A nano compound for treating bronchopulmonary dysplasia and its preparation method and application

Abstract

The invention discloses a nano compound for treating bronchopulmonary dysplasia, and a preparation method and application thereof, comprising the following steps: S1: dissolving CuCl2·2H2O and gardenia glycoside in ethanol at a molar ratio of 3-5:1 to obtain a reaction solution 1; S2: adjusting the pH value of the reaction solution 1 in step S1 to 7 to obtain a reaction solution 2 for standby use; S3: reacting the reaction solution 2 in step S2 at a preset temperature for a preset time to obtain a reaction solution 3 for standby use; S4: centrifuging, washing and drying the reaction solution 3 in step S3 to obtain a nano compound. The nano compound prepared by the invention can significantly improve the survival rate and body weight of mice, inhibit inflammation and apoptosis caused by hyperoxia, promote lung development, and improve lung function of mice, indicating that the gardenia glycoside metal chelate has a potential therapeutic effect on BPD.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nano compound for treating bronchopulmonary dysplasia and a preparation method and application thereof. Background Art

[0002] Bronchopulmonary dysplasia (BPD) is a chronic respiratory disease and a lung injury in premature infants caused by mechanical ventilation (barotrauma) and oxygen therapy. The main characteristics of BPD are airway damage, fibrosis, emphysema and inflammation.

[0003] In recent years, with the antenatal application of glucocorticoids, the widespread use of pulmonary surfactant, and the improvement of oxygen therapy technology and neonatal management technology, the survival rate of premature infants has been significantly improved. However, at the same time, the incidence of a new type of BPD, which is characterized by simplified alveolar structure and impaired lung development, has been increasing year by year.

[0004] At present, the onset of BPD may be related to the following factors:

[0005] (1) The groups included in the study are different. For example, the younger the gestational age and the lower the birth weight, the higher the incidence rate;

[0006] (2) Different definitions: the incidence of the need for oxygen supplementation at 36 weeks of corrected gestational age is much lower than that of the need for oxygen supplementation at 28 days after birth;

[0007] (3) Differences in treatment methods, such as whether the oxygen administration method is correct and whether the fluid replacement is excessive.

[0008] Among them, small gestational age, low birth weight, mechanical ventilation, and long-term oxygen therapy are high-risk factors for BPD. Related reports show that the incidence of BPD in extremely low birth weight infants is as high as 25%-35%, which seriously affects the survival rate of premature infants.

[0009] However, there is no exact unified standard for the treatment of BPD. Most of them are aimed at avoiding secondary lung damage, promoting lung damage repair and lung development. Most of them are based on comprehensive treatment strategies such as lung protective ventilation strategy, appropriate oxygen therapy, NO administration, infection control, etc. There are no specific drugs or other treatment methods. Therefore, finding drugs that can effectively treat BPD is the focus and difficulty of BPD treatment. Summary of the invention

[0010] In view of the deficiencies in the prior art, the present invention provides a nano compound for treating bronchopulmonary dysplasia, a preparation method and application thereof, and the nano compound has a good therapeutic effect on bronchopulmonary dysplasia.

[0011] To achieve the above object, the present invention adopts the following technical solution: a method for preparing a nano compound for treating bronchopulmonary dysplasia, comprising the following steps:

[0012] S1: dissolving CuCl2·2H2O and gardenia glycoside in a solvent at a molar ratio of 3-5:1 to obtain a reaction solution 1;

[0013] S2: adjusting the pH value of the reaction solution 1 in step S1 to 7 to obtain a reaction solution 2 for standby use;

[0014] S3: reacting the reaction solution 2 in step S2 at a preset temperature for a preset time to obtain a reaction solution 3 for standby use;

[0015] S4: centrifugally separate, wash and dry the reaction solution in step S3 to obtain a nano compound.

[0016] Furthermore, in step S2, the pH value of the reaction solution 1 is adjusted using sodium carbonate solution.

[0017] Furthermore, in step S4, ethanol washing is used.

[0018] Furthermore, in step S4, the washed solid is vacuum dried.

[0019] Furthermore, in step S1, the solvent includes any one of deionized water or ethanol.

[0020] Furthermore, in step S3, the preset temperature is 25°C or 37°C.

[0021] Furthermore, in step S3, the reaction solution 2 is reacted at a preset temperature for 12-24 hours.

[0022] The present invention also provides a nano compound obtained according to the above preparation method.

[0023] On the other hand, the present invention also provides a use of a nano compound in treating bronchopulmonary dysplasia.

