Treatment effect of baicalein on acute lung injury induced by combination of S protein and LPS

Abstract

The invention belongs to the technical field of medicines, and discloses application of baicalein in drugs related to the treatment effect of S protein and LPS induced acute lung injury. Specifically, the invention discloses an application of baicalein in preparation of drugs playing a protective role in treating pulmonary edema, inflammatory cell infiltration and cytokine storm caused by acute lung injury induced by S protein combined with LPS. Pharmacological experiments prove that baicalein has an anti-inflammatory effect in treatment of acute lung injury induced by S protein combined with LPS, and can improve pulmonary edema and promote lesion lung tissue repair.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology. Specifically, it relates to the use of baicalein in the preparation of a therapeutic drug for acute lung injury pulmonary edema, inflammatory cell infiltration and cytokine storm induced by S protein combined with LPS. Background Art

[0002] After severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) invades the body, it causes novel coronavirus infection. According to the infection course, it is divided into the incubation period, the clinical symptom period and the recovery period. The clinical symptom period is mainly divided into the early stage, the middle stage and the severe stage. [1] When the virus invades the body, it enters the bronchi and alveoli through the upper respiratory tract. The body produces an autoimmune response, and macrophages produce cytokines and inflammatory chemokines to the infection site, inducing the activation of neutrophils and lymphocytes to phagocytose the virus. However, the excessive immune response leads to the overexpression of inflammatory factors in the body, resulting in a "cytokine storm", leading to diffuse alveolar damage, causing pneumonia and acute lung injury, and further causing multiple organ dysfunction throughout the body. This is also one of the main causes of death in severe patients. [2,3]

[0003] Lipopolysaccharide (LPS) is the main component of endotoxin. After invading the body, it releases a large number of inflammatory factors that damage alveolar epithelial cells, resulting in lung tissue damage and causing acute lung injury. LPS is a widely used modeling agent for preparing acute lung injury. [4,6]

[0004] SARS-CoV-2 has a Spike (S) protein layer, which is composed of S1 and S2 subunits. S1 mediates the adhesion of the virus to host cells, and S2 mediates the fusion of the virus with the host cell membrane. Therefore, the S protein is considered the main protein for the virus to infect the body. [7]

[0005] Studies have found that SARS-CoV-2 Spike RBD does not cause obvious pathological tissue changes in lung tissue. S-RBD promotes the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in mice in a Toll-like receptor 4-dependent manner. [8] ​​​Therefore, at present, the S protein combined with LPS is used to study acute lung injury after novel coronavirus infection. The research found that the combination of the S protein and LPS can exacerbate the degree of lung injury caused by LPS, aggravate pulmonary edema, cause infiltration of inflammatory cells in lung tissue, accompanied by diffuse congestion or hemorrhagic necrosis in some lung tissues, pulmonary interstitial fibrosis, increased expression of inflammatory factors, and exacerbate oxidative stress in lung tissue. [9,11] The research found that the SARS-CoV-2 S protein can bind to LPS, and the S protein can regulate the aggregation state of lipopolysaccharide and increase the inflammatory response in vivo and in vitro. [12,13] The S protein can significantly enhance the excessive inflammation induced by lipopolysaccharide in mice, increase pro-inflammatory cytokines and reduce the level of anti-inflammatory factors, exacerbate the damage of local inflammatory cell infiltration to the lungs and liver, and cause imbalance of L-tryptophan balance to exacerbate digestive symptoms.

[14] Therefore, the combination of the S protein and LPS is used to simulate acute lung injury induced by severe-stage novel coronavirus infection.

[0006] Baicalein is one of the flavonoid compounds in the roots of Scutellaria baicalensis, and has pharmacological effects such as anti-inflammatory, antibacterial, antiviral, antioxidant, and anti-tumor. Modern pharmacological research shows that baicalein can reduce lung injury and pulmonary edema caused by Streptococcus pneumoniae infection, reduce the level of inflammatory factors, and improve the survival rate of animal models.

[15] Baicalein can inhibit the thickening of the vascular wall and pulmonary tissue fibrosis in rats with pulmonary hypertension induced by monocrotaline.

[16] Chinese Patent CN 116327743 reported the application of a baicalein inhalation preparation in the treatment of LPS-induced acute lung injury.

[17] In summary, there has been no research report on the therapeutic effect of baicalein in the treatment of acute lung injury induced by the combination of the S protein and LPS so far. In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a new use of baicalein in the preparation for treating acute lung injury induced by the combination of the S protein and LPS.

[0008] One of the purposes of the present invention: to provide the use of baicalein in related drugs and other products for the treatment of acute lung injury induced by the combination of the S protein and LPS.

