NL2032827B1 - Application of mpla in preparing drug for preventing and treating radiation-induced lung injury - Google Patents
Application of mpla in preparing drug for preventing and treating radiation-induced lung injury Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/7024—Esters of saccharides
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The present invention provides an application of MPLA in preparing a drug for preventing and treating radiation-induced lung injury, and belongs to the technical field 5 of medicines. The drug for preventing and treating radiation-induced lung injury prepared from the MPLA of the present invention, in addition to small toxic and side effects, can remarkably alleviate lung inflammation of a mouse with a breast irradiated by a 20Gy 60Coy ray, and alleviate fibrosis induced by radiation and reduce apoptosis of pulmonary epithelial cells after radiation, showing a remarkable curative effect and 10 a wide application prospect.
Description
APPLICATION OF MPLA IN PREPARING DRUG FOR PREVENTING AND
TREATING RADIATION-INDUCED LUNG INJURY
[01] The present invention belongs to the technical field of medicines, and particularly relates to an application of MPLA in preparing a drug for preventing and treating radiation-induced lung injury.
[02] Lung is one of the most sensitive tissues to ionizing radiation. When lung tissues suffer from radiation injury, more surface active materials of type II cells are released, so that alveolar collapse and atelectasis occur. Meanwhile, endothelial substances of blood capillaries seep into alveolar spaces to further lead to hyperemia in alveolar walls and alveolar interstitial edema, resulting in a gas exchange disorder and the like. Then infiltration of inflammatory cells (mainly lymphocytes) follows, thereby forming lymphocytic alveolitis. Lamellar bodies in the cytoplasm of alveolar type II cells are decreased, the cells deform, swell and fall into the alveolar spaces; at the time, it is at an acute radiation pneumonitis stage. Several weeks later, fibroblasts and the type II cells proliferate due to repair effect to injury, and pulmonary interstitial edema is converted into collagenous fibers, collagens are deposited, the blood capillaries are occluded, the alveolar septa are thickened, and it enters into a fibrosis stage gradually to form radiation pulmonary flbrosis. To prevent and treat radiation-induced lung injury, domestic and foreign researches now mainly concentrate on comprehensive treatment by glucocorticoid, antioxidant, cell factors, immunoregulation, stem cells and the like, but ideal preventing and treating drugs have not been found till now.
[03] MPLA (Monophosphoryl lipid A) is a chemical derivative of LPS isolated from salmonella R595, and is a high-efficiency specific TLR4 agonist. Now, it has been widely applied in the field of vaccine research and development as an immunologic adjuvant and has not showed obvious toxic and side effects in clinical tests exceeding 300,000 person-time. In the prior art, there are no related reports on application of MPLA in preventing and treating radiation-induced lung injury.
[04] The present invention is intended to provide an application of a
Monophosphoryl lipid A (MPLA) in preparing a drug for preventing and treating radiation-induced lung injury, which features remarkable curative effect and small toxic and side effects.
[05] In order to solve the technical problem, the present invention provides the following technical solution:
[06] The present invention provides an application of a Monophosphoryl lipid A (MPLA) in preparing a drug for preventing and treating radiation-induced lung injury.
[07] Preferably, the MPLA prevents and treats the radiation-induced lung injury by activating TLR4.
[08] Preferably, the MPLA is intragastrically administrated.
[09] Preferably, an administration dosage of the MPLA is 0.5-1.5 ug/ 0.1 ml DMSO, and the MPLA is administrated 8-14 h before ionizing radiation.
[10] Preferably, radioactive irradiation is performed with a ®®Coy ray.
[11] Compared with the prior art, the present invention has the following beneficial effects:
[12] The MPLA is applied to preventing and treating radiation-induced lung injury for the first time. It can be known from the experimental results of mice that the MPLA administrated 12 h before irradiation at a dosage of 1 ug/0.1 ml DMSO can remarkably alleviate lung inflammation of the mice with a breast irradiated by a 20Gy S°Coy ray, alleviate fibrosis induced by radiation and reduce apoptosis of pulmonary epithelial cells after radiation; it does not cause drug loss to the lung of the mice, has remarkable curative effect and small toxic and side effects, demonstrats unique feature of the
MPLA in protecting ionizing radiation-induced lung injury, and has a wide application prospect.