[0024] The invention also provides a nano compound, and the antibacterial gel is obtained by the above-mentioned preparation method.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The invention cross-links inorganic copper and gardenia glycoside to form organic copper, and successfully constructs a gardenia glycoside metal chelate (Cu-GE). The polyphenolic components contained in gardenia glycoside and the synergistic effect of metal ions can form a polyphenol network, which improves the stability and bioavailability of gardenia glycoside, thereby enhancing the biological activity of gardenia glycoside.

[0027] At the same time, through biological experiments, it can be seen that the gardenia glycoside metal chelate prepared by the present invention can significantly improve the survival rate and body weight of mice, inhibit inflammation and apoptosis caused by hyperoxia, promote lung development, and improve lung function of mice, indicating that the gardenia glycoside metal chelate has a potential therapeutic effect on BPD. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing the effects of hyperoxia-induced weight loss on mice under different conditions;

[0029] Figure 2 This is a graph showing the effects of different conditions on the survival rate of mice induced by hyperoxia;

[0030] Figure 3 The results of the effects of different conditions on mouse lung tissue morphology (100×);

[0031] Figure 4 This is the result of TUNEL detection of the effects of different conditions on apoptosis of lung tissue cells in 14-day newborn mice (200×). DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0033] The main reagents and equipment information involved in this application are as follows:

[0034] Main reagents:

[0035]

[0036]

[0037] Main instruments:

[0038] Instrument Name model factory Surgical instruments / Shanghai Surgical Instrument Factory Optical Microscope DM2000 Leica Corporation of Germany Clean bench 1384 Thermo Fisher Scientific, USA Pipette / Eppendorf, Germany 100% medical oxygen / Chongqing Dadukou District Jieda Gas Business Department Oxygen meter CY-12C Jiande Meicheng Electrochemical Analytical Instrument Factory Electronic balance TZC-(B)TSE-3 / 0-1 Fuzhou Kedi Electronic Technology Co., Ltd. Desktop high speed refrigerated centrifuge FRESCO21 ThermoScientific, Inc. Tissue / cell homogenizer DS1000 Hubei Xinzongke Viral Disease Engineering Technology Co., Ltd. Multifunctional ELISA reader 1510 ThermoScientific, Inc. Fluorescence quantitative PCR instrument CFXConnect Bio-Rad Corporation Western blot developer SyngeneGnomeXRQ American Genetics Ltd. Non-invasive animal lung function tester <![CDATA[FinePointe TM MALE]]> Buxco

[0039] The present application provides a method for preparing a nanocompound for treating bronchopulmonary dysplasia, comprising the following steps:

[0040] S1: dissolving CuCl2·2H2O and gardenia glycoside in a solvent at a molar ratio of 3-5:1 to obtain a reaction solution 1;

[0041] S2: adjusting the pH value of the reaction solution 1 in step S1 to 7 to obtain a reaction solution 2 for standby use;

[0042] S3: reacting the reaction solution 2 in step S2 at a preset temperature for a preset time to obtain a reaction solution 3 for standby use;

[0043] S4: centrifugally separate, wash and dry the reaction solution in step S3 to obtain a nano compound.

[0044] Furthermore, in step S2, the pH value of the reaction solution 1 is adjusted using sodium carbonate solution.

[0045] Furthermore, in step S4, ethanol washing is used.

[0046] Furthermore, in step S4, the washed solid is vacuum dried.

[0047] Furthermore, in step S1, the solvent includes any one of deionized water or ethanol.

[0048] Furthermore, in step S3, the preset temperature is 25°C or 37°C.

[0049] Furthermore, in step S3, the reaction solution 2 is reacted at a preset temperature for 12-24 hours.

[0050] Example 1

[0051] The present application provides a nano compound 1, which is prepared by the following method:

[0052] CuCl2·2H2O and GE (Gardenia glycoside) were dissolved in ethanol (100% ethanol solution) at a molar ratio of 4:1, and the mixture was stirred until the solid was completely dissolved.

[0053] The homogeneous solution was adjusted to pH 7.0 with carefully dripped sodium carbonate solution and further reacted at 37° C. for 24 h. Then, the complex in the mixture was collected by centrifugation, washed several times with ethanol (100% ethanol solution), and vacuum dried to obtain the target product.

[0054] Example 2

[0055] The present application provides a nanocompound 2, which is prepared by the following method:

[0056] CuCl2·2H2O and GE were dissolved simultaneously in deionized water at a molar ratio of 3:1, and the mixture was stirred until the solid was completely dissolved.

