Application of akermanite in preparation of medicine for treating / preventing acute lung injury

Abstract

The invention discloses an application of akermanite in preparation of a medicine for treating / preventing acute lung injury, the akermanite is used for treating the acute lung injury, and the akermanite is used in a therapeutically effective dose to realize treatment of the acute lung injury. According to the application of the akermanite in preparation of the medicine for treating / preventing the acute lung injury, provided by the invention, experimental results show that the akermanite leach liquor can influence lung epithelium repair of ALI, effectively relieve the lung injury, repair a respiratory barrier structure by promoting lung epithelium cell regeneration, and finally effectively prevent and treat occurrence and development of ALI / ARDS by improving lung functions.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an application of magnesia chalcedony in preparing a medicine for treating / preventing acute lung injury. Background Art

[0002] Acute lung injury (ALI) is caused by various direct and indirect pathogenic factors that damage alveolar epithelial cells and capillary endothelial cells, which leads to diffuse alveolar damage, followed by diffuse interstitial and alveolar edema and infiltration of inflammatory cells such as neutrophils, and ultimately acute hypoxic respiratory insufficiency. If the disease progresses to an oxygen and oxygen index below 200, it enters the severe stage of acute respiratory distress syndrome (ARDS).

[0003] ALI / ARDS is a common critical respiratory disease in clinical practice. Its main clinical manifestations are difficult-to-correct hypoxemia and respiratory distress, and its incidence is high.

[0004] In the prior art, the treatment of ALI / ARDS is mainly divided into two categories: supportive physical therapy and drug therapy. In terms of drug therapy, a variety of drugs have been used to treat ALI / ARDS, but only some drugs have shown more obvious efficacy in the patient population. Many other therapeutic drugs with good preclinical results, such as statins, β-agonists, nonsteroidal anti-inflammatory drugs, antioxidants, exogenous surfactants, neutrophil elastase inhibitors, anticoagulants, anti-TNF biologics, etc., have failed in clinical trials despite strong pathophysiological principles and preclinical data to prove their effectiveness. Similarly, despite the use of lung protective ventilation, neuromuscular blockers and prone positioning, the mortality rate of this syndrome remains at around 40%. Therefore, new treatments are urgently needed to improve the clinical outcomes of ALI / ARDS. Summary of the invention

[0005] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the main purpose of the present invention is to provide a use of magnesia chalcedony in the preparation of a drug for treating / preventing acute lung injury.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The invention discloses an application of magnesia chalcedony in the preparation of a drug for treating / preventing acute lung injury. The magnesia chalcedony is used for treating acute lung injury. The magnesia chalcedony is used in a therapeutically effective amount to achieve the treatment of acute lung injury.

[0008] In a specific embodiment, the acute lung injury includes but is not limited to LPS-induced lung injury.

[0009] In a specific embodiment, the magnesia chalcedony is a magnesia chalcedony extract, and the volume concentration of the magnesia chalcedony extract is 1 / 64.

[0010] In a specific embodiment, the magnesia feldspar is a liquid medicine.

[0011] In a specific embodiment, the administration route of the magnesia chalcedony is aerosol inhalation.

[0012] In a specific embodiment, the drug further comprises a pharmaceutically acceptable carrier.

[0013] Compared with the prior art, the present invention has at least the following advantages:

[0014] The use of magnesia chalcedony provided by the present invention in the preparation of a drug for treating / preventing acute lung injury. The experimental results of the present invention show that the magnesia chalcedony extract can affect the lung epithelial repair of ALI, effectively reduce lung damage, and repair the respiratory barrier structure by promoting the regeneration of lung epithelial cells, and ultimately effectively prevent and treat the occurrence and development of ALI / ARDS by improving lung function. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0016] Figure 1 The promoting effect of different doses of magnesia chalcedony materials in the examples on the proliferation of primary mouse lung type II epithelial cells (AT2);

[0017] Figure 2 The results of gross pathological observation of the lungs of mice after the treatment of ALI with different concentrations of magnesia feldspar in the examples;

[0018] Figure 3 The HE staining results of the lung tissue of mice after ALI treatment with different concentrations of magnesia feldspar in the example;

[0019] Figure 4 The results of the gross pathological observation of the lungs at different times after ALI treatment by aerosol inhalation of magnesia chalcitonite extract (concentration 1 / 64);