[13] FIG. 1 shows comparison of HE staining sections of lung tissues of mice in each group with breasts radiated by a 20Gy *°Coy ray.
[14] FIG. 2 shows comparison of HE staining Ashcroft scores of lung tissue sections of mice in each group with breasts radiated by a 20Gy “°Coy ray (** representing P<<0.01).
[15] FIG. 3 shows comparison of Masson staining sections of lung tissues of mice in each group with breasts radiated by a 20Gy ‘0Coy ray.
[16] FIG. 4 shows comparison of Masson staining positive rates of lung tissue sections of mice in each group with breasts radiated by a 20Gy ®’Coy ray (** representing P<<0.01).
[17] FIG. 5 shows comparison of TUNEL staining sections of lung tissues of mice in each group with breasts radiated by a 20Gy ‘0Coy ray.
[18] FIG. 6 shows comparison of TUNEL staining positive rates of lung tissue sections of mice in each group with breasts radiated by a 20Gy ®’Coy ray (** representing P<0.01).
[19] The present invention provides an application of MPLA in preparing a drug for preventing and treating radiation-induced lung injury. The MPLA is a chemical derivative of LPS isolated from salmonella R595, and is a high-efficiency specific
TLR4 agonist.
[20] In the present invention, the MPLA prevents and treats the radiation-induced lung injury by activating TLR4. The TLR4 of the present invention belongs to a pattern recognition molecular receptor family, expressed on surfaces of nonspecific lymphocytes (such as macrophages, neutrophile granulocytes and mastocytes) and T and B lymphocytes mediating specific immune reactions.
[21] In the present invention, the MPLA is intragastrically administrated. An administration dosage of the MPLA of the present invention is preferably 0.5-1.5 ug/0.1 ml DMSO, and more preferably 1 ug/0.1 ml DMSO, and the administration time is preferably 8-14 h before ionizing radiation, and more preferably 12 h before ionizing radiation. The administration dosage of the MPLA of the present invention is an administration dosage for one mouse.
[22] In the present invention, radioactive irradiation is performed with a $%Coy ray.
An irradiation dosage of the 9Coy ray in the present invention is preferably 20 Gy, a dosage rate is preferably 61.75cGy/min, and the irradiated part is a breast.
[23] The technical solution in the present invention will be described clearly and completely below in combination with specific embodiments in the present invention.
[24] Example 1 Construction of a mouse model with radiation-induced lung injury and HE staining for lung tissues
[25] 6-8-week-old male C57BL/6 mice (obtained from Beijing Vital River
Laboratory Animal Technology Co., Ltd.) were selected and divided into four groups randomly: 12 mice in an irradiation group (IR), 12 mice in an 12-h-beforepre-irradiation MPLA administration group (MPLA+IR), 12 mice in a pure
MPLA administration group (MPLA) and 12 mice in a blank control group (NC); the mice were irradiated at breast at a single time with a ‘°Coy of a radiation center (Institute of Military Medicine), with a single dosage being 20 Gy and a dosage rate being 61.75cGy/min. The MPLA (1 pg/mouse in 0.1 ml of dimethyl sulfoxide (DMSQ)) or DMSO (0.1 ml/mouse) ws delivered to corresponding groups by entogastric administration, the MPLA was administrated to the MPLA+IR group and the MPLA group, and DMSO was administrated to the IR group and the NC group.
Mouse feeding: the mice were placed in a cage with daily replaced packing at 25°CH+/-1°C, and sufficient water and food were ensured.
[26] The mice were put to death at different time points after radiation exposure (week 1, week 4 and week 8), lung tissues were taken, fixed and embedded with wax blocks, and subjected to HE staining after being sliced, and Ashcroft scores of HE staining of lung sections were counted. t-test was performed on related data by using a
SPSS25.0 statistical software, and there was remarkable difference if P<0.05.
[27] HE staining steps:
[28] 1. A section dewaxed to water was placed in a hematoxylin aqueous solution and stained for several minutes. 5 [29] 2. The section was separated in color in acid water and ammonia water for several seconds, respectively.
[30] 3. The section was placed in distilled water for a moment after being washed with flowing water for 1 h.
[31] 4. The section was dehydrated in 70% alcohol and 90% alcohol for 10 min.
[32] 5. The section was stained with an alcohol-eosin staining solution for 2-3 min.
[33] 6. The stained section was dehydrated with alcohol and became transparent through xylene.