[0057] The homogeneous solution was adjusted to pH 7.0 with carefully dripped sodium carbonate solution and further reacted for 18 h at 25 ° C. Then, the complex in the mixture was collected by centrifugation, washed with ethanol several times, and vacuum dried to obtain the target product.

[0058] Example 3

[0059] The present application provides a nano compound 3, which is prepared by the following method:

[0060] Dissolve CuCl2·2H2O and GE in ethanol at a molar ratio of 5:1, and stir the mixture until the solid is completely dissolved.

[0061] Adjust the homogeneous solution to pH 7.0 with a carefully dropped sodium carbonate solution, and further react at 37 °C for 12 h. Then, collect the complex in the mixture by centrifugation, wash it several times with ethanol, and obtain the target product after vacuum drying.

[0062] Example 4

[0063] To verify the therapeutic effect of the nanocompound prepared by the present invention on BPD, an animal experiment was conducted using the nanocompound in Example 1 as an example, and the nanocompounds in other examples had similar experimental effects to those in Example 1.

[0064] The following animal experiments were carried out using the nanocompound in Example 1 (corresponding to the Cu-GE group) and geniposide (corresponding to the GE group) respectively to verify the application effect of the nanocompound of the present invention in the treatment of bronchopulmonary dysplasia.

[0065] Animal experiment

[0066] I. Experimental procedure

[0067] Twenty-eight SPF-grade C57BL / 6J neonatal mice within 24 h, with an average body weight of 1.27 ± 0.13 g, and 4 naturally delivered female mice were purchased from the Experimental Animal Center of Chongqing Medical University. The experimental animal production license number is SCXK(Yu)2022-0017. This study was approved by the Experimental Animal Welfare and Ethics Committee. The experimental animals were raised and the experimental operations were carried out in the Experimental Animal Center of Chongqing Medical University, at a temperature of 22-26 °C, a relative humidity of 40%-70%, and a 12 / 12 h light / dark alternating illumination, ensuring the free access to food and water for the experimental animals. The experimental animal use license number is SYXK(Yu)2022-0010.

[0068] Twenty-one SPF C57BL / 6J mice within 24 hours after birth were randomly divided into air group, hyperoxia + PBS group (i.e., BPD group), hyperoxia + GE group (Gardenia glycoside group), and hyperoxia + Cu-GE group (Nanocompound 1 group). There were 7 mice in each group. The mice in the hyperoxia + PBS group, hyperoxia + GE group, and oxygen + Cu-GE group were placed in a self-made closed oxygen box (40cm×30cm×25cm), and 100% oxygen was continuously and slowly infused to maintain FIO2 at about 85% (monitored by an oxygen meter). The mother mice in the air group and the hyperoxia group were exchanged every day to prevent the mother mice from being poisoned by continuous inhalation of high-concentration oxygen, which would reduce their feeding ability. Soda lime was laid on the bottom of the oxygen box to absorb CO2, and the CO2 concentration in the oxygen box was maintained at <0.5%, the relative humidity was 40%-70%, and the temperature was 22-26℃. The air group was placed in the same room. The box was opened for 1 hour every day to add water and feed, record the weight of the mice, change the bedding, and intervene in the hyperoxia group. Hyperoxia + GE group and oxygen + Cu-GE group: GE equivalent concentration of 10 mg / ml, 5 μl / g was intraperitoneally injected 30 minutes before the first hyperoxia exposure, and then injected once a day for 13 days. The hyperoxia + PBS group and the air group were injected with an equal volume of PBS solution. The left lung of the newborn mice at 14 days after birth was fixed in 4% paraformaldehyde, and the right lung was stored in a -80℃ refrigerator for later use.

[0069] 2. Experimental Results

[0070] (1) Effects of Cu-GE on body weight and mortality of BPD mice induced by hyperoxia

[0071] pass Figure 1 , Figure 2 The data showed that the mice in the air group were in good condition, and their weight gradually increased with age. Compared with the GE group and the Cu-GE group, the growth of mice in the BPD group was improved, and their weight increased significantly on the 14th day (P<0.05), indicating that GE and Cu-GE have a certain therapeutic effect on bronchopulmonary dysplasia.

[0072] Combination Figure 1 , Figure 2 From the data in , it can be seen that the growth of the Cu-GE group was better than that of the GE group mice, indicating that the drug treatment of Cu-GE is more effective in treating BPD than GE alone.

