Application of procyanidine B2 in preparation of product for relieving animal lung injury caused by chronic cold exposure

Abstract

The invention relates to the technical field of biological medicine, in particular to application of procyanidine B2 in preparation of a product for relieving animal lung injury caused by chronic cold exposure. The procyanidine B2 is found to be capable of relieving oxidative stress injury of the lung and enhancing the oxidation resistance of the lung. Technical support is provided for application of procyanidine B2 as a natural lung protection medicine under the condition of chronic cold exposure of animals, and a new thought is provided for comprehensive treatment of animal lung injury caused by chronic cold exposure.

Description

TECHNICAL FIELD

[0001] The application relates to the field of biological medicine, in particular to application of procyanidin B2 in preparation of products for relieving lung injury of animals caused by chronic cold exposure. BACKGROUND

[0002] Cold is one of the common stressors in the north. Animals in cold regions are affected in different degrees in growth, immune performance and reproductive capacity due to long-term or repeated exposure to cold stimulation environment lower than their thermal neutral zone, which is an important factor restricting the development of animal husbandry, especially in cold regions or seasonal low-temperature environment. According to the duration and intensity of cold exposure, continuous cold exposure for less than 1 day is called acute cold exposure, and more than 1 day is called chronic cold exposure. Cold can accelerate the heat loss of mammals. In order to resist cold, the body will decompose nutrients to provide heat, and the essence is oxidation reaction. Excessive oxidation reaction will cause imbalance between oxidation and antioxidant in the body, causing oxidative stress and endangering animal health. After cold exposure, studies have shown that the energy metabolism level in the liver, heart and skeletal muscle of animals increases, and the total antioxidant capacity decreases, indicating that the enhancement of oxidation reaction is necessary for the survival of animals in low-temperature environment, but it will cause oxidative damage to tissues.

[0003] The lung is the most important organ of the respiratory system, and the gas exchange with the outside world is mainly realized through alveoli. Compared with other parts, the direct contact of alveoli with cold air makes the temperature loss more severe, and it is more likely to cause oxidative stress reaction in the lung, inducing acute and progressive damage to the lung. With the development of the field of biological medicine, people pay more and more attention to the safety of drugs, and the development and utilization of natural additives have become the general trend of the development and use of additives.

[0004] In summary, how to find a new lung protective agent to relieve oxidative damage to the lung of animals in cold regions, especially active ingredients of natural origin, has important scientific value and clinical application prospect, and is also a difficult problem to be broken through by those skilled in the art. SUMMARY

[0005] In order to solve the above problems, the application provides application of procyanidin B2 in preparation of products for relieving lung injury of animals caused by chronic cold exposure. The application finds that procyanidin B2 (PB2) can relieve oxidative stress injury of the lung and enhance the antioxidant capacity of the lung, thereby providing technical support for comprehensive treatment of multiple lung injuries caused by chronic cold exposure.

[0006] In order to achieve the above purpose, the application provides the following technical scheme: The application provides application of procyanidin B2 in preparation of a product for relieving lung injury of an animal caused by chronic cold exposure.

[0007] Preferably, the relieving lung injury of the animal caused by chronic cold exposure comprises relieving oxidative stress injury of the lung of the animal and / or enhancing the antioxidant capacity of the lung of the animal.

[0008] Preferably, the relieving oxidative stress injury of the lung of the animal comprises one or more of the following: reducing the level of active oxygen in the lung, reducing the content of malondialdehyde (MDA) in the lung, reducing the expression level of heat shock protein 60 (HSP60) in the lung, reducing the expression level of heat shock protein 70 (HSP70) in the lung and reducing the expression level of heat shock protein 90 (HSP90) in the lung.

[0009] Preferably, the enhancing the antioxidant capacity of the lung of the animal comprises one or more of the following: improving the activity of glutathione (GSH) in the lung, up-regulating the expression level of superoxide dismutase 1 (SOD-1) in the lung, down-regulating the expression level of catalase (CAT) in the lung and down-regulating the expression level of heme oxygenase 1 (HO-1) in the lung.