[34] 7. Canada balsam was dripped onto the transparent section, and the section was sealed with cover glass.
[35] 8 Microscopic examination was performed and image acquisition and analysis are conducted.
[36] As shown in FIG. 1 and FIG. 2, at week 1 after the breast was irradiated by 20
Gy, the lung tissues of the mice in the IR group showed obvious inflammatory changes, vessels of alveolar walls expanded, and inflammatory exudation was visible in alveolar spaces; at week 4 after the breast was irradiated, pneumonic pathological features of the mice were aggravated, collapse of part of pulmonary alveoli was visible, the normal structure of the lung was destroyed, and the alveolar walls were thickened; and at week 8 after the breast was irradiated, the alveolar walls were further thickened and started to show fibrosis. Compared with the irradiation control group, the early inflammatory degree of the MPLA administration irradiation group (MPLA+IR) is lighter, and the degree of later fibrosis was alleviated to some extent.
[37] Example 2 Masson staining for lung tissues of mice
[38] Lung wax blocks of mice in each group in example 1 were taken, and sliced tor Masson staining to determine fibrosis of lung tissues, and the staining positive areas of collagens of the lung were counted. t-test was performed on related data by using a SPSS25.0 statistical software, and there was remarkable difference if P<0.05.
[39] Masson staining steps:
[40] 1. A paraffin section was dewaxed to water: the section was put in xylene I for 20 min, xylene II for 20 min, absolute ethyl alcohol I for 10 min, absolute ethyl alcohol II for 10 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min and 70% alcohol for 5 min successively, and washed with distilled water.
[41] 2. Straining of nuclei with hematoxylin: nuclei were stained with Weigert iron hematoxylin in a Masson staining kit for 5 min, washed with tap water, differentiated with 1% hydrochloric acid alcohol for several seconds, washed with tap water, washed with flowing water for several minutes, and returned to blue.
[42] 3. Ponceau staining: the section was stained with a ponceau acid azaleine solution in the Massion staining kit for 5-10 min and rinsed rapidly with distilled water.
[43] 4. Phosphomolybdic acid treatment: the section was treated with a phosphomolybdic acid aqueous solution in the Masson staining kit for about 3-5 min.
[44] 5. Aniline blue staining: the section was not washed but directly re-stained with an aniline blue solution in the Masson staining kit for 5 min.
[45] 6. Differentiation: the section was treated with 1% ice CH3COOH for 1 min.
[46] 7. Dehydration and sealing: the section was put in 95% alcohol I for 5 min, 95% alcohol II for 5 min, absolute ethyl alcohol I for S min, absolute ethyl alcohol II for 5 min, xylene I for 5 min and xylene II for 5 min successively for being dehydrated until it became transparent, taken out from xylene and aired slightly, and sealed with neutral gum.
[47] 8. Microscopic examination was performed and image acquisition and analysis were conducted.
[48] As shown in FIG. 3 and FIG. 4, a Masson staining result is similar to a HE staining result. At week 1 after the breast was irradiated, the lungs of the mice (IR) in the irradiation group mainly had inflammatory changes; at week 4 after the breast was irradiated, blue stained collagen fibers started to show; and at week 8 after the breast was irradiated, pulmonary fibrosis was aggravated gradually. It is found by counting the collagen staining positive areas that the degree of pulmonary fibrosis of the mice in the MPLA administration plus irradiation group (MPLA+IR) is lighter than that of the pure irradiation group (IR). It indicates that the MPLA alleviates ionizing radiation-induced pulmonary fibrosis.
[49] Example 3 TUNEL staining for lung tissues of mice
[50] Lung wax blocks of mice in each group in example 1 were taken, and sliced for TUNEL staining to determine apoptosis of lung cells, and the number of TUNEL positive cells in the lung cells was counted. t-test was performed on related data by using a SPSS25.0 statistical software, and there was remarkable difference if P<0.05. [S1] TUNEL staining steps:
[52] 1. Dewaxing of section: a paraffin section was dewaxed with xylene I for 5-10 min, dewaxed with xylene II for 5-10 min, washed with absolute ethyl alcohol for 5 min, washed with 90% ethanol for 2 min, washed with 70% ethanol for 2 min and washed with distilled water for 2 min.