[0073] Combined with the diagram, the survival rate of mice in the BPD group was about 40%, the survival rate of mice in the GE group was 57%, and the survival rate of mice in the Cu-GE group was significantly increased to 71%, indicating that Cu-GE and GE both have potential therapeutic effects on BPD and can improve the survival rate. Combining the survival rate of mice in the Cu-GE group and the survival rate of mice in the GE group, it can be explained that since metal Cu is introduced into the nanocompound of the present application, a metal coordination compound (Cu-GE) formed by metal Cu and GE is constructed, which can significantly improve the stability of GE, thereby enhancing the biological activity of GE, so that the survival rate of mice in the Cu-GE group is greater than that of mice in the GE group.

[0074] (2) Effects of Cu-GE on lung tissue morphology of BPD mice induced by hyperoxia

[0075] pass Figure 2 The results showed that at 14 days, the alveolar structure of mice in the air group was intact and regularly arranged, with clear secondary ridges, thinning alveolar septa and uniform size; the lung tissue of the BPD group was congested, edematous, and inflammatory infiltrated, with obvious expansion of the alveolar cavity, disordered lung tissue structure, and simplified alveolar structure; compared with the BPD group, the secondary ridges of the lung tissue in the Cu-GE group increased, and the alveolar structure tended to be normal ( Figure 3 A).

[0076] RAC is the radial alveolar count, which is an indicator for evaluating the number of alveoli. At 14d1, the RAC value of the BPD group was significantly lower than that of the air group (P<0.01). After Cu-GE was given, the RAC value increased significantly (P<0.01). The results showed that Cu-GE significantly increased the number of alveoli under hyperoxia ( Figure 3 B).

[0077] MLI is the mean alveolar intercept, which is used to evaluate the size of the alveolar inner diameter. At 14 days, the MLI value of the BPD group was significantly higher than that of the air group (P<0.01). After the administration of Cu-GE, the MLI value was significantly reduced (P<0.01), indicating that Cu-GE can promote the development of the lung under hyperoxia ( Figure 3 C).

[0078] (3) TUNEL detection of the effect of Cu-GE on apoptosis of lung tissue cells

[0079] pass Figure 4 The data show that: TUNEL staining results showed that there was no significant apoptosis in the air group, hyperoxia induced an increase in alveolar epithelial cell apoptosis, and after Cu-GE intervention, alveolar epithelial cell apoptosis decreased. It can be seen that Cu-GE has a significant inhibitory effect on alveolar epithelial cell apoptosis when treated with inflammatory factors in vitro.

[0080] In summary, compared with the simple drug (GE) treatment, there are shortcomings such as short biological half-life, which leads to systemic toxicity and low biological effect caused by multiple administration. The metal chelate of gardenia glycoside constructed by the present invention can effectively improve the stability of GE, improve the stability and bioavailability of GE. Compared with the simple drug group (GE group), Cu-GE can significantly improve the survival rate and body weight of mice, inhibit inflammation and apoptosis caused by hyperoxia, promote lung development, and improve the lung function of mice, indicating that Cu-GE has a potential therapeutic effect on BPD.

[0081] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A method for preparing a nanocompound for treating bronchopulmonary dysplasia, characterized in that: The steps include: S1: dissolving CuCl2·2H2O and gardenia glycoside in a solvent at a molar ratio of 3-5:1 to obtain a reaction solution 1; S2: adjusting the pH value of the reaction solution 1 in step S1 to 7 to obtain a reaction solution 2 for standby use; S3: reacting the reaction solution 2 in step S2 at a preset temperature for a preset time to obtain a reaction solution 3 for standby use; S4: centrifugally separate, wash and dry the reaction solution in step S3 to obtain a nano compound.

2. The preparation method according to claim 1, characterized in that: In step S2, the pH value of the reaction solution 1 is adjusted using a sodium carbonate solution.

3. The preparation method according to claim 1, characterized in that: In step S4, ethanol is used for washing.

4. The preparation method according to claim 1, characterized in that: In step S4, the washed solid is vacuum dried.

5. The preparation method according to claim 1, characterized in that: In step S1, the solvent includes any one of deionized water or ethanol.

6. The preparation method according to claim 1, characterized in that: In step S3, the preset temperature is 25°C or 37°C.

7. The preparation method according to claim 1, characterized in that: In step S3, the reaction solution 2 is reacted at a preset temperature for 12-24 hours.

8. A nano compound, characterized in that: Obtained according to the preparation method according to any one of claims 1 to 7.

9. Use of the nanocompound according to claim 8 in the preparation of a drug for treating bronchopulmonary dysplasia.

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