[0010] Preferably, the animal is a mouse.

[0011] Preferably, the product comprises a drug or a feed additive.

[0012] The application provides a drug for relieving lung injury of an animal caused by chronic cold exposure, wherein the effective component comprises procyanidin B2; and the unit dose of the drug is 45-105 mg / kg body weight of the mouse.

[0013] Preferably, the unit dose of the drug is 49-98 mg / kg body weight.

[0014] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0015] Beneficial effects: This invention discovers that proanthocyanidin B2 can alleviate oxidative stress damage to the lungs of animals and enhance their antioxidant capacity. Specifically, it includes: reducing the level of reactive oxygen species in the lungs; reducing the content of malondialdehyde in the lungs; reducing the expression levels of heat shock protein 60, 70, and 90 in the lungs; increasing the activity of glutathione in the lungs; upregulating the expression level of superoxide dismutase 1 in the lungs; downregulating the expression levels of catalase and heme oxygenase 1 in the lungs. This invention provides technical support for the application of proanthocyanidin B2 as a natural lung-protective agent in animals under chronic cold exposure and offers a new approach to the comprehensive treatment of lung damage caused by chronic cold exposure. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 The structural formula for PB2; Figure 2 HE staining results of lung tissue from mice subjected to chronic cold exposure; Figure 3 The results show the effects of different doses of PB2 on the safety of mouse lungs; Figure 4 The results show the effect of PB2 on pathological damage to lung tissue in mice caused by chronic cold exposure. Figure 5 The results show the effects of chronic cold exposure on ROS, MDA content and GSH activity in mouse lungs. Figure 6 The results show the effect of PB2 on the expression levels of HSP60, HSP70, and HSP90 in the liver of mice with chronic cold exposure. Figure 7 The results show the effects of PB2 on the expression levels of CAT, GST, SOD-1 and HO-1 in the liver of chronically cold-exposed mice. Detailed Implementation

[0018] This invention provides the application of proanthocyanidin B2 in the preparation of products that alleviate lung damage in animals caused by chronic cold exposure.

[0019] In one embodiment, the animal is a mouse. In another embodiment, the product includes a drug or feed additive.

[0020] As one implementation, alleviating lung damage caused by chronic cold exposure in animals includes: alleviating oxidative stress damage in the lungs and / or enhancing the antioxidant capacity of the lungs. As one implementation, alleviating oxidative stress damage in the lungs includes: reducing the level of reactive oxygen species in the lungs, reducing the content of malondialdehyde in the lungs, reducing the expression level of heat shock protein 60 in the lungs, reducing the expression level of heat shock protein 70 in the lungs, and reducing the expression level of heat shock protein 90 in the lungs, or one or more of these. As one implementation, enhancing the antioxidant capacity of the lungs includes: increasing the activity of glutathione in the lungs, upregulating the expression level of superoxide dismutase 1 in the lungs, downregulating the expression level of catalase in the lungs, and downregulating the expression level of heme oxygenase 1 in the lungs, or one or more of these.

[0021] PB2 is an important member of plant polyphenols, belonging to the proanthocyanidin family. It has a dimer structure and is composed of two epicatechin or catechin monomers linked by specific chemical bonds. Figure 1 These natural compounds have advantages such as low price, non-toxicity, and easy accessibility. This invention discovers that proanthocyanidins B2 can alleviate oxidative stress damage to the lungs of animals and enhance their antioxidant capacity. Specifically, this includes: reducing the level of reactive oxygen species in the lungs; reducing the content of malondialdehyde in the lungs; reducing the expression levels of heat shock protein 60, 70, and 90 in the lungs; increasing the activity of glutathione in the lungs; upregulating the expression level of superoxide dismutase 1 in the lungs; downregulating the expression levels of catalase and heme oxygenase 1 in the lungs. This invention provides technical support for the application of proanthocyanidins B2 as a natural lung-protective drug in animals under chronic cold exposure and offers a new approach to the comprehensive treatment of lung damage caused by chronic cold exposure.