[0020] Figure 5 This is the result of the evaluation of the gross lung injury after the treatment of ALI with magnesia chalcedony extract (concentration 1 / 64). M2: magnesia chalcedony extract;

[0022] Figure 6The results of lung histological changes after ALI was treated with magnesia chalcitonite extract (concentration 1 / 64) 3 and 5 days later;

[0023] Figure 7 The results of immunofluorescence double staining of SPC (lung AT2 cells) and T1α (lung AT1 cells) in the lung tissue of mice in the treatment group in the example;

[0024] Figure 8 The results of the changes in lung function indexes after ALI was treated with magnesia feldspar extract (concentration 1 / 64);

[0025] Fig. 9 The expiratory frequency and minute ventilation of the ventilation indexes in the pulmonary bronchoalveolar lavage fluid sediment smear of each group in the embodiment at 24h, 3, 5 and 7 days after injury;

[0026] Fig.10 The apnea PAU and airway stenosis index of airway obstruction index in the pulmonary bronchoalveolar lavage fluid sediment smear of each group in the embodiment at 24h, 3, 5 and 7 days after injury;

[0027] Fig.11 The maximum inspiratory flow rate and the maximum expiratory flow rate of the conductivity index in the pulmonary bronchoalveolar lavage fluid sediment smear of each group in the embodiment at 24h, 3, 5 and 7 days after injury;

[0028] Fig.12 The tidal volume TNb of the volume index in the pulmonary bronchoalveolar lavage fluid sediment smear of each group in the embodiment at 24h, 3, 5 and 7 days after injury;

[0029] Fig.13 The total number of PMN cells counted in the pulmonary bronchoalveolar lavage fluid sediment smears of each group at 24h, 3, 5 and 7 days after injury in the embodiment;

[0030] Fig.14 The results of the changes in protein content in the bronchoalveolar lavage fluid of the ALI group and the CMS group at 24h, 3, 5 and 7 days after injury in the example;

[0031] Fig.15 The expression changes of IL-6 and TNF-α in lung tissue of ALI group and CMS group 3 days after injury in the example. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments and drawings. However, the following embodiments are only preferred embodiments of the present invention and are not exhaustive.

[0033] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained from commercial channels.

[0034] In the following examples, the magnesia chalcite extract used was prepared by the following method:

[0035] Weigh 10.0 g of magnesia chalcedony powder and add it to 50 ml of serum-free DMEM / F12 culture medium, shake and mix thoroughly, then incubate in a 37°C, 5% carbon dioxide incubator, and extract on a shaker at 120 r / min for 24 hours. Then, centrifuge the suspension at 10,000 r / min for 15 minutes, take the supernatant, and then adjust the pH value to 7.4±0.5 with 0.1 mol / L HCl solution, and then filter with a 0.22 μm microporous filter membrane to obtain a sterile magnesia chalcedony extraction solution, and finally dilute it with serum-free DMEM / F12 culture medium in proportion to a concentration of 1 / 32 and 1 / 64 for in vivo and in vitro experiments.

[0036] Before cell experiments, 10% fetal bovine serum and 1% double antibody were added to the culture medium.

[0037] Example 1: Study on the effect of different concentrations of CMS extract on the proliferation of primary alveolar type 2 cells (AT2) in mouse lung

[0038] 8-10 week old C57BL / 6 male mice weighing 20-25 g were obtained from the Institute of Zoology, Chinese Academy of Sciences, Beijing [License No. SYXK (Beijing) 2007-0004]. The mice were raised for about 1 week to adapt to the new environment and given sufficient feed and drinking water. Mouse lung AT2 cells were obtained by lavage, enzyme digestion, and magnetic bead sorting. They were inoculated into culture bottles and stimulated with different concentrations of CMS extracts. After 24 hours, the cell proliferation was detected using the CCK-8 kit. The results are shown in Figure 2. Figure 1 As shown (*p<0.05, compared with group 0. M2 material: chalcedony-containing mineral extract (CMS)), it can be seen from the figure that the OD value of 1 / 64 CMS extract is significantly higher than that of the control group (0), indicating that 1 / 64 CMS extract has a pro-proliferation effect on AT2 cells.

