug dose increased from low to high, the amount of collagen deposition in the lung tissue gradually decreased, and the alveolar structure repair effect gradually improved, exhibiting a significant concentration-dependent protective effect. In conclusion, the combined intervention of the two drugs significantly outperformed single-drug intervention in inhibiting radiation-induced pulmonary fibrosis, more effectively delaying fibrosis progression, repairing radiation-induced lung tissue damage, and maintaining lung tissue morphology and structural stability.

[0070] (ii) Lung wet / dry ratio and vascular endothelial cell apoptosis rate

[0071] After the ionizing radiation treatment was completed, bilateral lungs of mice in each group were harvested on days 7, 14, and 20. The lungs were washed with pre-cooled physiological saline, and the fresh wet weight and dry weight were measured. The wet / dry weight ratio was calculated. The apoptosis rate of vascular endothelial cells in the lungs of each mouse was determined using a Tunel assay kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.). The specific operation was performed according to the Tunel assay kit instructions.

[0072] Wet / Dry density = Fresh wet weight of left lung / Dry weight of left lung after drying

[0073] Table 1. Wet / dry ratio of mouse lungs in each group

[0074] Table 2 Apoptosis rate of vascular endothelial cells in each group of mice

[0075] Experimental data showed that, compared with the blank control group, the irradiation-only group had an increased lung wet / dry ratio and a significantly higher level of pulmonary vascular endothelial cell apoptosis. This result confirms that ionizing radiation can severely damage the normal structure of pulmonary blood vessels and lung tissue in mice, exacerbating pulmonary exudative edema and causing significant radiation-induced lung injury. After intervention with different drugs, the lung wet / dry ratio and vascular endothelial cell apoptosis rate were reduced in mice in the fluvastatin group, tanshinone group, low-dose combination group, medium-dose combination group, high-dose combination group, and positive control group. The reduction in lung wet / dry ratio and vascular endothelial cell apoptosis rate was more significant in the medium-dose combination group and the high-dose combination group. Fluvastatin and tanshinone IIA, administered alone, could effectively alleviate radiation-induced lung injury in mice, reduce pulmonary edema and vascular endothelial cell apoptosis, and their single-drug intervention effects were basically equivalent to those of the clinical positive drug amifostine. Compared with single-drug intervention, the combined use of two drugs can exert a better protective effect against lung injury, and the repair and prevention of radiation-induced lung injury are more significant. At the same time, the experimental results show that there is no significant difference in the protective efficacy between medium and high doses of combined drugs, and the effects are similar.

[0076] (III) Detection of inflammatory factor levels

[0077] Blood samples were taken from each group of mice, and the serum IL-8 and TGF-β1 levels were measured by enzyme-linked immunosorbent assay (ELISA) as disclosed in the literature (Zhou Yanping, Qiu Mingyi, Hu Zuowei, et al. Effects of Sha Shen Mai Dong Tang on plasma IL-6, TNF-α, and TGF-β1 in rats with radiation pneumonia [J]. Chinese Journal of Experimental Traditional Medical Formulae, 2014.).

[0078] Table 3. Serum IL-8 and TGF-β1 levels in each group of mice

[0079] The results showed that the expression levels of TNF-α and TGF-β1 in mice in the irradiation-only group were significantly higher than those in other groups; the expression levels of TNF-α and TGF-β1 in mice in the fluvastatin group, tanshinone group, and positive control group were significantly lower than those in the irradiation-only group, and the differences were not significant compared with the low-dose combination group; however, the expression levels of TNF-α and TGF-β1 in mice in the medium-dose combination group and the high-dose combination group were significantly different from those in the positive control group. This indicates that the fluvastatin combined with tanshinone of the present invention can more effectively inhibit the expression of TGF-β1 and the production and release of TNF-α, thereby alleviating the inflammatory response.