[0022] Based on the above advantages, this invention provides a drug for alleviating lung damage caused by chronic cold exposure in animals, the active ingredient of which includes proanthocyanidin B2; the unit dose of the drug, based on mouse body weight, is 45-105 mg / kg body weight. The dosage of proanthocyanidin B2 in the drug of this invention is based on the original dosage of proanthocyanidin B2. The unit dose described in this invention is the safe dose for mice to alleviate lung damage caused by chronic cold exposure, and it is also the unit package dose of the product.

[0023] In one embodiment, the unit dose of the drug is 49-98 mg / kg body weight.

[0024] As one implementation, the drug also includes pharmaceutically acceptable excipients.

[0025] To further illustrate the present invention, the application of proanthocyanidin B2 provided by the present invention in the preparation of products that alleviate lung damage in animals caused by chronic cold exposure is described in detail below with reference to embodiments and accompanying drawings, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0026] Preparation Example The main instruments and reagents used in the embodiments of this invention are as follows: artificial climate chamber (Jinan Xubang, Jinan, China); proanthocyanidin B2 (PB2, purity 98%, Chengdu Ruifensi Biotechnology Co., Ltd., CAS NO: 29106-49-8); hematoxylin staining solution (Beyotime, China); eosin staining solution (Beyotime, China); formaldehyde solution (Beyotime, China); paraformaldehyde (Yongjin, China); ethanol (Beyotime, China); xylene (Beyotime, China); GSH detection kit (Beyotime, China); MDA detection kit (Beyotime, China); DHE fluorescent probe (MCE, HY-D0079); BCA kit (Beyotime, China); SDS-PAGE (Solepro, China).

[0027] Example 1. Establish a mouse model of oxidative lung injury induced by chronic cold exposure and a model of treatment with different concentrations of PB2. The experimental animals were 5-week-old male C57BL / 6 mice weighing 22-24 g (purchased from Charles River Laboratories). They were pre-fed for one week under conditions of 24±2℃, 40% humidity, 12h light / dark cycle, free access to food and sterile water. After the pre-feding period, the mice were randomly divided into 6 groups of 6 mice each: control group, chronic cold exposure group, PB2 (50mg / kg) group, PB2 (100mg / kg) group, PB2 (50mg / kg) + chronic cold exposure group, and PB2 (100mg / kg) + chronic cold exposure group (PB2 administered by gavage). The control group was fed normally at room temperature; the chronic cold exposure group was exposed to 4℃ for 3 hours daily at random times for a total of 3 weeks.

[0028] 2. Effects of chronic cold exposure on pathological damage to lung tissue in mice This invention assesses pathological changes in lung tissue from chronically cold-exposed mice by HE staining and observation under an optical microscope.

[0029] After the chronic cold exposure was completed, mice in both the control and chronic cold exposure groups were removed from the climate chamber. Mice were euthanized by cervical dislocation, and their thoracic cavities were cut open to collect lung tissue. A portion of the collected lung tissue was fixed in a 15 mL centrifuge tube containing 4% paraformaldehyde for subsequent tissue section preparation.

[0030] The specific steps for HE staining are as follows: (1) Fixed Fresh lung tissue from mice in the control group and the chronic cold exposure group was fixed in 4% formaldehyde solution for 24 h.

[0031] (2) Washing and dehydration After fixation, the fixative was rinsed off with running water, and the tissue was then immersed in ethanol with gradually increasing concentration gradients for 2 hours each time.

[0032] (3) Transparent The tissue was soaked in 50% and 100% xylene for 2 hours, respectively. (1) Paraffin impregnation and embedding Melted paraffin is introduced into an embedding cassette containing tissue. After the paraffin cools, a wax block containing tissue is obtained.

[0033] (2) Slicing The paraffin-embedded lung tissue sample was cut into 5 μm thick sections using a microtome.