[0039] Example 2: Study on the effect of CMS extract in treating acute lung injury

[0040] This example tests and evaluates the effect of CMS extract in treating acute lung injury. The specific test method is as follows:

[0041] C57BL / 6 male mice aged 8-10 weeks and weighing 20-25g were selected. The mice were randomly divided into three groups, namely, saline control group (NS group), LPS injury group (ALI group), and CMS material treatment group (CMS group). In the ALI group and CMS group, mice inhaled LPS (O55:B5, sigma) at a concentration of 5mg / ml through a single ultrasonic nebulization, and the nebulization lasted for 30min. The mice in the CMS group were given ultrasonic nebulization inhalation treatment of CMS extract (concentration 1 / 16, concentration 1 / 32 and concentration 1 / 64) 12h and 24h after LPS injury, and each nebulization lasted for 30 minutes. Each animal received 2 treatments. The control group only received 2 saline nebulization inhalations, each lasting 30 minutes. Pulmonary function was measured before the animals were sampled. FinePointe WBP whole body volumetric system (BUXCO Research Systems, Incorporated, Wilmington, NC; Data Sciences International, INC USA) was used to detect the lung function of mice. Samples were taken on the 3rd, 5th and 7th days after LPS injury. The left lung was used for lavage, and the protein concentration, inflammatory cell count and inflammatory cytokine concentration of the lavage fluid were measured. The right lung was routinely paraffin-embedded, sectioned and stained with HE, as well as immunofluorescence staining of SPC (lung AT2 specific marker) and T1α (lung AT1 specific marker). The results were as follows:

[0042] 1) Test of different doses of CMS extract on the gross pathology, injury and lung histology of ALI patients

[0043] The therapeutic effects of different doses of CMS extract on ALI were observed in gross pathology. Figure 2 As shown, from Figure 2 It can be seen that compared with the control group, only the 1 / 64 dose of CMS group had the mildest degree of bilateral lung damage (hemorrhage and congestion) 3 days after ALI. The 1 / 32 and 1 / 16 doses of CMS groups did not show improvement in ALI damage, but instead tended to aggravate the damage. These results suggest that the 1 / 64 dose of CMS material may have a therapeutic effect on ALI. Among them, histological observations such as Figure 3 As shown, from Figure 3 It can be seen that the 1 / 64 dose of CMS material can significantly reduce the infiltration of inflammatory cells in lung tissue and the shedding of lung epithelial cells, as well as reduce the increase in the number of cells in the alveolar cavity. This shows that CMS material can significantly reduce the degree of LPS-induced lung injury.

[0044] Compared with the ALI group, the LPS mice were treated with 1 / 64 concentration of magnesia chalcedony extract. Figure 4 As shown, Figure 4 The results of gross pathological observation of the lungs at different times after ALI treatment by nebulized inhalation of magnesia chalcitonite extract (concentration 1 / 64). As can be seen from the figure, the areas of pulmonary hemorrhage, congestion and consolidation on the 3rd and 5th days after injury were significantly reduced. On the 5th day after injury, the gross observation of the lungs in the CMS group was close to the appearance of normal lungs. These results show that CMS extract is effective in treating acute lung injury in mice and can significantly reduce acute lung injury. In addition, the degree of lung injury was quantitatively analyzed, and the proportion of the injured area of ​​each lung was calculated, that is, the percentage (%) of the gross lung consolidation and / or congestion area in the entire lung area. Image J software was used to distinguish the colors of the injured and normal parts of the lungs, and the proportion of the injured area was calculated. The results are shown in Figure 5 As shown (M2 is the leaching solution of magnesia chalcite), from Figure 5 It can be seen that on the 3rd and 5th days of treatment, the proportion of gross pulmonary consolidation and / or congestion area in the CMS group was significantly reduced, indicating that the CMS extract significantly improved the lung injury condition compared with the ALI group.

