Use of astilbe in preparation of drugs for preventing and treating acute lung injury

Abstract

The application discloses an application of Rosa roxburghii in preparation of a medicine for preventing and treating acute lung injury and belongs to the field of biological pharmacy. The water extract of the Rosa roxburghii is prepared, the intervention effect on a mouse model of acute lung injury is determined, the Rosa roxburghii is confirmed to have the effect of inhibiting acute lung injury, a new medicine use of the water extract of the Rosa roxburghii is provided, and the application has important clinical significance.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to the use of astilbe in the preparation of drugs for the prevention and treatment of acute lung injury. Background Technology

[0002] Acute lung injury (ALI) is a respiratory emergency characterized by acute hypoxic insufficiency or respiratory failure, increased alveolar permeability, and severe alveolar edema. In severe cases, it can lead to acute respiratory distress syndrome (ARDS), which is characterized by severe lung inflammation, alveolar-capillary barrier disruption, and pulmonary edema. ALI can lead to the development of chronic diseases, such as idiopathic pulmonary fibrosis (IPF) [Kligerman S. Pathogenesis, Imaging, and Evolution of Acute Lung Injury. Radiol Clin North Am. 2022 Nov; 60(6): 925-939.], chronic obstructive pulmonary disease (COPD), asthma, and lung cancer [Ayilya BL, Balde A, Ramya M, Benjakul S, Kim SK, Nazeer RA. Insights on the mechanism of bleomycin to induce lung injury and associated in vivo models: A review. Int Immunopharmacol. 2023 Aug; 121: 110493.]. ALI is a common acute and critical illness in clinical practice, with a mortality rate of up to 40%. However, there is currently no specific treatment strategy for ALI in clinical practice.

[0003] Astilbe, scientifically known as Astilbe chinensis (Maxim.) Franch. et Savat., belongs to the genus Astilbe in the family Saxifragaceae. In Zhejiang, Astilbe is a commonly used medicinal material with the effects of clearing heat and relieving exterior syndromes, relieving cough, eliminating phlegm, and relieving asthma. It is widely used in the treatment of colds, coughs, and other diseases. Astilbe contains a variety of active ingredients, such as bergenin, which exhibit various pharmacological effects including anti-inflammatory, antibacterial, antiviral, expectorant, and antitussive properties [Tang Q, Wang Q, Sun Z, et al. Bergenin Monohydrate Attenuates Inflammatory Response via MAPK and NF-κB Pathways Against Klebsiella pneumonia Infection. Front Pharmacol. 2021 May 4; 12: 651-664.][Yang L, Zheng Y, Miao YM, et al. Bergenin, a PPARγagonist, inhibits Th17 differentiation and subsequent neutrophilic asthma by preventing GLS1-dependent glutaminolysis. Acta Pharmacol Sin. 2022 Apr; 43(4): 963-976.]. These mechanisms of action provide theoretical support for the use of astilbe in the treatment of pulmonary inflammation. Pharmacodynamic tests showed that the aqueous extract of Astilbe significantly inhibited ear swelling in mice and counteracted carrageenan-induced paw edema in mice [Jiang Shipeng, Lai Pingfan, Yang Yiling. Experimental study on the antipyretic, anti-inflammatory, antibacterial and anti-endotoxin effects of Astilbe root [J]. Chinese Medicinal Herbs, 2007, (01): 77-80.], indicating that it has significant anti-inflammatory effects. In addition, the Chinese Pharmacopoeia points out that bergenin in Astilbe is an effective drug for treating bronchitis. Experimental studies have confirmed that Astilbe can significantly prolong the cough latency period in mice, reduce the number of coughs, and increase the excretion of phenol red, indicating that it has good expectorant and antitussive effects [Lai Pingfan, Jiang Shipeng, Yang Yiling. Study on the analgesic, blood-activating, antitussive and expectorant effects of Astilbe root. Chinese Journal of Traditional Chinese Medicine. 24 (2006): 2061-2064.]. This has a significant effect on relieving symptoms such as cough and phlegm caused by lung inflammation. In summary, astilbe has anti-inflammatory, analgesic, antitussive, expectorant, and bronchitis-treating effects, but there are no reports on the effects of astilbe granules on acute lung injury and lung inflammation, and no studies on the mechanism of action of astilbe in the direct treatment of ALI have been reported. Summary of the Invention

[0004] Based on the deficiencies in the prior art, the purpose of this invention is to provide a new pharmaceutical use of astilbe in the treatment, relief or prevention of ALI, namely, the use of astilbe in the preparation of a medicament for the prevention and treatment of acute lung injury.