[0080] Experimental Example 3

[0081] C57BL / 6 mice were anesthetized with 50 mg / kg 1% sodium pentobarbital solution via intraperitoneal injection and fixed in a supine position. Local gamma-ray ionizing radiation was applied to the chest of the C57BL / 6 mice using 60Co as the radiation source at a dose of 30 Gy and a radiation rate of 200 cGy / min. Lead plates were used to shield other parts of the C57BL / 6 mice to prevent ionizing radiation damage. Following ionizing radiation, the C57BL / 6 mice exhibited acute radiation-induced pneumonia within 1–2 weeks, and bilateral lung fibrosis symptoms appeared within 3–5 weeks, largely consistent with the course of radiation-induced lung injury in humans, indicating successful establishment of a mouse model of radiation-induced lung injury.

[0082] One hundred mice with the radiation-induced lung injury model constructed above were selected and divided into 5 groups: irradiation group, fluvastatin group, tanshinone group, combination therapy group, and positive control group, with 20 mice in each group. Six hours after ionizing radiation, mice in the fluvastatin group were intraperitoneally injected with 10 mg / kg / day of fluvastatin, mice in the tanshinone group were intraperitoneally injected with 20 mg / kg / day of tanshinone IIA, mice in the combination therapy group were intraperitoneally injected with 10 mg / kg / day of fluvastatin and 20 mg / kg / day of tanshinone IIA, and mice in the positive control group were intraperitoneally injected with 20 mg / kg of amifostine. The corresponding drugs were administered once every 1 day thereafter, while mice in the irradiation group were not given any drugs. The survival rate of mice in each group was recorded within 20 days.

[0083] The results are as follows Figure 4As shown, mice in the irradiation group began to die on day 5 after ionizing radiation, with a survival rate of only 23.33% on day 20; mice in the fluvastatin group and the positive control group died on days 6-13, with survival rates of 36.67% and 38.33% on day 20, respectively; mice in the tanshinone group continued to die on day 15, with a survival rate of 35% on day 20; mice in the combination therapy group only experienced a small number of deaths on days 8-12, with a survival rate of 81.67% on day 20. This indicates that fluvastatin combined with tanshinone can significantly improve the survival rate of mice with radiation-induced lung injury. Compared with amifostine or fluvastatin alone, fluvastatin combined with tanshinone improves the survival rate of mice with radiation-induced lung injury; compared with fluvastatin or tanshinone alone, fluvastatin combined with tanshinone significantly reduces the dosage of both fluvastatin and tanshinone.

[0084] In summary, the combination of fluvastatin and tanshinone in this invention can significantly reduce lung tissue inflammation and collagen fiber deposition in mice with radiation-induced lung injury, and reduce the level of inflammatory factors. Its preventive and therapeutic effects are significantly better than those of fluvastatin alone. The two have a significant synergistic effect, and no obvious side effects have been found, indicating good safety.

[0085] 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. A pharmaceutical composition, characterized by, The active ingredients of the pharmaceutical composition include fluvastatin and tanshinone.

2. The pharmaceutical composition of claim 1, wherein, The tanshinone is any one or more of tanshinone I, tanshinone IIA, dihydrotanshinone, and cryptotanshinone.

3. The pharmaceutical composition according to claim 1, characterized in that, The mass ratio of fluvastatin to tanshinone is 1:(0.5-4).

4. Use of the pharmaceutical composition according to any one of claims 1-3 in the preparation of an agent for the prevention and / or treatment of radiation-induced lung injury.

5. The application according to claim 4, characterized in that, The effective dose of fluvastatin in the pharmaceutical composition is 5-30 mg / kg / day, and the effective dose of tanshinone is 10-50 mg / kg / day.

6. The application according to claim 4, characterized in that, The radiation-induced lung injury includes radiation-induced pneumonia or radiation-induced pulmonary fibrosis.

7. An agent for the prevention and / or treatment of radiation-induced lung injury, characterized in that, It includes the pharmaceutical composition according to any one of claims 1-3 and pharmaceutically acceptable excipients.

8. The formulation according to claim 7, characterized in that, The dosage forms of the preparation include tablets, capsules, granules, suspensions, injections, or lyophilized powder injections.

9. The formulation according to claim 7, characterized in that, The excipients are selected from any one or more of the following: diluents, disintegrants, binders, lubricants, solubilizers, preservatives, suspending agents, and flavoring agents.

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