[0009] Another purpose of the present invention: to provide the use of baicalein in the treatment of symptoms such as inflammatory response and lung tissue lesions caused by acute lung injury induced by the combination of the S protein and LPS.

[0010] The third object of the present invention: Baicalein of the present invention can achieve the therapeutic effect on acute lung injury induced by S protein combined with LPS by reducing pulmonary edema and pulmonary hemorrhage caused by acute lung injury induced by S protein combined with LPS, restoring the cellular vacuolar state of damaged lung tissue, and promoting the repair of diseased lung tissue.

[0011] The fourth object of the present invention: Baicalein of the present invention can achieve an anti-inflammatory effect by reducing the number of neutrophils and monocytes in the blood and the expression levels of inflammatory factors such as IL-6, TNF-α, and chemokine ligand 1 (KC / GRO) in the serum, so as to achieve the therapeutic purpose of acute lung injury induced by S protein combined with LPS.

[0012] The present invention provides the use of baicalein in the treatment of acute lung injury induced by S protein combined with LPS. Male C57BL / 6J mice were anesthetized with isoflurane, and a mixed solution of S protein + LPS (0.1 mg / mL S protein solution, 0.04 mg / mL LPS solution) 50 μL was instilled into the trachea to establish an acute lung injury model. Mice in the LPS negative control group were instilled with LPS (0.04 mg / mL) solution 50 μL into the trachea, and mice in the S protein negative control group were instilled with S protein (0.1 mg / mL) solution 50 μL into the trachea. The normal group was given the same volume of normal saline. After injection, the mouse's nose was pinched for 5 - 10 s immediately and then put back into the cage. One hour after modeling, intragastric administration was carried out. Mice in the administration group were given different doses of baicalein (50 mg / kg, 100 mg / kg, 200 mg / kg), and mice in the other groups were given the same volume of 0.5% CMC-Na. Modeling and administration were continued for 3 days. Three days after drug treatment, blood was collected to detect the five-category count of white blood cells; the whole lungs of mice were weighed, and the lung index (%) = lung weight (g) / body weight (g) × 100%; a part of the left lung tissue of mice was fixed and sectioned, and stained with H&E to observe the pathological state; serum was taken to detect the expression level of inflammatory factors to judge the therapeutic effect of baicalein on acute lung injury induced by S protein combined with LPS.

[0013] The compounds of the present invention can be administered in unit dosage form, and the administration route can be enteral or parenteral, such as oral, intramuscular, subcutaneous, nasal, oral mucosa, skin, peritoneal or rectal, etc.

[0014] The dosage forms for administration can be liquid dosage forms and solid dosage forms. For example, the liquid dosage forms can be true solution types, colloid types, particulate dosage forms, emulsion dosage forms, suspension dosage forms. Other dosage forms such as tablets, capsules, dripping pills, aerosols, pills, powders, solution agents, suspensions, emulsions, granules, suppositories, freeze-dried powder injections, etc.

[0015] The compounds of the present invention can be made into conventional preparations, or can also be sustained-release preparations, controlled-release preparations, targeted preparations and various particulate drug delivery systems.

[0016] Beneficial technical effects: Baicalein described in the present invention has prominent pharmacological activities. By improving pulmonary edema, reducing inflammatory cell infiltration in lung tissue, and promoting the repair of diseased lung tissue, it realizes the therapeutic effect on acute lung injury; by increasing the percentage of lymphocytes in the blood, reducing the percentages of monocytes and neutrophils in the blood, and decreasing the expression levels of inflammatory factors such as IL-6, KC / GRO, and TNF-α, it realizes the anti-inflammatory effect. Therefore, it is applicable to the preparation of drugs for preventing, alleviating, and / or treating acute lung injury induced by S protein combined with LPS, as well as acute lung injury pulmonary edema, inflammatory cell infiltration, and cytokine storm. Description of the Drawings

[0017] Figure 1 Effect of baicalein on the lung index of mice with acute lung injury induced by S protein combined with LPS;

[0018] Figure 2 Effect of baicalein on the damaged lung tissue of mice with acute lung injury induced by S protein combined with LPS;

[0019] Figure 3 Effect of baicalein on the five-category white blood cell count of mice with acute lung injury induced by S protein combined with LPS;

[0020] Figure 4 Effect of baicalein on serum inflammatory cytokines TNF-α, IL-6, and KC / GRO in mice with acute lung injury induced by S protein combined with LPS; Detailed Embodiments

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the drawings.