[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0006] This invention provides the use of astilbe in the preparation of a medicament for the prevention and treatment of acute lung injury.

[0007] Specifically, the astilbe is an aqueous extract of astilbe, and the preparation method is as follows: astilbe is boiled in water to obtain the aqueous extract of astilbe.

[0008] Preferably, each milliliter of the astilbe water extract contains 5g of raw herb.

[0009] In some embodiments of the present invention, the dosage form of the drug is a liquid, powder for injection, tablet, capsule, powder, pill, ointment, granule or dressing.

[0010] Preferably, when the drug is in liquid form, the dosage is 1.56–15.6 g / kg. The drug is administered continuously for 7 days.

[0011] Mice were randomly divided into a normal group, a model group, a dexamethasone group (DXM), a high-dose astilbe group (LXF H), a medium-dose astilbe group (LXF M), and a low-dose astilbe group (LXF L). The treatment groups were given prophylactic medication for 7 consecutive days. After 7 days of administration, the model group and the four treatment groups were induced by intratracheal infusion of LPS.

[0012] After LPS modeling in mice, the mice were anesthetized, intubated, and placed in the chamber of a forced pulmonary function instrument for lung function testing. Astilbe can reduce functional residual capacity (FRC) in ALI mice and alleviate lung function damage.

[0013] Immediately after mouse sacrifice, lung tissue was extracted and cultured in 10% serum to form a single-cell suspension. Macrophage polarization in the lung tissue cells was then examined. High doses of astilbe significantly increased the proportion of M2 cells in the lung tissue of ALI mice, promoting lung injury repair.

[0014] After mice were euthanized, a portion of lung tissue was collected and mixed with PBS to prepare a 10% homogenate. The supernatant was then centrifuged, and the levels of inflammatory factors IL-6, IL-1β, and TNFα were measured. Low and medium doses of astilbe significantly reduced the levels of inflammatory factors in the lung tissue homogenate.

[0015] Partial lung tissue samples were fixed in neutral formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The pathological morphological changes in the lung tissue of mice in each group were observed. High-dose and medium-dose astilbene treatments improved bronchial inflammatory cell infiltration, reduced airway wall thickness, and alleviated airway inflammation in ALI mice.

[0016] After partial lung tissue was harvested, it was rapidly frozen in liquid nitrogen. Total RNA was extracted from the lung tissue using a rapid RNA extraction kit, and then reverse transcribed into cDNA. The relative expression levels of CCR1 and CCR5 target genes in the chemokine signaling pathway were analyzed using qRT-PCR. GAPDH was selected as an endogenous control. All doses of astilbe significantly reduced the relative expression levels of CCR1 and CCR5 genes in the lung tissue of ALI mice.

[0017] Animal model studies have found that astilbe can significantly increase the M2 ratio, reduce the FRC index of forced lung function in animals, alleviate pathological changes of lung inflammation, reduce inflammatory factors TNF-α and IL-6 in lung tissue, and reduce the expression of CCR1 and CCR5 genes.

[0018] The present invention also provides a drug for preventing and treating acute lung injury, wherein the drug is astilbe itself or an aqueous extract of astilbe, and a pharmaceutically acceptable salt.

[0019] Preferably, the drug is a liquid. The dosage of the drug is 1.56–15.6 g / kg. The drug is administered continuously for 7 days.

[0020] The beneficial effects of this invention are:

[0021] This invention prepares an aqueous extract of astilbe and, through intervention in an ALI mouse model, clarifies that astilbe has an inhibitory effect on ALI, providing a new pharmaceutical use for the aqueous extract of astilbe and having significant clinical implications. Attached Figure Description

[0022] Figure 1 The effect of Astilbene on lung function in ALI mice. Compared with the normal group, "Δ" p < 0.05, "ΔΔ" p < 0.01; compared with the model group, "*" p < 0.05.