[0045] 2) Changes of lung histology after treatment of ALI with CMS extract

[0046] The histological changes of the lungs were observed under a microscope (magnification: HE×1000) on the 3rd and 5th days after the treatment of ALI with the extract of magnesia feldspar (concentration 1 / 64). Figure 6 As shown in the figure, on the third day after lung injury, congestion and a large number of inflammatory cells infiltrated the lung tissues of mice in the ALI group, part of the alveolar septa thickened (indicating pulmonary interstitial edema), and many shed AT1 (pulmonary type I epithelial cells) and some AT2 (pulmonary type II epithelial cells) cells were seen in the alveolar cavity. The number of AT1 and AT2 cells on the alveolar septa decreased significantly, among which AT1 was more obvious. After CMS treatment, the number of inflammatory cells and shed AT1 in the alveolar cavity was significantly reduced, and pulmonary interstitial edema was significantly alleviated. Under the oil microscope (HE staining, ×1000), it can be seen that on the third day after injury in the LPS group, some alveolar structures were significantly destroyed, the number of AT cells shed in the alveolar cavity and the alveolar septum increased, and the number of inflammatory cells increased; while in the CMS treatment group, AT cells shed in the alveolar cavity and the alveolar septum were rare, and the lung histological changes were close to the normal lung tissue structure. The results suggest that CMS treatment can significantly reduce lung inflammation and AT cell damage, thereby effectively alleviating the pathological damage of acute lung injury.

[0047] 3) Effect of SPC and T1α immunofluorescence double staining of lung tissue after ALI treatment with CMS extract

[0048] The lung tissues of the ALI group and the CMS group were subjected to immunofluorescence staining of SPC (AT2, lung type II cells) and T1a (AT1, lung type I cells) using paraffin sections. Figure 7 As shown in the figure, the results show that in the ALI group (upper row), the number of AT2 cells (green mark) in the lung tissue was significantly reduced, while a large number of AT1 cells (red mark) fell off in the alveolar cavity, and the number of DAPI positive (blue, stained nuclei) signals increased, indicating that the number of cells in the lung increased, mainly caused by the increase in inflammatory cells; on the contrary, in the CMS group (lower row), the number of AT2 and AT1 in the lung tissue was relatively large, and the number of AT1 cells located on the alveolar septum increased, the number of cells in the alveolar cavity was significantly reduced, and the number of DAPI positive signals was significantly less than that in the ALI group, indicating that the infiltration of inflammatory cells decreased and the number of AT2 increased significantly. The results suggest that CMS treatment can significantly reduce the inflammatory response of ALI pneumonia, reduce the damage of AT1 and AT2, and have a good protective effect on acute lung injury.

[0049] 4) Changes in lung function indicators after CMS extract treatment of ALI

[0050] After LPS aerosolization, mice were treated with 1 / 64 CMS aerosolization for two times at 12h and 24h, respectively. The lung function indexes of mice in each group were detected by non-invasive means (FinePointe WBP whole body plethysmography system (BUXCU Research Systems, Incorporated, Wilmington, NC; Data Sciences International, INC USA). The results are shown in Figure 8-12 As shown in the figure, the results show that:

[0051] LPS-induced ALI mice have significantly impaired lung function. Respiratory rate f (BPM) and minute ventilation (MVb) are ventilation indicators. After ALI occurs, the entire airway is damaged, inflammatory edema and exudation exist in the airway epithelium, and airway compliance decreases, which leads to a slowing of respiratory rate (BPM) and a decrease in ventilation volume (MVb); apnea (PAU) and airway stenosis index (Penh) are indicators of pulmonary airway obstruction. After ALI occurs, the airway is narrowed and obstructed due to edema, inflammatory response, and tracheal epithelial shedding, resulting in increased PAU and Penh indicators; treatment with magnesia chalcitriol extract has a significant improvement effect on these changes; maximum inspiratory flow rate (PIFb, ml / sec) and maximum expiratory flow rate (PEFb, ml / sec) are conductivity indicators. After ALI, the damage to the upper and lower respiratory tract leads to pulmonary edema and decreased lung compliance, which in turn causes obstruction of inspiration and exhalation, resulting in decreased PIFb and PEFb indicators. Magnesia chalcitriol treatment can significantly improve these two indicators; tidal volume (TVb) is a volume indicator. After ALI, TVb decreases significantly due to respiratory restriction, but CMS treatment of ALI can significantly increase lung TVb. Overall, compared with the control group, ALI mice have significant differences in lung function indicators such as ventilation, airway obstruction, conductivity, and volume (p < 0.05), and magnesium chalcitriol treatment of ALI can significantly improve these lung function indicators ( Figure 8-12 ).