[0022] Example 1

[0023] Effect of baicalein on the lung index of mice with acute lung injury induced by S protein combined with LPS

[0024] 1. Experimental method

[0025] In Example 1, 63 male SPF-grade C57BL / 6J mice weighing 18 - 20 g were randomly divided into 7 groups, with 9 mice in each group, and were respectively set as the normal group, S protein + LPS group, S protein group, LPS group, and three low, medium, and high-dose groups of S protein + LPS + baicalein (50 mg / kg, 100 mg / kg, 200 mg / kg). The corresponding doses of drugs were administered by gavage for 3 consecutive days. The whole lungs of mice in each group were taken, rinsed with physiological saline to remove excess blood, and then the excess moisture was blotted with filter paper and weighed, recorded as the lung weight. The lung index (%) = lung weight (g) / body weight (g) × 100%.

[0026] 2. Experimental results

[0027] The lung index can evaluate the severity of pulmonary edema. Compared with the mice in the blank group, the lung index of the mice in the model group increased significantly (P < 0.001), indicating that obvious pulmonary edema occurred in the mice in the model group. Compared with the model group, baicalein at 100 mg / kg significantly decreased the lung index (P < 0.05) and improved pulmonary edema( Figure 1 ). The lung indices of the animals in each group are shown in Table 1.

[0028] Table 1 Effects of baicalein on the lung index of mice with acute lung injury induced by S protein combined with LPS (Mean±SD, n = 6)

[0029]

[0030] Mean±SD, n = 6; # P < 0.05, ### P < 0.001 vs normal group, * P < 0.05, *** P < 0.001 vs S protein + LPS model group

[0031] Example 2

[0032] Effects of baicalein on the damaged lung tissue of mice with acute lung injury induced by S protein combined with LPS

[0033] 1. Experimental method

[0034] For the animals in the example, 63 male SPF-grade C57BL / 6J mice with a body weight of 18 - 20 g were randomly divided into 7 groups with 9 mice in each group, and were respectively set as the normal group, S protein + LPS group, S protein group, LPS group, and low, medium, and high dose groups of S protein + LPS + baicalein (50 mg / kg, 100 mg / kg, 200 mg / kg). The corresponding dose of the drug was administered by gavage for 3 consecutive days. The left lungs of the mice in each group were partially dissected, fixed with fixative, dehydrated, paraffin-embedded and sectioned, and stained with H&E.

[0035] 2. Experimental results

[0036] Compared with the normal group, the alveolar structure in the model group was significantly absent, with alveolar hemorrhage, pulmonary interstitial edema, and massive infiltration of inflammatory cells. Baicalein at 50 mg / kg, 100 mg / kg, and 200 mg / kg could significantly improve the pathological damage of the lung tissue and reduce the degree of lung hemorrhage and pulmonary edema( Figure 2 ).

[0037] Example 3

[0038] Effects of baicalein on the five-category white blood cell count of mice with acute lung injury induced by S protein combined with LPS 1. Experimental method

[0039] In the examples, 63 male SPF-grade C57BL / 6J mice weighing 18 - 20 g were selected and randomly divided into 7 groups with 9 mice in each group, which were set as the normal group, S protein + LPS group, S protein group, LPS group, and low, medium, and high-dose groups of S protein + LPS + baicalein (50 mg / kg, 100 mg / kg, 200 mg / kg). The corresponding doses of drugs were administered by gavage for 3 consecutive days, and then blood was collected. 20 μL of blood was placed in a 10 ml plastic hose containing blood cell diluent, and a blood routine analyzer was used to detect the percentages of lymphocytes, neutrophils, and monocytes in the capillary blood of the mice.

[0040] 2. Experimental results

[0041] Compared with the normal group, after the first day of modeling in the model group, the capillary blood of the mice was continuously detected for three days. It was found that the percentage of lymphocytes decreased significantly from the first day to the third day (all P < 0.001), the percentage of neutrophils increased significantly (all P < 0.001), and the percentage of monocytes increased significantly (P < 0.05, P < 0.001, P < 0.001), indicating that the mice in the model group had inflammation and the body's immune function decreased. Compared with the model group, there were no significant differences in each drug administration group after the first and second days of drug administration. On the third day of drug administration, baicalein at 50 mg / kg and 200 mg / kg could significantly increase the percentage of lymphocytes (all P < 0.001), and baicalein at 50 mg / kg, 100 mg / kg, and 200 mg / kg could significantly decrease the percentages of neutrophils and monocytes (all P < 0.001), reducing inflammation and regulating immune cells ( Figure 3 ). The percentages of lymphocytes, neutrophils, and monocytes in each group of animals are shown in Tables 2, 3, and 4.