[0034] (6) Spreading, scooping, and roasting the slices. Float the wax strip on water at 40°C to flatten it. Remove the wax strip and bake at 60°C for 1 hour to remove the paraffin from the tissue.

[0035] (7) Dewaxing and hydration of sections The sections were dewaxed by soaking in xylene. Then they were hydrated sequentially in ethanol at gradually decreasing concentrations, and finally soaked in distilled water.

[0036] (8) Staining Follow the instructions for hematoxylin staining solution and eosin staining solution.

[0037] (9) Sectioning, dehydration, clearing and mounting The sections were soaked in ethanol at gradually increasing concentrations, then soaked in xylene, and finally mounted with neutral resin.

[0038] See results Figure 2 Compared with the control group, the lung tissue structure of the chronic cold exposure group was disordered, such as hemorrhage, neutrophil infiltration and alveolar cell necrosis, which significantly induced pathological changes. It can be seen that chronic cold exposure leads to pathological damage in the lung tissue of mice.

[0039] 3. Effects of different doses of PB2 on the safety of mouse lungs After chronic cold exposure, mice in the control group, PB2 (50 mg / kg) group, and PB2 (100 mg / kg) group were removed from the artificial climate chamber. Mice were euthanized by cervical dislocation, and their thoracic cavities were cut open to collect lung tissue. A portion of the lung tissue was fixed in a 15 mL centrifuge tube containing 4% paraformaldehyde for subsequent tissue sectioning and HE staining; another portion of the lung tissue was homogenized using a ball mill, and the GSH and MDA levels were detected using GSH and MDA detection kits, respectively.

[0040] Detection of lung MDA levels: Lung tissues from mice in each group were randomly selected and homogenized using a ball mill. The MDA content was then measured according to the instructions of the MDA detection kit.

[0041] Detection of lung GSH activity levels: Fresh lung tissue was randomly selected from each group of mice, and the GSH activity level was measured according to the instructions of the GSH detection kit. Based on the literature "Study on Lung Injury Model in Mice with Sepsis", HE staining results were observed under a microscope, and an overall score was calculated in conjunction with the opinions of a pathology professor. The core indicators were alveolar wall integrity, pulmonary edema, and inflammatory cell infiltration. Normal lung tissue was scored from 0 to 3 points, with 0-1 points indicating no damage and 2-3 points indicating varying degrees of damage.

[0042] See results Figure 3 In the figures, A represents HE staining results (scale bar: 50 μm); B represents histological scoring results; and C represents GSH and MDA content detection results. HE staining results showed that, compared with the control group, treatment with different doses of proanthocyanidin B2 resulted in clearer lung tissue structure, well-preserved alveolar cells, clear cytoplasm, and prominent nuclei and nucleoli. Compared with the control group, different doses of PB2 had no effect on GSH and MDH levels in lung tissue. These results indicate that PB2 does not affect the safety of mouse lung tissue.

[0043] 4. Effects of PB2 on pathological damage to lung tissue in mice induced by chronic cold exposure The test results for different groups of mice are shown in the figure. Figure 4 In the figure, A represents the HE staining result, with a scale bar of 50 μm; B represents the histological scoring result. To be considered statistically significant compared to the control group ( P <0.01), ## indicates a significant difference compared to the chronic cold exposure group ( P<0.01). Histological analysis showed that the lung tissue structure of the control group was good, with clear alveolar cell cytoplasm and prominent nuclei. However, significant structural disturbances in the lungs were observed in the chronic cold exposure group, such as hemorrhage, neutrophil infiltration, and alveolar cell necrosis. PB2 (50 mg / kg) treatment alleviated the pathological changes in the lungs of mice induced by chronic cold exposure, and PB2 (100 mg / kg) treatment had a more significant therapeutic effect on lung injury induced by chronic cold exposure in mice. These results clearly demonstrate that the therapeutic effect of PB2 treatment on lung injury induced by chronic cold exposure is dose-dependent.