[0023] Figure 2 The effects of Astilbene on inflammatory factors (IL-6 and TNFα) in lung tissue of ALI mice were investigated. Compared with the normal group, "Δ" p < 0.05, "ΔΔ" p < 0.01; compared with the model group, "*" p < 0.05.

[0024] Figure 3The effect of astilbene on the histopathological changes of lung airway tissue in ALI mice was investigated. A represents airway inflammation score; B represents airway wall thickness (μm); C represents the normal group, D the model group, E the dexamethasone group, F the high-dose astilbene group, G the medium-dose astilbene group, and H the low-dose astilbene group. Scale bar: 100 μm. Compared with the normal group, "Δ" p < 0.05, "ΔΔ" p < 0.01; compared with the model group, "*" p < 0.05, "**" p < 0.01.

[0025] Figure 4 The effect of Astilbene on macrophage polarization in the lung tissue of ALI mice. "ΔΔ" indicates p < 0.01 compared to the normal group; "**" indicates p < 0.01 compared to the model group.

[0026] Figure 5 The effect of Astilbene on the expression levels of CCR1 and CCR5 genes in the lung tissue of ALI mice. Compared with the normal group, "Δ" p < 0.05, "ΔΔ" p < 0.01; compared with the model group, "*" p < 0.05, "**" p < 0.01. Detailed Implementation

[0027] Example 1: Preparation method of astilbe drug

[0028] Take 5000g of raw herbs and crush them. Add 5 times the amount of water to the herbs and soak for 30 minutes. Decoction at 100℃ for 1 hour. Add water and decoct again. Filter the decoction to obtain filtrate. Concentrate the filtrate to a clear extract to obtain an aqueous extract containing 5g of raw herbs per milliliter.

[0029] Example 2: Astilbene treatment of acute lung injury in mice

[0030] 1 Experimental Methods

[0031] (1) Grouping of animals

[0032] Mice were randomly divided into a normal group, a model group, a dexamethasone group (DXM), a high-dose astilbe group (LXF H), a medium-dose astilbe group (LXF M), and a low-dose astilbe group (LXF L), with 10 mice in each group.

[0033] (2) Animal modeling and drug dosage

[0034] LPS (lipopolysaccharide) modeling: 4 mg / kg LPS was instilled into the trachea of ​​mice, with an LPS concentration of 4 mg / ml.

[0035] Dosage: Dexamethasone is administered intraperitoneally at a dose of 5 mg / kg; astilbe is administered by gavage at a dose of 1.56 g / kg (low dose), 7.8 g / kg (5 times the clinical dose), and 15.6 g / kg (10 times the clinical dose).

[0036] (3) Animal modeling and drug administration

[0037] Except for the normal group and the model group, mice were given prophylactic medication for 7 days. After 7 days of medication, the model group and the four medication groups were induced by tracheal infusion with LPS.

[0038] (4) Forced lung function test in mice

[0039] After LPS modeling in mice, they were anesthetized with 1% sodium pentobarbital at a dose of 50 mg / kg, endotracheal intubation was performed, and the mice were placed in the cavity of a forced pulmonary function instrument for pulmonary function testing.

[0040] (5) Flow cytometry detection of macrophage polarization in lung tissue

[0041] Immediately after mouse sacrifice, a portion of lung tissue was extracted and added to 10% serum culture medium to prepare a single-cell suspension. The single-cell suspension was centrifuged (300g / 5min), the supernatant was discarded, and the cells were washed 1-2 times with a certain amount of PBS, the supernatant was discarded, the cells were dispersed, and the cells were resuspended at 5×10⁶ cells / mL. 6 -10×10 6 Between. Resuspend cells in 500 μL of PBS in EP tubes or flow cytometry tubes, taking 100 μL from each tube, and add 5 μL of surface staining antibody to each. Add CD11b, CD86, CD206, and F4-80 antibodies respectively, mix well, and detect macrophage polarization in lung tissue cells.