[0042] Table 2 Effects of baicalein on the percentage of lymphocytes in mice with acute lung injury induced by S protein combined with LPS (Mean ± SD, n = 6)

[0043]

[0044]

[0045] Mean ± SD, n = 6; # P < 0.05, ### P < 0.001 vs normal group, *** P < 0.001 vs S protein + LPS model group

[0046] Table 3 Effects of baicalein on the percentage of neutrophils in mice with acute lung injury induced by S protein combined with LPS (Mean ± SD, n = 6)

[0047]

[0048] Mean±SD, n = 6; # P < 0.05, ## P < 0.01, ### P < 0.001 vs normal group, *** P < 0.001 vs S protein + LPS model

[0049] Group

[0050] Table 4 Effects of baicalein on the percentage of monocytes in mice with acute lung injury induced by S protein combined with LPS (Mean±SD, n = 6)

[0051]

[0052] Mean±SD, n = 6; # P < 0.05, ### P < 0.001 vs normal group, *** P < 0.001 vs Example 4 of S protein + LPS model group

[0053] Effects of baicalein on serum inflammatory cytokines TNF-α, IL-6, and KC / GRO in mice with acute lung injury induced by S protein combined with LPS

[0054] 1. Experimental method

[0055] In Example 4, 63 male SPF-grade C57BL / 6J mice, weighing 18 - 20 g, were randomly divided into 7 groups with 9 mice in each group, and were respectively set as normal group, S protein + LPS group, S protein group, LPS group, and three low, medium, and high dose groups of S protein + LPS + baicalein (50 mg / kg, 100 mg / kg, 200 mg / kg). The corresponding dose of the drug was given by gavage for 3 consecutive days, and then blood was collected. The blood was aliquoted into 1.5 mL EP tubes, placed at room temperature for 2 h, and then put into a pre-cooled 4°C low-temperature centrifuge and centrifuged at 3000 rpm for 15 min. 25 μL of serum was added to the sample diluent and diluted twofold, and the MUTI-SPOT Assay System was used to detect the expression levels of inflammatory factors. MUTI-SPOT Assay System to detect the expression levels of inflammatory factors.

[0056] 2. Experimental results

[0057] Compared with the normal group, the expression levels of IL-6, KC / GRO, and TNF-α in the model group mice were significantly increased (all P<0.001), indicating that an inflammatory response occurred in the model. Compared with the model group, baicalein at 50 mg / kg could significantly reduce the expression levels of IL-6, KC / GRO, and TNF-α (P<0.001, P<0.5, P<0.01), baicalein at 100 mg / kg significantly reduced the expression levels of IL-6, KC / GRO, and TNF-α (P<0.001, P<0.01, P<0.01), and baicalein at 200 mg / kg significantly reduced the expression levels of IL-6 and KC / GRO (both P<0.05), alleviating lung inflammation( Figure 4 ). The expression levels of IL-6, KC / GRO, and TNF-α in animals of each group are shown in Table 5.

[0058] Table 5 Effects of baicalein on the expression levels of IL-6, KC / GRO, and TNF-α in mice with acute lung injury induced by S protein combined with LPS (Mean±SD, n=6)

[0059]

[0060] Mean±SD, n=6; # P<0.05, ### P<0.001 vs normal group, * P<0.05, ** P<0.01, *** P<0.001 vs S protein

[0061] +LPS model group

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[0086]

[17] Chinese Patent CN 116327743.

Claims

1. Use of baicalein in the preparation of a drug for treating acute lung injury and its complications induced by S protein combined with LPS.

2. The application according to claim 1, wherein Baicalein can achieve the therapeutic effect on acute lung injury by promoting the repair of the diseased lung tissue.

3. The application according to claim 1, characterized in that, Baicalein achieves the therapeutic effect on acute lung injury through its anti-inflammatory effect.

4. The application according to claim 3, characterized in that Baicalein achieves its anti-inflammatory effect by increasing the percentage of lymphocytes in the blood and decreasing the percentages of monocytes and neutrophils in the blood.

5. The application according to claim 3, characterized in that, Baicalein achieves its anti-inflammatory effect by reducing the levels of IL-6, KC / GRO, and TNF-α.

6. The application according to any one of claims 1 to 5, characterized in that, The single-dose application of baicalein for treating body injuries in adults is 1 - 1000 mg / kg, preferably 5 - 500 mg / kg.

7. Use of a pharmaceutical composition in the preparation of a medicament for treating acute lung injury induced by the combination of S protein and LPS, characterized in that, The pharmaceutical composition comprises baicalein and a pharmaceutically acceptable carrier or excipient.

8. The application according to claim 7, characterized in that, The pharmaceutical composition is selected from: oral preparations, injectable dosage forms, or dosage forms for administration via the skin and mucosa.

9. The application according to claim 7, characterized in that, The oral preparations include tablets, sustained-release agents, capsules, controlled-release agents, enteric-coated agents, dripping pills, or liquid preparations, and the injectable dosage forms include intramuscular injection, intravenous injection, or intravenous drip.

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