[0044] 5. Effects of PB2 on ROS, MDA content and GSH activity in the lungs of chronically cold-exposed mice To determine the effect of chronic cold exposure on oxidative stress levels in mouse lungs, the content of ROS in mouse lung tissue was detected using immunofluorescence; the effect of chronic cold exposure on MDA content and GSH activity in mouse lungs was detected using ELISA.

[0045] After the cold exposure was completed, the mice were removed from the artificial climate chamber. The mice were euthanized by cervical dislocation, and the lungs were collected. One portion of the lungs was immediately flash-frozen in liquid nitrogen and then placed in 1.5 mL centrifuge tubes for subsequent ROS detection experiments; the other portion of the lungs was immediately flash-frozen in liquid nitrogen and stored at -80°C, labeled, and used for subsequent ELISA experiments.

[0046] Detection of lung ROS levels: (1) Preparation of frozen sections: Fresh lungs of mice in each group were taken and frozen sections were prepared.

[0047] (2) Fluorescent probe imaging Following the ROS kit instructions, observe the results using a fluorescence microscope after transfection.

[0048] Detection of lung MDA levels: Lung tissues from mice in each group were randomly selected and homogenized using a ball mill. The MDA content was then measured according to the instructions of the MDA detection kit.

[0049] Detection of lung GSH activity levels: Fresh lung tissue was randomly selected from each group of mice, and the GSH activity level was detected according to the instructions of the GSH detection kit. See results Figure 5 And Table 1, where A is the ROS fluorescence microscopy observation result, scale bar is 50μm; B is the ROS detection result; C is the MDA content detection result; D is the GSH activity detection result; To be considered statistically significant compared to the control group ( P<0.01), ## indicates a significant difference compared to the chronic cold exposure group ( P <0.01).

[0050] Table 1. Relative contents of different indicators in different groups of mice.

[0051] The results showed that the level of ROS in the lungs of mice in the chronic cold exposure group was significantly higher than that in the control group, while PB2 treatment significantly reduced the level of ROS in the lungs in a dose-dependent manner. Simultaneously, the level of MDA and the activity of GSH in the lungs of mice were detected. ELISA results showed that, compared with the control group, the content of MDA in the lungs of mice in the chronic cold exposure group was increased, while proanthocyanidin B2 treatment significantly reduced the content of MDA in the lungs in a dose-dependent manner. Conversely, the activity of GSH in the lungs of mice in the chronic cold exposure group was decreased, while proanthocyanidin B2 treatment significantly increased the activity of GSH in the lungs in a dose-dependent manner. These results further demonstrate that proanthocyanidin B2 can alleviate oxidative stress damage to the lungs of mice induced by chronic cold exposure and enhance the antioxidant capacity of the mouse lungs.

[0052] 6. The effect of PB2 on the expression levels of HSP60, HSP70, HSP90, CAT, GST, SOD-1 and HO-1 in the liver of chronically cold-exposed mice was detected by Western blot. After chronic cold exposure, the mice were removed from the climate chamber. The mice were euthanized by cervical dislocation, and their lungs were collected. A portion of the lungs was immediately flash-frozen in liquid nitrogen and stored at -80°C, clearly labeled, for subsequent Western blot experiments.

[0053] The specific steps for Western blot are as follows: Three mice were randomly selected from each group, and lung tissue was harvested and placed in centrifuge tubes. RIPA lysis buffer (containing 1% PMSF) was added to the centrifuge tubes, and the tissue was homogenized using a ball mill. The homogenate was centrifuged at 4℃ for 10 min, and the supernatant was collected. The protein concentration was determined by the BCA method (BCA kit purchased from Beyotime). Protein samples were dropped onto an SDS-PAGE gel for electrophoresis and then transferred to a PVDF membrane (10 μl loading, 2 μg / μl concentration). The membrane was blocked with 5% skim emulsion at room temperature for 1.5 h, followed by overnight incubation at 4°C with the following primary antibodies: HSP60 (1:2000; Proteintech), HSP70 (1:1000; Proteintech), and HSP90 (1:1000; Proteintech); CAT (1:2000; Proteintech), SOD-1 (1:2000; Proteintech), Nrf-2 (1:2000; Proteintech), HO-1 (1:2000; Proteintech), and β-actin (1:50000; Proteintech). The primary antibody was washed 5 times with TBST, 6 min each time. The cells were then incubated with HRP-labeled secondary antibody (1:10,000; Proteintech) at room temperature for 1 h, followed by washing with TBST 5 times, 6 min each time. Bands were visualized using an enhanced chemiluminescent substrate (Thermo Fischer Scientific) and imaged on a multifunctional imaging system. Image analysis was performed using Image Pro Plus 5.0 software.