[0052] 5) Changes in protein concentration, inflammatory cell counts, and inflammatory cytokines in bronchoalveolar lavage fluid sediment smears after CMS extract treatment of ALI

[0053] Bronchoalveolar lavage (BALF) of mice: After the animal experiment, bronchoalveolar lavage was performed. The specific method was: lavage with 1 ml of normal saline three times through tracheal intubation. The collected alveolar lavage fluid was centrifuged at 3000 rpm / min and 4°C for 10 minutes; the sediment was smeared and Swiss stained, and the total number of neutrophils (PMN) in 5 fields (×200) was randomly counted; the protein concentration of the supernatant was determined by BCA method and ELISA; finally, the expression levels of inflammatory cytokines TNF-α and IL-6 in lung tissue were detected by Western Blot. After centrifugation of the lung lavage fluid, the sediment was smeared and Swiss stained, and the total number of neutrophils (PMN) was counted in five randomly selected fields. 24 hours after injury, the number of PMN in the lung caused by LPS increased significantly and reached a peak, and it was still significantly higher than the normal control group on the 7th day after injury. In the 1 / 64 magnesia chalcite extract treatment group, except for the 3rd day, the total number of PMN at all other time points was significantly lower than that of the ALI group ( Fig.13 As shown, *P<0.05, **P<0.01, compared with the control group; # P<0.05, ##P < 0.01, compared with the LPS group. C: normal control group, LPS: LPS group, M2: magnesia chalcedony treatment group). This suggests that magnesia chalcedony can exert its anti-inflammatory effect by inhibiting the infiltration of inflammatory cells, thereby alleviating lung injury.

[0054] Except for the 7th day after ALI, the BALF protein concentrations in the ALI group and the CMS treatment group were higher than those in the normal control group; compared with the ALI group, the BALF protein content in the CMS group was significantly reduced at each time point. By the 7th day after injury, the protein concentration basically returned to normal levels, but the protein concentration in the LPS group was still significantly higher than that in the normal control group ( Fig.14 , where *P<0.05, **P<0.01, compared with the control group; # P<0.05, ## P < 0.01, compared with the LPS group. C: normal control group, LPS: LPS group, M2: magnesia chrysogenite treatment group). The increase in protein concentration in BALF indicates lung damage, especially the destruction of the pulmonary blood-gas barrier, which further leads to increased pulmonary vascular permeability and protein exudation. Magnesia chrysogenite treatment can significantly reduce protein exudation in BALF, indicating that CMS materials may reduce inflammatory damage to lung endothelial and alveolar epithelial cells by inhibiting inflammatory response, and may promote alveolar epithelial cell proliferation (fluorescence staining showed that there were more SPC-positive AT2 cells with green fluorescence in lung tissue than in the LPS group), which ultimately helps to repair the damaged alveolar barrier structure. In summary, magnesia chrysogenite has a good therapeutic effect on acute lung injury.

[0055] On the third day after ALI, Western Blot showed that the expression levels of inflammatory cytokines TNF-α and IL-6 in lung tissue increased significantly; after treatment with magnesia chalcopyrite, the levels of TNF-α and IL-6 in lung tissue decreased significantly ( Fig.15 , where **P<0.01, compared with the control group; ## P < 0.01, compared with LPS group. C: normal control group, LPS: LPS group, M2: magnesia chalcedony treatment group), indicating that magnesia chalcedony treatment can effectively reduce the degree of inflammatory response in lung tissue.

[0056] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. The use of magnesia chalcedony in the preparation of a drug for treating / preventing acute lung injury, characterized in that: The magnesia chalcedony is used for treating acute lung injury, and the magnesia chalcedony is used in a therapeutically effective amount to achieve the treatment of acute lung injury.

2. The use according to claim 1, characterized in that: The acute lung injury includes but is not limited to LPS-induced lung injury.

3. The use according to claim 1, characterized in that: The magnesia chalcedony is a magnesia chalcedony leaching solution, and the volume concentration of the magnesia chalcedony leaching solution is 1 / 64.

4. The use according to claim 1, characterized in that: The magnesia chalcedony is a liquid medicine.

5. The use according to claim 4, characterized in that: The administration route of the magnesia chalcedony is atomization inhalation.

6. The use according to claim 5, characterized in that: The drug further includes a pharmaceutically acceptable carrier.

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