[0042] (6) ELISA detection of inflammatory factors in lung tissue

[0043] After the mice were euthanized, a portion of lung tissue was taken and mixed with PBS to prepare a 10% homogenate. The supernatant was collected by centrifugation and the levels of IL-6, IL-1β, and TNFα inflammatory factors were detected.

[0044] (7) Pathological morphological changes in lung tissue

[0045] Partial lung tissue was fixed in neutral formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and the pathological morphological changes of lung tissue in each group of mice were observed.

[0046] (8) Target gene expression in the lung tissue chemokine signaling pathway

[0047] After partial lung tissue harvesting, it was rapidly frozen in liquid nitrogen. Total RNA was extracted from the lung tissue using a rapid RNA extraction kit, and then reverse transcribed into cDNA. The relative expression levels of CCR1 and CCR5 target genes in the chemokine signaling pathway were analyzed using qRT-PCR. GAPDH was selected as an endogenous reference. Primer sequences for the target genes are shown in Table 1. The relative expression levels of the target genes were calculated using the 2-Δct method.

[0048] Table 1 Primer sequences

[0049]

[0050]

[0051] 2-head test results

[0052] (1) Effects of astilbe on lung function in ALI mice: After LPS modeling, the forced vital capacity (FVC) of ALI mice was significantly reduced (p < 0.05), while the functional residual capacity (FRC) was significantly increased (p < 0.05). The positive control group, high-dose astilbe, and medium-dose groups significantly reduced FRC (p < 0.05). The results are shown in the table below. Figure 1 .

[0053] (2) Effects of astilbe on inflammatory factors in lung tissue of ALI mice: After LPS modeling, the levels of inflammatory factors IL-6 and TNFα in lung tissue homogenates of ALI mice were significantly increased. Medium and low doses of astilbe significantly reduced the content of inflammatory factors in lung tissue homogenates (p < 0.05). The results are shown in the table below. Figure 2 .

[0054] (3) Pathological morphological observation of astilbe in the lung tissue of ALI mice: After LPS modeling, the lung tissue of ALI mice showed significant peribronchial inflammatory cell infiltration and significant airway wall thickening. High-dose and medium-dose astilbe in the lungs of ALI mice could improve bronchial inflammatory cell infiltration, reduce airway wall thickness, and alleviate airway inflammation (p<0.05). The results are shown in the table below. Figure 3 .

[0055] (4) Effects of astilbe on macrophage polarization in the lung tissue of ALI mice: After LPS modeling, M2 cells in ALI mice were significantly reduced (p < 0.01), while M1 cells increased, but there was no significant difference between the model group and the normal group. High doses of astilbe significantly increased the proportion of M2 cells (p < 0.01), as shown in the following figures. Figure 4 .

[0056] (5) Effects of astilbe on genes related to the chemokine signaling pathway in lung tissue: Results are as follows Figure 5As shown, administration of astilbe significantly reduced the gene expression levels of chemokine receptors CCR1 and CCR5 (p < 0.01).

[0057] 3. Conclusion

[0058] Astilbe can protect lung function in mice with acute lung injury, reduce the levels of inflammatory factors IL-6 and TNFα in the lung tissue of mice with acute lung injury, promote the differentiation ratio of M2 anti-inflammatory macrophages in lung tissue, alleviate inflammatory cell infiltration in the pathological structure of lung tissue, and reduce airway wall thickness. Astilbe can significantly reduce the gene expression levels of chemokines CCR1 and CCR5, suggesting that the therapeutic effect of astilbe on acute lung injury may be through chemokine receptors acting on the chemokine signaling pathway, regulating the chemotaxis of inflammatory cells, and thus alleviating lung inflammation.

Claims

1. The application of astilbe as the sole active ingredient in the preparation of a drug for the prevention of acute lung injury, wherein the astilbe is an aqueous extract of astilbe, and the preparation method is as follows: astilbe is boiled in water to obtain the aqueous extract of astilbe.

2. The application as described in claim 1, characterized in that, Each milliliter of the astilbe water extract contains 5g of raw herb.

3. The application as described in claim 1, characterized in that, The dosage form of the drug is tablets, capsules, powders, pills, ointments, or granules.

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