[0054] See results Figure 6-7 And Table 2, where A represents the results of Western blot imaging, and B represents the relative expression levels of proteins. To be considered statistically significant compared to the control group ( P <0.01), ## indicates a significant difference compared to the chronic cold exposure group ( P <0.01).

[0055] Table 2. Relative contents of different indicators in different groups of mice.

[0056] Heat shock proteins are important indicators for determining whether an animal is under stress. Western blot was used to detect the expression levels of heat shock-related proteins in mouse lung tissue. The results showed that, compared with the control group, the expression levels of HSP60, HSP70, and HSP90 in the lungs of mice in the chronic cold exposure group were significantly increased, further confirming that cold exposure induces oxidative damage in lung tissue. PB2 treatment significantly reduced the expression levels of HSP60, HSP70, and HSP90 in the lungs in a dose-dependent manner, indicating that PB2 can inhibit stress induced by chronic cold exposure.

[0057] To further determine the effect of PB2 on the antioxidant capacity of the lungs of chronically cold-exposed mice, the protein expression levels of GSH, SOD-1, CAT, and HO-1 in mouse lung tissue were detected using Western blot. The results showed that, compared with the control group, the expression levels of GSH and SOD-1 in the lungs of mice in the chronically cold-exposed group were significantly reduced, and PB2 treatment significantly upregulated the expression levels of GSH and SOD-1 in the lungs in a dose-dependent manner. Compared with the control group, the expression levels of CAT and HO-1 in the lungs of mice in the chronically cold-exposed group were significantly upregulated, and PB2 treatment significantly downregulated the expression levels of CAT and HO-1 in the lungs in a dose-dependent manner.

[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of proanthocyanidin B2 in the preparation of products that alleviate lung damage in animals caused by chronic cold exposure.

2. The application according to claim 1, characterized in that, The methods for alleviating chronic cold exposure-induced lung damage in animals include: alleviating oxidative stress damage to the lungs and / or enhancing the antioxidant capacity of the lungs.

3. The application according to claim 2, characterized in that, The method for alleviating oxidative stress damage in animal lungs includes reducing the level of reactive oxygen species in the lungs, reducing the content of malondialdehyde in the lungs, reducing the expression level of heat shock protein 60 in the lungs, reducing the expression level of heat shock protein 70 in the lungs, and reducing the expression level of heat shock protein 90 in the lungs, or one or more of these methods.

4. The application according to claim 2, characterized in that, The enhancement of the antioxidant capacity of animal lungs includes one or more of the following: increasing the activity of glutathione in the lungs, upregulating the expression level of superoxide dismutase 1 in the lungs, downregulating the expression level of catalase in the lungs, and downregulating the expression level of heme oxygenase 1 in the lungs.

5. The application according to any one of claims 1 to 4, characterized in that, The animal in question is a mouse.

6. The application according to claim 1, characterized in that, The products include pharmaceuticals or feed additives.

7. A drug for alleviating lung damage in animals caused by chronic cold exposure, characterized in that, The active ingredient includes proanthocyanidin B2; the unit dose of the drug, based on mouse body weight, is 45-105 mg / kg body weight.

8. The medicament according to claim 7, characterized in that, The unit dose of the drug is 49-98 mg / kg body weight.

9. The drug according to claim 7 or 8, characterized in that, The drug also includes pharmaceutically acceptable excipients.

Images