[53] 2. Transparency: 100 ul of Proteinase K working solution was dropwise added onto the tissue section to react at 37°C for 30 min. [S4] 3. Confining: the sample section was immersed in a confining liquid and confined at room temperature (15-25°C) for 10 min; and the sample section was immersed in 1#PBS and rinsed three times, 5 min every time.
[55] 4. Preparation of positive section: 100 pl of DNase I reaction solution containing different active units U was prepared, 100 ul of prepared DNase I reaction solution was dropwise added onto the sample section, the sample section was treated at room temperature for 10-30 min, and the positive section was immersed in IxPBS and rinsed three times, 5 min every time.
[56] 5. Ligation: a TdT enzyme reaction solution was prepared, 50 ul of TdT enzyme reaction solution was dropwise added to each sample, the sample was covered with cover glass and put in a warm box, lucifugal reaction was kept at 37°C for 60 min,
and the reacted sample section was immersed in 1xPBS and rinsed three times, 5 min every time. [S7] 6. Labeling: 50 ul of Streptavidin-HRP working solution was dropwise added to each sample, the sample was covered with cover glass and put in the warm box, and lucifugal reaction was kept at 37°C for 30 min.
[58] 7. Developing: the periphery of the sample was sucked dry with absorbent paper, and 50 pl of DAB working solution was dropwise added to each sample, and developing reaction was kept at room temperature for 30 s-5 min; and the developed sample section was immersed in 1::PBS and rinsed three times, 5 min every time.
[59] 8. Re-staining: the section was stained with hematoxylin for 30 s-5 min (observed and determined under microscope), and washed with water three times.
[60] 9. Dehydration and sealing: the section was dehydrated with alcohol gradiently until the section became transparent, transparentized with xylene, and sealed with neutral gum.
[61] 10. Shooting under microscope: apoptosis of myoloid tissues of mice in each group were observed, 5 sights of view were selected randomly for each section, proportions of positive cells in total cells (calculated based on 100 cells) in each sight of view were counted under an optical microscope, and an average value was taken. A calculation formula was apoptosis rate=positive cell number/total cell numberx100%
[62] As shown in FIG. 5 and FIG. 6, in an acute injury phase at week 1 after radiation, apoptosis occurred to a lot of cells of lung tissues of mice in the pure irradiation (IR) group, and the apoptosis level was declined to some extent at week 8.
At week 1 and week 8 after irradiation, the apoptosis level of the MPLA administration plus irradiation (MPLA+IR) group was obviously lower than that of the pure irradiation group, indicating that the MPLA alleviated the apoptosis level of lung cells after irradiation.
[63] The above-mentioned examples are only the preferred embodiments of the present invention. It should be noted that improvements and modifications may be made without departing the principle of the present invention by those of ordinary skill in the art, and such improvements and modifications should also be regarded to fall into the protection scope of the present invention.
Claims (5)
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210762811.XA CN114832003A (en) | 2022-07-01 | 2022-07-01 | Application of MPLA in preparation of medicine for preventing and treating radioactive lung injury |
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| CN107669692A (en) * | 2017-09-07 | 2018-02-09 | 中国人民解放军第二军医大学 | Applications of the MPLA in preparing ionising radiation and causing intestinal tract injury protective agents |
| CN109260211A (en) * | 2018-09-17 | 2019-01-25 | 中国人民解放军第二军医大学 | MPLA causes the application in injury of testis protective agents in preparation ionising radiation |
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| CA3113552A1 (en) * | 2017-09-29 | 2019-04-04 | Jeroen MELIEF | Immunogenic composition for the treatment of cancer |
| WO2022087483A1 (en) * | 2020-10-22 | 2022-04-28 | Wisconsin Alumni Research Foundation | Use of toll-like receptor 4 agonists to treat inflammation and tissue injury |
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| CN102258527A (en) * | 2010-05-28 | 2011-11-30 | 中国医学科学院药物研究所 | Application of stimulating agent CRX-675 of Toll-like receiver in resisting pulmonary fibrosis |
| CN107669692A (en) * | 2017-09-07 | 2018-02-09 | 中国人民解放军第二军医大学 | Applications of the MPLA in preparing ionising radiation and causing intestinal tract injury protective agents |
| CN109260211A (en) * | 2018-09-17 | 2019-01-25 | 中国人民解放军第二军医大学 | MPLA causes the application in injury of testis protective agents in preparation ionising radiation |
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